Supplementary Tables 1-12

Supplementary Table 1: Demographic data (backups and holdovers). Supplementary Table 2: The total number of mutations found in different lines and the sublines. Supplementary Table 3: All homozygous variant sites between the ancestor (G0) and the N2 reference genome (ws292) identified at the 3X coverage criterion. Supplementary Table 4: All dummy sites that were randomly inserted to the N2 reference genome (ws292) using GATK FastaAlternateReferenceMaker. Supplementary Table 5: Random SNPs and indels were inserted into the reference genome twice to create two pseudo-references (D1 and D2). All MA lines were re-aligned and genotyped against each pseudo-reference. At each dummy site, the expected genotype is 1/1. We record which sites did not return 1/1. False negatives are the sum of 0/0 and 0/1; no recall includes NA, 0/1, and 0/0. Supplementary Table 6: Mean FtR rates from data in Suppl. Table 5. Supplementary Table 7: the complete set of mutations, with O1 line, O2 subline, their location, genotype, gene name, annotation and their putative impact. Supplementary Table 8: information on multi-nucleotide variants (MNVs). Supplementary Table 9: Estimates of mutation rates per-site (x109). Variables and calculations are the same as in Table 1 in the main text except estimates for SNVs and indels do not include mutations in MNVs. Supplementary Table 10: Mutational variances. Supplementary Table 11: Block means and SEM of fitness assays. Supplementary Table 12: Fitness data.