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

Maternally inherited <i>Wolbachia</i> alphaproteobacteria are the most common arthropod endosymbionts. Often <i>Wolbachia</i> spread to high frequencies through cytoplasmic incompatibility, in which <i>cif</i> loci act through sperm to kill embryos lacking <i>Wolbachia</i>. Closely related <i>Wolbachia</i> with diverse <i>cif</i> 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 <i>Wolbachia</i> that codiverge with their hosts. Applying these calibrations to <i>Wolbachia</i> variants closely related to pathogen-blocking <i>w</i>Mel from <i>Drosophila melanogaster</i>, we demonstrate that over a timescale of 1-2 million years, a core set of single-copy <i>Wolbachia</i> loci evolve largely through bifurcation rather than by gene exchange with distant <i>Wolbachia</i>. Dating bifurcating core genomes, we show that "<i>w</i>Mel-like" <i>Wolbachia</i> diverged 2.1×10⁵-2.4×10⁶ years inhabit dipteran and hymenopteran hosts diverged more than 10⁸ years. Previous published analysis of variants related to <i>w</i>Ri from <i>D. simulans</i>, the first <i>Wolbachia</i> found in a drosophilid, concluded that "<i>w</i>Ri-like" <i>Wolbachia</i> spread among different <i>Drosophila</i> in tens of thousands of years. However, our new calibrations suggest these estimates from a mutation-based calibration underestimated <i>w</i>Ri-like spread by about a factor of seven. In addition, <i>cif</i> exchanges between <i>w</i>Mel-like and <i>w</i>Ri-like <i>Wolbachia</i> genomes have occurred over ~10⁴-10⁶ years. Comparing intact <i>cif</i> loci found in various <i>Wolbachia</i>, 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 <i>w</i>Mel variants analyzed may offer new options for <i>Wolbachia</i>-based biocontrol efforts.