# Long-Term Nitrogen Addition Eliminates the Cooling Effect on Climate in a Temperate Peatland
## 4 Materials and Methods
### Measuring Greenhouse Gas Fluxes and Environmental Parameters  
Greenhouse gas fluxes were measured twice a month from May to September in 2019, following 12 years of continuous N fertilization. A PVC collar (14 cm high, 25 cm in diameter) with a groove for chamber placement was inserted into each plot to a depth of 5 cm in October 2017. After two years, any root damage and peat disturbance caused by the collar installation should have been restored. Greenhouse gas fluxes were measured on a sunny day from 10:00 to 15:00 local time using a portable greenhouse gas analyzer (LGR-915-0011, Los Gatos Research Inc, San Jose, CA, USA), which was connected to a transparent or a dark chamber (25 cm in diameter and 50 cm in height), fitted into the groove of the PVC collar. The dark chamber blocked light from entering, preventing photosynthesis during the measurement. As a result, the CO2 flux measured by the dark chamber represented ecosystem respiration (ER). In contrast, light could penetrate the transparent chamber, allowing photosynthesis to occur during the measurement. Consequently, the CO2 flux measured by the transparent chamber represented net ecosystem CO2 exchange (NEE). There was only about a 1-min break between measurements from the transparent chamber (for NEE) and the dark chamber (for ER). Therefore, it was reasonable and accurate to estimate gross primary productivity (GPP) based on the NEE and ER measurements taken at the same plot [67]. To ensure an airtight connection with the chamber, each PVC collar was supplied with water sealing in the groove during the measurements. A fan was fixed on top of the chambers to mix and cool down the air inside. The aluminum foil was used to cover the whole chamber exterior to reflect sunlight for heat reduction for the dark chamber. 180 consecutive values of CO2 concentration were recorded at 1 Hz time frequency during each 3-min measurement period. The chamber was ventilated for approximately 1 min after each measurement to ensure that the CO2 concentration returned to ambient levels before beginning the next measurement. CH4 flux and ER were measured simultaneously. For the specific calculation method of greenhouse gas flux, please refer to another article by the author [68]. During each gas sampling campaign, soil temperatures at 5 and 20 cm below the moss surface were measured using a Delta TRAK digital thermometer, while the soil moisture (SM) was assessed with a Stevens moisture meter (TZS-1K, Zhejiang Top Cloud-Agri Technology Co., Ltd., Hangzhou, China) in each plot. Additionally, the photosynthetically active radiation (PAR) in each plot was measured using an Apogee quantum meter (MQ-200, Apogee Inc., Logan, UT, USA) simultaneously with the NEE sampling. NEE was accepted when PAR \(\geq\) 1000 \(\upmu\)mol photons m\({}^{-2}\) s\({}^{-1}\). The water table depth (WTD) was also measured in PVC tubes installed at each plot during NEE measurements. The vegetation and plant cover within each collar were assessed by visually estimating the percentage cover of graminoids, shrubs, and _Sphagntum_ mosses based on their vertical projection area. All the estimations were carried out by the same observer to minimize between-observer errors. To assure accuracy, vertical photographs of each collar were taken from 20 cm above the ground, and vegetation cover estimates were corrected in the laboratory if necessary. Furthermore, peat pore water from a depth of approximately 20 cm in each sampling plot was collected to measure dissolved organic carbon (DOC). The DOC concentration was analyzed using a TOC analyzer (TOC-LCPH/TN, Shimadzu Inc., Kyoto, Japan). Before measurement, the peat pore water was filtered using an oil-free diaphragm vacuum pump and a 0.45 um micro-filtration membrane. Soil samples were collected at the end of July 2019, prior to fertilization and in the absence of recent precipitation, to avoid confounding effects from nutrient leaching caused by heavy rainfall.