Spike-in quantification reveals biogeography, hotspots, deterministic assembly, and network stability in South China Sea bacterioplankton.

Marine bacterioplankton are critical for biogeochemical cycles and ecosystem health, but traditional relative abundance data fail to capture true microbial loads and their environmental relevance. Using spike-in calibrated absolute quantification and high-throughput sequencing, we investigated surface bacterioplankton communities across the open South China Sea (SCS). Our results showed strong spatial heterogeneity in absolute bacterial abundance. The northern SCS functioned as a biogeochemical hotspot for carbon, nitrogen, and sulfur cycling genes due to nutrient inputs from the Pearl River plume and adjacent coastal human activities. Community assembly was dominated by deterministic processes (homogeneous selection, 64.17%), indicating that environmental filtering largely controls community structure. Co-occurrence network analysis revealed contrasting stability regimes: the eastern SCS, with strong hydrodynamic mixing, supported robust networks, whereas the northern region exhibited high vulnerability, and the southern island-reef system depended heavily on keystone taxa, implying greater fragility under species loss. Despite taxonomic turnover, core metabolic functions showed high redundancy, providing a buffer against environmental perturbations. These findings demonstrate that absolute quantification is essential for identifying nutrient-enriched or eutrophication-sensitive hotspots, assessing regional ecosystem vulnerability, and guiding targeted environmental monitoring. We advocate integrating spike-in calibrated sequencing into routine marine environmental assessments to enable more accurate, mechanism-based evaluations of microbial responses to nutrient enrichment, anthropogenic disturbance, and climatic pressures in marginal seas.