Nitrogen pulse drives a prolonged microcystin event by increasing the proportion of toxigenic Microcystis genotypes

Microcystins (MCs) pose a major risk to drinking water safety, yet their production drivers remain poorly understood. We conducted a year-long, high-frequency monitoring program in a large subtropical reservoir to identify environmental triggers and regulatory mechanisms of Microcystis blooms and toxin production. Microcystis blooms occurred in May and July, followed by a six-month MC contamination event from August to January. An extreme nitrogen pulse was detected on August 1 and was followed by a sharp increase in MC concentrations that peaked in October, suggesting that the N pulse was the primary trigger of the prolonged contamination event. However, MC concentrations were negatively correlated with chlorophyll a, Microcystis abundance, and absolute mcyB gene copies, revealing a "MC production paradox." Notably, MC concentrations also exhibited an inverse temporal relationship with phosphorus (TP), despite TP being positively associated with mcyG-carrying Microcystis. Spearman correlation showed that MC levels were strongly linked to the proportion of mcyG-carrying Microcystis (mcyG/Micro) and phycocyanin. Multiple regression (adjusted R² = 0.693) identified mcyG/Micro and phycocyanin as the strongest positive predictors, while water temperature influenced competitive dynamics and toxigenic potential. Spatial heterogeneity was evident, with strong genotype-toxin relationships occurring near the nutrient inlet but being absent downstream. These findings challenge biomass-based risk assessment and highlight the need for mechanism-driven monitoring. We propose a practical monitoring framework integrating mcyG/Micro and phycocyanin as complementary indicators, providing a monitoring tool for more accurate and timely early warning of cyanotoxin hazards in drinking water reservoirs.