The range of sampling times affects Zika virus evolutionary rates and divergence times.

The rate of evolution of viral genomes is a fundamental parameter for understanding the origin and spread of epidemics. For instance, molecular dating is one of the many practical outcomes of evolutionary rate estimation. In this sense, the rate of evolution of ZIKV merits attention, because it has been shown to be higher than the average rate reported for other flaviviruses. It has been hypothesized that the higher rate of ZIKV evolution is due to a bias related to the analysis of sequences collected within a short time range, which would increase the chance of sampling slightly deleterious nucleotide polymorphisms. To investigate this hypothesis, we assembled datasets with different ranges of sampling times and also decomposed the ZIKV evolutionary rate into synonymous and non-synonymous rates. Our results demonstrated that the rate of ZIKV evolution is time dependent and that the observed increase in short-term rates is largely accounted for by a higher non-synonymous rate, suggesting the presence of slightly deleterious variants not yet eliminated by purifying selection. On the other hand, we show that synonymous rates were less impacted by the range of sampling times, generating timescales with reduced uncertainty. We conclude that, for inferring the ZIKV timescale and reconstructing the history of epidemics, synonymous changes are the most appropriate substitution type to be examined. We were able to obtain ZIKV divergence times that were time independent and exhibited greater precision than previous estimates. This observation should also hold for other serially sampled fast-evolving pathogens with evidence of time dependence of evolutionary rates.