In contrast to most other reconstructions, particularly of P450 enzymes, we did not observe a general trend towards greater thermostability in the deeper nodes. Instead, specific lineages appeared to have lost and then sometimes developed thermostability de novo during CYP116B evolution (Figure , Supplementary Fig. ). P450-AX, -TT, and -TB were the most thermostable extant forms, in agreement with previous studies 41 with 15 T50 values of 54, 53, and 52 °C, respectively, over 10°C higher than those for P450-JT and -AT, forms that were markedly less thermostable and derived from organisms with generally lower optimal growth temperatures 41 . Amongst the ancestors, N3, which theoretically gave rise to mesophilic and uncharacterised extant CYP116B sequences, was the most stable with a 15 T50 of 51 °C (Table , Supplementary Fig. ).