Abstract Mutation is one of the most fundamental evolutionary forces. Studying variation in the mutation rate within and among closely-related species can help reveal mechanisms of genome divergence, but such variation is unstudied in the vast majority of organisms. Previous studies on ciliated protozoa have found extremely low mutation rates. In this study, using mutation-accumulation techniques combined with deep whole-genome sequencing, we explore the germline base-substitution mutation-rate variation of three cryptic species in the Paramecium aurelia species complex—P. biaurelia, P. sexaurelia, and P. tetraurelia. We find that there is extremely limited variation of the mutation rate and spectrum in the three species and confirm the extremely low mutation rate of ciliates.
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