Significance The mutualistic association between legumes and rhizobia has ecological and agronomical relevance because of its contribution to the global nitrogen cycle by biological nitrogen fixation. Legumes from the Inverted Repeat Lacking Clade (IRLC) impose irreversible differentiation to their endosymbionts through nodule-specific cysteine-rich (NCR) peptides. This study indicates that NCR gene families evolved via different pathways in IRLC species, in which their size and composition directly impacted the morphotype of their bacterial partners. The positive correlation between the diversity of NCRs with their physiological effects on bacteria provides a better understanding of the multiple roles played by this large family in nodule functioning.