Effect of calibration choice on the estimated divergence time. (A) Comparison of divergence time estimates of a tree calculated using a root of 3.5 Ga and a minimum age for the MRCA of Cyanobacteria of 2.45 Ga (x axis) against a tree calculated using a minimum age for the MRCA of 3.00 Ga (y axis). There is no deviation from the slope (red dashed line) meaning that the calibration did not have a strong effect on the estimated divergence time. The orange dot represents the divergence time of the duplication that led to PsaA and PsaB. (B) Divergence times of a tree calculated using a root of 3.5 Ga and a minimum age for the MRCA of Cyanobacteria of 2.45 Ga (x axis) against a tree calculated without a calibration on this point (y axis). (C) Divergence times of a tree calculated using a root of 3.5 Ga and a minimum age for the MRCA of Cyanobacteria of 2.45 Ga (x axis) against a tree calculated without a calibration on points 8, 7, 9 (y axis). (D), (E), and (F) were calculated as (A), (B), and (C) respectively, but employing a root of 3.8 Ga instead. In all scenarios the duplication that led to PsaA and PsaB is the oldest node after the root. Removing the oldest calibration constraints led to younger ages on a number of nodes, however the estimated age of the PsaA/PsaB duplication event (orange dot) is set in the early Archean in every scenario.