Changes in natural 15N abundance highlight warming-induced stimulation of soil nitrate losses by coupled nitrification–denitrification in an old-growth montane forest

Ambient soil nitrogen cycle with six soil nitrogen pools (bulk nitrogen, ammonium, nitrate, microbial biomass nitrogen, fine root nitrogen, and nitrogen loss). Coloring of the pools indicates positive, negative or neutral δ 15 N values. Long term-soil warming causes the plant-soil N cycle to open, with increased δ 15 N values of soil ammonium (+4.0‰) and fine roots (+0.8‰). While roots reflect inorganic soil N taken up, increased ammonium δ 15 N suggests increased nitrification, the product of which (nitrate) is lost more by denitrification than by nitrate leaching. Soil microbes (MBN) are drivers of mineralization, nitrification and denitrification (source of inorganic N) but also a sink of organic N, ammonium, and nitrate. • Long-term soil warming increases δ 15 N in soil ammonium and roots. • Warming-driven 15 N enrichment in roots and NH 4 + reveals enhanced nitrification–denitrification. • Coupled plant–soil δ 15 N shows denitrification dominates long-term N losses. Climate warming alters biogeochemical cycles, especially in high-altitude forests where warming accelerates soil organic matter decomposition and CO 2 efflux. Faster nitrogen (N) mineralization can enhance N availability to plants but may also increase N losses if soil microbial N use efficiency declines. However, long-term data on soil N loss mechanisms remain scarce. Key N cycling processes affect the natural 15 N: 14 N isotope ratio (δ 15 N) differentially, with (de)nitrification yielding 15 N-depleted products and leaving residual pools 15 N-enriched. We investigated belowground N cycling after 14 years of soil warming (+4 °C) in a temperate old-growth forest in Achenkirch, Austria, by measuring δ 15 N values in belowground N pools (root N, bulk soil N, microbial biomass N, ammonium, nitrate) through isotope ratio mass spectrometry. Warming had no effect on δ 15 N of bulk soil N, microbial biomass N, and nitrate, but significantly increased δ 15 N in root N (−5.0 to −4.1‰) and in soil ammonium (−2.9 to 1.1‰). Root δ 15 N, reflecting inorganic soil N, indicates that warming-induced N losses caused 15 N enrichment of inorganic soil N. Elevated ammonium δ 15 N points to increased rates of nitrification, while nitrate δ 15 N patterns imply denitrification (60–65% of nitrate sink) exceeding leaching as the main loss pathway, which aligns with available field observations. Coupled plant–soil δ 15 N analysis thus revealed decadal warming-driven changes in N cycling and identified coupled nitrification–denitrification as a key pathway of soil N loss, which is otherwise difficult to measure directly.