Ecological constraints and evolutionary trade-offs shape nitrogen fixation across habitats

From its earliest beginnings, lifes expansion into new habitats has been profoundly shaped by its reciprocal interactions with Earths changing environments. Understanding how ancient metabolisms co-evolved with their environments requires uncovering the ecological and evolutionary processes that structured the functionally linked genes and networks underlying these metabolisms. Here, we focus on nitrogen (N2) fixation, one of lifes most critical metabolisms, and investigate the drivers of complexity in its associated gene machinery today. We used a large-scale comparative genomics framework to construct a comprehensive catalog of extant 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 composition and organization of the genes supporting the overarching N2 metabolism, driven by feedback between genome evolution and shifting environmental and ecological conditions.