Assuming that mutations evolve de novo, are neutral, and are uniformly distributed across the genome, every genomic position has the same probability of acquiring a mutation. Under these assumptions, observing a mutation in our experiments is equivalent to randomly drawing marbles from a vase without replacement. The size of the vase is equivalent to the number of genomic positions we evaluated with our sequencing approach, and the sample size (number of marbles drawn) equals the number of mutations observed per replicate. To assess how likely the empirically observed overlap is, we simulated such a sampling procedure using R and found that the probability of the observed overlap (fig. S4, A and B) is extremely small (P < 10−5). Because of, i.a., random variation in sequencing coverage and complex genomic regions, not every position of the reference genome can be reliably evaluated; hence, reference genome size is an overestimate of vase size. To get an idea of how large the pool of mutable sites need to be to reproduce the empirically observed overlap, we repeated the simulations for a grid of vase sizes using the empirical number of observed mutations (minimum across three replicates) as the total number of marbles drawn and reported the number of marbles drawn more than once (i.e., SNPs observed in multiple replicates) in table S2.

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