Homologous recombination plays an important role in the structuring of genetic variation of many bacteria; however, its importance in adaptive evolution is not well established. We investigated the association of intersubspecific homologous recombination (IHR) with the shift to a novel host (mulberry) by the plant-pathogenic bacterium Xylella fastidiosa. Mulberry leaf scorch was identified about 25 years ago in native red mulberry in the eastern United States and has spread to introduced white mulberry in California. Comparing a sequence of 8 genes (4,706 bp) from 21 mulberry-type isolates to published data (352 isolates representing all subspecies), we confirmed previous indications that the mulberry isolates define a group distinct from the 4 subspecies, and we propose naming the taxon X. fastidiosa subsp. morus. The ancestry of its gene sequences was mixed, with 4 derived from X. fastidiosa subsp. fastidiosa (introduced from Central America), 3 from X. fastidiosa subsp. multiplex (considered native to the United States), and 1 chimeric, demonstrating that this group originated by large-scale IHR. The very low within-type genetic variation (0.08% site polymorphism), plus the apparent inability of native X. fastidiosa subsp. multiplex to infect mulberry, suggests that this host shift was achieved after strong selection acted on genetic variants created by IHR. Sequence data indicate that a single ancestral IHR event gave rise not only to X. fastidiosa subsp. morus but also to the X. fastidiosa subsp. multiplex recombinant group which infects several hosts but is the only type naturally infecting blueberry, thus implicating this IHR in the invasion of at least two novel native hosts, mulberry and blueberry.

Enrichment methodologies enable the analysis of minor members in multi-species transcriptomic data. We compared the standard enrichment of bacterial and eukaryotic mRNA to a targeted enrichment using an Agilent SureSelect (AgSS) capture for Brugia malayi, Aspergillus fumigatus, and the Wolbachia endosymbiont of B. malayi (wBm). Without introducing significant systematic bias, the AgSS quantitatively enriched samples, resulting in more reads mapping to the target organism. The AgSS-enriched libraries consistently had a positive linear correlation with their unenriched counterparts (r² = 0.559-0.867). Up to a 2,242-fold enrichment of RNA from the target organism was obtained following a power law (r² = 0.90), with the greatest fold enrichment achieved in samples with the largest ratio difference between the major and minor members. While using a single total library for prokaryote and eukaryote enrichment from a single RNA sample could be beneficial for samples where RNA is limiting, we observed a decrease in reads mapping to protein coding genes and an increase in multi-mapping reads to rRNAs in AgSS enrichments from eukaryotic total RNA libraries compared to eukaryotic poly(A)-enriched libraries. Our results support a recommendation of using AgSS targeted enrichment on poly(A)-enriched libraries for eukaryotic captures, and total RNA libraries for prokaryotic captures, to increase the robustness of multi-species transcriptomic studies.