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Mutualism is of fundamental importance in ecosystems. Which factors help to keep the relationship mutually beneficial and evolutionarily successful is a central question. We addressed this issue for one of the most significant mutualistic interactions on Earth, which associates plants of the leguminosae family and hundreds of nitrogen (N 2 )-fixing bacterial species. Here we analyze the spatio-temporal dynamics of fixers and non-fixers along the symbiotic process in the Cupriavidus taiwanensis-Mimosa pudica system. N 2 -fixing symbionts progressively outcompete isogenic non-fixers within root nodules, where N 2 -fixation occurs, even when they share the same nodule. Numerical simulations, supported by experimental validation, predict that rare fixers will invade a population dominated by non-fixing bacteria during serial nodulation cycles with a probability that is function of initial inoculum, plant population size and nodulation cycle length. Our findings provide insights into the selective forces and ecological factors that may have driven the spread of the N 2 -fixation mutualistic trait.