Coral-associated nitrogen fixation rates and diazotrophic diversity on a nutrient-replete equatorial reef.

The role of diazotrophs in coral physiology and reef biogeochemistry remains poorly understood, in part because N2 fixation rates and diazotrophic community composition have only been jointly analyzed in the tissue of one tropical coral species. We performed field-based 15N2 tracer incubations during nutrient-replete conditions to measure diazotroph-derived nitrogen (DDN) assimilation into three species of scleractinian coral (Pocillopora acuta, Goniopora columna, Platygyra sinensis). Using multi-marker metabarcoding (16S rRNA, nifH, 18S rRNA), we analyzed DNA- and RNA-based communities in coral tissue and skeleton. Despite low N2 fixation rates, DDN assimilation supplied up to 6% of the holobiont's N demand. Active coral-associated diazotrophs were chiefly Cluster I (aerobes or facultative anaerobes), suggesting that oxygen may control coral-associated diazotrophy. Highest N2 fixation rates were observed in the endolithic community (0.20 µg N cm-2 per day). While the diazotrophic community was similar between the tissue and skeleton, RNA:DNA ratios indicate potential differences in relative diazotrophic activity between these compartments. In Pocillopora, DDN was found in endolithic, host, and symbiont compartments, while diazotrophic nifH sequences were only observed in the endolithic layer, suggesting a possible DDN exchange between the endolithic community and the overlying coral tissue. Our findings demonstrate that coral-associated diazotrophy is significant, even in nutrient-rich waters, and suggest that endolithic microbes are major contributors to coral nitrogen cycling on reefs.