Strong selection and high mutation supply characterize experimental Chlorovirus evolution

Viruses are a taxonomic group relevant to both society and ecology. Characterizing their patterns of molecular evolution helps us understand how viruses evolve and expands our understanding of the underlying fundamental processes such as mutation, selection and drift. One group of viruses whose evolution has not yet been extensively studied are the Phycodnaviridae, a globally abundant family of freshwater large dsDNA viruses. Here we studied the evolutionary change of this family's type specimen Paramecium bursaria Chlorella Virus 1 (PBCV-1), during experimental coevolution with its algal host. We used pooled genome sequencing of six independently evolved populations to characterize genomic change over five time points. Across six experimental replicates involving either strong or weak demographic fluctuations, we found single nucleotide polymorphisms (SNPs) at 67 sites. Variation was highly repeatable, with just two of the SNPs found in only a single experimental replicate. Across the genome, we did not find evidence that non-synonymous SNPs were found in more (or less) replicates than synonymous SNPs, but non-synonymous SNPs did on average reach higher allele frequencies. One SNP was located in an intergenic region, the other 66 were located in twenty open reading frames (ORFs), providing new information about potential targets of selection during the Chlorella-Chlorovirus coevolution. Three genes A122/123R, A140/145R and A540L showed an excess of variable sites. Following the allele frequency trajectories at these sites over time revealed that repeated mutations often did not exhibit similar patterns of frequency change in independent evolutionary replicates. In summary, our investigation showed that the studied populations were not mutation-limited and experienced strong positive selection. We identified several genes putatively involved in the evolution of increased host range. Our investigation increased our understanding of the processes governing evolution of freshwater large dsDNA viruses, which is ultimately necessary to understand the functioning of natural aquatic ecosystems.