Parallel evolution of full-length genomes in a long-term evolution experiment with phage ΦX174

The study of evolution in organisms with high mutation rates requires detailed data on the genomic composition of the population. Here we report on an innovative use of high-throughput sequencing technology combined with rapid experimental evolution that allowed us to follow the genetic diversification of four independent bacteriophage populations that we evolved without imposing any direct selection pressure for 412 generations in unprecedented detail. Tracing over 80000 bacteriophage genomes, of which 884 are unique, we find that evolution is largely neutral but document multiple instances of parallel evolution. Unlike previous observations of parallelism, we do not just see the parallel evolution of mutations at single sites, but on the level of full-length genomes. Computer simulations that recapitulate our experiments in great detail show that this degree of paralellism is inconsistent with neutral evolution. We further show that the observed extent of parallel evolution biases phylodynamic analysis of migration rates, and wrongly estimates significant migration between the independent evolution lines. Our approach is applicable to many other viruses and paves the way to advanced population genetic investigations that aim to shed light on phenomena that relate to genetic linkage across the genome. Significance StatementGenetic sequences of organisms contain imprints of their population biology and migration history. To recover this history, phylodynamic inference has been developed that combines phylogenetics with population biological models. Although phylodynamic methods are being applied widely, they have not yet been experimentally validated. To do this, we evolved a bacteriophage in four independent evolution lines for over 400 generations. We did not impose any direct selection pressure in these evolution experiments because phylodynamic inference works best in neutrally evolving systems. We found that often the same mutations and combinations of mutations evolve in the independent evolution lines. As a result, phylodynamics wrongly estimates migration between the lines. We conclude that the effects of selection should be included into phylodynamic inference.