Response to Tcherkez and Farquhar: Rubisco adaptation is more limited by phylogenetic constraint than by catalytic trade-off.

Rubisco is the primary entry point for carbon into the biosphere. It has been widely proposed that rubisco is highly constrained by catalytic trade-offs due to correlations between the enzyme's kinetic traits across species. In previous work, we have shown that the strength of these correlations, and thus the strength of catalytic trade-offs, have been overestimated due to the presence of phylogenetic signal in the kinetic trait data (Bouvier et al., 2021). We demonstrated that only the trade-offs between the Michaelis constant for CO and carboxylase turnover, and between the Michaelis constants for CO and O were robust to phylogenetic effects. We further demonstrated that phylogenetic constraints have limited rubisco adaptation to a greater extent than the combined action of catalytic trade-offs. Recently, however, our claims have been contested by Tcherkez and Farquhar (2021), who have argued that the phylogenetic signal we detect in rubisco kinetic traits is an artefact of species sampling, the use of rbcL-based trees for phylogenetic inference, laboratory-to-laboratory variability in kinetic measurements, and homoplasy of the C trait. In the present article, we respond to these criticisms on a point-by-point basis and conclusively show that all are unfounded. As such, we stand by our original conclusions. Namely, although rubisco kinetic evolution has been limited by biochemical trade-offs, these are not absolute and have been previously overestimated due to phylogenetic biases. Instead, rubisco adaptation has in fact been more limited by phylogenetic constraint.