Sobol, M.; Ane, C.; McMahon, K.; Kacar, B.

Life s transition into new habitats has profoundly shaped its trajectory through a reciprocal relationship with Earth s changing environments. To understand how ancient metabolisms co-evolved with these environments, it is essential to identify the ecological and evolutionary processes that structured their underlying genetic networks. Here, we focus on nitrogen (N2) fixation, one of life s most critical and ancient metabolisms, to investigate the drivers of complexity in its associated genetic network. We used a large-scale comparative genomics framework to construct a comprehensive catalog of today s N2 fixation-associated genes and assessed their distribution across diverse microbial genomes and environmental backgrounds. Genomes enriched in N2 fixation genes generally have larger genome sizes, broader metabolic capabilities, wider habitat ranges, and are predominantly associated with mesophilic and aerobic lifestyles. Evolutionary reconstructions reveal a pattern of early gene gains in ancestral diazotrophs followed by lineage specific gene losses in later diverging taxa, suggesting evolutionary trade-offs shaped by changing environments. These findings demonstrate that the evolution of N2 fixation has been intertwined with the restructuring of its genetic network, driven by feedbacks between microbial evolution and changing environmental and ecological conditions.