1.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. 2.Eight European semi-natural shrubland ecosystems were exposed to warming and drought manipulations. One field site was additionally exposed to elevated atmospheric CO2. We evaluate 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 lab-based studies. 3.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 mineralization by soil microorganisms enabled to determine the long-term effect of climate change on microbial carbon uptake kinetics and turnover. 4.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.

PLANT_ISOTOPESIsotopic signal (13C and 15N) of plant responses to precipitation across the INCREASE experimental network (Data in Box 1A from Andresen et al., 2018).14C_RESPIRATIONValues of the 14C signature (natural abundance) of CO2 in soil efflux samples, collected at the Peaknaze site (UK) (Data in Box 2B from Andresen et al., 2018).13CPLFA_DKResults of a 13C-CO2 pulse experiment conducted at the Brandbjerg site (Denmark). Values of recovery of 13C (% from added 13C) in PLFA of soil Gram-negative bacteria, at different times since 13C pulse (Data in Box 3C from Andresen et al., 2018).14C_kineticsResults of an experiment on microbial mineralisation of carbon compounds, using low-molecular weight carbon substrates labelled with 14C. Values in this file show the exponential decay kinetics for 14C-CO2 evolution during microbial 14C substrate mineralisation (Data in Box 4A from Andresen et al., 2018).14C_halflifeResults of an experiment on microbial mineralisation of carbon compounds, using low-molecular weight carbon substrates labelled with 14C. Values correspond to half-life of added 14C-sugars in the soil solution of samples collected across the INCREASE network. Mean annual temperature of each site indicated (Data in Box 4B from Andresen et al., 2018).CALLUNA_ISOTOPESValues of δ13C in Calluna vulgaris leaves and annual precipitation of year before sample collection (Data in Box 1B from Andresen et al., 2018).13CPLFA_UKResults of a 13C-CO2 pulse experiment conducted along a gradient of soil moisture at the Clocaenog site (UK). Values correspond to 13C content in different phospholipid-derived fatty-acid methyl esters (FAMEs) extracted from soils after the 13C-pulse (Data in Box 3B from Andresen et al., 2018).