1. Atmospheric nitrogen (N) deposition, generally, has been simulated through a single or relatively few N applications per year for its ecological effect study. Despite the importance of timing in ecosystem processes, ecological experiments with more realistic N addition frequencies are rare.
2. We employed a novel design with typical twice (2X) vs. atypical monthly (12X) N applications per year to explore effects of N addition frequency on above- and below-ground biodiversity and function.
3. Each year, several response variables from either belowground or aboveground growth, N status and cycling, or plant and bacterial diversity differed as a result of N addition frequency. BNPP showed a large frequency effect in the relatively moist year but not in the dry year. Nitrogen addition decreased root growth in the monthly relative to the biannual applications, which could be highly consequential for predicting changes in global carbon and nitrogen cycling. Simulated N deposition tended to perturb biodiversity, but it is noteworthy that 12X applications that spread N deposition more evenly through a year have much less negative impacts on plant and bacterial diversities than 2X amendments per year. Soil N mineralization rate in year 6 was much lower when N additions were monthly compared with a biannual amendment, especially when simulated N deposition was high.
4. We have established that amendment frequency matters for understanding ecosystem response to N deposition. Experiments that more closely mimic the anthropogenic process of N deposition are needed to best assess ecosystem and potential global biogeochemical changes.

The total plant biomass and species number were used to estimate ANPP and plant biodiversity. Belowground net primary productivity (BNPP) was estimated by an in-growth core method. Soil temperature was automatically recorded at 1.5 h intervals using the iButton digital temperature logger (DS1922L, Maxim Integrated, CA USA) that was buried at 10-cm depth in the soil of each plot. Soil pH was measured in a 1:2.5 soil: water (w/v) solution by a pH meter (FE20-FiveEasy, Switzerland). The concentrations of NH₄⁺-N, NO₃^--N and dissolved N in soil extract were analysed with an autoanalyzer (AA3, SEAL Analytical GmbH, Hanau, Germany). Soil DON concentration was calculated by the equation: DON=DN/0.871- (NH₄⁺-N + NO₃^--N). Soil microbial biomass N (MBN) was measured using the chloroform fumigation-extraction method. Soil potential net N mineralization rate (or net nitrification rate) was estimated as the difference between initial and final inorganic N (or NO₃^--N) during 30-day incubation under standard conditions. For more methods and details, please refer to the paper..