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bioRxiv · 10.64898/2026.02.13.705778

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

Shropshire, J. D.·Conner, W. R.·Vanderpool, D.·Hoffmann, A. A.·Turelli, M.·Cooper, B. S.
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Abstract

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 1-2 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 2.1x105-2.4x106 years inhabit dipteran and hymenopteran hosts diverged more than 108 years. Previous published analysis of variants related to wRi from D. 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 seven. In addition, cif exchanges between wMel-like and wRi-like Wolbachia genomes have occurred over [~]104-106 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. Article SummaryMaternally transmitted Wolbachia endosymbionts associate with many arthropods. Closely related Wolbachia are known to occupy anciently diverged hosts, but the timescale of associations remains uncertain. Using genomic data from obligate Wolbachia codiverging with filarial nematodes, we calibrate Wolbachia molecular evolution. wMel-like variants that diverged about 104-106 years ago occur in hosts that diverged more than 108 years ago. Incompatibility loci move even more rapidly among these Wolbachia genomes. As these variants diverge, selection occurred on loci involved in both inducing and protecting against cytoplasmic incompatibility. These results elucidate Wolbachia host-range and genome dynamics and provide potential material for applications.

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Shropshire, J. D., Conner, W. R., Vanderpool, D., Hoffmann, A. A., Turelli, M., Cooper, B. S.. 2026-02-14. Calibrating and documenting host-switching and evolution of incompatibility loci for two closely related Wolbachia clades. https://doi.org/10.64898/2026.02.13.705778

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