To infer the age of the 65 recombinant fragments, we first reasoned that most of the mutations found were contributed by recombination and not by mutation once the fragment had been integrated in the genome. Thus, before dating the fragments, we first removed all the homoplastic variants with other MCAN strains found in the fragments. The final alignments for the 65 fragments consisted of only those variants accumulated after the recombination event. We then used the nonrecombinant part of the genome to infer a substitution rate assuming two different dating scenarios published for the tMRCA (5, 54). We ran BEAST (55) for each fragment prespecifying monophyletic groups and substitution rate based on the nonrecombinant genome phylogenetic reconstruction. We used an uncorrelated log-normal distribution for the substitution rate in all cases and a skyline model for population size changes. We ran several chains of up to 10 × 106 generations sampling every 1 × 103 generations to ensure independent convergence of the parameters. For both evolutionary scenarios, the results obtained were largely congruent and proportional to the age limit imposed for the MTBC ancestor. The 5-ka scenario (54) was selected for plotting the ages in Fig. 3A and fig. S3, as there is now more evidence for this time frame.

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