Homologous recombination typically involves relatively small pieces of DNA , which suggests entry of donor DNA into the bacterial cell via transformation. However, not all species with significant levels of recombination, such as Escherichia coli, are typically competent for transformation , and it is also possible that large pieces of DNA entering the cell via conjugation may be cleaved into smaller pieces before recombination occurs. Regardless of the processes that make donor DNA available, it is clear that homologous recombination in bacteria is an important mechanism of genetic exchange. Like sex in eukaryotes, recombination blends genetic variation and as such acts as a cohesive process that can inhibit the subdivision of taxa , but when the donor DNA comes from a genetically differentiated population, recombination can dramatically increase genetic diversity. However, the critical issue raised by Smith et al. of why the relative rate of recombination is so variable across taxa remains unresolved. This issue is unlikely to be resolved until we understand more about the benefits of homologous recombination. The possible benefits remain largely speculative, since adaptive consequences of recombination between close relatives are diffi- cult to detect. Adaptive shifts may be important in maintenance of transformation , and there is some evidence that homologous recombination may be important in promoting a number of adaptive responses. These include maintaining variability of surface proteins in animal pathogens to avoid host defenses , 25 liter pot plastic transfer of virulence genes in plant-pathogenic bacteria , and evolution of new taxa by, for example, facilitating adaptation to novel hosts .

Homologous recombination can most easily be studied when genetic transfer has occurred between genetically distinct but closely related taxa. Under such circumstances, it may be possible to detect not only recombinant events but also sources of incorporated DNA and, potentially, to identify adaptive consequences of the exchange. This scenario is found in the plant-pathogenic bacterium Xylella fastidiosa, where intersub specific homologous recombination is well documented . X. fastidiosa infects xylem vessels of a wide range of plant host species in the Americas . X. fastidiosa has been divided into four subspecies, three of which are found in the United States . These groups are genetically distinct, with values of DNADNA hybridization between them of less than 70% , sequence differences of 2% or more at synonymous sites , and distinct 16S rRNA gene and 16S-23S rRNA gene spacer sequences . These differences reflect estimated divergence times of more than 15,000 years . Furthermore, each subspecies has a distinct and largely non-overlapping set of plant hosts : in the UnitedStates, X. fastidiosa subsp. fastidiosa causes Pierce’s disease of grape, X. fastidiosa subsp. sandyi causes oleander leaf scorch, and X. fastidiosa subsp. multiplex causes leaf scorch disease on a range of trees, including oak, elm, and peach. In South America, X. fastidiosa subsp. pauca infects citrus and coffee . X. fastidiosa is competent for transformation , and some isolates carry conjugative plasmids , so sympatry of subspecies potentially creates conditions conducive for both the occurrence and detection of IHR.

Sympatry of X. fastidiosa subspecies appears to be relatively recent: while X. fastidiosa subsp. multiplex is probably native to the United States, there is compelling evidence that the other two subspecies found in the United States were introduced . X. fastidiosa subsp. sandyi has been known in the United States for only about 30 years, while X. fastidiosa subsp. fastidiosa has presumably been present since the first known outbreak of Pierce’s disease ca. 130 years ago. Furthermore, it appears that a similar situation exists in South America. While X. fastidiosa subsp. pauca is native to South America, there is evidence of the introduction of a second subspecies into Argentina and/or Brazil causing plum leaf scald, first observed in 1935 . Analysis of sequences indeed demonstrated large-scale recombination of X. fastidiosa subsp. fastidiosa sequences into X. fastidiosa subsp. multiplex in the United States , and, in Brazil, there has been substantial recombination into X. fastidiosa subsp. pauca of sequences from a distinct taxon, tentatively identified as X. fastidiosa subsp. multiplex . However, large-scale introgression is not the rule. Analyses of genomes of U.S. isolates of X. fastidiosa subsp. fastidiosa show very limited introgression of X. fastidiosa subsp. multiplex ; moreover, large-scale introgression into X. fastidiosa subsp. multiplex is restricted to a welldefined set of genotypes, suggesting that it may have been initiated by very few events . The majority of X. fastidiosa subsp. multiplex isolates show little evidence of IHR, and the data available suggest that even intra subspecific recombination is limited . Thus, the picture emerging in X. fastidiosa is one of limited successful homologous recombination on a short time scale, with bursts of large-scale exchange occurring very infrequently.

This raises the possibility that, by substantially increasing the available genetic variability, these large-scale events facilitate rapid evolutionary change that can result in colonization of new plant hosts. This scenario has been proposed as the mechanism underpinning the invasion of blueberry by recombinant forms of X. fastidiosa subsp. multiplexand, more speculatively, the infection of citrus and coffee by X. fastidiosa subsp. pauca in Brazil . Another candidate for this scenario is the form of X. fastidiosa infecting mulberry, a form that does not appear to fit within the framework of the four subspecies so far identified. Kostka et al. first observed the disease of mulberry leaf scorch in the Washington, DC, area on the native red mulberry , and further study revealed infected trees along the east coast as far north as New York City, NY. Since that time, MLS has been observed in Nebraska and also in California on the introduced white mulberry . Previous genetic assessment showed that, although the 16S rRNA gene sequence of mulberry isolates is consistent with that of X. fastidiosa subsp. fastidiosa, based on analyses of randomly amplified polymorphic DNAs and 16S-23S rRNA gene spacer sequences, these types cluster as a distinct group . Here we used multilocus sequence typing to evaluate the genetic relationship of the mulberry isolates to the 4 subspecies and to establish its hybrid ancestry via IHR, supporting the hypothesis that IHR facilitates host shifts. We show that this ancestry is shared with the recombinant group of X. fastidiosa subsp. multiplex; however, the recombinant group has largely introgressed into X. fastidiosa subsp. multiplex, and we propose that the continued genetic distinctiveness of the mulberry type merits recognition as a new subspecies, X. fastidiosa subsp. morus.Three of the mulberry-type alleles have been previously observed in the recombinant group of X. fastidiosa subsp. multiplex but nowhere else . The recombinant group is a set of STs that are genetically similar to the rest of X. fastidiosa subsp. multiplex , but they carry alleles that include some X. fastidiosa subsp. fastidiosa sequence . All recombinant-group STs have X. fastidiosa subsp. multiplex sequence at 4 loci but have some recognizable X. fastidiosa subsp. fastidiosa sequence in 1 to 3 of the remaining 4 loci, leuA, cysG, holC, and pilU. Notably, all X. fastidiosa subsp. morusisolates carry X. fastidiosa subsp. fastidiosa sequence at these same 4 loci plus malF . A defining feature of the recombinant-group STs is that some of their X. fastidiosa subsp. fastidiosa sequence did not originate from the X. fastidiosa subsp. fastidiosa strains currently found in the United States. Instead,25 litre plant pot the sequence appears to be derived from X. fastidiosa subsp. fastidiosa variants found in Central America . The mulberry types show this same characteristic . Of the five loci in mulberry-type STs that include X. fastidiosa subsp. fastidiosa sequence, four show a closer relationship to Costa Rica sequence than to any allelic sequence found in the United States, while there is no difference at pilU, since the most similar allele is found in both locations . For leuA allele 4 and cysG allele 18 , the Costa Rica X. fastidiosa subsp. fastidiosa alleles provide a fit that is total of 4 bp better than that seen with alleles found in the United States . Even more compelling is the example of holC allele 5, where the difference is 7 bp; the U.S. allele is 8 bp different, while the best-fit Costa Rica allele is only 1 bp different . Further tests of this ancestry are provided by variation at holC and malF. First, holC allele 5 and the allele derived from it by one base change are unique to the mulberry isolates.

However, holC allele 5 appears to be the template from which recombinant-group holCallele 7 and allele 9 originated via recombination, as can be seen by noting the correspondence of their differing lightface data in Table 4 with those of holC 5. Second, in the recombinant group, malF is invariant for a widespread X. fastidiosa subsp. multiplex allele , but the mulberry types are invariant for a unique X. fastidiosa subsp. fastidiosa allele . This provides a new test of the link of the X. fastidiosa subsp. fastidiosa to Central American rather than U.S. sequence. Consistent with expectations, malF allele 6 is only 1 bp distant from Costa Rica malF allele 19 but is 3 bp distant from the most similar U.S. allele . Adding up these differences for the most basal allele found in the mulberry type at each of the 5 loci reveals only 4-bp differences from Costa Rica alleles, whereas the differences from the U.S. X. fastidiosa subsp. fastidiosa alleles total 17 bp. In summary, X. fastidiosa subsp. morus is similar to the recombinant group of X. fastidiosa subsp. multiplex in the loci carrying X. fastidiosa subsp. fastidiosa sequence, the alleles occurring at those loci, and the close relationship of the alleles to X. fastidiosa subsp. fastidiosa sequence found only in Central America. Previously, Nunney et al. proposed that the recombinant group originated by a transfer of DNA from an X. fastidiosa subsp. fastidiosa donor to an X. fastidiosa subsp. multiplex recipient. The direction of the transfer was assumed on the basis of the close relationship of the recombinant group to the rest of X. fastidiosa subsp. multiplex . The sequence data from X. fastidiosa subsp. morus bring this assumption of direction into question. Instead, it is probable that the common origin involved introgression of X. fastidiosa subsp. multiplex into a unique X. fastidiosa subsp. fastidiosa strain that had been introduced into the United States from Central America. Under this hypothesis, X. fastidiosa subsp.morusis the relatively unaltered descendant of the ancestral hybrid, while repeated introgression from X. fastidiosa subsp.multiplex gave rise to the recombinant group ofX. fastidiosa subsp. multiplex. We examined the plausibility of this hypothesis through parsimony analysis, using alleles as characters, and the result is broadly supportive of the idea of a common origin of the mulberry-type and recombinant-group STs . The initial analysis produced 7 equally parsimonious trees, but by assuming that the four X. fastidiosa subsp. fastidiosa alleles inconsistent with the current U.S. strains were themselves ancestral , the total was reduced to 2 trees. The only difference between these two trees involved the position of the clade corresponding to ST27, ST28, and ST40. In one tree , the required recombination transfer of cysG allele 18 is minimized to one event ; alternatively, in the second tree, the clade branches from the ST58 lineage, which removes the necessity of a recombination transfer of leuA allele 6 , which is an X. fastidiosa subsp. multiplex allele unique to the recombinant group. Consistent with the common-origin hypothesis, parsimony requires very little postorigin modification within X. fastidiosa subsp. morus. Specifically, it requires only the basal acquisition of cysG allele 18, derived by recombination from presumed ancestral cysG allele 12 . All other allelic changes are single base substitutions. The genesis of the recombinant group is more complex, consistent with the conclusions of Nunney et al. and with the assumption of a history of continued introgression. Thus, the data suggest that the recombinant group has undergone suffi- cient additional recombination with X. fastidiosa subsp. multiplex that it has ceased to be a separate taxon. On the other hand, the mulberry isolates show no evidence of such introgression and thus have remained a distinct taxon meriting subspecific status. To this point, there is no evidence of intermediate genotypes that bridge the genetic space that now exists between the recombinant group and X. fastidiosa subsp. morus .