Isotopic methods for non-destructive assessment of carbon dynamics in shrublands under long-term climate change manipulation

© 2018 The Authors. Methods in Ecology and Evolution © 2018 British Ecological Society This version available http://nora.nerc.ac.uk/519018/ NERC has developed NORA to enable users to access research outputs wholly or partially funded by NERC. Copyright and other rights for material on this site are retained by the rights owners. Users should read the terms and conditions of use of this material at http://nora.nerc.ac.uk/policies.html#access This document is the author’s final manuscript version of the journal article, incorporating any revisions agreed during the peer review process. There may be differences between this and the publisher’s version. You are advised to consult the publisher’s version if you wish to cite from this article. The definitive version is available at http://onlinelibrary.wiley.com/ Article (refereed) - postprint This is the peer reviewed version of the following article: Andresen, Louise C.; Dominguez, Maria T.; Reinsch, Sabine; Smith, Andrew R.; Schmidt, Inger K.; Ambus, Per; Beier, Claus; Boeckx, Pascal; Bol, Roland; de Dato, Giovanbattista; Emmett, Bridget A.; Estiarte, Marc; Garnett, Mark H.; Kröel-Dulay, György; Mason, Sharon L.; Nielsen, Cecilie S.; Peñuelas, Josep; Tietema, Albert. 2018. Isotopic methods for non-destructive assessment of carbon dynamics in shrublands under long-term climate change manipulation. Methods in Ecology and Evolution, 9 (4). 866-880, which has been published in final form at https://doi.org/10.1111/2041-210X.12963 This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. Contact CEH NORA team at [email protected] The NERC and CEH trademarks and logos (‘the Trademarks’) are registered trademarks of NERC in the UK and other countries, and may not be used without the prior written consent of the Trademark owner. Accepted Article This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/2041-210X.12963 This article is protected by copyright. All rights reserved. DR. LOUISE C. ANDRESEN (Orcid ID : 0000-0002-4890-889X) DR. SABINE REINSCH (Orcid ID : 0000-0003-4649-0677) Article type : Review Handling editor: Prof. Robert Freckleton Corresponding author mail id : [email protected] Isotopic methods for non-destructive assessment of carbon dynamics in shrublands under long-term climate change manipulation L.C. Andresen1*), M.T. Domínguez2 **), S. Reinsch2, A. R. Smith4,2, I.K. Schmidt3, P. Ambus3, C. Beier3, P. Boeckx6, R. Bol7,1, G. de Dato8**), B.A. Emmett2, M. Estiarte11,12, M.H. Garnett13, G. Kröel-Dulay14, S.L. Mason1, C.S. Nielsen15, J. Peñuelas11,12, and A. Tietema1. Running head: Isotope techniques for ecosystem C science Tweet: The pros and cons of carbon assessment methods using isotopes across climate change experiments in shrublands. Affiliations: 1Institute for Biodiversity and Ecosystem Dynamics (IBED), University of Amsterdam, NL; 2Centre for Ecology and Hydrology, Bangor, UK; 3University of Copenhagen, Dept. Geosciences and Natural Resource Management, DK; 4School of Environment, Natural Resources & Geography, Bangor University, Bangor, UK. 5Institute of Forest Ecology, BOKU, AU; 6Isotope Bioscience Laboratory - ISOFYS, Ghent University, BE; 7Institute of Bio- and Geosciences, IBG-3: Agrosphere, Forschungszentrum Jülich, Jülich, DE; 8Department for Innovation in Biological, Agro-food and Forest systems, University of Tuscia, Viterbo, IT; 9Forest & Nature Lab, Ghent University, BE; 10National Research Accepted Article This article is protected by copyright. All rights reserved. Council, Institute of Biometeorology, Sassari, IT; 11CSIC, Global Ecology Unit CREAFCSIC-UAB, Cerdanyola del Vallès (Catalonia), SP; 12CREAF, Cerdanyola del Vallès, Barcelona, Catalonia, SP; 13NERC Radiocarbon Facility, Scottish Enterprise Technology Park, Rankine Avenue, East Kilbride, Scotland, UK; 14Centre for Ecological Research, Institute of Ecology and Botany, Hungarian Academy of Sciences, HU; 15Department of Forest Ecology and Management, Swedish University of Agricultural Sciences, SE. *) Current address: Department of Earth Sciences, University of Gothenburg, PO Box 460, 405 30 Göteborg, SE. **) Current address: Department of Crystallography, Mineralogy and Agricultural Chemistry, Edaphology Unit, University of Seville, SP (Corresponding author). ***) Current address: Council for Agricultural Research and Economics (CREA) - Research Centre for Forestry and Wood, IT. (giovanbattista[email protected].it). Summary 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 nondestructive 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. Accepted Article This article is protected by copyright. All rights reserved. 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. Key-words: warming; drought; bomb-C; FACE; pulse-labelling; stable isotopes; 14C Introduction Global climate change scenarios predict that increased greenhouse gas (e.g. CO2, CH4 and N2O) concentrations in the atmosphere will alter the periodicity and magnitude of drought events and will increase mean global temperatures by approximately 0.2 °C per decade (IPCC 2013). For the European continent this will manifest as drier summers in the South and increased precipitation in the North (IPCC 2013). Elucidating the consequences of such Accepted Article This article is protected by copyright. All rights reserved. atmospheric changes for biogenic carbon fluxes is one of the main challenges for the scientific community. Some models have predicted a positive feedback to climate change, resulting from higher increases in respiratory fluxes from ecosystems (e.g. carbon release through soil respiration) than in net primary productivity, which would lead to further increases in atmospheric CO2 (Friedlingstein et al. 2006; Denman et al. 2007). To assess the likelihood of this positive feedback, experimental studies that analyse the long-term adaptations of ecosystem carbon fluxes to climate change are critically needed. However, climate change experiments are often conducted at short or medium time scales due to funding constraints, or due to the limited life-span of the experimental plots, as repeated removal of samples often leads to disturbances and experimental artefacts in the studied system. Hence, there is a necessity for the maintenance of long-term experiments using non-destructive methods. Carbon fluxes through the plant-soil-air continuum play a central role in soil carbon cycling (Zak et al. 2000; Phillips et al. 2006). Consequently, aboveground to belowground fluxes might largely determine carbon emissions from ecosystems under the different climate change scenarios (Chapin et al. 2009). Stable carbon isotope studies can give important insights into carbon fluxes through the plant-soil-air continuum with the minimal disturbance to the system. The isotopic carbon composition of compartments in this continuum is a result of the different isotope fractionation processes along the pathway from CO2 fixation by plants to carbon allocation to soil (reviewed in Brüggemann et al. 2011). Thus, the analysis of the natural abundance of carbon isotopes in these compartments can give information about some processes related to photosynthesis and carbon losses through plant or soil respiration. In addition, in-situ pulse labelling with the heavy stable carbon isotope (13C) is a powerful tool to analyse short-term dynamics of carbon allocation to the soil with high resolution (Högberg et al. 2008; Epron et al. 2012; Reinsch & Ambus 2013). The application of these isotopic Accepted Article This article is protected by copyright. All rights reserved. methods can therefore provide unique information about aboveground-belowground linkages and their alterations in response to climate changes. In order to investigate long-term effects of climate change on shrubland ecosystems, an experimental network was established across Europe (the INCREASE network). Studying the response of shrublands to climate change is important, since they are representative ecosystems in Mediterranean and North European countries, where they play an important ecological role in preserving biodiversity (Wessel et al. 2004). In addition, land area covered by shrublands has dramatically decreased in temperate Europe during the past century, due to land use changes, increased pollution and eutrophication, and climate change (Fagúndez 2013). In Mediterranean regions, however, shrublands have increased their extension due to land abandonment (Fagúndez 2013). Within the climate change network, common non-destructive methods were used across sites to ensure the comparison of treatment effects across different climatic regions. Evaluating the impact of climate change treatments on shrubland carbon dynamics was one of the main objectives of this experimental network, and thus a range of methodologies to quantify and trace distinct carbon pools and their fluxes have been applied since 1999. Priority was given to those techniques that minimise disturbances to vegetation and soil to guarantee long-term research. Here, we review isotope methods that have been applied across this climate change experimental network to study ecosystem carbon dynamics in the plant-soil-air continuum. In particular, we focus on methodologies that: 1) analyse the abundance of naturally rare isotopes of carbon (13C and 14C) and nitrogen (15N) in the different ecosystem compartments, 2) trace experimentally-induced changes in the isotopic signatures to assess rhizodeposition utilisation by soil biota, and 3) manipulate and trace the isotopic composition of C-compounds to analyse C mineralisation by soil microorganisms in laboratory studies. Along-side the Accepted Article This article is protected by copyright. All rights reserved. methods, data from the field studies are presented as accompanying illustrative boxes, and practical recommendations for the applications of these methodologies at large-scale climate change experiments are outlined in Table 1. The experimental climate change network INCREASE The experimental network for the study of climate change impacts on European shrublands (INCREASE, ‘An Integrated Network on Climate Research Activities on Shrubland Ecosystems’) was established in 1998. The network is comprised of eight shrublands situated across a natural temperature gradient of mean annual temperature from c. 8 °C in the North to c. 16 °C in the South, and a rainfall gradient ranging from 510 mm to 1741 mm from East to West (Fig. 1). These sites represent Continental, Atlantic and Mediterranean shrublands. At each site, whole-ecosystem warming and drought treatments were applied in triplicates of 20 m2 plots, by using automated retractable curtain constructions (see Beier et al. 2004 and Mikkelsen et al. 2008 for a full description). At one of the Danish sites (DK-BRA), a FACE treatment was installed, and combinations of the climate treatments were established and resulted in a plot size of 9 m2. Climatic conditions at the plot level (air temperature, humidity, soil temperature and moisture) were recorded in half-hour or hourly intervals, and main carbon pools and fluxes have been periodically monitored, including aboveground plant biomass (Kröel-Dulay et al. 2015), litter production, soil respiration and net ecosystem carbon exchange (Beier et al. 2008; Lellei-Kovács et al. 2016). Methodologies using natural abundance of carbon isotopes Accepted Article This article is protected by copyright. All rights reserved. 1. Ecosystem processes reflected by stable isotope fractionation (13C and 15N) The relative abundance of the rare and heavy stable isotopes of nitrogen (15N) and carbon (13C) compared to the most abundant stable isotope, 14N and 12C respectively, is expressed as the delta (δ) notation (e.g. δ13C and δ15N in ‰), which is the deviation of the 13C or 15N abundance in the sample compared to a reference material (Brand & Coplen 2014). Most natural processes (chemical, physical or enzymatically catalysed) discriminate against heavy isotopes (e.g. 13C, 15N, 18O), which in open systems results in an isotopically depleted product with comparably smaller concentration of the heavy isotopes than its corresponding substrate (Fry 2006). If the dominant process rate changes, or if the substrate is exhausted, then the δ value of the product (such as the plant leaf) may significantly change, due to the underlying fractionation. Decreases in soil water availability due to drought can alter the isotope signature of both carbon and nitrogen in the aboveground plant biomass. During drought stress, leaves reduce stomatal opening to preserve water. As this happens, the space that confines the air as an immediate source of CO2 for photosynthesis (the sub-stomatal cavity) becomes a more closed system due to the restriction of the renewal of CO2, and as a result a higher proportion of the heavy 13C in CO2 is fixed by Rubisco (C3 plants; Tcherkez et al. 2011). Hereby the discrimination against the heavy 13C isotope is decreased. As a consequence, in plants with a C3 photosynthetic pathway a 13C enrichment in the leaf occurs during drought stress (Cernusak et al. 2013). Indeed, the 13C enrichment at the leaf level is related to an increased intrinsic water use efficiency (WUEi), the ratio of assimilation to stomatal conductance (Farquhar & Richards 1984). Changes in soil water availability may also alter the leaf nitrogen isotope signature by changing the nitrogen availability with soil depth, and thereby the 15N signature of the plant nitrogen source (Lloret et al. 2004). In general, an increase in the Accepted Article This article is protected by copyright. All rights reserved. δ15N signature in the leaves indicates a progressive N saturation and/or N losses in the surrounding system because all major pathways of N loss (denitrification, ammonia volatilization and nitrate leaching) cause δ15N enrichment of the remaining nitrogen (Peñuelas et al 2000). Interpretation of changes in leaf δ15N, however, is not straightforward since leaf δ15N signatures might largely depend on mycorrhizal associations, and shifts in nitrogen sources between organic and inorganic compounds under a drought or warming could influence the leaf δ15N as well (Michelsen et al. 1998; Andresen et al. 2009). For instance, the increase in plant δ15N values with aridity may also result from increasing reliance on recycled organic N sources as opposed to new inputs. Across the INCREASE network the effects of warming and drought on plant 13C and 15N natural abundance was monitored over four years, starting two years after onset of the climate manipulation. Current year shoots or leaves were analysed for δ13C and δ15N immediately after each artificially prolonged drought. Plant material was dried at 70°C and ground to a fine powder before of δ13C and δ15N using isotope ratio mass spectrometry (IRMS). We expected to find higher δ13C values: i) in drought treated plants (compared to control plots) and, ii) in plants growing at drier locations across the precipitation gradient (for a given common plant species). Furthermore, we expected iii) the δ15N to change in response to drought, as the nitrogen source (depth) is changed (at one location, within-species). Some significant effects of the drought treatment were observed on plant tissue δ13C and δ15N (Box 1). Differences between years (effect of time) were more pronounced than the effect of the drought treatment for Populus alba δ13C (HU), Erica multiflora δ15N (SP) and Globularium alypum δ15N and δ13C (SP). Only Calluna vulgaris showed a significant response to the drought treatment for δ13C as hypothesized (Box 1A). For C. vulgaris, which was growing at several locations (UKCL, NL and DK-MOLS), the δ13C was higher at drier locations, when compared along the precipitation gradient, and also higher in the drought treatment at the NL and UK-CL sites Accepted Article This article is protected by copyright. All rights reserved. et al. 2011). However, when applied properly, the insights into terrestrial carbon allocation can be detailed and novel (Box 3). 2. Free Air Carbon dioxide Enrichment (FACE)-labelling An alternative method for 13C labelling of vegetation and whole-ecosystems is to use 13C- depleted CO2 in FACE experiments. The FACE technique has through decades been used within cropping systems (Kimball 2016), grasslands (Hovenden et al. 2014; Reich et al. 2014; Mueller et al. 2016) and forests (Terrer et al. 2016) experiments, with the primary goal of assessing potential carbon dynamics and enhancement of plant growth (Andresen et al. 2016). As a side effect, the change in carbon isotopic composition of vegetation exposed to the FACE-treatment can be used to trace freshly assimilated carbon into soil microbial biomass, fauna and organic carbon pools. This approach was used at the Brandbjerg site (DK-BRA). The CO2 used to elevate concentrations of atmospheric CO2 to 510 ppm had δ13C values ranging from –3.0 to –36.7 ‰ throughout 8 years of experimental treatment, with an overall mean of -26.1 ‰. The source of the CO2 supplied was brewery surplus CO2 as a chemically obtained side product. The mixing of the added CO2 via FACE with ambient CO2 in the moving air mass resulted in a 13C depletion ranging from -6.7 to -15.6 ‰. On average, this equals a depletion of CO2 in FACE plots of -4.8 ‰ relative to the atmospheric -8 ‰ average. Ecosystem carbon pools became depleted accordingly, and the FACE-13C depletion acted as a long-term persistent isotope labelling. As a result, soil fauna (Enchytraeids) sampled from each of the climate-treated plots was significantly depleted in δ13C by -0.5 to -2.0 ‰ in the CO2 treatments (Andresen et al. 2011). This was due to translocated 13C substrate through the food web, starting with plant assimilation of 13C-depleted CO2, followed by plant root exudation and microbial utilization of the 13C depleted substrate and eventual digestion of Accepted Article This article is protected by copyright. All rights reserved. microbes by enchytraeids. Hereby the freshly supplied carbon source was recognized to be transferred in the natural setting, within a given time scale. Also microbial biomass and PLFAs had a different baseline of 13C content in ambient (not-treated) plots compared to CO2 treated plots (Andresen et al. 2014). This was used for the calculation of 13C enrichments in each PLFA biomarker, also illustrating the pathway of newly-assimilated carbon into microbial biomass A general drawback of the 13C-FACE label is again the contamination of the surroundings, as even short and small un-planned draft winds can carry the depleted label onto ‘ambient’ plots, and these will most likely be ‘contaminated’ with 13C (though not markedly exposed to high CO2 concentrations) after some years of FACE activity. Therefore, one needs to collect reference material for the ‘natural abundance’ level well away from the FACE experiment. Also, FACE-CO2 can only be used as tracer if the isotopic composition of the FACE-CO2 is considerably different than the isotopic composition of the atmospheric CO2. 3. In-situ injection of 13C-enriched substrate solutions As a much more localized approach, in-situ injection of 13C- and 15N-enriched substrates directly below the soil surface can be used to assess the competition for the substrate between i) plants and soil microbes, ii) microbial groups, and iii) the effects of the climate change treatments upon the competition for carbon or nitrogen substrates. Much research has focused on the sharing of nitrogen sources between plant and microbes (Kuzyakov & Xu 2013) using in-situ soil injections of 15N labelled inorganic nitrogen (ammonium and nitrate) or organic nitrogen (amino acids) (Sorensen et al., 2008). Once amino acids with dual labelled compounds (15N and 13C) were available for experimental use, double-labelled substrate was used to explore e.g. plant uptake of intact amino acids (Näsholm et al. 2009; Rasmussen et al. Accepted Article This article is protected by copyright. All rights reserved. 2010), and microbial utilization of carbon substrates (Dungait et al. 2013; Rinnan & Baath 2009). In a labelling experiment at the DK-BRA site, amino acid injections into the soil were conducted to analyse the impact of the climate treatments on the uptake of free amino acid nitrogen by plants and soil microbes. Dual-labelled glycine (13C215N-glycine: 99 atom% 13C - of both carbon atoms - and 99 atom% 15N) was added to 20 × 20 cm2 sub-plots (Andresen et al. 2009). Each sub-plot received 0.1 L of re-demineralised water labelled with 0.027 g glycine, corresponding to 687 mg glycine m-2 (223 mg C m-2 or 0.016 mg glycine g-1 dry weight soil). The label was injected into the soil just below the soil surface with a syringe moved among 16 evenly spaced points of a template, placed on top of the vegetation (Andresen et al. 2009). One day (c. 24 h) after labelling with glycine, soil cores were sampled from the soil surface to 15 cm depth, for determining the relative uptake of the amino acid in plant roots (IRMS solid sample) and soil microbes. As in many other soil labelling experiments, the largest label recovery (measured by 15N recovery since respiratory losses of 13C remain unknown) was found in the total microbial biomass compared to total plant biomass (Kuzyakov & Xu 2013). A subsample of fresh soil was extracted with redemineralised water, and another set of subsamples was first vacuum-incubated with chloroform for 24 hours to release microbial carbon and nitrogen (Joergensen & Mueller 1996; Brookes et al. 1985), before extraction with re-demineralised water. A third subsample of soil was freeze-dried and later used for PLFA extractions. The 13C enrichment in PLFA markers thus indicated the activity (vitality) of the specific microbial group (Watzinger 2015). We found that bacteria opportunistically utilised the freshly added glycine substrate, i.e. incorporated 13C, whereas fungi showed only minor or no glycine derived 13C-enrichment (Andresen et al. 2014). In comparison, 13C traced into the microbial community via the 13C- CO2 pulse label at the same site (DK-BRA) also reached the bacterial community first. Accepted Article This article is protected by copyright. All rights reserved. Bacteria showed high 13C enrichment compared to fungal groups (Reinsch et al. 2014). This suggests that in-situ injection of 13C substrates might be a plausible alternative to mimic rhizodeposition effects. With the direct addition of 13C label to the soil a strong labelling of the microbial community was more easily achieved than with the indirect 13C labelling of microbes via plant assimilated 13C-CO2 (Box 3). Use of labelled carbon-compounds to analyse carbon mineralisation by soil microorganisms Since soil microorganisms have an important role in controlling the availability of nutrients via mineralisation of SOM, our understanding of how microbial functioning in the ecosystem is altered by global change must be improved (Grayston et al. 1997). Microbial catabolic diversity of a soil is directly related to the carbon decomposition function within a soil and potentially provides a sensitive and ecologically relevant measure of the microbial community functional structure (Garland & Mills 1991). Subsequently, multiple assays have been developed to generate community level physiological profiles (CLPP) that can act as fingerprints of microbial function. Three approaches for measuring CLPP in soils are reported in the literature: (i) Biolog (Garland & Mills 1991); (ii) a substrate-induced respiration (SIR) technique (Degens & Harris 1997); and (iii) MicroResp (Campbell et al. 2003). These methods are all based on quantifying CO2 respired during the mineralisation of organic carbon compounds that vary in size, charge and structural complexity. The first approach, Biolog MicroPlateTM (Biolog), assesses the catabolic diversity of soil organisms using a microtitre plate by incubating a soil culture in the presence of nutrients and 95 different carbon substrates; respired CO2 is used to reduce a tetrazolium violet salt, which results in a colour change that can be quantified colorimetrically (Garland & Mills 1991). This approach, Accepted Article This article is protected by copyright. All rights reserved. however, has been criticized for bias towards fast growing organisms that thrive in culture (Preston-Mafham et al. 2002). In response to the criticism of the Biolog method, Degens & Harris (1997) developed a method based on SIR where individual substrates are added to intact soil and evolved CO2 is sampled and quantified. Finally, Campbell et al. (2003) combined aspects of both methods (MicroRespTM) where the response to carbon substrate addition to soil is measured colorimetrically using a cresol red indicator dye in a microtitre plate format. Community level physiological profiling of soils samples collected from all treatments across the network was conducted to determine the catabolic utilisation profile, turnover and pool allocation of low molecular weight (LMW) carbon compounds by using a selection of 14C- labelled substrates. This method enabled the attribution of respired CO2 to specific metabolic processes that facilitates the quantification and qualification of microbial mineralisation kinetics of substrates varying in structural complexity and recalcitrance. The kinetics of microbial 14C-CO2 evolution can be described using a first order exponential decay model (Box 4). The number of terms used in the exponential decay model can be used to explain how microbial kinetics relates time, substrate complexity and carbon pool allocation to, for example, rapidly cycled labile soil solution carbon, microbial structural carbon and recalcitrant extracellular soil organic carbon (Kuzyakov & Demin 1998; Nguyen & Guckert 2001; Boddy et al. 2007). Attribution of modelled carbon pool sizes and turnover rates to biological function are not only time and substrate dependent. Therefore, soil physical, biological and chemical interactions may be miss-attributed to biological function. Indeed, current knowledge and techniques available might not be enough to examine the interaction between discrete carbon pools (Glanville et al, 2016). Using the half-life of 14C labelled carbon in soil solution we were able to examine the environmental gradient of the warming treatment across the climate change network and identified that temperature becomes rate Accepted Article This article is protected by copyright. All rights reserved. limiting for microbial uptake of carbon from the soil solution pool at < 10.5 °C. We also showed that experimentally manipulated warming simply speeds up the catabolic utilisation of labile LMW carbon in a predictable pattern (Box 4). Conclusions and recommendations Stable isotope studies provide insightful information about carbon (and nitrogen) fluxes through the plant-soil-atmosphere continuum with minimal disturbance to the system. The value of the different isotope techniques depends on the specific research questions. The analysis of the natural abundance of the heavy isotopes is only useful when isotope signatures in the different carbon or nitrogen pools are clearly distinct as a result of important fractionation processes. In practice, the application of this technique is limited to the study of the effects of changing abiotic conditions on processes that operate over a relative broad period of time, for instance to study changes in plant water use efficiency or N sources in a drought experiment over the growing season or different years. In contrast, the radiocarbon analysis (“bomb-C” technique) of instantaneous fluxes (soil or ecosystem respiration) has been proved to be very useful to evaluate whether different factors of climate change provoke the release of older carbon sources through soil or ecosystem respiration, a central question in relation to the proposed positive feedback between climate change and SOM decomposition. However, the progressive dilution of the bomb-C signature of the atmosphere will limit the application of this technique in the upcoming decades. If the analysis of climate change effects on plant carbon belowground and cycling through the microbial community is the main research interest, then 13C labelling approaches are the most appropriated tools. Coupled with the analysis of 13C in specific microbial compounds, this Accepted Article This article is protected by copyright. All rights reserved. technique constituted a remarkable advance in the study of processes occurring at the rhizosphere level. A significant challenge of the application of this technique is the achievement of sufficient 13C enrichment in microbial biomass where the pools of background carbon in the studied compartments are high and hence dilute the 13C signal. As an alternative, direct injection of 13C-enriched substrates into soil can be applied to mimic rhizodeposition and to achieve a higher 13C signal in the microbial community. Fumigation with FACE-CO2 can be used to achieve a longer-term labelling of soil microbes and fauna. The application of these techniques, however, is not exempt from difficulties and disadvantages. To keep a high caution and avoid mistakes, our collective recommendations for applying the described methods are provided and addressed in Table 1. For in-situ pulse-labelling studies there are major seasonality constraints to the distribution of the label throughout the ecosystem compartments, i.e. the seasonality of carbon allocation belowground due to changing plant activity, or the plant health status which determines the amount of tracer entering the system. Importantly, field plots previously ‘contaminated’ by highly enriched isotope labelling should be considered potentially inoperable for further scientific isotope studies using the natural abundance approach. However, plant and soil structures remain largely undisturbed. In outlook for setting up a large-scale climate manipulation, areas that have not been previously used for experimental work with isotopes should consequently be selected. In particular, the ‘bomb-C’ method is very sensitive to the contamination of soil or plant samples with 14C-enriched material, and thus its application should be limited to sites and facilities where no 14C-labelling work has been conducted. Additionally, it should be noted that any history of fertilization might also alter the natural isotope abundance of ecosystem compartments (in particular 15N signatures), potentially confounding experimental results. The surroundings of a FACE experiment can be also “contaminated” by draft winds carrying the depleted label onto ambient plots. Accepted Article This article is protected by copyright. All rights reserved. Acknowledgements This paper is a WP3 and WP15 of the INCREASE EU-infrastructure. Financial support by the European Commission (FTP7-ICT-2008-1, Grant Agreement no. 227628) is gratefully acknowledged. LCA, IKS, PA and CB received support from the VKR foundation through the CLIMAITE project. ME and JP acknowledge support from the Spanish Government grant CGL2016-79835-P, the Catalan Government grant SGR2014- 274, and the European Research Council grant ERC-SyG-610028 IMBALANCE-P. GKD was supported by the Hungarian Scientific Research Fund (OTKA K112576) and by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences. The ClimMani (ESSEM COST Action ES1308, Climate Change Manipulation Experiments in Terrestrial) is acknowledeged. Authors state that they do not have any conflicts of interest to declare. Data Accessibility Datasets associated with this article are available through Dryad Digital Repository, DOI: doi:10.5061/dryad.mc460 (Andresen et al., 2018). Author contributions statement All authors contributed to the collection of the data included as illustration of the methodologies. LCA, MTD, SR, and ARS wrote the manuscript. All authors contributed critically to the drafts and gave final approval for publication. Accepted Article This article is protected by copyright. All rights reserved. References Andresen, L.C., Michelsen, A., Jonasson, S., Beier, C. & Ambus, P. (2009) Glycine uptake in heath plants and soil microbes s responds to elevated temperature, CO2 and drought. Acta OEcologica, 313, 283-295. Andresen, L.C., Konestabo, H.S., Maraldo, K., Holmstrup, M., Ambus, P., Beier, C. & Michelsen, A. (2011) Organic matter flow in the food web at a temperate heath under multifactorial climate change. 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(2011) 12C/13C fractionations in plant primary metabolism. Trends in Plant Science, 16, 499-506. Terrer, C., Vicca, S., Hungate, B.A., Phillips, R.P. & Prentice, I.C., 2016. Mycorrhizal association as a primary control of the CO2 fertilization effect. Science, 353, 72–74. Treonis, A.M., Ostle, N.J., Stott, A.W., Primrose, R., Grayston, S.J. & Ineson, P. (2004) Identification of groups of metabolically-active rhizosphere microorganisms by stable isotope probing of PLFAs. Soil Biology and Biochemistry, 36, 533-537 Trumbore, S.E. (2009) Radiocarbon and Soil Carbon Dynamics. Annual Reviews on Earth Planetary Sciences, 37, 47–66. Watzinger, A. (2015) Microbial phospholipid biomarkers and stable isotope methods help reveal soil functions. Soil Biology and Biochemistry, 86, 98-107. Wessel, W.W., Tietema, A., Beier, C., Emmett, B.A., Peñuelas, J., & Riis–Nielsen, T. (2004) A Qualitative Ecosystem Assessment for Different Shrublands in Western Europe under Impact of Climate Change. Ecosystems, 7, 662–671. Zak, D.R., Pregitzer, K.S., King, J.S. & Holmes, W.E. (2000) Elevated atmospheric CO2, fine roots and the response of soil micro-organisms: a review and hypothesis. New Phytologist, 147, 201-222. Accepted Article This article is protected by copyright. All rights reserved. Table 1. Suggestions and advice to consider when applying isotopic methods for the study of carbon fluxes in the plant-soil system. Method Expenses (cost) Advice (do’s and don’ts) Before you start Data analysis hint Time spent Bomb-C (natural 14C abundance) High (AMS analysis); Equipment for CO2 sampling is cheap (closed chambers, carbon-free pump, batteries, and molecular sieve system). An IRGA is also required. - Avoid materials and labs with possible 14C contamination. - If soil CO2 is to be analysed in the field, long incubation times are required to get sufficient CO2 for AMS analysis (typically >1 ml). - Think carefully about the soil depths to be analysed, and take the sample consistently. 14C signatures might vary strongly along few cm in the soil. - If bulk soil 14C is to be analysed, try to remove the roots as much as possible, because of their contrasted 14C signature. - If you are not sure about potential 14C contamination in your lab, use another lab or make a swipe test. - Make previous trials to assess the incubation times required to get a sufficient CO2 sample - Go through the whole process of sample preparation with a trial sample. - Discuss your results with the Radiocarbon facility staff. - Processing time depends on the type of sample, although is usually low; determination by AMS may take several months depending on the facility. In situ 13C-CO2 pulselabelling 13C- enriched compounds used for labelling and as standards are usually expensive; 13C determination in specific compounds is expensive, although cheaper than AMS - Consider the target pools to be analysed and the potential dilution of the label by the unlabelled root system or soil carbon pool. - If your study requires a high 13C enrichment, mind the potential risk of contaminating the site. - Avoid above ambient CO2 concentrations in the chamber. - If you need to monitor CO2 during your pulse, remember that IRGAs are rather insensitive to 13CO2. - Test your chamber and tubing materials for adsorption / desorption effects, and ensure these are without carbon content (use PTFE (Teflon) tape, not gluing paper-based). - Make a previous trial if possible and go through the whole process of sample preparation. Report the label addition per area: g 13C m-2. - Pulse labelling experiments are usually short, but intensive (high sampling frequency immediately after the pulse). - Experiments requiring root washing or microbial compound extraction are time consuming. Natural abundance of isotopes (13C and 15N) IRMS analysis is relatively cheap - Make sure the history of sampling site is known (previous labelling experiments?) - Be aware that FACE can dilute the isotopic signal, most CO2 enriched systems use 13C depleted sources,because this is - Sampling time and grinding / weighing of sample. - Analysis usually done at a dedicated Accepted Article This article is protected by copyright. All rights reserved. cheaper. natural abundance facility. 14C-substrates mineralisation Analysis of the trapped 14C-CO2 is relatively cheap. - High risk of contaminating lab equipment. - You need to work in a dedicated 14C lab safely away from the natural abundance facility. - Continue sampling until decline in emission is level, this ensures better model fit. 13C-injection in situ Similar to 13C-CO2 pulselabelling. - Contamination risk of 13C leaching is present, but smaller to our judgement than from 13C- CO2 experiments. - Do not use areas dedicated to natural abundance work. - Labelling intended for soil microbial components is more intense from 13C liquid substrate in-situ injection than from 13C-CO2 pulse labelling. - Soil sampling is destructive, consider to have several parallel plots to harvest an undisturbed plot at each sampling event. - Sample handling from field work until the extraction takes a few days so plan only one sampling event per week if possible. Accepted Article This article is protected by copyright. All rights reserved. Figure Captions Figure 1. Map of the European INCREASE network, with the shrubland field sites and annual temperature (red line, right axis) and precipitation (bars, left axis) norm. Sites in Denmark: Mols (DK-MOLS) and Brandbjerg (DK-BRA); in United Kingdom: Clocaenog (UK-CL) and Peaknaze (UK-PK); in The Netherlands (NL): Oldebroek; in Spain (SP): Garraf; in Italy (IT): Porte Conte, and in Hungary (HU): Kiskunsàg. Box 1. Isotopic signal of plant leaf responses to precipitation. Stable isotopes (δ13C and δ15N) in aboveground plant material collected across the network was analysed by isotopic ratio mass spectrometry (IRMS). A: Leaves and twigs (t) from P. alba (HU), E. multiflora L. (SP), G. alypum L. (SP) and C. vulgaris (NL); filled circle ● is control, open circle ○ is drought treatment, ▼ is warming treatment. P-values indicate effects of treatment, year, and the interaction of these factors on 13C or 15N, analysed by two-way ANOVA; ns is non-significant effect. Number indicates year (2001=1, 2002=2, 2003=3 or 2004=4). Species (site) differences and annual differences are stronger than treatment effects. B: δ13C of C. vulgaris leaves versus annual precipitation of the previous year. Accepted Article This article is protected by copyright. All rights reserved. Box 2. Impact of warming and drought on the 14C signature of soil respiration. A: Records of atmospheric 14C over the 20th century. The unit for 14C signature (% Modern) is a measurement of the deviation of the 14C/12C ratio of a sample from the "Modern" standard, which is defined as 95% of the radiocarbon concentration (in AD 1950) of a reference material (NBS Oxalic Acid I, SRM 4990B), adjusted to a 13C reference value of –19 ‰. B: At the UK-PK site, the 14C signature of the soil efflux was measured (bars, left axis). 14C values were highly heterogeneous (ranging from 105.49 to 110.13 % Modern; values of > 100 % Modern suggest that a substantial component -and potentially all- of the carbon was trapped by photosynthesis during the post-bomb era i.e. since ~AD 1955).There were no significant effects of the warming treatment, while there was a trend towards the release of older carbon in the drought plots. On average, the carbon being released from the plots had been fixed from the atmosphere between six and eight years earlier (line, right axis). C: Detail of a closed static chamber used to collect CO2 from the soil efflux. Box 3. Analysis of rhizodeposit utilisation by microbes using in-situ 13C-CO2 pulse-labelling experiments. A: At the Clocaenog site (UK-CL) this technique was applied along a peat layer gradient. Repeated pulses of 13C-CO2 were applied during eight hours to C. vulgaris using sealed transparent domes attached to a core inserted into the ground. B: The incorporation of 13C into soil microbial PLFAs was analysed. Despite a high applied dose of 13C (99 atom %), the dilution of the tracer within the large pool of unlabelled root biomass was remarkable, and as a consequence most of the analysed PLFAs showed no 13C enrichment. C: 13C recovery in Gram negative bacteria after a 13C-CO2 pulse at the Brandbjerg site (DK-BRA). The enrichment pattern in PLFAs attributed to Gram negative bacteria in soils exposed to drought and elevated CO2 concentration (+120 ppm) for 8 years show different carbon utilization patterns and magnitudes under imposed climatic conditions implying changed carbon cycle Accepted Article This article is protected by copyright. All rights reserved. dynamics. D: Flow-through pulse-labelling equipment showing the gas reservoir containing 13C-CO2 for up to eight hours of labelling connected to transparent Plexiglas chambers via tubing. Box 4. Exponential decay kinetics for 14CO2 evolution during microbial 14C substrate mineralisation. The catabolic utilisation profile, turnover and pool allocation of low molecular weight (LMW) carbon substrates was determined in soils collected across the experimental network. Sixteen 14C labelled amino acids and sugars varying in structural complexity and recalcitrance were used in a multiple substrate induced respiration (SIR) assay on soil. Evolved CO2 was collected using NaOH traps and absorbed 14CO2 was measured with a scintillation counter. A: For substrate mineralisation a double-term first order decay model with an asymptote fitted the data with an r2 of 0.99. Using the coefficients from the fitted equation, estimated half-life of the substrate in the first phase (soil solution uptake) was 30 h, and in the second slower phase (microbial turnover) 408 h. Approximately 40 % of the substrate was immobilised in the soil, 48.3 % respired during the first phase, and 13.2 % respired during the slower second phase. B: Half-life of the substrate in the soil solution versus mean annual temperature, in control (triangle) and warming (circle) treatments, data points are mean ± SE (n=3). Warming treatment and relative warmer site, simply increases the catabolic utilisation of labile LMW-carbon until a threshold mean annual temperature of 11.5 ºC. Accepted Article This article is protected by copyright. All rights reserved. Accepted Article This article is protected by copyright. All rights reserved.