Virus-free O. mediterraneus lines were obtained by end-point dilution, and virus absence was confirmed by PCR using degenerate primers for the viral DNA polymerase and major capsid protein gene (table S3). We started 204 clonal cultures from one single cell in 1-ml microwells and tested for virus susceptibility after 7 days. Virus susceptibility was tested using cell-free, virus-producing culture (VPC) medium.

To obtain cell-free VPC medium (i.e., separate potential virions from cell lysates), medium from growing or lysed cultures was centrifuged at 25,000g for 10 min at room temperature and the supernatant was filtered (<0.45 μm). To test whether a cell line was susceptible to lysis, VPC medium was introduced into the growing test culture, with a control culture grown in parallel inoculated with the same volume of sterile medium, and visually inspected for lysis up to 5 days after inoculation. This visual inspection detected when most cells are lysed, and thus, the ancestral cell was inferred to be S. The validity of this inference relied on two assumptions: a low switch rate between the S and R phenotypes and a low difference in growth rates between the R and susceptible phenotypes.

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