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Nitrogen fixation, the process that converts atmospheric dinitrogen gas (N2) into biologically reactive ammonia (NH3), is an important biogeochemical process that influences the bioavailability of nitrogen (N) on a global scale. Diazotrophs, the highly specialized microbes that carry out this energy-demanding process, populate a variety of habitats in marine, terrestrial, and freshwater ecosystems. However, the patterns and drivers of diazotroph biodiversity in freshwater and coastal habitats remain poorly studied compared to open ocean and terrestrial ecosystems. The molybdenum dependent nitrogenase is encoded by three protein subunits the NifH, NifD and NifK. The nifH gene is widely used as a marker for nitrogen fixation, however, the presence of pseudo-nifH genes (eg, homologs of the nifH that have been adapted for other functions) can greatly overestimate the presence of nitrogen fixation, especially in …