Repetition and order of exposure to salinization and nutrient enrichment drive freshwater microbial community responses.

Biological systems face multiple stressors that impact biodiversity and ecosystem functions, with complex interactions that vary spatially and temporally leading to unpredictable outcomes. In freshwater ecosystems, benthic microbial communities underpin vital functions like decomposition and primary productivity, but are threatened by stressors such as salinization and nutrient enrichment, which are intensifying and increasingly co-occurring due to climate change. We experimentally tested how stressor repetition and different orders of stressor exposure affect freshwater benthic microbial community responses over time. Communities established on tiles in 1000 L open freshwater ponds established more than 10 years ago in the field were exposed to elevated salinity and nutrient enrichment, either once or repeatedly, independently or in combination, and under different orders. Repeated exposure to nutrient enrichment led to stronger functional changes than the single exposure, while repeated exposure to elevated salinity resulted in weaker changes compared to a single exposure. Critically, we found that the sequence in which stressors occurred was a major determinant of microbial responses, driving interaction outcomes in opposing directions. When exposure to nutrient enrichment preceded elevated salinity, gross primary productivity was halved and carbon metabolic rates increased by 50% compared to communities treated in the reverse order. This study is among the first in a complex, outdoor freshwater system to demonstrate that stressor sequence can strongly shape multiple stressor effects, highlighting the order of stressor exposure as a key but often overlooked dimension of global change ecology. These findings suggest that microbial functions, including productivity and carbon cycling, will fluctuate more dramatically as stressors increasingly occur in different sequences under global change.