In contrast the acquisition of novel alleles is analogous to the effect of sexual reproduction in eukaryotes: it increases the genetic variance that natural selection can act on but does not, in itself, result in a qualitative change in the ecology of the recipient . Due to these fundamental differences, we favor reserving the term “LGT” for the transfer of novel genes, using the term “interspecific” or “intersubspecific homologous recombination” for the transfer of alleles; however, both processes, if successful, lead to genetic “introgression,” a term commonly used to describe the spread of genetic material across taxonomic boundaries in plants and animals and now increasingly used to describe the analogous process in bacteria . Homologous recombination is almost ubiquitous among bacteria, although the degree to which it occurs varies widely among species . It involves the replacement of a stretch of DNA sequence in one individual’s genome by a homologous sequence from another individual of the same species following any of the 3 mechanisms of DNA transfer . It typically involves short pieces of DNA . Given the prevalence of homologous recombination, it is generally assumed that it is beneficial,best grow pots in some cases enabling bacteria to enhance their resistance to antibiotics and avoid host defenses or perhaps promoting adaptation to novel environments . Analogy with the assumed benefits maintaining sexual recombination in metazoans strongly supports this view. Documenting the adaptive benefit of homologous recombination in bacteria has proved difficult. This is to be expected even if the benefits are large and common.

Homologous recombination typically falls off rapidly with genetic distance , so a well-established population will usually reflect the mixing of relatively similar alleles. This mixing can be easily detected by the lack of clonality between genes and quantified using evolutionary models ; however, detection of recombination breaks within genes is more problematic. The approaches currently used have very limited power; although the introgression test has improved this situation . Another approach is to test loci sequenced from 2 or more taxa and use the genetic partitioning program STRUCTURE . Alleles that cannot be confidently allocated to one or more of the taxa are likely to be mosaics generated by recombination . To link recombination to adaptive change, it is useful to study a system in which recombination is limited, recognizable, and likely to lead to novel adaptation. Arnold et al. recently made an interesting link between the acquisition of novel adaptations in bacteria via LGT and that via hybridization in metazoans. Excellent examples of how interspecific introgression can result in adaptation to new environments in higher plants are given in the work of Rieseberg and colleagues on the effects of introgression in sunflower species . However, it is not only metazoans that hybridize: bacterial homologous recombination can sometimes result in interspecific introgression . Interspecific hybridization of this kind is likely to be relatively rare, suggesting that the ideal study system is one with a significant frequency of homologous recombination between well-defined groups within a species . This level of study appears most likely to provide valuable insights into recombination-related adaptive change in pathogens.

For example, Didelot et al. showed that two human-pathogenic forms of Salmonella entericaare relatively dissimilar across about 75% of their genomes but show marked convergence across the rest. They concluded that this similarity reflects adaptation to the human host, driven by homologous recombination and selection. Similarly, Sheppard et al. proposed that human activity has probably led to an increase in recombination between Campylobacter jejuni and Campylobacter coli and may have also created novel environments that have favored the evolution of hybrids. Another species in which homologous recombination between closely related but distinct taxa has been documented is the plant pathogenic bacterium Xylella fastidiosa . X. fastidiosa is a xylem-limited bacterium that is transmitted by xylem-feeding insects, typically leafhoppers, and is divided into four subspecies: fastidiosa, sandyi, multiplex, and pauca . These subspecies have diverged genetically by 1 to 3%, apparently due to their geographical isolation over about the last 20,000 to 50,000 years . This isolation has now broken down, due presumably to human activity . The cooccurrence of the previously allopatric subspecies has resulted in intersubspecific homologous recombination , recombination that can be detected relatively easily due to the preexisting genetic divergence of the subspecies . Consistent with these observations, recent experimental work has confirmed that X. fastidiosa is transformationally competent and that some isolates carry a conjugative plasmid . X. fastidiosa is known to infect a wide range of hosts, causing scorch and dwarfing diseases . In citrus, it causes citrus variegated chlorosis , a disease restricted to South America, and in grapevines in the United States and Central America, it causes Pierce’s disease.

In the United States, it also causes disease in almond, apricot, plum, peach, alfalfa, pecan, and blueberry. However, individual X. fastidiosa strains are not generalists. The different subspecies infect a characteristic and largely non-overlapping range of plant hosts, and even within subspecies, different genotypes show differences in host specificity . For example, in the South American X. fastidiosa subsp. pauca, citrus isolates do not typically grow in coffee and vice versa , and in X. fastidiosa subsp. multiplex, Nunney et al. found associations between the genotype and host plant. In their study of X. fastidiosa subsp. multiplex, Nunney et al. used the multilocus sequence typing protocol of Yuan et al. to categorize 143 isolates. The MLST protocol is valuable for gaining insight into the evolutionary history and genetic diversity of taxa . MLST groups isolates into sequence types , where each ST defines a unique set of alleles across the loci used . Based on 8 loci, 31 of these isolates were identified as IHR forms , and 2 isolates were considered “intermediate” , while the remaining 110 non-IHR isolates showed no evidence of introgression. The IHR and intermediate types together were considered to define the “recombinant” group of X. fastidiosa subsp. multiplex isolates . Most were observed more than once, and 5 were found in two different U.S. states or districts . The analysis of Nunney et al. was focused on the evolution and host range ofX. fastidiosa subsp. multiplex. For this purpose, it was necessary to identify and exclude isolates whose recent evolution was influenced by intersubspecific recombination. As such, once the 23 non-IHR STswere identified, there was no further analysis of the remaining recombinant group STs. In particular, no evidence was presented for classifying some alleles as atypical of X. fastidiosa subsp. multiplex beyond the observation that they were never found in the non-IHR group . Nunney et al. did observe one intriguing pattern when they compared their results to those of Parker et al. . Of the 143 isolates, 13 were also used in the study by Parker et al. , in which typing was based on a different set of 9 loci. Unexpectedly, these 13 isolates maintained the same grouping with the IHR and non-IHR types corresponding, respectively,plants in pots ideas to the clade A and clade B groupings . This highly statistically significant concordance strongly suggested that IHR is not distributed randomly across all X. fastidiosa subsp. multiplex isolates but instead is restricted to a small subset, while the remainder is little influenced by IHR. However, Parker et al. failed to find evidence of intersubspecific recombination within any of the X. fastidiosa subsp. multiplex isolates, despite applying a series of 9 tests designed to detect recombination contained within the RDP4 program and the PHI program . This result presented a strong argument against our hypothesis that clade A members cluster because they are recombinant types carrying alleles derived from IHR . Here we reexamined the sequence data obtained in their study by using the more sensitive introgression test to determine if their tests missed evidence of IHR and, if so, whether it was confined to clade A. A second related question concerned the relationship among the recombinant IHR group members. In particular, what could be concluded about the origin of the group given the observation from 2 independent studies that the members appear to form a well-defined cluster of genotypes? Third, we used the sequence data to examine the hypothesis that the introgressed DNA was from X. fastidiosa subsp. fastidiosa, the subspecies that causes Pierce’s disease. X. fastidiosa subsp. fastidiosa is native to Central America, and all known isolates in the United States and northern Mexico can be traced back to a single introduced genotype .

IHR would be of limited interest if it simply randomized the genetic differences among the subspecies but had a minimal effect on pathogenesis. For this reason, we were particularly interested in documenting any possible invasion of new plant hosts associated with IHR. The hypothesis is that IHR creates a range of novel genotypes that are far more variable than can arise from a lineage diversifying through point mutations, and this diversity facilitates adaptive evolution of a kind not possible for a clonal lineage. This kind of probabilistic evolutionary hypothesis can rarely be directly proven based on an individual case; however, it makes predictions that, if generally supported, would cause the hypothesis to be accepted. In the case of X. fastidiosa, compelling evidence supporting the hypothesis would be the invasion of a new native host plant that is uniquely associated with IHR. Our data support this hypothesis: in X. fastidiosa subsp. multiplex, IHR is indeed associated with the invasion of at least 2 new native plant hosts, blueberry and blackberry.To investigate intersubspecific homologous recombination , we analyzed 31 isolates previously identified as IHR-type and 2 isolates previously identified as intermediate-type X. fastidiosa subsp. multiplex , based on sequence of the 7 housekeeping loci used in the MLST scheme defined by Yuan et al. plus a region of the pilUgene. Together, these 33 isolates made up the recombinant group. Details regarding the isolation and typing of the 33 isolates were provided by Nunney et al. , and a summary of salient features is provided in Table S1 in the supplemental material in that article. All sequences used have previously been published and are available both in GenBank and the MLST website . To detect IHR, we employed a modified version of the introgression test developed by Nunney et al. . In its original form, the test compares a set of target sequences, some of which may have been involved in IHR, to a set of potential donor sequences. Each variable site is classified as F, a fixed difference between the target sequences and the donor sequences, or P, a polymorphic site within the target sequences where at least one variant base is shared with the donor set. In the modified version of the test, the targeted introgression test, the target sequence is known a prioriand is compared to two references, the donor group, D , and the ancestral group, A . The minimum number of nucleotide differences between the target and the two references defines a ratio of D to A equivalent to the ratio of F to P and can be tested in the same way . In some cases, there is no breakpoint because the whole locus appears to be an introgressed sequence . Although the signal of introgression across the entire sequenced region may be clear, it is valuable to have a statistical test that documents the strength of the signal. In this case, the null expectation is the ratio that reflects the pairwise differences between the donor and ancestral group versus the pairwise differences within the ancestral group . We used this ratio to define the expectation of the D/A ratio for a chi-square test of complete introgression. Gene diversity and distance trees were calculated using MEGA5 , and the maximum parsimony tree was created using the PARS program in Phylip . Distance trees and the maximum parsimony tree were used rather than other methods, given the known occurrence of intersubspecific recombination in the data. ClonalFrame was used to provide an independent estimate of the relative importance of recombination versus mutation in the recombinant group.Based on 8 loci sequenced , Nunney et al. identified 9 sequence types belonging to the recombinant group of X. fastidiosa subsp. multiplex. These STs all showed evidence of intersubspecific homologous recombination at one or more of the 8 loci and were characterized by 18 alleles, 10 of which were never found in non-IHR X. fastidiosa subsp. multiplex strains .