Viral gain-of-function experiments uncover residues under diversifying selection in nature

Viral gain-of-function mutations are commonly observed in the laboratory; however, it is unknown whether those mutations also evolve in nature. We identify two key residues in the host recognition protein of bacteriophage lambda that are necessary to exploit a new receptor; both residues repeatedly evolved among homologs from environmental samples. Our results provide evidence for widespread host-shift evolution in nature and a proof of concept for integrating experiments with genomic epidemiology.