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

From its earliest beginnings, life's expansion into new habitats has been profoundly shaped by its reciprocal interactions with a changing Earth. To understand the evolution of life's metabolic engines, we must therefore uncover the ecological and evolutionary processes that shaped their underlying genes and networks. Here, we focus on nitrogen (N₂) fixation, one of life's most critical metabolisms, and investigate the ecological and evolutionary drivers of complexity within its associated gene machinery. We used a large-scale comparative genomics framework to construct a comprehensive catalog of extant N₂ fixation-associated genes and to assess their distribution across diverse microbial genomes and environments. Genomes enriched in N₂ fixation genes are generally larger, encode broader metabolic capabilities, span wider habitat ranges, and are predominantly associated with mesophilic and aerobic lifestyles. Evolutionary reconstructions reveal early gene gains in ancestral diazotrophs followed by lineage-specific gene losses in later-diverging taxa, consistent with evolutionary trade-offs driven by changing environments. Together, these findings show that the evolution of N₂ fixation is tightly intertwined with the composition and organization of its supporting gene machinery, reflecting feedbacks between genome evolution and shifting environmental and ecological contexts.