Drift, selection and recombination bias drive the evolution of a non-ribosomal peptide

Non-ribosomal peptides play important roles in microbial ecology and medicine, for example as toxins and antibiotics such as penicillin. Like ordinary proteins, non-ribosomal peptides are subject to evolutionary change, which is driven by rampant recombinations of their synthetases. The evolutionary dynamics and causes of non-ribosomal peptide diversification are not well understood, because recombinations complicate phylogenetic analysis and statistical tests for natural selection and drift. Here we study the non-ribosomal GameXPeptides, which entomopathogenic bacteria use to kill their insect prey. We use recombination-aware phylogenetics, ancestral sequence reconstruction and statistical analysis of historical recombination patterns to retrace the evolution of GameXPeptides. This uncovers dozens of new GameXPeptides, including striking patterns of structural convergence. We provide statistical and experimental evidence that this diversity is shaped by selection, but also that GameXPeptides experience substantial genetic drift and biased recombination rates that limit the space of accessible peptide variants. Our work illuminates the complex interplay of adaptive and non-adaptive evolutionary forces acting on some of the most versatile chemical weapons in nature.