Estimating evolutionary rates in giant viruses using ancient genomes

Pithovirus sibericum is a giant (610 Kpb) double-stranded DNA virus discovered in a purportedly 30,000-year-old permafrost sample. A closely related virus, Pithovirus massiliensis, was recently isolated from a sewer in southern France. An initial comparison of these two virus genomes assumed that P. sibericum was directly ancestral to P. massiliensis and gave a maximum evolutionary rate of 2.60 × 10(−5) nucleotide substitutions per site per year (subs/site/year). If correct, this would make pithoviruses among the fastest-evolving DNA viruses, with rates close to those seen in some RNA viruses. To help determine whether this unusually high rate is accurate we utilized the well-known negative association between evolutionary rate and genome size in DNA microbes. This revealed that a more plausible rate estimate for Pithovirus evolution is ∼2.23 × 10(−6) subs/site/year, with even lower estimates obtained if evolutionary rates are assumed to be time-dependent. Hence, we estimate that Pithovirus has evolved at least an order of magnitude more slowly than previously suggested. We then used our new rate estimates to infer a time-scale for Pithovirus evolution. Strikingly, this suggests that these viruses could have diverged at least hundreds of thousands of years ago, and hence have evolved over longer time-scales than previously suggested. We propose that the evolutionary rate and time-scale of pithovirus evolution should be reconsidered in the light of these observations and that future estimates of the rate of giant virus evolution should be carefully examined in the context of their biological plausibility.