Reconstructing ancient protein alphabets: what can we truly infer?

The canonical amino acid alphabet has remained remarkably stable since life's early stages, yet the factors that shaped its emergence remain debated. Early views emphasized prebiotic availability and the expansion of metabolic pathways, but recent advances (particularly from protein biophysics and deep phylogenetics) have brought new perspectives to this question. Together, these views agree that the canonical alphabet emerged from a chemically restricted repertoire and was gradually tightened by metabolic innovation and selection for foldable, functional proteins. However, these approaches can yield inconsistent trajectories, underscoring how much remains unresolved. Here, we compare insights from four lines of evidence, highlight their limitations, and argue that our chronology of amino acid recruitment should be based on where the approaches converge.