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Long-term climate change experiments are extremely valuable for studying ecosystem responses to environmental change. Examination of the vegetation and the soil should be non-destructive to guarantee long-term research. In this paper, we review field methods using isotope techniques for assessing carbon dynamics in the plant–soil–air continuum, based on recent field experience and examples from a European climate change manipulation network. Eight European semi-natural shrubland ecosystems were exposed to warming and drought manipulations. One field site was additionally exposed to elevated atmospheric CO2. We discuss the isotope methods that were used across the network to evaluate carbon fluxes and ecosystem responses, including: (1) analysis of the naturally rare isotopes of carbon (13C and 14C) and nitrogen (15N); (2) use of in situ pulse labelling with 13CO2, soil injections of 13C- and 15N-enriched substrates, or continuous labelling by free air carbon dioxide enrichment (FACE) and (3) manipulation of isotopic composition of soil substrates (14C) in laboratory-based studies. The natural 14C signature of soil respiration gave insight into a possible long-term shift in the partitioning between the decomposition of young and old soil carbon sources. Contrastingly, the stable isotopes 13C and 15N were used for shorter-term processes, as the residence time in a certain compartment of the stable isotope label signal is limited. The use of labelled carbon-compounds to study carbon mineralisation by soil micro-organisms enabled to determine the long-term effect of climate change on microbial carbon uptake kinetics and turnover. Based on the experience with the experimental work, we provide recommendations for the application of the reviewed methods to study carbon fluxes in the plant–soil–air continuum in climate change experiments. 13C-labelling techniques exert minimal physical disturbances, however, the dilution of the applied isotopic signal can be challenging. In addition, the contamination of the field site with excess 13C or 14C can be a problem for subsequent natural abundance (14C and 13C) or label studies. The use of slight changes in carbon and nitrogen natural abundance does not present problems related to potential dilution or contamination risks, but the usefulness depends on the fractionation rate of the studied processes. Comisión Europea-FTP7- ICT- 2008-1/227628 Gobierno español-CGL2016-79835-P Gobierno catalán-SGR2014-274 European Research Council (ERC)-SyG-610028 IMBALANCE Hungarian Scientific Research Fund-OTKA K112576

Litter decomposition is a key process for carbon and nutrient cycling in terrestrial ecosystems and is mainly controlled by environmental conditions, substrate quantity and quality as well as microbial community abundance and composition. In particular, the effects of climate and atmospheric nitrogen (N) deposition on litter decomposition and its temporal dynamics are of significant importance, since their effects might change over the course of the decomposition process. Within the TeaComposition initiative, we incubated Green and Rooibos teas at 524 sites across nine biomes. We assessed how macroclimate and atmospheric inorganic N deposition under current and predicted scenarios (RCP 2.6, RCP 8.5) might affect litter mass loss measured after 3 and 12 months. Our study shows that the early to mid-term mass loss at the global scale was affected predominantly by litter quality (explaining 73% and 62% of the total variance after 3 and 12 months, respectively) followed by climate and N deposition. The effects of climate were not litter-specific and became increasingly significant as decomposition progressed, with MAP explaining 2% and MAT 4% of the variation after 12 months of incubation. The effect of N deposition was litter-specific, and significant only for 12-month decomposition of Rooibos tea at the global scale. However, in the temperate biome where atmospheric N deposition rates are relatively high, the 12-month mass loss of Green and Rooibos teas decreased significantly with increasing N deposition, explaining 9.5% and 1.1% of the variance, respectively. The expected changes in macroclimate and N deposition at the global scale by the end of this century are estimated to increase the 12-month mass loss of easily decomposable litter by 1.1-3.5% and of the more stable substrates by 3.8-10.6%, relative to current mass loss. In contrast, expected changes in atmospheric N deposition will decrease the mid-term mass loss of high-quality litter by 1.4-2.2% and that of low-quality litter by 0.9-1.5% in the temperate biome. Our results suggest that projected increases in N deposition may have the capacity to dampen the climate-driven increases in litter decomposition depending on the biome and decomposition stage of substrate.

Long‐term climate change experiments are extremely valuable for studying ecosystem responses to environmental change. Examination of the vegetation and the soil should be non‐destructive to guarantee long‐term research. In this paper, we review field methods using isotope techniques for assessing carbon dynamics in the plant–soil–air continuum, based on recent field experience and examples from a European climate change manipulation network.\ud Eight European semi‐natural shrubland ecosystems were exposed to warming and drought manipulations. One field site was additionally exposed to elevated atmospheric CO2. We discuss the isotope methods that were used across the network to evaluate carbon fluxes and ecosystem responses, including: (1) analysis of the naturally rare isotopes of carbon (13C and 14C) and nitrogen (15N); (2) use of in situ pulse labelling with 13CO2, soil injections of 13C‐ and 15N‐enriched substrates, or continuous labelling by free air carbon dioxide enrichment (FACE) and (3) manipulation of isotopic composition of soil substrates (14C) in laboratory‐based studies.\ud The natural 14C signature of soil respiration gave insight into a possible long‐term shift in the partitioning between the decomposition of young and old soil carbon sources. Contrastingly, the stable isotopes 13C and 15N were used for shorter‐term processes, as the residence time in a certain compartment of the stable isotope label signal is limited. The use of labelled carbon‐compounds to study carbon mineralisation by soil micro‐organisms enabled to determine the long‐term effect of climate change on microbial carbon uptake kinetics and turnover.\ud Based on the experience with the experimental work, we provide recommendations for the application of the reviewed methods to study carbon fluxes in the plant–soil–air continuum in climate change experiments. 13C‐labelling techniques exert minimal physical disturbances, however, the dilution of the applied isotopic signal can be challenging. In addition, the contamination of the field site with excess 13C or 14C can be a problem for subsequent natural abundance (14C and 13C) or label studies. The use of slight changes in carbon and nitrogen natural abundance does not present problems related to potential dilution or contamination risks, but the usefulness depends on the fractionation rate of the studied processes.

International audience; Field-scale experiments simulating realistic future climate scenarios are important tools for investigating the effects of current and future climate changes on ecosystem functioning and biogeochemical cycling. We exposed a semi-natural Danish heathland ecosystem to elevated atmospheric carbon dioxide (CO2), warming, and extended summer drought in all combinations. Here, we report on the short-term responses of the nitrogen (N) cycle after two years of treatments. Elevated CO2 significantly affected above-ground stoichiometry by increasing the carbon to nitrogen (C/N) ratios in the leaves of both co-dominant species (Calluna vulgaris and Deschampsia flexuosa), as well as the C/N ratios of Calluna flowers and by reducing the N concentration of Deschampsia litter. Below-ground, elevated CO2 had only minor effects, whereas warming increased N turnover, as indicated by increased rates of microbial NH4+-N consumption, gross mineralization, potential nitrification, denitrification and N2O emissions. Drought reduced below-ground gross N mineralization and decreased fauna N mass and N mineralization. Leaching was unaffected by treatments but was significantly higher across all treatments in the second year than in the much drier first year indicating that ecosystem N loss is highly sensitive to changes and variability in amount and timing of precipitation. Interactions between treatments were common and although some synergistic effects were observed, antagonism dominated the interactive responses in treatment combinations, i.e. responses were smaller in combinations than in single treatments. Nonetheless, increased C/N ratios of photosynthetic tissue, decreased litter N production, and decreased fauna N mineralization prevailed in the full treatment combination indicating reduced N turnover in the simulated future climate scenario, which could act to reduce the potential growth response of plants to elevated atmospheric CO2 concentration. In conclusion, effects observed in single-factor studies should be evaluated with caution. Multi-factor climate experiments are needed for improving realistic estimation of the combined ecosystem responses to future climate changes.