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 utilized Nanopore sequencing to characterize wCin2 Wolbachia genome divergence across recent to more distant evolutionary timescales. We report rapid evolution of population-level differences in gene content (including cif loci and MGEs) and genome structure, with differentiation increasing with time since host divergence. Notably, structural variants were the first to appear both between and among wCin2 populations. Our results also indicated that the CI phenotype previously attributed to a distinct Wolbachia strain, wCin3, that coinfects flies in the southwestern USA and Mexico, may instead 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. Author SummaryIn our study, we explore the genome evolution of Wolbachia, a common bacterial endosymbiont found in many insects that can manipulate its hosts reproduction to help it spread more effectively. Wolbachias genome is shown to change dramatically over long evolutionary periods due to mobile genetic elements. However, little is known about how Wolbachias genome evolves over shorter ecological timeframes. Here, we utilized cherry-infesting fruit flies and their Wolbachia strain known as wCin2, both of which have spread across North America for the past 150,000 years. Using long-read sequencing, we assembled multiple wCin2 Wolbachia genomes from across North America and made comparisons between wCin2 Wolbachia genomes that have recently diverged and comparisons to those that have been isolated for over 150,000 years. Our findings reveal that Wolbachias genome can evolve swiftly, with differences in gene content and most prominently genome structure arising between recently separated Wolbachia populations and increasing as populations diverge over time. Interestingly, these changes also impact the key genes responsible for Wolbachias reproductive manipulation. This research highlights the rapid and dynamic nature of Wolbachia genome evolution across different evolutionary timescales and offers insights into endosymbiont-host relationships as well as their potential impact on host speciation and vector control.