They contrast markedly with the mean estimate of / 0.02 with an upper bound of 0.06 obtained for the non-IHR group . Recombination in clade A. As noted earlier, Nunney et al. proposed that the two clades A and B in the X. fastidiosa subsp. multiplex tree of Parker et al. corresponded to isolates that had been subject to IHR versus those largely free of IHR . However, this hypothesis is undermined by the absence of evidence of intersubspecific introgression from X. fastidiosa subsp. fastidiosa to X. fastidiosa subsp. multiplex in the analysis of clade A by Parker et al. . However, in our reanalysis of these data using the targeted introgression test , we found that out of the 9 loci, 6 showed statistically significant evidence of IHR in alleles found in the clade A isolates . Furthermore, in this reanalysis, all of the 6 clade A genotypes carried recombinant alleles in at least 4 of these loci, while none of the 9 clade B genotypes showed any similar evidence of IHR. As noted previously, the 6 recombinant group isolates common to both studies were all classified into group A . They encompassed 4 of the genotypes identified by Parker et al. : Almond2 , the Blueberry1 group , the Blueberry2 group , 25 liter pot plastic and the Ragweed group . They showed significant evidence of IHR at 4 or 5 of the 9 loci , so that in total they showed significant evidence of IHR in 6 or 7 out of the 17 genes examined.
Plant hosts of the recombinant group. Given the identification of a discrete recombinant subgroup within X. fastidiosa subsp. multiplex, we investigated the possibility that it corresponded with a shift in the plant hosts that were infected. Among the 33 isolates defining 9 STs, there were 5 plant hosts represented more than once: ST27 on almond , ST27 and ST40 on purple leaf plum , ST22, ST28, ST42, and ST58 on giant ragweed , ST32 on blackberry , and ST42 and ST43 on blueberry , to which we can add American elm by including the 4 additional clade A isolates from Parker et al. . Closer examination of X. fastidiosa subsp. multiplex isolates from these host plants, using the 143 isolates subjected to MLST , showed that almond and purple leaf plum isolates were recombinant types only about one-quarter of the time . The ratios in American elm from Washington, DC , and western ragweed from Texas were substantially higher , but it was only in blackberry , and blueberry that 100% of the isolates were recombinant. While the sample size for blackberry was very small, the isolates were geographically separate and defined a single sequence type that was found on no other host. ST32 differed from all other STs in the recombinant group, except ST41, in carrying the nonrecombinant pilU3 allele . Blueberry isolates were better represented and again were isolated from two different states, Florida and Georgia. The 7 isolates that we typed defined two STs: ST43, which was found in both Florida and Georgia and was unique to these blueberry isolates, and ST42, which was isolated in Georgia on blueberry but also in Texas on western ragweed .
Analysis of the recombinant group ofX. fastidiosa subsp. multiplex showed three important results. First, intersubspecific recombination was shown to have occurred in 50% of 8 loci scatThered throughout the genome that were chosen independently of the data . Second, it was shown that the donor of the introgressed sequence was X. fastidiosa subsp. fastidiosa, a subspecies introduced from Central America into the United States as a single strain . However, the introgressed sequence at two of the loci did not come from any of the X. fastidiosa subsp. fastidiosa genotypes that have been found in the United States. This result suggests that another introduction of X. fastidiosa subsp. fastidiosa must have occurred, an introduction that resulted in successful IHR, after which the donor genotype seems to have disappeared. This involvement of an unexpected X. fastidiosa subsp. fastidiosa strain supports the hypothesis that the members of the recombinant group share a single ancestral IHR event. Third, the hypothesis that IHR has facilitated a shift to new hosts is strongly supported by the example of blueberry, where 10 isolates have been typed and potentially supportedby the example of blackberry . A link between the shift to a novel plant host and homologous recombination has not been previously identified. Of course, the direct causation of this link can never be proved without knowledge of the genetic changes driving this shift. It can always be argued that the link is fortuitous and that one or more point mutations in the nonrecombined X. fastidiosa subsp. multiplex genome are causal in the host shift. First, both blueberry and blackberry are native to the United States, so if only a simple genetic change was required to infect these species, why did the native nonrecombinant X. fastidiosa subsp. multiplex apparently never acquire these changes? Second, a similar but even more extensive mixing of the genomes of X. fastidiosa subspp. fastidiosa and multiplex is found in the only form of X. fastidiosa that infects another U.S. native plant, mulberry . Furthermore, in other bacterial species, it has been demonstrated that recombination can drive rapid evolution, both in the laboratory and, in the case of Helicobacter pylori, in mice .
Similarly, McCarthy et al. concluded that lineages of Campylobacter jejuni in chickens versus cattle and sheep were able to shift host type, because rapid adaptation was facilitated by recombination with the resident host population. In the study by Nunney et al. , it was shown that the recombinant genotypes formed a well-defined group , demonstrating that intersubspecific homologous recombination was not randomly distributed across the X. fastidiosa subsp. multiplex isolates. This work was based on a survey of 143X. fastidiosa subsp. multiplex isolates using just 8 loci. There were 33 isolates that showed some evidence of IHR in at least 1 locus: all but 2 showed statistically significant evidence in at least 2 loci, while the remaining 110 showed no such evidence . The generality of this discrete group of recombinant forms was supported by our analysis presented here of the sequence data from 9 more loci sequenced by Parker et al. . These loci divided isolates into 2 groups that appeared to correspond to the recombinant and non-IHR groups, respectively , even though Parker et al. found no evidence of IHR. Upon reanalysis, we found statistically significant IHR in 6 of the 9 loci in the clade A data but no evidence of IHR in the clade B data. Clade A included 6 isolates that we had typed in the present study, and each of these showed evidence of IHR in 4 or 5 of the additional 9 loci. Thus, 25 litre plant pot in two independent samplings that together examined 17 loci, there was clear evidence of substantial genomewide IHR in the recombinant group iso-lates, amounting to 50% of the genes showing IHR across the MLST locis plus the pilU locus . The average was higher when based on the loci sequenced by Parker et al. ; however, this was probably biased upwards by the manner in which the loci were chosen . None of the IHR events in 6 of the 9 loci identified using the targeted introgression test, or in the case of complete introgression, a chi-square test, were detected by Parker et al. using PHI and the 9 tests implemented in RDP . This failure of the standard tests of recombination to detect IHR was previously noted by Nunney et al. , motivating the development of their introgression test. We examined the hypothesis that the recombinant group STs were derived from a single IHR event involving a X. fastidiosa subsp. multiplex recipient and an X. fastidiosa subsp. fastidiosa donor. The distribution of allelic differences among the recombinant STs was consistent with them all being derived from a single initial event, but a small number of other intersubspecific and intrasubspecific recombination events would also be needed . More importantly, the genotypes seen in the recombinant group can be accounted for entirely, or very nearly so, based on a single X. fastidiosa subsp. fastidiosa donor genotype. For example, the substantial variation in cysG can all be accounted for by an ancestral introgression of X. fastidiosa subsp. fastidiosa allele 12 followed by subsequent intrasubspecific recombination of X. fastidiosa subsp. multiplex sequence to form the other two alleles . In contrast, variation at pilU could be accounted for by a second donor contributing the X. fastidiosa subsp. fastidiosa pilU9 allele, but it could also have arisen by a single mutation in pilU1 unique to the recombinant group.
A possible single X. fastidiosa subsp. multiplex recipient genotype was also identified . This genotype is consistent with a known ST: setting cysG to allele 3 makes the recipient identical to ST45, which was sampled from the states of California, Kentucky, and Texas . Elsewhere, we consider a slightly different hypothesis regarding the origin of the recombinant group in which the donor and recipient subspecies are reversed—i.e., that it was derived from a single IHR event, but involving an X. fastidiosa subsp. multiplex donor and an X. fastidiosa subsp. fastidiosa recipient; however, apart from the role reversal, the conclusions are unalThered . The ancestral reconstruction allows us to consider the second question posed earlier: is the donor consistent with the X. fastidiosa subsp. fastidiosa genotypes found in the United States? The answer is very clearly “no.” The original donor carried cysG12 and holC19 . These alleles are both found in isolates from Central America, but no X. fastidiosa subsp. fastidiosa isolate found in the United States comes close to matching this criterion: the most similar U.S. ST has a 12-bp mismatch. There has been extensive sampling of X. fastidiosa subsp. fastidiosa within the United States, based on 85 isolates sampled across the United States from 15 different host plants . There is very little variation within X. fastidiosa subsp. fastidiosa isolates from the United States, consistent with the hypothesis that all X. fastidiosa subsp. fastidiosa isolates currently found in the United States are derived from a single strain introduced from Central America . Based on these data, we conclude that the X. fastidiosa subsp. fastidiosa donor was introduced into the United States from Central America and recombined with a native X. fastidiosa subsp. multiplex genotype similar to ST45; however, this donor lineage of X. fastidiosa subsp. fastidiosa was ultimately unsuccessful and died out. We can never conclusively prove the absence of this genotype from North America. However, X. fastidiosa has been extensively sampled from many plant species throughout the United States, and no isolates of X. fastidiosa subsp. fastidiosa have been found that carry alleles similar to the inferred donor alleles cysG12 and holC19 ; indeed all X. fastidiosa subsp. fastidiosa isolates so far found in the United States are consistent with the introduction into the United States of just a single genotype . The transient presence of the donor genotype is consistent with a single large-scale introgression event founding the recombinant group. This raises the possibility that conjugation might have been involved; however, if this was the case, the genomic DNA was broken into pieces prior to homologous recombination, since the data show short regions of recombination. The data from the MLST loci plus pilU show 7 significant recombination events , and 3 of them included at least one recombination breakpoint. Since these loci range in length from roughly 400 to 700 bp, this result would be consistent with an average recombination length of no more than a few kb, similar to the 2.6-kb average length observed by Nunney et al. in a comparison of two X. fastidiosa subsp. fastidiosa genomes . Similarly, the regions identified from the data of Parker et al. showed the same pattern, with a high proportion of recombination breakpoints identified within the sequenced loci . In this context, it is important to note that Rogers and Stenger have found a conjugative plasmid in X. fastidiosa. Furthermore, a high rate of transformation has been demonstrated in the lab , and it has been shown that this process can result in efficient recombination with only a few hundred bases of homologous sequence .