Calibrating and documenting host-switching and evolution of incompatibility loci for two closely related <i>Wolbachia</i> clades

Maternally inherited Wolbachia alphaproteobacteria are the most common arthropod endosymbionts. Often, Wolbachia spread to high frequencies through cytoplasmic incompatibility, in which cif loci act through sperm to kill embryos lacking Wolbachia. Closely related Wolbachia with diverse cif loci often associate with anciently diverged hosts, but the timescale of associations remains uncertain. We produce new calibrations based on filarial nematodes with vertically inherited Wolbachia that codiverge with their hosts. Applying these calibrations to Wolbachia variants closely related to pathogen-blocking wMel from Drosophila melanogaster, we demonstrate that over a timescale of 1 to 2 million years, a core set of single-copy Wolbachia loci evolve largely through bifurcation rather than by gene exchange with distant Wolbachia. Dating bifurcating core genomes, we show that "wMel-like" Wolbachia diverged 2.1 × 105 to 2.4 × 106 years inhabit dipteran and hymenopteran hosts diverged more than 108 years. Previous published analysis of variants related to wRi from Drosophila simulans, the first Wolbachia found in a drosophilid, concluded that "wRi-like" Wolbachia spread among different Drosophila in tens of thousands of years. However, our new calibrations suggest these estimates from a mutation-based calibration underestimated wRi-like spread by about a factor of 7. In addition, cif exchanges between wMel-like and wRi-like Wolbachia genomes have occurred over ∼104 to 106 years. Comparing intact cif loci found in various Wolbachia, we find function-preserving selection in their evolution. We discuss these results in light of theoretical predictions concerning selection on cytoplasmic incompatibility phenotypes within and among host lineages. The wMel variants analyzed may offer new options for Wolbachia-based biocontrol efforts.