Rapid evolution of Wolbachia in cherry fruit flies

Wolbachia is a widespread bacterial endosymbiont in arthropods known for its ability to manipulate host reproduction through cytoplasmic incompatibility (CI), thereby promoting its spread. However, Wolbachia harbors an array of mobile genetic elements (MGEs) that can rapidly alter its genomic structure and content, including the cif loci responsible for CI. At the phylogenetic scale, Wolbachia genomes are shown to be dynamic with MGEs causing widespread genome rearrangements. But on ecological time scales, the evolution of Wolbachia remains largely unknown. In this study, we leverage the natural history of Wolbachia in cherry-infesting fruit flies in the Rhagoletis cingulata sibling species group. Members of this species group share a common Wolbachia strain, wCin2, and their divergence spans from 1,000 to 150,000 years ago. We utilize long-read sequencing to assemble and characterize wCin2 Wolbachia genome divergence across recent to more distant evolutionary timescales. We report rapid evolution of population-level differences in gene content (notably within cif loci), synteny, and structural variation linked to MGEs with differentiation increasing with time since host divergence. Moreover, our results indicate that the CI phenotype previously attributed to a distinct Wolbachia strain, wCin3, that coinfects flies in the southwestern USA and Mexico, may be caused by cif genes that were horizontally transferred into wCin2. Finally, we discovered a novel Wolbachia strain, wInd, which appears to have been recently horizontally acquired by cherry fly populations in the Pacific Northwest of the USA. Our findings underscore the fluidity and rapid genome evolution of Wolbachia, with significant implications for cif gene dynamics and potential impacts on host evolutionary trajectories.