C Werner
email: c.werner@uni-bayreuth.de

H Schnyder

M Cuntz

C Keitel

M J Zeeman

T E Dawson

F.-W Badeck

E Brugnoli

J Ghashghaie

T E E Grams

Z E Kayler

M Lakatos

X Lee

C Máguas

J Ogée

K G Rascher

R T W Siegwolf

S Unger

J Welker

L Wingate

A Gessler

Progress and challenges in using stable isotopes to trace plant carbon and water relations across scales

Stable isotope analysis is a powerful tool for assessing plant carbon and water relations and their impact on biogeochemical processes at different scales. Our processbased understanding of stable isotope signals, as well as technological developments, has progressed significantly, opening new frontiers in ecological and interdisciplinary research. This has promoted the broad utilisation of carbon, oxygen and hydrogen isotope applications to gain insight into plant carbon and water cycling and their interaction with the atmosphere and pedosphere. Here, we highlight specific areas of recent progress and new research challenges in plant carbon and water relations, using selected examples covering scales from the leaf to the regional scale. Further, we discuss strengths and limitations of recent technological

Introduction

Stable isotopes are a powerful tool for tracing biogeochemical processes across spatio-temporal scales [START_REF] Yakir | The use of stable isotopes to study ecosystem gas exchange[END_REF]. The stable isotope composition of plant material, animal tissues, sediments and trace gases can be used as indicators of ecological change (Dawson and Siegwolf, 2007). The assessment of the circulation of isotopes in the biosphere allows characterisation and quantification of biogeochemical cycles as well as exploration of food webs [START_REF] Fry | Stable Isotope Ecology[END_REF]. Stable isotope studies give insights into key reactions of plant metabolism [START_REF] Schmidt | Carbon isotope effects on key reactions in plant metabolism and 13 C-patterns in natural compounds[END_REF], can increase our understanding of water movement along the soilplant-atmosphere continuum [START_REF] Dawson | The role of hydrogen and oxygen isotopes in understanding water movement along the soil-plant-atmosphere continuum[END_REF], and allows palaeoclimatic/-physiological reconstructions [START_REF] Beerling | Modelling changes in plant functions over the Phanerozioc[END_REF]. Moreover, the analysis of the isotopic composition of trace gases exchanged between ecosystems and the atmosphere gives insights in the underlying processes driving the source and sink strength of biomes for CO 2 , CH 4 and/or N 2 O (Flanagan et al., 2005). Stable carbon, oxygen and hydrogen isotope composition of organic matter and inorganic compounds such as CO 2 and H 2 O is altered during vegetation-soil-atmosphere exchange processes, such as evapotranspiration, carbon assimilation and respiration. This leaves an isotopic imprint on soil, plant and atmospheric carbon and water pools and associated fluxes. These isotopic fingerprints can then be used to trace different processes involved in the transfer of carbon and water across the plant-soil-atmosphere continuum. Particularly the multiple-isotope approach, i.e. the simultaneous measurements of stable isotope composition of different elements (δ 2 H, δ 18 O and/or δ 13 C, for definition see Tables 1 and 2), provides a unique way to investigate the interrelation between water and carbon fluxes [START_REF] Ehleringer | Variation in leaf carbon isotope discrimination in Encelia farinosa: implications for growth, competition, and drought survival[END_REF][START_REF] Griffiths | Stable Isotopes -integration of biochemical, ecological and geochemical processes[END_REF]Flanagan et al., 2005;[START_REF] Yakir | The use of stable isotopes to study ecosystem gas exchange[END_REF]. The use of biological archives may enable extrapolation of this information to longer time scales, such as the Anthropocene. Methodological advances allow isotopologue and compound-specific analyses at unprecedented resolution, providing new insight into isotope fractionation processes in metabolic pathways and in biogeochemical processes. Further, a more advanced mechanistic understanding of processes affecting the stable isotope composition in various ecosystem compartments allows modelling and prediction of water and carbon fluxes based on stable isotope information. In turn, new findings open new research frontiers and challenges. Here, we highlight recent progress and developments in carbon and oxygen isotope research and discuss the potential for further extending our knowledge about water and carbon fluxes and cycling. We present advances and challenges on various scales from the leaf (Sect. 2.1), plant (Sect. 2.2), community (Sect. 2.3), ecosystem (Sect. 2.4), and regional scales (Sect. 2.5) as well as of different types of temporal (historical) isotopic archives (Sect. 2.6). At each scale, pertinent reviews are indicated which survey the published literature and pioneering work; thereafter, we focus on selected examples from the last decade. Finally, we highlight strengths and limitations of new technological developments (Sect. 3) and present an outlook (Sect. 4) on what we identify as main goals of the stable isotope research in carbon and water biogeochemistry.

2 Isotope effects across temporal and spatial scales 2.1 Leaf-level processes

CO 2 and H 2 O exchange

Leaf CO 2 and H 2 O fluxes have unique and distinct isotope signals that carry useful physiological and biogeochemical information. For example, environmental stresses, such as drought, cause systematic variation in carbon isotope discrimination during photosynthesis ( 13 C, see Table 1), shedding light on different steps of CO 2 transfer from the atmosphere to the chloroplasts [START_REF] Evans | Carbon isotope discrimination measured concurrently with gas exchange to investigate CO 2 diffusion in leaves of higher plants[END_REF]Farquhar et al., 1989a, b;[START_REF] Brugnoli | Photosynthetic fractionation of carbon isotopes[END_REF]. On the other hand, stomatal opening and, hence, transpiration, cause 18 O and deuterium ( 2 H) enrichment of water at the sites of evaporation ( ev , see Table 2), which lead to the enrichment of the total leaf water [START_REF] Dongmann | On the enrichment of H 18 2 O in the leaves of transpiring plants, Radiat[END_REF]Farquhar and Lloyd, 1993). The oxygen of leaf-dissolved CO 2 exchanges with the 18 O-enriched leaf water, entraining distinct 18 O discrimination ( 18 O) during photosynthetic CO 2 exchange (Farquhar et al., 1993). The knowledge of 13 C and 18 O together can then be used to assess the limitations to CO 2 transfer between the intercellular air space and the chloroplasts (Gillon and Yakir, 2000b).

The theoretical understanding of the individual 13 C and 18 O fractionation phenomena (including transport/diffusion, transformations and exchange processes) in CO 2 and H 2 O is well established for systems in steady-state [START_REF] Dongmann | On the enrichment of H 18 2 O in the leaves of transpiring plants, Radiat[END_REF][START_REF] Farquhar | On the relationship between carbon isotope discrimination and the intercellular carbon dioxide concentration in leaves[END_REF][START_REF] Evans | Carbon isotope discrimination measured concurrently with gas exchange to investigate CO 2 diffusion in leaves of higher plants[END_REF]Farquhar et al., 1993). However, we are only at the beginning of gaining theoretical understanding for those in non-steadystate. On-line isotope discrimination studies, i.e. instantaneous measurements of leaf/plant gas exchange and the associated isotopic signals, during transient conditions and shortterm dynamics bare the potential to expand our understanding beyond steady-state.

Even though our mechanistic understanding of photosynthetic carbon isotope discrimination and evaporative oxygen isotope enrichment has been increasing within the last decade, there are still open questions and methodological Table 1. Introduction to terms and equations of carbon isotopes, photosynthetic discrimination and post-carboxylation fractionation.

Carbon isotopes, photosynthetic discrimination and post-carboxylation fractionation

Equations

The delta notation for carbon isotopes Carbon has two stable isotopes, 12 C and 13 C, with natural abundances of 98.9 and 1.1 %, respectively. The relative abundance of 13 C in any sample is conventionally expressed in the δ notation (Eq. 1) which is defined as the relative deviation of the isotope ratio R (R = 13 C / 12 C) of a sample relative to that of an international standard (and is often expressed in ‰). The international standard is the R of CO 2 from a fossil belemnite in the Pee Dee formation of South Carolina. Today, 13 C standards are obtained from the IAEA in Vienna and are referred to as V-PDB [START_REF] Coplen | Reporting of stable carbon, hydrogen, and oxygen isotopic abundances, in: Reference and intercomparison materials for stable isotopes of light elements[END_REF][START_REF] Coplen | Guidelines and recommended terms for expression of stable-isotope-ratio and gas-ratio measurement results[END_REF].

δ y X = ( R sample R standard -1) (1)
where in the case of carbon isotopes, y X is replaced by 13 C, and R sample and R standard are the measured 13 C / 12 C ratios in the sample and standard, respectively

Carbon isotope discrimination

The change in relative abundance of 13 C between an educt and product is called discrimination, often denoted with . In the case of CO 2 as the source and the plant material as the product of photo-and biosynthesis, carbon isotope discrimination is described in Eq. (2).

13 C = (δ 13 C aδ 13 C p )/(1 + δ 13 C p )

(2) where δ 13 C a and δ 13 C p are the δ 13 C values of the CO 2 in air and the plant, respectively.

Photosynthetic carbon isotope discrimination

Isotope discrimination during carbon assimilation has been modelled by [START_REF] Farquhar | On the relationship between carbon isotope discrimination and the intercellular carbon dioxide concentration in leaves[END_REF]Farquhar et al. ( , 1989a) ) for C 3 plants by Eq. (3). This equation has been developed to describe leaf-level photosynthetic discrimination during the light period, where e denotes the fractionation of mitochondrial respiration in the light, i.e. day respiration [START_REF] Tcherkez | On the 13 C/ 12 C isotopic signal of day and night respiration at the mesocosm level[END_REF] and * the compensation point in the absence of day respiration. When Eq. ( 3) is applied to analyse 13 C of bulk tissue as an integrative parameter for preceding photosynthetic discrimination during formation of this material, e denotes the integrated respiratory discrimination both during light and dark-respiration. However, additional factors such as fractionation during carbon allocation, tissue turnover or carbon partitioning into different plant organs may affect the observed discrimination. To date, we still lack a quantitative description of these processes (see Sects. 2.1.2 and 2.2). 13

C = a b p a -p s p a + a p s -p i p a + (e s + a 1 ) p i -p c p a + b p c p a - e R d k +f * p a (3)
where a b is the fractionation during diffusion in the boundary layer (2.9 ‰); a is fractionation during binary diffusion in air (4.4 ‰); e s is discrimination during CO 2 dissolution (1.1 ‰ at 25 • C); a l is fractionation during diffusion in the liquid phase (0.7 ‰); b is fractionation during carboxylation in C 3 plants (≈ 29.5 ‰); p a is atmospheric CO 2 partial pressure; p s is CO 2 partial pressure at the leaf surface; p i is sub-stomatal CO 2 partial pressure; p c is CO 2 partial pressure at the site of carboxylation; e is fractionation during mitochondrial respiration; f is fractionation during photorespiration; k is carboxylation efficiency; and * is compensation point in the absence of mitochondrial respiration (R d ).

Simplified model of C 3 photosynthetic isotope discrimination Few empirical/experimental studies have used Eq. ( 3), partly due to lack of needed input data. Instead, a simplified version [START_REF] Farquhar | On the relationship between carbon isotope discrimination and the intercellular carbon dioxide concentration in leaves[END_REF] has been used extensively (Eq. 4). This equation is valid on the condition that effects of boundary layer, internal conductance, photorespiration, day respiration and allocation are negligible. In strict terms, these conditions are met if boundary layer and internal conductance are infinitely high, photorespiration and respiration are infinitely low or nondiscriminating, and isotope discrimination during allocation and partitioning does not happen. To account for effects of the neglected terms in Eq. ( 4), the value of b is often slightly reduced (≈ 28 ‰) [START_REF] Brugnoli | Photosynthetic fractionation of carbon isotopes[END_REF].

13 C = a + (b -a) p i p a
(4) where a is the fractionation during binary diffusion in air (4.4 ‰); b is fractionation during carboxylation in C 3 plants; and p a and p i are the atmospheric and sub-stomatal CO 2 partial pressures, respectively.

Intrinsic water use efficiency (WUE i )

In Eq. (4), 13 C is directly proportional to p i /p a , which is determined by the relationship between photosynthetic assimilation (A) and stomatal conductance (g s ). Therefore, 13 C is a measure of intrinsic water use efficiency WUE i (or transpiration efficiency), the ratio of assimilation to transpiration, which can be estimated as WUE i / VPD [START_REF] Farquhar | Isotopic composition of plant carbon correlates with water-use efficiency of wheat genotypes[END_REF]Farquhar et al., 1989b).

WUE i = A g s = p a (1- p i pa ) 1.6 = p a (1- 13 C-a b-a ) 1.6
(5) where A is photosynthetic assimilation, g s is stomatal conductance; a is the fractionation during binary diffusion in air (4.4 ‰); b is fractionation during carboxylation in C 3 plants; and p a and p i are the atmospheric and sub-stomatal CO 2 partial pressures, respectively. The factor 1.6 denotes the ratio of diffusivities of water vapour and CO 2 in air.

Table 2. Introduction to terms and equations of oxygen and hydrogen isotopes and evaporative enrichment.

Oxygen and hydrogen isotopes and evaporative enrichment Equations

The delta notation for oxygen and hydrogen isotopes Whereas hydrogen has two stable isotopes, 1 H and 2 H (or D; deuterium), oxygen possesses the isotopes 16 O, 17 O and 18 O. Since the natural abundance of 17 O is very low (approx. 0.038 atom %), mostly the ratio between 18 O and 16 O and thus the relative abundance expressed as δ 18 O (calculated as shown in Eq. ( 1) with the standard of Vienna standard mean ocean water (VSMOW) in case of water and VPDB-CO 2 in case of CO 2 ) is considered.

δ y X = ( R sample R standard -1) (1)
where y X is replaced by 18 O or 2H in the case of oxygen and hydrogen isotopes and R sample and R standard are the measured 18 O / 16 O or 2 H / 1 H ratios in the sample and standard, respectively.

Evaporative enrichment

The (evaporative) oxygen ( 18 O ev ) or hydrogen ( 2 H ev ) isotope enrichment of leaf water or plant organic matter is expressed as enrichment above source water (often assumed to be soil or xylem water) by Eq. ( 6).

18 O ev = where δ 18 O p and δ 2 H p is the oxygen and hydrogen isotopic composition, respectively, of leaf water or plant organic matter and δ 18 O sw and δ 2 H sw are the respective isotope compositions of the source water.

Leaf water enrichment

The enrichment of the leaf water has been modelled with approaches of increasing complexity (e.g. [START_REF] Cuntz | Modelling advection and diffusion of water isotopologues in leaves[END_REF]. Steady-state isotopic enrichment of oxygen or hydrogen over source water at the site of evaporation in the leaf ( e ) can be calculated by the Craig & Gordon model [START_REF] Craig | Deuterium and oxygen-18 variations in the ocean and the marine atmosphere[END_REF][START_REF] Dongmann | On the enrichment of H 18 2 O in the leaves of transpiring plants, Radiat[END_REF] by Eq. ( 7). In steady state conditions (i.e. source water isotopic composition is equal to the one of transpired water), the isotopic enrichment of water vapour relative to the source water taken up by the plant ( v ) can be approximated by -ε + . This model was developed for open water surfaces and only applies to the water composition at the site of evaporation, and not the whole leaf (mean lamina mesophyll water).

e = ε + + ε k + ( v -ε k ) ea e i
(7) ε + is the equilibrium fractionation between liquid water and water vapour; ε k is the kinetic fractionation as vapour diffuses from leaf intercellular spaces to the atmosphere (Farquhar et al., 1989a), v is the isotopic enrichment of water vapour relative to the source water taken up by the plant, and e a /e i is the ratio of ambient to intercellular vapour pressures.

Steady-state isotopic enrichment of leaf water

The steady-state isotopic enrichment of mean lamina mesophyll water ( LsP ) can be described by correcting Eq. ( 7) for the so-called Péclet effect (Farquhar and Lloyd, 1993), as shown in Eq. ( 8). The Péclet effect is the net effect of the convection of unenriched source water to the leaf evaporative sites via the transpiration stream as opposed by the diffusion of evaporatively enriched water away from the sites of evaporation.

LsP = e 1-e -℘ ℘ with ℘ = E•L C•D (8)
where ℘ is the Péclet number, E the leaf transpiration rate (mol m -2 s -1 ), L is the scaled effective path length (m) for water movement from the xylem to the site of evaporation, C the molar concentration of water (mol m -3 ), and D the tracer-diffusivity (m 2 s -1 ) of heavy water isotopologues (either H 18 2 O or 2 H 1 HO) in "normal" water. L is a fitted parameter (using Eq. 8; [START_REF] Flanagan | Photosynthetic gas exchange and the stable isotope composition of leaf water: comparison of a xylem-tapping mistletoe and its host[END_REF] as it cannot be measured directly.

Non-steady-state isotopic enrichment of leaf water Non-steady-state effects in lamina mesophyll water enrichment ( LnP ) have been added by [START_REF] Farquhar | On the isotopic composition of leaf water in the non-steady state[END_REF] by Eq. ( 9). This equation has an analytical solution and can be calculated with the "Solver" function in Excel.

LnP = LsP -α + α k gtw i 1-e ℘ ℘ d(Vm LnP ) dt
(9) where α = 1 + ε, (α + and α k are corresponding to ε + and ε k , respectively), V m is lamina leaf water molar concentration (mol m -2 ), t is time (s), g t is the combined conductance of stomata and boundary layer for water vapour (mol m -2 s -1 ), and w i is the mole fraction of water vapour in the leaf intercellular air spaces (mol mol -1 ).

Advection-diffusion model of leaf water enrichment

The non-steady-state model of leaf water enrichment as given by Eq. ( 9) is a simplification of the advection-diffusion description of leaf water enrichment ( LnAD ), as given by [START_REF] Cuntz | Modelling advection and diffusion of water isotopologues in leaves[END_REF] and [START_REF] Ogée | Non-steady-state, non-uniform transpiration rate and leaf anatomy effects on the progressive stable isotope enrichment of leaf water along monocot leaves[END_REF] in Eq. ( 10). Steady-state approaches often accurately describe leaf water isotopic enrichment (e.g. [START_REF] Welp | δ 18 O of water vapour, evapotranspiration and the sites of leaf water evaporation in a soybean canopy[END_REF], especially for longer times (weeks, months or years) or spatial scales (ecosystem studies). If shorter times and spatial scales are considered (diel measurements or gradients across a leaf), non steady-state approaches are more suitable, especially for modelling leaf water enrichment during the night [START_REF] Cernusak | Environmental and physiological controls over oxygen and carbon isotope composition of Tasmanian blue gum, Eucalyptus globules[END_REF].

∂ LnAD dt = -vr m ∂ LnAD dr + Dr m ∂ 2 LnAD dr 2
(10) where r denotes the distance from the xylem to the evaporating site (m), v r is the advection speed of water in the mesophyll (m s -1 ), m the volumetric water content of the mesophyll, and D r = m κ m D the effective diffusivity of the water isotopologues (m 2 s -1 ), with κ m (< 1) the tortuosity factor of the water path through the mesophyll. The volumetric water content in the leaf mesophyll m is related to the water volume V m (per unit leaf area) and the mesophyll thickness r m through m = V m /(Cr m ) [START_REF] Cuntz | Modelling advection and diffusion of water isotopologues in leaves[END_REF].

Enrichment of organic matter Newly produced assimilates are assumed to obtain an imprint of the signature of the average bulk mesophyll leaf water at the time when they were produced. For oxygen, an equilibrium fractionation factor (ε wc ) results in carbonyl oxygen being ca. 27 ‰ more enriched than water [START_REF] Sternberg | Biogeochemical implications of the isotopic equilibrium fractionation factor between the oxygen atoms of acetone and water[END_REF], which has been confirmed for cellulose (e.g. [START_REF] Yakir | Oxygen and hydrogen isotope fractionation during cellulose metabolism in Lemna gibba L[END_REF], leaf soluble organic matter (e.g. [START_REF] Barnard | Evaporative enrichment and time lags between δ 18 O of leaf water and organic pools in a pine stand[END_REF] and phloem sap sucrose (e.g. Cernusak et al., 2003b, Gessler et al., 2007a).

issues which need to be addressed to better understand the physiological information imprinted on plant material.

Progress and challenges

Mesophyll conductance

Mesophyll or leaf internal conductance (often referred to as g m or g i ) has emerged as a significant (co-)limitation for CO 2 transport to the chloroplast, with large variation between species and environmental scenarios of light, temperature, drought and salinity [START_REF] Warren | Internal conductance does not scale with photosynthetic capacity: implications for carbon isotope discrimination and the economics of water and nitrogen use C[END_REF][START_REF] Flexas | Mesophyll conductance to CO 2 : current knowledge and future prospects[END_REF]. On-line measurements of 13 C in conjunction with gas exchange have been instrumental in detecting these variations of g i . Variation in g i is related to developmental changes and morphological/structural features of leaves, such as cell wall thickness, chloroplast arrangement, and leaf porosity [START_REF] Flexas | Mesophyll conductance to CO 2 : current knowledge and future prospects[END_REF][START_REF] Evans | Resistances along the CO 2 diffusion pathway inside leaves[END_REF]. Moreover, g i may be regulated via the expression of particular aquaporins capable of transporting CO 2 across plasma membranes (cooporins) [START_REF] Hanba | Overexpression of the barley aquaporin HvPIP2;1 increases internal CO 2 conductance and CO 2 assimilation in the leaves of transgenic rice plants[END_REF][START_REF] Flexas | Rapid variations in mesophyll conductance in response to changes in CO 2 concentration around leaves[END_REF]. Strong dynamic responses of g i to various environmental factors at the scale of minutes to days have been reported [START_REF] Flexas | Mesophyll conductance to CO 2 : current knowledge and future prospects[END_REF][START_REF] Bickford | High-frequency field measurements of diurnal carbon isotope discrimination and internal conductance in a semi-arid species, Juniperus monosperma[END_REF], and such variation has also been observed at the canopy-scale [START_REF] Schäufele | Dynamic changes of canopy-scale mesophyll conductance to CO 2 diffusion of sunflower as affected by CO 2 concentration and abscisic acid[END_REF]. So far, the metabolic basis of these short-term adjustments of g i is unknown.

Contribution of day respiration to 13 C dynamics

Recent high-resolution on-line 13 C measurements [START_REF] Bickford | High-frequency field measurements of diurnal carbon isotope discrimination and internal conductance in a semi-arid species, Juniperus monosperma[END_REF] as well as labelling and modelling approaches [START_REF] Tcherkez | On the 13 C/ 12 C isotopic signal of day and night respiration at the mesocosm level[END_REF] indicate that the isotopic composition of day respiration is not the same as that of concurrently fixed carbon dioxide. In part, the respiratory carbon isotope fractionation during daytime [START_REF] Tcherkez | On the 13 C/ 12 C isotopic signal of day and night respiration at the mesocosm level[END_REF] is related to fuelling of respiration by old carbon pools [START_REF] Nogués | Respiratory carbon metabolism following illumination in intact french bean leaves using 13 C/ 12 C isotope labelling[END_REF]. This calls for further experimental studies, a more detailed theoretical description of whole-leaf 13 C during daytime gas exchange [START_REF] Wingate | Variations in 13 C discrimination during CO 2 exchange in Picea sitchensis branches in the field[END_REF][START_REF] Tcherkez | Theoretical considerations about carbon isotope distribution in glucose of C 3 -plants[END_REF], and consideration of this effect in carbon isotope-based estimations of g i .

Water isotope enrichment in leaves

Isotopic enrichment in leaf water is reasonably well understood [START_REF] Craig | Deuterium and oxygen-18 variations in the ocean and the marine atmosphere[END_REF][START_REF] Dongmann | On the enrichment of H 18 2 O in the leaves of transpiring plants, Radiat[END_REF]Farquhar and Lloyd, 1993;[START_REF] Cuntz | Modelling advection and diffusion of water isotopologues in leaves[END_REF][START_REF] Ogée | Non-steady-state, non-uniform transpiration rate and leaf anatomy effects on the progressive stable isotope enrichment of leaf water along monocot leaves[END_REF], except for the parameter that characterises the effective path length for water movement from the xylem to the site of evaporation (see Péclet effect, Table 2). This parameter is especially important for modelling leaf water enrichment in non-steady state. Understanding how the effective length is adjusted by environmental conditions requires knowledge of how water transport inside the leaf is changing, for example, with the leaf's water status [START_REF] Barbour | Expressing leaf water and cellulose oxygen isotope ratios as enrichment above source water reveals evidence of a Péclet effect[END_REF][START_REF] Barbour | A new measurement technique reveals temporal variation in δ 18 O of leaf-respired CO 2[END_REF][START_REF] Ferrio | Effect of water availability on leaf water isotopic enrichment in beech seedlings shows limitations of current fractionation models[END_REF][START_REF] Kahmen | The influence of species and growing conditions on oxygen isotope leaf water enrichment and its impact on "effective path length[END_REF][START_REF] Ferrio | The Péclet effect on leaf water enrichment correlates with leaf hydraulic conductance and mesophyll conductance for CO 2[END_REF]. It is likely that in leaves, all water pools are involved during water transport [START_REF] Yakir | Oxygen-18 of leaf water: a crossroad for plant-associated isotopic signals[END_REF]; however, the leaf water pools might not be considered as perfectly mixed (e.g. [START_REF] Helliker | Establishing a grassland signature in veins: 18 O in the leaf water of C 3 and C 4 grasses[END_REF]. [START_REF] Gan | O-18 spatial patterns of vein xylem water, leaf water, and dry matter in cotton leaves[END_REF] compared different leaf water evaporative enrichment models (i.e. the twopool model, the Péclet effect model and the string-of-lakes model), which assume different water isotopic gradients and different mixing of leaf water pools. The different models all described large parts of the observed dynamics of leaf water enrichment but not all facets were captured by a single model. The non-steady state Péclet model of [START_REF] Farquhar | On the isotopic composition of leaf water in the non-steady state[END_REF] see Table 2, Eq. 9) is the simplification of the diffusion-advection model of [START_REF] Cuntz | Modelling advection and diffusion of water isotopologues in leaves[END_REF]Eq. 10), which assumes that the leaf represents a continuum of unenriched (source) and enriched (evaporative sites) water. The latter, more complex model is less sensitive to noise in the input data and gives smoother results. [START_REF] Cuntz | Modelling advection and diffusion of water isotopologues in leaves[END_REF], however, state that comparably good results could also be achieved with different well mixed metabolic pools of water. For epiphytic and non-vascular plants which lack permanent access to soil water, it has been shown that a description of water isotope dynamics requires consideration of distinct water pools as well as water potentials [START_REF] Helliker | Toward a plant-based proxy for the isotope ratio of atmospheric water vapor[END_REF][START_REF] Hartard | Water isotopes in desiccating lichens[END_REF][START_REF] Helliker | On the controls of leaf-water oxygen isotope ratios in the atmospheric Crassulacean Acid Metabolism epiphyte Tillandsia usneoides[END_REF].

Exchange of H 2 O and CO 2 oxygen isotopes

The enzyme carbonic anhydrase (CA) catalyzes oxygen exchange between water and CO 2 via (reversible) interconversions of CO 2 and H 2 O to bicarbonate (HCO - 3 ) and protons [START_REF] Badger | The role of carbonic-anhydrase in photosynthesis[END_REF]. This exchange underlies 18 O discrimination during CO 2 exchange ( 18 O), and retroflux to the atmosphere of CO 2 that has previously equilibrated with leaf water, which has a strong effect on the 18 O content of atmospheric CO 2 (Farquhar et al., 1993). Also, this signal provides a measure of photosynthetic activity of the terrestrial biosphere (Farquhar et al., 1993). At the leaf level, measurements of gas exchange, CA, 18 O and 13 C can help to partition mesophyll conductance into a cell wall and a chloroplast component (Gillon and Yakir, 2000a). However, work by [START_REF] Cousins | C 4 photosynthetic isotope exchange in NAD-ME-and NADP-ME-type grasses[END_REF] indicates that CA activity may not be a good predictor for CO 2 -H 2 O isotopic exchange, endorsing the view that more work is needed to fully understand the control of 18 O and its physiological implications.

Ternary effects on CO 2 isotopes during gas exchange

Ternary effects, i.e. effects of concurrent water vapor diffusion on CO 2 diffusion through stomata, are taken into account for CO 2 exchange [START_REF] Von Caemmerer | Some relationships between the biochemistry of photosynthesis and the gas-exchange of leaves[END_REF] but not for isotopes. Mesophyll conductance is a parameter greatly influenced by ternary effects. [START_REF] Farquhar | Ternary effects on the gas exchange of isotopologues of carbon dioxide[END_REF] recently showed that by applying the ternary correction, oxygen isotope composition of CO 2 in the www.biogeosciences.net/9/3083/2012/ Biogeosciences, 9, 3083-3111, 2012

chloroplast and mitochondria better match the oxygen isotopic composition of water at the sites of evaporation. The ternary effect has been observed to be greatest when the leafto-air water vapor pressure deficit is large. Farquhar and Cernusak ( 2012) also observed that a large impact of ternary corrections occurred when the difference in the isotopic composition of CO 2 between the leaf interior and the ambient air was large. The precision of current isotope fractionation models can be improved by applying the ternary correction equations for isotope fractionation and isotope exchange during gas exchange measurements.

Post-carboxylation and respiratory fractionation

The carbon isotope signal, imprinted through photosynthetic 13 C discrimination (the sum of terms one to four on the right hand side of Eq. 3, Table 1), can be altered by multiple processes in down-stream metabolic pathways (termed postcarboxylation fractionation), which will be reflected in different carbon pools and respired CO 2 . Despite early evidence by [START_REF] Park | Metabolic fractionation of 13 C and 12 C in plants[END_REF], carbon isotope fractionation during dark respiration has long been considered negligible [START_REF] Lin | Carbon isotopic fractionation does not occur during dark respiration in C3 and C4 plants[END_REF]. Systematic studies by [START_REF] Duranceau | δ 13 C of CO 2 respired in the dark in relation to δ 13 C of leaf carbohydrates in Phaseolus vulgaris L. under progressive drought[END_REF] and [START_REF] Ghashghaie | δ 13 C of CO 2 respired in the dark in relation to δ 13 C of leaf metabolites: comparison between Nicotiana sylvestris and Helianthus annuus under drought[END_REF] with a range of C 3 species again provided clear evidence for substantial and systematic variation in carbon isotope ratios of leaf dark respiration (see review by [START_REF] Ghashghaie | Carbon isotope fractionation during dark respiration and photorespiration in C3 plants[END_REF]. These authors introduced the term "apparent respiratory fractionation" to describe the manifested differences between carbon isotope compositions of leaf dark-respired CO 2 and its putative substrates (mainly carbohydrates), caused by multiple processes in the respiratory pathways (see below). The work of Ghashghaie and coworkers promoted a significant number of studies on post-carboxylation fractionations in downstream metabolic processes [START_REF] Klumpp | C-isotope composition of CO 2 respired by shoots and roots: fractionation during dark respiration?[END_REF][START_REF] Badeck | Post-photosynthetic fractionation of stable carbon isotopes between plant organs -a widespread phenomenon[END_REF][START_REF] Cernusak | Viewpoint: Why are non-photosynthetic tissues generally 13 C enriched compared with leaves in C 3 plants?, Review and synthesis of current hypotheses[END_REF][START_REF] Tcherkez | 12 C/ 13 C fractionations in plant primary metabolism[END_REF]Werner and Gessler, 2011).

Progress and challenges

Post-carboxylation fractionation

Already within the Calvin cycle, isotopic fractionation occurs mainly due to metabolic branching points and the use of triose phosphates that can either be exported to the cytosol or continue to be used within the Calvin cycle. The triose phosphates that are not exported are subject to certain enzyme catalyzed reactions (aldolisation and transketolisation) which involve position-specific discrimination during C-C bond making. As a result, the C-3 and C-4 positions within glucose are enriched in 13 C and thus a nonhomogeneous intra-molecular distribution of 13 C within carbohydrates is established [START_REF] Rossmann | Evidence for a nonstatistical carbon isotope distribution in natural glucose[END_REF][START_REF] Tcherkez | Theoretical considerations about carbon isotope distribution in glucose of C 3 -plants[END_REF][START_REF] Gilbert | Accurate quantitative isotopic 13 C NMR spectroscopy for the determination of the intramolecular distribution of 13 C in glucose at natural abundance[END_REF]. Subsequently, photorespiration and starch-sucrose partitioning result in diel changes in isotopic signatures of phloem sugars, with day sucrose being 13 C-depleted, while night exported sucrose is 13 C-enriched [START_REF] Tcherkez | Theoretical considerations about carbon isotope distribution in glucose of C 3 -plants[END_REF][START_REF] Gessler | Experimental evidence for diel variations of the carbon isotope composition in leaf, stem and phloem sap organic matter in Ricinus communis[END_REF]. Analyses of sugar δ 13 C and its diurnal variations offer potential for improved tracing of these changes in these metabolic activities.

Apparent respiratory fractionation

During the last decade the knowledge of relevant apparent fractionation in the respiratory pathways has significantly advanced, demonstrating substantial variability in respiratory fractionation among species, organs and functional groups, as well as with environmental conditions (see reviews by [START_REF] Ghashghaie | Carbon isotope fractionation during dark respiration and photorespiration in C3 plants[END_REF]Werner and Gessler, 2011).

The observed apparent respiratory fractionation and its variability are mainly attributed to (i) non-homogeneous 13 Cdistribution within hexose molecules reported by [START_REF] Rossmann | Evidence for a nonstatistical carbon isotope distribution in natural glucose[END_REF] and modelled by [START_REF] Tcherkez | Theoretical considerations about carbon isotope distribution in glucose of C 3 -plants[END_REF], (ii) relative contributions of different pathways to respiration (reviewed by [START_REF] Ghashghaie | Carbon isotope fractionation during dark respiration and photorespiration in C3 plants[END_REF], as well as (iii) enzymatic isotope effects during decarboxylation reactions (recently reviewed by [START_REF] Tcherkez | 12 C/ 13 C fractionations in plant primary metabolism[END_REF].

Fragmentation fractionation

The non-homogeneous intra-molecular distribution of 13 C in carbohydrates, results in so-called "fragmentation fractionation" [START_REF] Tcherkez | Theoretical considerations about carbon isotope distribution in glucose of C 3 -plants[END_REF], leaving its imprint on synthesized metabolites. If one of these products is decarboxylated, then respired CO 2 will carry an isotopic signature different from the average sugar signature. New data indicate that the heterogeneous 13 C distribution in carbohydrates may vary among species and with environmental conditions [START_REF] Gilbert | The intramolecular 13C-distribution in ethanol reveals the influence of the CO 2 -fixation pathway and environmental conditions on the sitespecific 13 C variation in glucose[END_REF]. Moreover, switches between substrates [START_REF] Tcherkez | Metabolic origin of carbon isotope composition of leaf dark-respired CO 2 in French bean[END_REF] during light-dark transition of leaves (i.e. light enhanced dark respiration due to decarboxylation of lightaccumulated malate, [START_REF] Barbour | A new measurement technique reveals temporal variation in δ 18 O of leaf-respired CO 2[END_REF] and the oxidative pentosephosphate pathway (PPP) were shown to markedly change δ 13 C, of respired CO 2 [START_REF] Bathellier | Divergence in δ 13 C of dark respired CO 2 and bulk organic matter occurs during the transition between heterotrophy and autotrophy in Phaseolus vulgaris plants[END_REF][START_REF] Bathellier | Metabolic origin of the δ 13 C of respired CO 2 in roots of Phaseolus vulgaris[END_REF]. The implication of these processes still needs to be explored.

Temporal dynamics and apparent respiratory fractionation

So far, a full quantitative understanding of apparent respiratory fractionation has not yet been achieved. However, measurements with a high temporal resolution indicated remarkable diel dynamics in leaf respiratory δ 13 CO 2 , which differed between functional plant groups [START_REF] Priault | Pronounced differences in diurnal variation of carbon isotope composition of leaf respired CO 2 among functional groups[END_REF][START_REF] Werner | Short-term dynamics of isotopic composition of leaf respired CO 2 upon darkening: measurements and implications[END_REF]Werner and Gessler, 2011). Feeding experiments with positionally labelled glucose or pyruvate can trace changes in carbon partitioning in the metabolic branching points of the respiratory pathways [START_REF] Tcherkez | Theoretical considerations about carbon isotope distribution in glucose of C 3 -plants[END_REF], which has been used at the organ level as evidence for an important contribution of PPP to root respiration [START_REF] Bathellier | Metabolic origin of the δ 13 C of respired CO 2 in roots of Phaseolus vulgaris[END_REF] as well as to identify differences between functional groups [START_REF] Priault | Pronounced differences in diurnal variation of carbon isotope composition of leaf respired CO 2 among functional groups[END_REF][START_REF] Wegener | The magnitude of diurnal variation in carbon isotopic composition of leaf dark respired CO 2 correlates with the difference between δ 13 C of leaf and root material[END_REF]. One challenge of labelling experiments is to find methods for channelling additional labelled metabolites into plant organs in vivo to shed further light on potential involvement of these metabolites and their metabolic pathways. Furthermore, the commitment of metabolites to alternative pathways at metabolic branching points needs to be quantified. This is particularly relevant where metabolic channelling evokes compartmentation in organelles with membranes, which are impermeable for intermediate products, as shown recently for the Krebs cycle (Werner et al., 2011).

Intra-molecular site-specific isotope fractionation

Recent results of [START_REF] Gilbert | The intramolecular 13C-distribution in ethanol reveals the influence of the CO 2 -fixation pathway and environmental conditions on the sitespecific 13 C variation in glucose[END_REF] demonstrated that access to site-specific isotope fractionation is now possible using 13 C NMR (see Sect. 3) to directly determine intramolecular 13 C distributions at natural abundance. The challenge in the application of this new technology is to ensure sufficient sample quantities from metabolic pools of interest. Of special interest is that obtained data can be interpreted directly in terms of isotope effects associated with specific enzymes.

Environmental effects

More studies of the sensitivity of respiratory δ 13 C to changes in environmental conditions and between organs are needed, which will allow for a better understanding of temporal variability in post-photosynthetic fractionation [START_REF] Dubbert | Temporal and spatial variations in carbon isotope fractionation of dark-respired CO 2 and different plant carbon pools in three Mediterranean species under different climatic conditions[END_REF] and could provide a basis for the use of respiratory δ 13 CO 2 as an indicator of physiological activity (e.g. Barbour et al., 2011a, b).

Bulk leaf tissue δ 13 C and δ 18 O and water use efficiency

The 13 C model by [START_REF] Farquhar | On the relationship between carbon isotope discrimination and the intercellular carbon dioxide concentration in leaves[END_REF]Eq. 3 in Table 1) predicts a linear relationship between 13 C and intrinsic water use efficiency (WUE i ; the ratio of net assimilation, A, to stomatal conductance, g s ), for conditions where mesophyll conductance is very high and (photo)respiratory 13 C discrimination is negligible (Eq. 4). Empirical studies in controlled conditions confirmed this linear relationship between 13 C, estimated from bulk biomass carbon isotope composition ( 13 C b ), and WUE i [START_REF] Farquhar | On the relationship between carbon isotope discrimination and the intercellular carbon dioxide concentration in leaves[END_REF](Farquhar et al., , 1989b;;[START_REF] Ehleringer | Variation in leaf carbon isotope discrimination in Encelia farinosa: implications for growth, competition, and drought survival[END_REF][START_REF] Griffiths | Stable Isotopes -integration of biochemical, ecological and geochemical processes[END_REF][START_REF] Brugnoli | Photosynthetic fractionation of carbon isotopes[END_REF]. In the following three decades, this linear (simplified) model of 13 C (Eq. 4) was used widely as an indicator of water use efficiency at the leaf, plant and ecosystem scale (e.g. [START_REF] Bonal | Interspecific variability of 13 C among trees in rainforests of French Guiana: functional groups and canopy integration[END_REF][START_REF] Lauteri | Genetic variation in carbon isotope discrimination in six European populations of Castanea sativa Mill. originating from contrasting localities[END_REF][START_REF] Saurer | Carbon isotope discrimination indicates improving water-use efficiency of trees in northern Eurasia over the last 100 years[END_REF][START_REF] Ponton | Comparison of ecosystem water-use efficiency among Douglas-fir forest, aspen forest and grassland using eddy covariance and carbon isotope techniques[END_REF] in retrospective studies of carbon-water relations based on biological archives (see Sect. 2.6), and in breeding crop varieties for improved yield under water-limited conditions [START_REF] Condon | Improving intrinsic water-use efficiency and crop yield[END_REF].

Progress and challenges

The dual-isotope approach Combined analyses of the carbon and oxygen isotopic composition of bulk leaf biomass provide a means to distinguish the separate effects of stomatal conductance and net photosynthesis on WUE i [START_REF] Scheidegger | Linking stable oxygen and carbon isotopes with stomatal conductance and photosynthetic capacity: A conceptual model[END_REF]. Preferably, however, carbon isotope discrimination 13 C b , and bulk biomass oxygen isotope discrimination, 18 O b , should be used in such an approach to account for effects of differences in δ 13 C of assimilated CO 2 and variations of δ 18 O of source water. A distinction between stomatal and photosynthetic influences cannot be made from analysis of 13 C b alone. The conceptual model of [START_REF] Scheidegger | Linking stable oxygen and carbon isotopes with stomatal conductance and photosynthetic capacity: A conceptual model[END_REF] was successfully applied in the field (Keitel et al., 2003, Sullivan andWelker, 2007) and further adapted for air pollution studies evaluating the effect of NO x on plant metabolism [START_REF] Siegwolf | Stable isotope analysis reveals differential effects of soil nitrogen and nitrogen dioxide on the water use efficiency in hybrid poplar leaves[END_REF][START_REF] Guerrieri | Impact of different nitrogen emission sources on tree physiology: a triple stable isotope approach[END_REF][START_REF] Guerrieri | Anthropogenic NO x emissions alter the intrinsic water-use efficiency (WUEi) for Quercus cerris stands under Mediterranean climate conditions[END_REF][START_REF] Savard | Tree-ring stable isotopes and historical perspectives on pollution -An overview[END_REF]. [START_REF] Grams | Combining δ 13 C and δ 18 O analyses to unravel competition, CO 2 and O 3 effects on the physiological performance of different-aged trees[END_REF] extended the model to estimate stomatal aperture directly for interpreting physiological changes. δ 18 O of bulk organic matter has also been used to determine whether a change in WUE i results from the increase in atmospheric CO 2 [START_REF] Saurer | Human impacts on tree-ring growth reconstructed from stable isotopes, in: Stable Isotopes as Indicators of Ecological Change, Terrestrial Ecology Series[END_REF]. Also, the effects of changes in vapor pressure deficit (VPD) resulting from increasing temperature or decreasing precipitation have been assessed along a Siberian transect [START_REF] Sidorova | Do centennial tree-ring and stable isotope trends of Larix gmelinii (Rupr.) Rupr. indicate increasing water shortage in the Siberian north?[END_REF][START_REF] Knorre | Twentieth century trends in tree ring stable isotopes (δ 13 C and δ 18 O) of Larix sibirica under dry conditions in the forest steppe in Siberia[END_REF]. The dual-isotope approach has proven a valuable concept for ecological applications. However, the interpretation of 13 C b in terms of WUE i under natural changing environments is complex (e.g. [START_REF] Seibt | Carbon isotopes and water use efficiency: sense and sensitivity[END_REF], as it provides a time-integrated record of photosynthetic discrimination over the period that the carbon forming the leaf was fixed, which can be derived from multiple sources, e.g. fresh assimilates, carbon exported from mature leaves or even older storage pools. Moreover, different leaf carbon pools have different residence and turnover times [START_REF] Nogués | Respiratory carbon metabolism following illumination in intact french bean leaves using 13 C/ 12 C isotope labelling[END_REF][START_REF] Lehmeier | Root and shoot respiration of perennial ryegrass are supplied by the same substrate pools: assessment by dynamic 13 C labeling and compartmental analysis of tracer kinetics[END_REF]Lehmeier et al., , 2010b)). Thus, during leaf formation, growth and maintenance leaf bulk material integrates isotopic information from different time periods and sources, which is weighted by the amount of carbon incorporated from each source/period. Therefore, interpretation of 13 C b in terms of WUE i under natural changing environments requires several precautions, as described below.

Interpretation of 13 C b and 18 O b in relation to leaf traits

A comparison of WUE i between different species based on 13 C in bulk leaf material is nontrivial. Differences in not only leaf structural, anatomical, but also physiological traits can modulate 13 C b [START_REF] Ehleringer | Variation in leaf carbon isotope discrimination in Encelia farinosa: implications for growth, competition, and drought survival[END_REF][START_REF] Werner | Carbon isotope discrimination as a tracer of functional traits in a Mediterranean macchia plant community[END_REF], as well as 18 O in lamina leaf water [START_REF] Barbour | Do pathways of water movement and leaf anatomical dimensions allow development of gradients in (H 2 O)-O-18 between veins and the sites of evaporation within leaves?[END_REF][START_REF] Kahmen | Physiological and morphological effects on leaf water δ 18 O enrichment in different Eucalyptus species[END_REF]) and 18 O b . Different leaf structures may affect mesophyll conductance (e.g. [START_REF] Kogami | CO 2 transfer conductance, leaf structure and carbon isotope composition of Polygonum cuspidatum leaves from low and high altitudes[END_REF][START_REF] Hanba | The effect of internal CO 2 conductance on leaf carbon isotope ratio[END_REF] and thus 13 C b (see Sect. 2.1.1). Mesophyll conductance is generally neglected when calculating WUE i from stable isotope discrimination (see Eq. 5). If there are varying influences of mesophyll conductance on 13 C among species, WUE i calculated from Eq. (5) will be not directly comparable. Leaf traits may also affect the scaled effective path length for water movement from the xylem to the site of evaporation [START_REF] Wang | Temporal and spatial variations in the oxygen-18 content of leaf water in different plant species[END_REF] and thus influence 18 O b (cf. Eq. 8, Table 2). The conceptual model of [START_REF] Scheidegger | Linking stable oxygen and carbon isotopes with stomatal conductance and photosynthetic capacity: A conceptual model[END_REF] does not account for such effects but strictly assumes oxygen isotope enrichment to be only affected by the ratio of ambient to intracellular water pressure (e a /e i ; cf. Eq. 7). Any other factor varying leaf water evaporative enrichment and thus 18 O b will thus constrain the interpretation of the impact of stomatal conductance versus net photosynthesis on WUE i . Moreover, due to differences in phenological phases and length of growing period leaf 13 C b and 18 O b of co-occurring species might provide an integrated signal over diverging environmental conditions (e.g. [START_REF] Werner | Carbon isotope discrimination as a tracer of functional traits in a Mediterranean macchia plant community[END_REF]. Thus, species-specific differences in phenology, growth pattern and leaf structures might constrain a direct comparison of bulk leaf 13 C and 18 O between different species [START_REF] Hanba | The effect of internal CO 2 conductance on leaf carbon isotope ratio[END_REF][START_REF] Warren | Internal conductance does not scale with photosynthetic capacity: implications for carbon isotope discrimination and the economics of water and nitrogen use C[END_REF]. Moreover, ontogeny can markedly alter the isotopic signature [START_REF] Terwilliger | Intrinsic water-use efficiency and heterotrophic investment in tropical leaf growth of two neotropical pioneer tree species as estimated from δ 13 C values[END_REF][START_REF] Bathellier | Divergence in δ 13 C of dark respired CO 2 and bulk organic matter occurs during the transition between heterotrophy and autotrophy in Phaseolus vulgaris plants[END_REF][START_REF] Salmon | Ontogeny and leaf gas exchange mediate the carbon isotopic signature of herbaceous plants through large non-photosynthetic discrimination[END_REF]. Repetitive sampling and isotope analysis of tissues and compounds which are known to integrate shorter and more defined time periods such as phloem sugars (e.g. [START_REF] Keitel | Carbon and oxygen isotope composition of organic compounds in the phloem sap provides a shortterm measure for stomatal conductance of European beech (Fagus sylvatica L.)[END_REF][START_REF] Dubbert | Temporal and spatial variations in carbon isotope fractionation of dark-respired CO 2 and different plant carbon pools in three Mediterranean species under different climatic conditions[END_REF] during the growing season, together with bulk leaf assessments, might help to better constrain the physiological meaning of 13 C b and 18 O b . For community scale stable isotope approaches (see Sect. 2.3), the potential limitations of using the bulk isotope signature need to be kept in mind and sampling strategies need to be adapted. For trees, the intra-annual analysis of tree ring, whole wood or cellulose can provide a tool to study periods during the growing season when the isotopic signature in this archive is directly coupled to leaf physiology [START_REF] Helle | Beyond CO 2 -fixation by Rubiscoan interpretation of 13 C/ 12 C variations in tree rings from novel intra-seasonal studies on broad-leaf trees[END_REF][START_REF] Offermann | The long way down-are carbon and oxygen isotope signals in the tree ring uncoupled from canopy physiological processes?[END_REF].

Interpretation of leaf 13 C b and 18 O b in relation to storage and remobilization

A part of the leaf structural organic matter of deciduous trees is made from remobilized starch (or other non-structural compounds) from overwinter storage pools in stems and roots (Kozlowski and Pallardy, 1997). That material was derived from the photosynthetic activity of previous year leaves, with different morpho-physiological characteristics in other environmental conditions, producing a previous year isotopic signal. Since starch can be 13 C-enriched by up to 4 ‰ as compared to newly assimilated sugars [START_REF] Gleixner | Stable isotope distribution in the major metabolites of source and sink organs of Solanum tuberosum L.: a powerful tool in the study of metabolic partitioning in intact plants[END_REF], growing leaves supplied from storage pools are often strongly 13 C enriched in spring (e.g. [START_REF] Terwilliger | Changes in the 13 C values of trees during a tropical rainy season: some effects in addition to diffusion and carboxylation by Rubisco?[END_REF][START_REF] Helle | Beyond CO 2 -fixation by Rubiscoan interpretation of 13 C/ 12 C variations in tree rings from novel intra-seasonal studies on broad-leaf trees[END_REF]. Moreover, during starch breakdown, carbonyl oxygen atoms are exchanged with unenriched water in stems, causing these incorporated starch-derived sugars to be 18 O depleted as compared to sugars formed in transpiring leaves (Gessler et al., 2007b). This "isotopic starch imprint" in the newly developed leaves is thought to be diluted during the growing season by carbon turnover and the incorporation of new assimilates into bulk leaf organic matter (see e.g. the seasonal course of bulk leaf δ 13 C of beech shown by [START_REF] Helle | Beyond CO 2 -fixation by Rubiscoan interpretation of 13 C/ 12 C variations in tree rings from novel intra-seasonal studies on broad-leaf trees[END_REF]. For interpreting the isotopic composition of a deciduous leaf, it is thus important to consider when the leaf was harvested during the season and that there might be species-specific differences in the extent to which the starch imprint or the influence of the assimilates incorporated during the current growing season dominate the bulk isotope signal. In turn, coniferous needles can accumulate large amounts of starch in spring, followed by mobilization towards the summer, and starch contents are generally low during the winter (e.g. [START_REF] Ericsson | Effects of fertilization and irrigation on the seasonal changes of carbohydrate reserves in different age-classes of needle on 20-year-old Scots pine trees (Pinus sylvestris)[END_REF]. As a consequence, at least part of the 13 C b during the growing season is related to the variation of starch content and isotopic composition [START_REF] Jäggi | The relationship between the stable carbon isotope composition of needle bulk material, starch, and tree rings in Picea abies[END_REF], a fact that also needs to be taken into account when calculating WUE i from 13 C b and comparing it among species.

Thus, the complexity of processes influencing 13 C b may constrain its use in ecological field studies. Carbon pools with shorter turnover times and thus a better-defined origin such as leaf soluble sugars [START_REF] Brugnoli | Correlation between the carbon isotope discrimination in leaf starch and sugars of C-3 plants and the ratio of intercellular and atmospheric partial pressures of carbon dioxide, editesd by[END_REF], phloem allocated carbon (e.g. [START_REF] Gessler | Stable isotope composition of organic compounds transported in the phloem of European beech -Evaluation of different methods of phloem sap collection and assessment of gradients in carbon isotope composition during leaf-to-stem transport[END_REF][START_REF] Scartazza | Comparisons of δ 13 C of photosynthetic products and ecosystem respiratory CO 2 and their responses to seasonal climate variability[END_REF] or even dark-respired δ 13 CO 2 of recent photosynthates (Barbour et al., 2011b) are therefore better indicators for recent changes in WUE ia , as outlined below (Sect. 2.2).

13 C and 18 O isotopes to trace plant integrated processes and plant-soil coupling

With their pioneering work on the phloem carbon isotopic composition of grasses [START_REF] Yoneyama | Variations of the natural abundances of nitrogen and carbon isotopes in Triticum aestivum, with special reference to phloem and xylem exudates[END_REF] and trees [START_REF] Pate | δ 13 C analysis of phloem sap carbon: novel means of evaluating seasonal water stress and interpreting carbon isotope signatures of foliage and trunk wood of Eucalyptus globules[END_REF], two groups paved the way to getting temporally and spatially (whole canopy) integrated information on leaf physiology. Pate and Arthur (1998) applied natural abundance stable isotope approaches to investigate transport and allocation of assimilates by combining sampling of phloem sap organic matter and source and sink organs. This work focused on transport directions and patterns of sourceto-sink transport, while tracing of carbon allocation with high temporal resolution in plants required the use of labelling experiments (e.g. [START_REF] Hansen | The fate and path of assimilation products in the stem of 8-year-old Scots pine (Pinus sylvestris L.) trees[END_REF]. Within the last ten years it was, however, shown that the transport of newly assimilated carbon within the plant and from the plant to the rhizosphere can also be followed by natural abundance stable isotope techniques (e.g. [START_REF] Scartazza | Comparisons of δ 13 C of photosynthetic products and ecosystem respiratory CO 2 and their responses to seasonal climate variability[END_REF][START_REF] Brandes | Short-term variation in the isotopic composition of organic matter allocated from the leaves to the stem of Pinus sylvestris: effects of photosynthetic and postphotosynthetic carbon isotope fractionation[END_REF]Wingate et al., 2010b).

Progress and challenges

Plant integrating information and phloem transport

The δ 13 C of phloem organic matter is mincreasingly being used to derive information on carbon allocation, canopy integrated WUE and canopy integrated mesophyll conductance in plants affected by environmental conditions (e.g. [START_REF] Keitel | Carbon and oxygen isotope composition of organic compounds in the phloem sap provides a shortterm measure for stomatal conductance of European beech (Fagus sylvatica L.)[END_REF][START_REF] Gessler | Stable isotope composition of organic compounds transported in the phloem of European beech -Evaluation of different methods of phloem sap collection and assessment of gradients in carbon isotope composition during leaf-to-stem transport[END_REF][START_REF] Scartazza | Comparisons of δ 13 C of photosynthetic products and ecosystem respiratory CO 2 and their responses to seasonal climate variability[END_REF][START_REF] Barbour | Variation in the degree of coupling between δ 13 C of phloem sap and ecosystem respiration in two mature Nothofagus forests[END_REF][START_REF] Rascher | On the use of phloem sap δ 13 C as an indicator of canopy carbon discrimination[END_REF][START_REF] Ubierna | Estimation of canopy average mesophyll conductance using δ 13 C of phloem contents[END_REF][START_REF] Dubbert | Temporal and spatial variations in carbon isotope fractionation of dark-respired CO 2 and different plant carbon pools in three Mediterranean species under different climatic conditions[END_REF]. A dual-isotope approach (δ 13 C and δ 18 O see Sect. 2.1.3) can also be successfully applied to phloem sugars to distinguish whether net assimilation and/or stomatal conductance is changing as a result of environmental conditions [START_REF] Keitel | Carbon and oxygen isotope composition of organic compounds in the phloem sap provides a shortterm measure for stomatal conductance of European beech (Fagus sylvatica L.)[END_REF], Cernusak et al., 2003, 2005;[START_REF] Brandes | Short-term variation in the isotopic composition of organic matter allocated from the leaves to the stem of Pinus sylvestris: effects of photosynthetic and postphotosynthetic carbon isotope fractionation[END_REF][START_REF] Keitel | Carbon isotopic composition and oxygen isotopic enrichment in phloem and total leaf organic matter of European beech (Fagus sylvatica L.) along a climate gradient[END_REF]. Even though the carbon and oxygen isotope composition of phloem organic matter can, in principle, integrate leaf physiology over the whole canopy and track transport of assimilates within the plant, it is now clear that several uncertainties constrain the interpretation of phloem isotopic information. These are related to (i) the temporal integration of the isotope signal in the phloem, (ii) potential changes of the isotope composition of phloem sugars in basipetal direction, and (iii) the chemical composition of phloem transported organic matter.

Phloem sugars and temporal integration

Short-term variations in the isotopic composition of leaf sugars -induced by either an environmental signal or plant internal processes -might or might not be reflected in the isotopic composition of phloem organic matter. Twig phloem organic matter of trees (e.g. Gessler et al., 2007a) and the stem phloem of herbaceous species (e.g. [START_REF] Gessler | Experimental evidence for diel variations of the carbon isotope composition in leaf, stem and phloem sap organic matter in Ricinus communis[END_REF] can be applied to monitor diel variation of evaporative 18 O and 2 H enrichment or carbon isotope fractionation. In the trunks of adult trees, however, the mixing of sugars of different metabolic origins can dampen the short-term variations and the isotope signatures provide time-integrated information on canopy processes instead [START_REF] Keitel | Carbon isotopic composition and oxygen isotopic enrichment in phloem and total leaf organic matter of European beech (Fagus sylvatica L.) along a climate gradient[END_REF][START_REF] Rascher | On the use of phloem sap δ 13 C as an indicator of canopy carbon discrimination[END_REF].

Change of the original isotope signal in phloem sugars

The original isotope signal imprinted on sugars in the leaf may be altered during basipetal transport in the phloem of trees (e.g. [START_REF] Rascher | On the use of phloem sap δ 13 C as an indicator of canopy carbon discrimination[END_REF]. The transport of sugar molecules itself does not fractionate to a measurable extent. However, carbon fixation by PEPc in the bark and oxygen atom exchange with stem water during metabolic processes in the stem tissue together with the continuous unloading and loading of sugars from and to the phloem might contribute to the observed isotope patterns [START_REF] Barnard | Evaporative enrichment and time lags between δ 18 O of leaf water and organic pools in a pine stand[END_REF][START_REF] Gessler | Within-canopy and ozone fumigation effects on δ 13 C and 18 O in adult beech (Fagus sylvatica) trees: relation to meteorological and gas exchange parameters[END_REF]. The change in δ 13 C along the transport path, however, varies strongly among species ranging from 13 C enrichment [START_REF] Brandes | Short-term variation in the isotopic composition of organic matter allocated from the leaves to the stem of Pinus sylvestris: effects of photosynthetic and postphotosynthetic carbon isotope fractionation[END_REF]Wingate et al., 2010a) and no change in δ 13 C [START_REF] Pate | δ 13 C analysis of phloem sap carbon: novel means of evaluating seasonal water stress and interpreting carbon isotope signatures of foliage and trunk wood of Eucalyptus globules[END_REF]Gessler et al., 2007a) to 13 C depletion [START_REF] Rascher | On the use of phloem sap δ 13 C as an indicator of canopy carbon discrimination[END_REF]. The nature of these species-specific differences remains to be clarified and might shed new light on mechanisms controlling assimilate partitioning in trees.

Chemical composition of phloem sugars

It is often assumed that only one major sugar, namely sucrose, is present in the phloem. However, besides sucrose, there are other transport carbohydrates -depending on species and phloem loading mechanisms -such as myoinositol and raffinose family sugars [START_REF] Karner | myo-Inositol and sucrose concentrations affect the accumulation of raffinose family oligosaccharides in seeds[END_REF]. In addition, it is still a matter of debate if hexoses are transported in the phloem or not [START_REF] Van Bel | Hexoses as phloem transport sugars: the end of a dogma?[END_REF][START_REF] Liu | Transport of sucrose, not hexose, in the phloem[END_REF]. Phloem sugar composition can vary with environmental conditions, which could be one factor for changes in phloem δ 13 C [START_REF] Merchant | Phloem sap and leaf δ 13 C, carbohydrates, and amino acid concentrations in Eucalyptus globulus change systematically according to flooding and water deficit treatment[END_REF], independent of the original leaf-borne isotope signal, since δ 13 C differs between different carbohydrate molecules [START_REF] Schmidt | Fundamentals and systematics of the nonstatistical distributions of isotopes in natural compounds[END_REF][START_REF] Devaux | Carbon stable isotope ratio of phloem sugars in mature pine trees throughout the growing season: comparison of two extraction methods[END_REF]. Compound-specific analysis, provided by modern LC-and GC-IRMS techniques (Sect. 3), will help to differentiate between changes in phloem δ 13 C that result from either changes in the chemical composition or changes in leaf level fractionation. In addition, comparable methods should be used to characterise the compound-specific oxygen isotope composition of phloem organic matter.

Only recently, the natural abundance stable isotope information in soil and ecosystem respired CO 2 cross-correlated to photosynthesis (or its proxies) has been used systematically to characterise the speed of link between canopy and soil processes (see review by [START_REF] Kuzyakov | Review: Time lag between photosynthesis and carbon dioxide efflux from soil: a review of mechanisms and controls[END_REF]Wingate et al., 2010b). Even though such approaches have significant potential, there is still debate about the physiological information conveyed by the isotope signal and of the processes involved (see review by [START_REF] Brüggemann | Carbon allocation and carbon isotope fluxes in the plant-soil-atmosphere continuum: a review[END_REF].

The link between above-and belowground processes

In their review, Kuzyakov and Gavrichkova (2010) postulated that approaches which quantify time lags between proxies of photosynthetic activity and natural abundance δ 13 C in soil respiration are (besides other techniques) appropriate to study the link between above-and belowground processes. Mencuccini and Holttä (2010) reviewed different approaches to assess the speed of link between assimilation and soil respiration and concluded, in contrast, "that isotopic approaches are not well suited to document whether changes in photosynthesis of tall trees can rapidly affect soil respiration". These different opinions may be related to uncertainties on the mechanisms involved, as described by Kayler et al. (2010a, b): on the one hand, there is evidence that pressure-concentration waves (Thompson and www.biogeosciences.net/9/3083/2012/ Biogeosciences, 9, 3083-3111, 2012

Holbrook, 2004), which travel rapidly through the phloem of plants (and not the supply of new assimilates transported via the phloem to the roots and the rhizosphere), are responsible for the fast response of soil respiration to changes in photosynthesis. On the other hand, the time-lag between the fixation of a carbon molecule during photosynthesis and its respiration belowground may contain real and important information about plant physiology and carbon use as well as the degree to which plant and soil are coupled (Kayler et al., 2010a). This information may be obtained by the assessment of δ 13 C in respired CO 2 but also in respiratory substrates when points listed above are taken into account. To unravel the importance of the different relevant processes, we need novel, pertinent experiments which combine (a) continuous measurements of the natural abundance stable isotope composition of soil respired CO 2 , as done by Wingate et al. (2010b), with (b) appropriate statistical approaches that are able to track time lags between photosynthesis and soil CO 2 efflux, as applied by [START_REF] Vargas | On the multitemporal correlation between photosynthesis and soil CO 2 efflux: reconciling lags and observations[END_REF]. Whereas (a) indicates how long it takes until a molecule with a given isotope composition imprinted during photosynthesis is transported from the leaves via the phloem to the roots where it is respired, (b) allows to detect the response time(s) of soil respiration towards changes in carbon assimilation, which might or might not be faster than the transport of a given molecule from the canopy to belowground.

Community-scale processes

Because different species living within the same habitat show marked differences in the isotopic composition of their leaf tissues, characterising community-wide variation in δ 13 C and/or δ 18 O, can provide potentially powerful tools for investigating the physiological basis for niche partitioning among community members [START_REF] Dawson | Stable isotopes in plant ecology[END_REF]. Good examples are utilization of different water sources and redistribution [START_REF] Caldwell | Hydraulic lift: consequences of water efflux from the roots of plants[END_REF][START_REF] Ryel | Rapid soil moisture recharge to depth by roots in a stand of Artemisia tridentate[END_REF], which can in turn be linked to community composition [START_REF] Ehleringer | Climate change and the evolution of C 4 photosynthesis[END_REF], niche partitioning and spatial and temporal variations in plant distributions (e.g. [START_REF] Dawson | Stable isotopes in plant ecology[END_REF][START_REF] Snyder | Water sources used by riparian trees varies among stream types on the San Pedro river, Arizona[END_REF][START_REF] Stratton | Temporal and spatial partitioning of water resources among eight woody species in a Hawaiian dry forest[END_REF][START_REF] Drake | Water resource partitioning, stem xylem hydraulic properties and water use strategies in a seasonally dry riparian tropical rainforest[END_REF][START_REF] Rose | Water source utilization by Pinus jeffreyi and Actostaphylos patula on thin soils over bedrock[END_REF][START_REF] Grams | Stable isotope signatures reflect competitiveness between trees under changed CO 2 /O 3 regimes[END_REF]. As stated above (Sect. 2.1.3), it must be kept in mind when comparing different species that the isotopic composition of bulk leaf material might be influenced by multiple factors such as structural, anatomical and physiological traits but also phenology.

There are only very few community-wide investigations on this topic (see [START_REF] Smedley | Seasonal carbon isotope discrimination in a grassland community[END_REF][START_REF] Guehl | Community-level diversity of carbon-water relations in rainforest trees, in: Ecology and Management of a Neotropical Rainforest, dited by[END_REF][START_REF] Kahmen | Addressing the functional value of biodiversity for ecosystem functioning using stable isotopes, in: Stable isotopes as indicators for ecological change[END_REF]. This scarcity may be in part related to difficulties in assigning cause and effect to observed variation from either physical (e.g. water availability, light) or biological (e.g. resource competition) factors.

Progress and challenges

Adding duel-isotope approaches to community ecology 13 C and 18 O signals can trace biotic and abiotic interactions within the plant community and may contribute to identifying what shapes community-scale processes. However, individual plants will not necessarily respond to environmental perturbations as "a community", but may respond according to species-specific traits and requirements and additionally depend on the interactions with the surrounding environment and other present species (e.g. [START_REF] Roscher | The role of biodiversity for element cycling and trophic interactions: an experimental approach in a grassland community[END_REF][START_REF] Gubsch | Foliar and soil delta 15 N values reveal increased nitrogen partitioning among species in diverse grassland communities[END_REF]. Competition and/or facilitation interactions between species, e.g. through depletion of a particular resource, may also be a source of isotopic variation, as shown for plant-plant competition for above-and belowground resources by combining δ 13 CO 2 and δ 18 H 2 O analyses [START_REF] Ramírez | Bulk leaf δ 18 O and δ 13 C reflect the intensity of intraspecific competition for water in a semi-arid tussock grassland[END_REF][START_REF] Grams | Stable isotope signatures reflect competitiveness between trees under changed CO 2 /O 3 regimes[END_REF]. Moreover, changes in community functioning, for example by alterations in nutrient, carbon and hydrological cycles after exotic plant invasion, can also be traced through stable isotopes (e.g. [START_REF] Rascher | Understory invasion by Acacia longifolia alters the water balance and carbon gain of a Mediterranean pine forest[END_REF].

Tracing spatial interaction between species within plant communities

Spatio-temporal variations in isotope ratios (i.e. isoscapes) contain a potential wealth of information regarding ecological processes [START_REF] West | A simplified GIS approach to modeling global leaf water isoscapes[END_REF][START_REF] Bowen | Isoscapes to address large-scale earth science challenges[END_REF], which have, so far been applied at larger spatial scales (see Sect. 2.5). At the community scale, spatial heterogeneity in resource availability, differential resource utilisation by neighbouring species and their interactions (competition and facilitation) occur in a spatially explicit dimension, which may contain crucial information regarding community functioning. For example, hydraulic redistribution of water sources is a key process which can shape plant communities (see review by [START_REF] Prieto | Water release through plant roots: new insights into its consequences at the plant and ecosystem level[END_REF]. Recently it has been shown that downscaling isoscapes to the community level allowed tracing the spatial impact of an invasive species on community functioning [START_REF] Rascher | Community scale 15 N isoscapes: tracing the spatial impact of an exotic N 2 -fixing invader[END_REF], and may therefore open new possibilities in resolving the spatial component of within-community interactions.

Tracing functional groups/community composition

During the last decade, the functional group approach has proved to be an efficient way to analyse plant functioning at the community scale. Leaf bulk 13 C allows the distinction of broad plant functional types, differing in structural, phenological and physiological leaf traits [START_REF] Brooks | Carbon isotope composition of boreal plants: functional grouping of life forms[END_REF][START_REF] Bonal | Interspecific variability of 13 C among trees in rainforests of French Guiana: functional groups and canopy integration[END_REF][START_REF] Werner | Carbon isotope discrimination as a tracer of functional traits in a Mediterranean macchia plant community[END_REF]. Functional traits such as water or nutrient use strategies, carbon acquisition, growth behaviours, and phenological cycles contribute significantly to the observed variation in isotope composition (e.g. [START_REF] Warren | Internal conductance does not scale with photosynthetic capacity: implications for carbon isotope discrimination and the economics of water and nitrogen use C[END_REF][START_REF] Gubsch | Foliar and soil delta 15 N values reveal increased nitrogen partitioning among species in diverse grassland communities[END_REF][START_REF] Salmon | Ontogeny and leaf gas exchange mediate the carbon isotopic signature of herbaceous plants through large non-photosynthetic discrimination[END_REF]Ramírez et al., 2012). However, the responsiveness of leaf 13 C as a functional tracer has to be verified for different communities and may differ with the predominant environmental constraints for plant growth and survival (e.g. [START_REF] Caldeira | Mechanisms of positive biodiversity-production relationships: insights provided by d13C analysis in experimental Mediterranean grassland plots[END_REF]. For example, in a tropical rainforest, 13 C was associated with differences in shade tolerance [START_REF] Bonal | Interspecific variability of 13 C among trees in rainforests of French Guiana: functional groups and canopy integration[END_REF][START_REF] Guehl | Community-level diversity of carbon-water relations in rainforest trees, in: Ecology and Management of a Neotropical Rainforest, dited by[END_REF], whereas in an upland water-limited grassland of Greece, a semi-arid Inner Mongolian steppe, and a Portuguese mediterranean macchia grouping according to 13 C was associated with species' competitive ability related to WUE i , nitrogen use efficiency, and structural adaptations to drought [START_REF] Tsialtas | Interspecific variation in potential wateruse efficiency and its relation to plant species abundance in a water-limited grassland[END_REF][START_REF] Gong | Tradeoffs between nitrogen-and water-use efficiency in dominant species of the semiarid steppe of Inner Mongolia[END_REF][START_REF] Werner | Carbon isotope discrimination as a tracer of functional traits in a Mediterranean macchia plant community[END_REF].

The role of water source partitioning on community functioning

Several mixing models have been used to determine the contribution of different water sources to plant and ecosystem evapotranspiration: Linear mixing models can be applied if the differences of δ 18 O among the water sources and xylem plant water are large enough; δD-δ 18 O plots can be used if the difference between water sources and xylem water is small (Ogle and Reynolds, 2004;[START_REF] Dawson | Forest Hydrology and Biogeochemistry: Synthesis of Research and Future Directions, Ecological Studies Series[END_REF]. The use of multiple source mass balance analyses can improve the capacity to quantitatively and objectively evaluate complex patterns in stable isotope data for determining possible contributions of different sources to total plant water uptake (see review by [START_REF] Hu | Partitioning of evapotranspiration and its controls in four grassland ecosystems: Application of a two-source model[END_REF]. Furthermore, combining water source partitioning with indicators of species functional responses (e.g. changes in leaf water potential and carbon isotope discrimination) lent insight regarding the degree of plasticity among individual members of a given plant community [START_REF] Máguas | Responses of woody species to spatial and temporal ground water changes in coastal sand dune systems[END_REF]. However, there is increasing awareness that the utilisation of simple linear mixing models to infer plant water uptake by comparing δD and δ 18 O of xylem or root crown, on the one hand, and soil water, on the other hand, does not adequately reflect the high heterogeneity of water sources that may be available for a plant. Given the importance of resource variability at the community level, the utilisation of more complex mixing models (for example, by [START_REF] Phillips | Mixing models in analyses of diet using multiple stable isotopes: a critique[END_REF][START_REF] Phillips | Mixing models in analyses of diet using multiple stable isotopes: a critique[END_REF]Gregg, 2001, 2003;[START_REF] Parnell | Source partitioning using stable isotopes: coping with too much variation[END_REF] as well as Bayesian models (Ogle et al., 2004) may be fruitful.

Use of stable isotopes to disentangle ecosystem exchange processes

At the ecosystem scale, stable isotopes can provide insight into the complex interaction between vegetation, soil and atmosphere exchange of carbon and water fluxes, including their responses and feed-backs to environmental drivers (e.g. [START_REF] Flanagan | Ecosystem-atmosphere CO 2 exchange: interpreting signals of change using stable isotope ratios[END_REF][START_REF] Dawson | Stable isotopes in plant ecology[END_REF][START_REF] Yakir | The use of stable isotopes to study ecosystem gas exchange[END_REF][START_REF] Yakir | The stable isotopic composition of atmospheric CO 2[END_REF][START_REF] Hemming | Pan-European delta C-13 values of air and organic matter from forest ecosystems[END_REF] please see [START_REF] Bowling | Carbon isotopes in terrestrial ecosystem pools and CO 2 fluxes[END_REF] for review of pioneer and recent literature). The core of the variation behind patterns in δ 13 C of ecosystem respiration (δ 13 C R ) lies in photosynthetic discrimination, the magnitude of metabolic fluxes and several post-carboxylation fractionation processes that differ between autotrophic and heterotrophic organs (see Sect. 2.1.2 and references therein). How these components manifest into integrative measures such as ecosystem respiration is fundamental to understanding ecosystem physiology and biogeochemistry. It is clear that ecosystem responses to climate and land use change, or perturbations, such as drought or fire, are an integrative signal from a network of carbon pools and organisms linking legacy conditions to current observations (e.g. Buchmann et al., 1997aBuchmann et al., , b, 1998;;[START_REF] Ehleringer | Carbon and oxygen isotope ratios in below-ground carbon-cycle processes[END_REF]. Thus, to properly account for ecosystem trace gas exchange and partitioning by stable isotopes, a detailed knowledge of the physical and biological basis of the isotopic signals for each of the fluxes and their dynamics across spatial and temporal scales in soil-biosphere-atmosphere interactions is required.

Progress and challenges

Recent findings on component sources and fluxes

Previous ecosystem 13 C and 18 O isotope research primarily focused on partitioning of soil and canopy sources, which are now a mainstay of ecosystem isotopic investigations (e.g. [START_REF] Buchmann | Carbon isotope dynamics in Abies amabilis stands in the Cascades[END_REF][START_REF] Kaplan | The stable carbon isotope composition of the terrestrial biosphere: Modeling at scales from the leaf to the globe[END_REF][START_REF] Yakir | The use of stable isotopes to study ecosystem gas exchange[END_REF] and literature therein). The inherent complexity behind ecosystem respiration lies behind the many contributing sources. Nowadays, studies of these components have expanded to include stem CO 2 flux, mycorrhizal and microbial contributions [START_REF] Esperschütz | Microbial response to exudates in the rhizosphere of young beech trees (Fagus sylvatica L.) after dormancy[END_REF], litter decomposition [START_REF] Bird | 13 C and 15 N stabilization dynamics in soil organic matter fractions during needle and fine root decomposition[END_REF][START_REF] Rubino | Carbon input belowground is the major C flux contributing to leaf litter mass loss: Evidences from a 13 C labelled-leaf litter experiment[END_REF], dissolved organic carbon [START_REF] Sanderman | A comparative study of dissolved organic carbon transport and stabilization in California forest and grassland soils[END_REF][START_REF] Müller | Effective retention of litter-derived dissolved organic carbon in organic layers[END_REF], erosion [START_REF] Schaub | Stable carbon isotopes as an indicator for soil degradation in an alpine environment (Urseren Valley, Switzerland)[END_REF], soil organic matter dynamics [START_REF] Klumpp | Long-term steady state 13C labelling to investigate soil carbon turnover in grasslands[END_REF][START_REF] Kayler | Application of δ 13 C and δ 15 N isotopic signatures of organic matter fractions sequentially separated from adjacent arable and forest soils to identify carbon stabilization mecha-nisms[END_REF] and CO 2 storage in soil air and solution [START_REF] Gamnitzer | Measuring and modelling the isotopic composition of soil respiration: insights from a grassland tracer experiment[END_REF]. Labelling has also played a central role in achieving a higher level of certainty in observing single source temporal patterns [START_REF] Ubierna | Storage and transpiration have negligible effects on δ 13 C of stem CO 2 efflux in large conifer trees[END_REF][START_REF] Powers | Pulse labeling of dissolved 13 Ccarbonate into tree xylem: developing a new method to determine the fate of recently fixed photosynthate[END_REF]. Similarly, the water oxygen and hydrogen isotope composition has been used as natural or artificial tracer of the ecosystem and component water fluxes and to partition evaporation and transpiration (e.g. [START_REF] Yakir | Fluxes of CO 2 and water between terrestrial vegetation and the atmosphere estimated from isotope measurements[END_REF][START_REF] Yepez | Dynamics of transpiration and evaporation following a moisture pulse in semiarid grassland: A chamber-based isotope method for partitioning flux components[END_REF][START_REF] Yepez | Intraseasonal variation in water and carbon dioxide flux components in a semiarid riparian woodland[END_REF][START_REF] Williams | Evapotranspiration components determined by stable isotope, sap flow and eddy covariance techniques[END_REF][START_REF] Lai | Contributions of evaporation, isotopic non-steady state transpiration and atmospheric mixing on the δ 18 O of water vapour in Pacific Northwest coniferous forests[END_REF][START_REF] Rothfuss | Partitioning evapotranspiration fluxes into soil evaporation and plant transpiration using water stable isotopes under controlled conditions[END_REF][START_REF] Wang | Partitioning evapotranspiration across gradients of woody plant cover: Assessment of a stable isotope technique[END_REF][START_REF] Kim | Isotopic enrichment of liquid water during evaporation from water surfaces[END_REF] to assess ecosystem water use efficiency (WUE) [START_REF] Ponton | Comparison of ecosystem water-use efficiency among Douglas-fir forest, aspen forest and grassland using eddy covariance and carbon isotope techniques[END_REF] and, e.g. the effects of hydraulic redistribution by roots and mycorrhiza (e.g. [START_REF] Ludwig | Below-ground competition between trees and grasses may overwhelm the facilitative effects of hydraulic lift[END_REF][START_REF] Kurz-Besson | Hydraulic lift in cork oak trees in a savannah-type Mediterranean ecosystem and its contribution to the local water balance[END_REF]see Sect. 2.3.2). These detailed studies are important because inferences can be drawn concerning carbon and water dynamics at larger time scales (e.g. erosion, soil organic matter transformations), and spatial variability across the ecosystem can be better described. The advantage of these studies is two-fold: (1) underlying connections between ecosystem carbon pools and fluxes and the influence of changes in environmental drivers can be characterised, and (2) results can be used in models designed to partition ecosystem respiration.

Canopy labelling

Whole ecosystem dynamics studied in situ using isotopes, at first pioneered through girdling [START_REF] Högberg | Large-scale forest girdling shows that current photosynthesis drives soil respiration[END_REF], have increased in number through whole canopy tracer application. Advances in our understanding of ecosystem processes through canopy labelling include assessing photosyntheticsoil-respiration coupling strength [START_REF] Steinmann | Carbon fluxes to the soil in a mature temperate forest assessed by 13 C isotope tracing[END_REF][START_REF] Högberg | High temporal resolution tracing of photosynthate carbon from the tree canopy to forest soil microorganisms[END_REF][START_REF] Bahn | Does photosynthesis affect grassland soil-respired CO 2 and its carbon isotope composition on a diurnal timescale?[END_REF][START_REF] Gamnitzer | Observing 13 C labelling kinetics in CO 2 respired by a temperate grassland ecosystem[END_REF][START_REF] Gamnitzer | Measuring and modelling the isotopic composition of soil respiration: insights from a grassland tracer experiment[END_REF], carbon allocation patterns [START_REF] Kuptz | Seasonal patterns of carbon allocation to respiratory pools in 60yr-old deciduous (Fagus sylvatica) and evergreen (Picea abies) trees assessed via whole-tree stable carbon isotope labeling[END_REF][START_REF] Epron | Seasonal variations of belowground carbon transfer assessed by in situ 13 CO 2 pulse labelling of trees[END_REF], and shading impacts [START_REF] Warren | Timing and magnitude of C partitioning through a young loblolly pine (Pinus taeda L.) stand using 13 C labeling and shade treatments[END_REF], to name a few. Quantitative methods of canopy labelling in connection with on-line tracer measurement techniques (Sect. 3) and modelling of the tracer distribution data (e.g. by compartmental analysis), holds the promise of testing hypotheses of ecosystem physiology, aboveground-belowground response to a changing climate, and the turnover times of seasonally dynamic carbon pools [START_REF] Epron | Pulse-labelling trees to study carbon allocation dynamics: a review of methods, current knowledge and future prospects[END_REF], studies that were previously limited to laboratory studies (e.g. [START_REF] Schnyder | Disentangling CO 2 fluxes: direct measurements of mesocosm-scale natural abundance 13 CO 2 / 12 CO 2 gas exchange, 13 C discrimination, and labelling of CO 2 exchange flux components in controlled environments[END_REF][START_REF] Lehmeier | Root and shoot respiration of perennial ryegrass are supplied by the same substrate pools: assessment by dynamic 13 C labeling and compartmental analysis of tracer kinetics[END_REF]Lehmeier et al., , 2010a, b) , b) or inferred from annual changes in biomass measured in the field. Recent findings have illustrated the complexity of dynamic processes that interact at the ecosystem scale. This calls into question the applicability of simple two-end member mixing models in complex systems with multiple sources (Kayler et al., 2010a) and poses a significant challenge for ecosystem studies, as outlined below.

Heterogeneous flux sources

Ecosystem respiration is a complex mixture of isofluxes from a range of biotic and abiotic sources that span the soil to vegetation canopy continuum (see [START_REF] Badeck | Post-photosynthetic fractionation of stable carbon isotopes between plant organs -a widespread phenomenon[END_REF][START_REF] Bowling | Carbon isotopes in terrestrial ecosystem pools and CO 2 fluxes[END_REF]literature therein). These sources contribute with distinct isotopic signatures at time scales from daily [START_REF] Bowling | Tunable diode laser absorption spectroscopy for stable isotope studies of ecosystem-atmosphere CO 2 exchange[END_REF][START_REF] Mortazavi | Influence of 13 Cenriched foliage respired CO 2 on δ 13 C of ecosystem-respired CO 2 , Global Biogeochem[END_REF]Werner et al., 2006;[START_REF] Kodama | Temporal dynamics of the carbon isotope composition in a Pinus sylvestris stand: from newly assimilated organic carbon to respired carbon dioxide[END_REF]Unger et al., 2010a;Wingate et al., 2010a) to seasonal cycles [START_REF] Griffis | Seasonal variation and partitioning of ecosystem respiration in a southern boreal aspen forest[END_REF][START_REF] Mcdowell | Response of the carbon isotopic content of ecosystem, leaf, and soil respiration to meteorological driving factors in a Pinus ponderosa ecosystem[END_REF][START_REF] Ponton | Comparison of ecosystem water-use efficiency among Douglas-fir forest, aspen forest and grassland using eddy covariance and carbon isotope techniques[END_REF][START_REF] Alstad | Environmental controls on the carbon isotope composition of ecosystem respired CO 2 in contrasting forest ecosystems in Canada and the USA[END_REF][START_REF] Schaeffer | Canopy structure and atmospheric flows in relation to the δ 13 C of respired CO 2 in a subalpine coniferous forest[END_REF]. These are based partly on phenology and disturbance regimes and all exhibit different effects on component fluxes. The challenge to advance our understanding of δ 13 C R lies in identifying and quantifying these fluxes and isotopic signatures of important ecosystem components (e.g. Unger et al., 2010a;[START_REF] Epron | Seasonal variations of belowground carbon transfer assessed by in situ 13 CO 2 pulse labelling of trees[END_REF]Barbour et al., 2011a). This is especially important to test hypotheses about temporal δ 13 C R patterns, for example, if δ 13 C R dynamics are heavily influenced by a sole component flux, resulting in a poorly mixed ecosystem source signal. Similarly, species-specific transport times of recent assimilates [START_REF] Epron | Seasonal variations of belowground carbon transfer assessed by in situ 13 CO 2 pulse labelling of trees[END_REF] can potentially delay the photosynthetic response signal in δ 13 C R , or abiotic phenomena (following section) can obscure component iso-fluxes (e.g. [START_REF] Ekblad | Forest soil respiration rate and δ 13 C is regulated by recent above ground weather conditions[END_REF][START_REF] Knohl | Shortterm variations in δ 13 C of ecosystem respiration reveals link between assimilation and respiration in a deciduous forest[END_REF]. In these cases, deciphering the drivers behind δ 13 C R may become increasingly difficult.

Abiotic influences

Analyses of δ 13 C R may lead to the identification of drivers and mechanisms underlying the dynamics of ecosystem metabolism; yet, other abiotic processes that are also affected by biological drivers (e.g. temperature) may amplify, dampen or time-lag responses in δ 13 C R , obfuscating the signal of biological respiration [START_REF] Brüggemann | Carbon allocation and carbon isotope fluxes in the plant-soil-atmosphere continuum: a review[END_REF]. Soil respiration, which can represent 20 to 70 % of total ecosystem respiration, is an integrative signal driven by many abiotic and biological processes. Recent studies have shown that factors such as diffusivity of soil CO 2 , dissolution of CO 2 from bicarbonates, and advection of soil gas may be responsible for strong 13 C-isotopic disequilibria between the CO 2 efflux at the soil surface and concurrent soil respiration [START_REF] Crow | Isotopic analysis of respired CO 2 during decomposition of separated soil organic matter pools[END_REF][START_REF] Kayler | A laboratory comparison of two methods used to estimate the isotopic composition of soil δ 13 CO 2 efflux at steady state[END_REF]Kayler et al., , 2010a;;[START_REF] Nickerson | A numerical evaluation of chamber methodologies used in measuring the δ 13 C of soil respiration[END_REF][START_REF] Ohlsson | Reduction of bias in static closed chamber measurement of δ 13 C in soil CO 2 efflux[END_REF][START_REF] Gamnitzer | Measuring and modelling the isotopic composition of soil respiration: insights from a grassland tracer experiment[END_REF]. Likewise, the oxygen isotope composition of soil respired CO 2 (δ 18 O S ) not only carries the isotopic signature of the soil water it interacted with, but also is influenced by the carbonic anhydrase in soil microorganisms that accelerate isotopic equilibration between CO 2 and soil water [START_REF] Wingate | The impact of soil microorganisms on the global budget of δ 18 O in atmospheric CO 2[END_REF](Wingate et al., , 2010b)).

Despite their potential to propagate uncertainties in isotopic information through the soil-canopy continuum, such processes merit inclusion in isotope ecosystem models, enhancing the interpretation of patterns and drivers of δ 13 C R .

Flux partitioning

Conventional partitioning methods based on eddy covariance methods typically require several days or weeks of data to cover key phenological periods in order to obtain robust regression parameters (e.g. [START_REF] Reichstein | On the separation of net ecosystem exchange into assimilation and ecosystem respiration: review and improved algorithm[END_REF], neglecting ecosystem responses at shorter time scales. These are, for example, "switches" of ecosystem states [START_REF] Baldocchi | How switches and lags in biophysical regulators affect spatial-temporal variation of soil respiration in an oak-grass savannah[END_REF][START_REF] Lee | Temporal variations of the isotopic signal of the whole-canopy transpiration in a temperate forest[END_REF] or the pulse-like response of soil respiration to strong rain events, occurring at time scales from minutes to hours (e.g. [START_REF] Xu | How soil moisture, rain pulses, and growth alter the response of ecosystem respiration to temperature[END_REF]Unger et al., 2010b[START_REF] Unger | Interpreting post-drought rewetting effects on soil and ecosystem carbon dynamics in a Mediterranean oak savannah[END_REF]. It would be helpful if the partitioning scheme could resolve episodic responses of this kind, because it is the transient, non-equilibrium responses that provide a rigorous test of model performance and validity. Assimilating continuous measurements of CO 2 and H 2 O fluxes and their isotopic compositions (e.g. δ 13 C, δ 18 O, δ 2 H) into process-based models should therefore provide a better-constrained solution.

Similarly, assimilating chamber-based flux measurements of these isotopic fluxes should help to explain and constrain our model predictions during metabolic switches, especially when photosynthetic products may become limiting such as during drought (Unger et al., 2010a), rainy periods (Wingate et al., 2010a) or when post-photosynthetic fractionation processes dominate the isofluxes, e.g. at dawn (Barbour et al., 2011a).

Regional scale isotope variation in precipitation and linkages to carbon cycling

High frequency, spatially dense precipitation isoscapes (i.e. spatial distribution maps of isotope records) over long time periods are continuing to assist our understanding of plant water relations, water sources and the extent to which they are driven by seasonally varying water sources and how these sources vary at the regional, inter-annual to inter-decadal scales [START_REF] Rozanski | Isotopic patterns in modem global precipitation[END_REF][START_REF] Welker | Isotopic (δ 18 O) characteristics of weekly precipitation collected across the United States: An initial analysis with application to water source studies[END_REF][START_REF] Vachon | Seasonality of precipitation affects the annual isotopic (δ 18 O) values across the United States: a sensitivity study[END_REF]. Knowledge on the spatial distribution of the isotope signatures of the source water taken up by plants is also prerequisite to disentangle the climatic and physiological information laid down as 18 O signal in plant organic matter and isotopic archives [START_REF] Augusti | The ins and outs of stable isotopes in plants[END_REF] on larger scales. At the regional scale, we now fully appreciate that seasonally snow covered systems provide meltwater to soils and river systems that reflect the highly depleted values of winter precipitation [START_REF] Dutton | Comparison of river water and precipitation δ 18 O across the 48 contiguous United States[END_REF][START_REF] Vachon | Monthly precipitation isoscapes (δ 18 O) of the United States: Connections with surface temperatures, moisture source conditions, and air mass trajectories[END_REF], and that this snow meltwater allows high rates of stomatal conductance and high rates of carbon fixation [START_REF] Alstad | Carbon and water relations of Salix monticola in response to winter browsing and changes in surface water hydrology: an isotopic study using δ 13 C and δ 18 O[END_REF]. The duration and extent to which snowmelt and summer precipitation sources are available to the vegetation may be critical to supporting higher plant water use, thus affecting stomatal conductance as well as carbon fixation and gross ecosystem production. The complexity of seasonal patterns of snow meltwater availability at the regional scale is reflected in the vegetation at higher latitudes where Arctic and North Atlantic Oscillation phase changes are recorded in the carbon and oxygen isotope composition [START_REF] Welker | Arctic and North Atlantic Oscillation phase changes are recorded in the isotopes (δ 18 O and δ 13 C) of Cassiope tetragona plants[END_REF].

Our emerging understanding of the temporal patterns of δ 18 O and δD during swings in the major climate oscillations provides a modern basis for calibrating storm track, climate oscillations and the source water of vegetation in conjunction with carbon fixation rates and variability.

Progress and challenges

Tracing climate phase variation

Understanding the role of moisture sourced from multiple regions (i.e. different storm tracks), and how those sources vary with climate phases (i.e. climate oscillations and modes, such as El Niño) as it affects vegetation carbon fixation is unknown [START_REF] Holmgren | El Niño effects on the dynamics of terrestrial ecosystems[END_REF][START_REF] Birks | Atmospheric circulation controls on precipitation isotope-climate relations in western Canada[END_REF][START_REF] Sjostrom | Storm-track trajectories control the isotope geochemistry of precipitation in the Eastern US[END_REF]. We continue to recognize that tree rings may be recorders of the general isotopic history of source water [START_REF] Briffa | Annual climate variability in the Holocene: interpreting the message of ancient trees[END_REF][START_REF] Csank | Climate variability in the Early Pliocene Arctic: Annually resolved evidence from stable isotope values of sub-fossil wood, Ellesmere Island, Canada[END_REF] regardless of geologic time period. However, understanding the extent to which these moisture sources and climate phases are recorded and how plant physiology alters the source water signal in the long-term growth record of trees is one of the great challenges today.

Isotope tracers in back trajectory analysis

Back trajectory analysis of weather and thus precipitation [START_REF] Draxler | Description of the HYSPLIT 4 modeling system[END_REF][START_REF] Sjostrom | Storm-track trajectories control the isotope geochemistry of precipitation in the Eastern US[END_REF]) is a modelling tool that has been used extensively to quantify long-distance transport of pollutants, and more recently for studies of isotopic characteristics of precipitation [START_REF] Burnett | Relationship between atmospheric circulation and winter precipitation δ 18 O in central New York State[END_REF]. Combining this tool with isotopic measurements of continental precipitation and water vapour (e.g. networks such as MIBA and GNIP) and carbon and water fluxes (e.g. networks such as Fluxnet) may be means by which almost real-time linkages between climate phases, moisture sources, plant water relations, carbon exchange and continental carbon cycling may be possible.

Isotopic archives and relevant aspects of spatio-temporal integration

Over the past decades, the use of stable isotope ratios in a wide range of materials -from tree, sediment and ice cores to corals, hair, cactus spines, the balleen of whales and fish odoliths -has provided some of the most important and novel insights into the patterns of past environmental changes and organismal response to these changes of almost any type of recorder (Dawson and Siegwolf, 2007). Such archives not only provide a way to look back in time but more recent examples show that one can also assign causes to responses to environmental changes on a mechanistic basis (e.g. [START_REF] Ogée | A single-substrate model to interpret intra-annual stable isotope signals in tree-ring cellulose[END_REF]. Stable isotope analysis of biological or abiotic archives can thus provide excellent tracers for spatial-and temporal-integration over different scales. Here we discuss progress and challenges of a few selected examples of biological archives.

Progress and challenges

Isotopic archives in trees

Tree rings enable retrospective analyses of intra-and interannual variation of carbon and oxygen isotope composition and the related ecophysiological drivers over many centuries [START_REF] Sidorova | Do centennial tree-ring and stable isotope trends of Larix gmelinii (Rupr.) Rupr. indicate increasing water shortage in the Siberian north?[END_REF][START_REF] Nock | Long-term increases in intrinsic water-use efficiency do not lead to increased stem growth in a tropical monsoon forest in western Thailand[END_REF][START_REF] Knorre | Twentieth century trends in tree ring stable isotopes (δ 13 C and δ 18 O) of Larix sibirica under dry conditions in the forest steppe in Siberia[END_REF][START_REF] Andreu-Hayles | Long tree-ring chronologies reveal 20th century increases in water-use efficiency but no enhancement of tree growth at five Iberian pine forests[END_REF][START_REF] Peñuelas | Increased water-use efficiency during the 20th century did not translate into enhanced tree growth[END_REF]. The advantages of tree rings are that they (i) can be reliably dated with a high temporal and spatial resolution; (ii) contain several proxies (stable C, H, O and N isotopes, tree ring width and tree ring density) in the same matrix (tree ring wood/cellulose), which was formed at the same time, location, and environmental conditions; and (iii) mostly the inclusion of a limited number of trees and species may provide a strong signal. However, single tree ring chronologies provide only limited spatial and community integration and report only local signals (ca. 10 -1 to 10 2 m). Signal strength is further reduced by species-specific responses to environmental impacts. Furthermore, the tree response is strongly affected by ontogeny (e.g. [START_REF] Monserud | Time-series analysis of δ 13 C from tree rings, I. Time trends and autocorrelation[END_REF] and site specific properties such as competition, soil type, water and nutrient availability, resulting in a considerable variability of the signal expression, even within the same species [START_REF] Saurer | Stable carbon isotopes in tree rings of beech: climatic versus site-related influences[END_REF]. Thus, constructions of ecosystem chronologies depend on the combination of several tree ring records from trees of different locations within the same site. This requires additional information, such as knowledge of past species dynamics.

Isotopic archives of herbaceous vegetation

The life span of herbaceous vegetation is much shorter than that of trees. However, isotopic reconstructions of climate change in herbaceous vegetation (crops and grassland) are possible if plants were sampled and preserved during the epoch. Such archives are relatively rare and are mainly represented by herbaria (e.g. Penuelas and Azcón-Bieto, 1992). In general, herbarium specimens have been sampled at different locations, so that long-term isotopic records from these involve a spatially disperse representation of a species' changing isotopic composition. Because of site differences, such isotopic records display relatively high variation. Rare opportunities for community-scale isotopic reconstructions are presented by long-term (agro-) ecological experiments with crops and grassland where biomass samples have been stored in dedicated archives [START_REF] Zhao | Trends in 13 C/ 12 C ratios and C isotope discrimination of wheat since 1845[END_REF][START_REF] Köhler | Intrinsic water-use efficiency of temperate semi-natural grassland has increased since 1857: an analysis of carbon isotope discrimination of herbage from the Park Grass Experiment[END_REF].

Grazer tissues as isotopic archives

For grassland, an analogy to tree rings is given by the yearly rings (annuli) of horns (or hoofs) of obligate grazers [START_REF] Barbosa | Analysing the isotopic life history of the alpine ungulates Capra ibex and Rupicapra rupicapra rupicapra through their horns[END_REF][START_REF] Barbosa | Last-century changes of alpine grassland water-use efficiency -a reconstruction through carbon isotope analysis of a time-series of Capra ibex horns[END_REF]. These can yield carbon isotopic records over many years, which reflect that of grassland vegetation [START_REF] Schnyder | Hair of grazing cattle provides an integrated measure of the effects of site conditions and interannual weather variability on δ13C of temperate humid grassland[END_REF]. The spatial integrations of tree and horn ring isotope compositions are quite contrasting: local and stationary for the tree, and vast and cyclic for horns, reflecting visits of the different parts of the year-round grazing grounds. Still, the use of grazer tissue for reconstructions of grassland isotopic chronologies usually rests on a number of assumptions, e.g. concerning the selectivity of grazing, the constancy of the relationships between isotopic composition of grazer tissues, and contributions of diet components of differential digestibility [START_REF] Wittmer | Do grazer hair and faeces reflect the carbon isotope composition of semi-arid C 3 /C 4 grassland?[END_REF]. Such assumptions can be and should be validated [START_REF] Wittmer | Do grazer hair and faeces reflect the carbon isotope composition of semi-arid C 3 /C 4 grassland?[END_REF]. A significant advantage of keratinous tissue (horn, hair/wool and hoofs) is given by its homogenous chemical composition, which reduces variation associated with metabolic isotope fractionation that can be significant in chemically heterogeneous tissue.

Micro-scale environmental record

A particular case of small-scale environmental records are carbon and oxygen isotope ratios of non-vascular plants (NVP). Cyanobacteria, algae, lichens, and bryophytes integrate local changes of CO 2 (e.g. [START_REF] Máguas | Spatial variation in carbon isotope discrimination across the thalli of several lichen species[END_REF] and water over long time periods due to their passive exchange with environmental conditions, low growth rates (ca. 0.02-30 mm a -1 ) and long life spans (hundreds to thousands of years). Therefore, NVP can be used, for example, for geochronologic aging (e.g. lichenometry), particularly dating deposited surfaces over the past 500 years with an accuracy of 10 % error [START_REF] Armstrong | Lichens, lichenometry and global warming[END_REF]). The δ 13 C of NVP archives environmental impacts over the whole life span in bulk organic material, and over several years if a chronosequence is sampled from the thallus margins or young shoots. Shortterm and online records can be obtained from analysing respired CO 2 and extracted bulk water. δ 13 C of NVP can be used to trace environmental CO 2 gradients [START_REF] Flanagan | Spatial and temporal variation in the carbon and oxygen stable isotope ratio of respired CO 2 in a boreal forest ecosystem[END_REF][START_REF] Lakatos | The stable isotopes δ 13 C and δ 18 O of lichens can be used as tracers of microenvironmental carbon and water sources, in: Stable isotopes as indicators of ecological change[END_REF][START_REF] Meyer | To concentrate or ventilate? Carbon acquisition, isotope discrimination and physiological ecology of early land plant life forms[END_REF], whereas fossil bryophytes record ancient CO 2 levels [START_REF] Fletcher | Fossil bryophytes as recorders of ancient CO 2 levels: Experimental evidence and a cretaceous case study, Global Biogeochem[END_REF][START_REF] Fletcher | Bryocarb: A process-based model of thallose liverwort carbon isotope fractionation in response to CO 2 , O 2 , light and temperature[END_REF]. Additionally, epiphytic plants function as atmospheric water traps [START_REF] Helliker | Toward a plant-based proxy for the isotope ratio of atmospheric water vapor[END_REF][START_REF] Helliker | On the controls of leaf-water oxygen isotope ratios in the atmospheric Crassulacean Acid Metabolism epiphyte Tillandsia usneoides[END_REF]). Because epiphytic NVP are commonly in equilibrium with water vapour, it is inferred that δ 18 O of bulk water and organic material might serve as a short and long-term recorder for atmospheric vapour, respectively [START_REF] Helliker | Toward a plant-based proxy for the isotope ratio of atmospheric water vapor[END_REF][START_REF] Hartard | δ 18 O characteristics of lichens and their effects on evaporative processes of the subjacent soil[END_REF][START_REF] Hartard | Water isotopes in desiccating lichens[END_REF]. In the same line, peat mosses serve as proxies for palaeoenvironmental changes [START_REF] Loader | Characterizing carbon isotopic variability in sphagnum[END_REF][START_REF] Lamentowicz | Last millennium palaeoenvironmental changes from a baltic bog (poland) inferred from stable isotopes, pollen, plant macrofossils and testate amoebae[END_REF][START_REF] Moschen | Stable carbon and oxygen isotopes in sub-fossil sphagnum: Assessment of their applicability for palaeoclimatology[END_REF][START_REF] Loisel | Sphagnum δ 13 C values as indicators of palaeohydrological changes in a peat bog[END_REF]. However, approaches that use oxygen isotopes as long-term recorder of environmental conditions need to account for the contributions of the different water signals from rain, dew and vapour, as well as physiological offsets which add considerable uncertainties [START_REF] Moschen | Stable carbon and oxygen isotopes in sub-fossil sphagnum: Assessment of their applicability for palaeoclimatology[END_REF].

New technical and methodological developments in stable isotope research

The past decade has seen tremendous progress in the development of new techniques that complement or rival traditional Isotope Ratio Mass Spectrometry (IRMS) for the determination of stable isotope abundances. This has lead to new dimensions in measurement speed, number of quantifiable isotopologues and sensitivity, increased the repeatability, precision and sample turn-over considerably, and offered new opportunities for in situ observations at natural abundance and in tracer experiments. Most important for carbon and water cycle research was the introduction of instruments using light absorption properties of small molecules for determination of stable isotope abundances, as well as the introduction of innovative techniques for compound-specific sample extraction.

Laser absorption spectroscopy (LAS)

The development of absorption spectroscopy instrumentation (LAS) provided new dimensions of measurement speed and number of quantifiable isotopologues offering data richness that had never been possible to achieve in fielddeployable instrumentation (e.g. [START_REF] Bowling | Tunable diode laser absorption spectroscopy for stable isotope studies of ecosystem-atmosphere CO 2 exchange[END_REF][START_REF] Kammer | Application of a quantum cascade laser-based spectrometer in a closed chamber system for real-time δ 13 C and δ 18 O measurements of soil-respired CO 2[END_REF][START_REF] Sturm | Eddy covariance measurements of CO 2 isotopologues with a quantum cascade laser absorption spectrometer[END_REF]; but see [START_REF] Schnyder | Mobile, outdoor continuous-flow isotope-ratio mass spectrometer system for automated high-frequency 13 C-and 18 O-CO 2 analysis for Keeling plot applications[END_REF]. The laser absorption spectroscopy is based on analysis of absorption of light in selected wavelengths in the near and mid-infrared to determine the mole fractions of individual isotopologues [START_REF] Kerstel | Isotope ratio infrared spectrometry[END_REF][START_REF] Kerstel | Advances in laser-based isotope ratio measurements: selected applications[END_REF][START_REF] Fried | Infrared absorption Spectroscopy[END_REF]. Optical measurement methods based on Fourier Transform Infrared Spectroscopy (FTIR), Cavity Ringdown Spectroscopy (CRDS), Integrated Cavity Output Spectroscopy (ICOS) and Tunable Diode Laser Absorption Spectroscopy (TDLAS) now approach levels of detection of small-molecule isotopologues comparable to laboratory-based isotope ratio mass spectrometers (IRMS). Measurement by absorption spectroscopy is non-destructive and can therefore be repeated to increase measurement precision [START_REF] Werle | Signal processing and calibration procedures for in situ diode-laser absorption spectroscopy[END_REF]. Furthermore, LAS enables a high temporal resolution of accurate isotope ratios, an ideal property for the visualisation of processes and temporal variability (e.g. [START_REF] Bowling | Tunable diode laser absorption spectroscopy for stable isotope studies of ecosystem-atmosphere CO 2 exchange[END_REF][START_REF] Lee | In-situ measurement of water vapor 18 O/ 16 O isotope ratio for atmospheric and ecological applications[END_REF][START_REF] Tuzson | High precision and continuous field measurements of δ 13 C and δ 18 O in carbon dioxide with a cryogen-free QCLAS[END_REF]. Further, new multi-species instruments that are becoming available enable so-called "clumped isotope" measurements [START_REF] Eiler | Clumped-isotope" geochemistry-The study of naturally-occurring, multiply-substituted isotopologues[END_REF], wherein the occurrence of two heavy isotopes in the same molecule can serve as a unique stable isotope tracer itself.

Compound Specific Isotope Analysis (CSIA)

The IRMS has experienced technological and methodological development, particularly Compound Specific Isotope Analysis (CSIA), which includes IRMS coupled to Gas Chromatography-Combustion (GC-C-IRMS; Maier-Augenstein, 1999) or Liquid Chromatography (LC-IRMS; [START_REF] Godin | Liquid chromatography combined with mass spectrometry for C-13 isotopic analysis in life science research[END_REF]. This facilitates the analysis of different compounds such as structural and labile carbohydrates extracted from plant organs, leaf wax alkanes, phloem sap and soil fractions (see review by [START_REF] Sachse | Molecular paleohydrology: interpreting the hydrogen isotopic composition of lipid biomarkers from photosynthetic organisms[END_REF]. For compound specific isotope analysis, the extraction method is crucial and might strongly affect the results obtained [START_REF] Richter | Preparation of starch and soluble sugars of plant material for the analysis of carbon isotope composition: a comparison of methods[END_REF]. Moreover, the need for derivatization of polar metabolites for GC-MS (gas chromatography-mass spectrometry) analysis and thus the introduction of additional carbon and oxygen via the derivatization agent to the analyte complicates the measurement of natural abundance stable isotope composition of these compound classes (e.g. [START_REF] Gross | Minimization of carbon addition during derivatization of monosaccharides for compound-specific delta C-13 analysis in environmental research[END_REF]. This problem does not occur with LC-IRMS systems, which are currently, however, restricted to carbon isotope analyses.

Nuclear Magnetic Resonance (NMR)

At the advent of development of new techniques for nuclear magnetic resonance spectroscopy (NMR), new options arise for studies of, e.g. starch-sugar partitioning and complementary information on (photo-)respiration by analyses of non-homogeneous distribution of 13 C within carbohydrate molecules (e.g. [START_REF] Gilbert | Accurate quantitative isotopic 13 C NMR spectroscopy for the determination of the intramolecular distribution of 13 C in glucose at natural abundance[END_REF][START_REF] Gilbert | The intramolecular 13C-distribution in ethanol reveals the influence of the CO 2 -fixation pathway and environmental conditions on the sitespecific 13 C variation in glucose[END_REF]. Analogously, options to distinguish between different water pools within the plant arise from new techniques for 18 O positional analyses by novel derivatiation approaches [START_REF] Sternberg | Variation in oxygen isotope fractionation during cellulose synthesis: intramolecular and biosynthetic effects[END_REF].

Nano-scale secondary ion mass spectrometers (NanoSIMS)

Linking isotopic analysis with high resolution microscopy has provided significant progress of spatially resolved information on the molecular and isotopic compositions of (biological) materials. New Nano-scale Secondary Ion Mass Spectrometers (NanoSIMS) represent a significant improvement in sensitivity and spatial resolution (down to 50 nm).

In a destructive manner, NanoSIMS analysis involves continuous bombardment of the sample surface with an ion beam and subsequent analysis of the released secondary ions according to their mass-to-charge ratios [START_REF] Herrmann | Nano-scale secondary ion mass spectrometry -A new analytical tool in biogeochemistry and soil ecology: A review article[END_REF]. Although adequate sample preparation remains challenging, imaging mass spectrometry via NanoSIMS represents a promising avenue for mapping the spatial organisation, metabolic pathways and resource fluxes within cells, plants and at the root-fungus-soil interface, in particular in labelling studies (e.g. [START_REF] Clode | In situ mapping of nutrient uptake in the rhizosphere using nanoscale secondary ion mass spectrometry[END_REF].

Progress and challenges

The pool of various new and improved techniques currently available for application of stable isotopes in environmental, physiological and ecological research is large. However, from the user's perspective, particularly for laser absorption spectroscopy, some general issues, as described below, should be resolved.

Instrument accuracy and calibration

Calibration biases for water vapour isotope laser spectrometers can result, for instance, from evaporation efficiency of the reference water, instrument nonlinearity and impurity of the carrier gas. Calibration of water vapour analysers is done, for example, using a liquid water injector ("dripper") into a flow of dry air [START_REF] Lee | In-situ measurement of water vapor 18 O/ 16 O isotope ratio for atmospheric and ecological applications[END_REF][START_REF] Wen | Continuous measurement of water vapor D/H and 18 O/ 16 O isotope ratios in the atmosphere[END_REF][START_REF] Baker | A simple, accurate, field-portable mixing ratio generator and rayleigh distillation device[END_REF][START_REF] Griffis | Determining the oxygen isotope composition of evapotranspiration using eddy covariance[END_REF][START_REF] Sturm | Water vapor δ 2 H and δ 18 O measurements using off-axis integrated cavity output spectroscopy[END_REF]. In addition, a heated vaporisation system is used wherein the liquid standard is completely vaporized without fractionation. Nevertheless, any concentration dependence in the analyser itself can bias the overall calibration of the instrument, especially when measuring ambient water vapour of widely varying mixing ratios [START_REF] Lee | In-situ measurement of water vapor 18 O/ 16 O isotope ratio for atmospheric and ecological applications[END_REF][START_REF] Wen | Continuous measurement of water vapor D/H and 18 O/ 16 O isotope ratios in the atmosphere[END_REF][START_REF] Schmidt | Concentration effects on laser-based δ 18 O and δ 2 H measurements and implications for the calibration of vapour measurements with liquid standards[END_REF][START_REF] Sturm | Water vapor δ 2 H and δ 18 O measurements using off-axis integrated cavity output spectroscopy[END_REF]. For CO 2 , calibration against two or more mixtures of CO 2 and dry air, which are tied to international reference standards, are critical. Impurities in the water sample to be analysed can cause a spectral interference with organic contaminants and have been observed in analysis of liquid samples extracted from biological sources, e.g. leaf water [START_REF] West | Discrepancies between isotope ratio infrared spectroscopy and isotope ratio mass spectrometry for the stable isotope analysis of plant and soil waters[END_REF][START_REF] Schultz | Identification and correction of spectral contamination in 2 H/ 1 H and 18 O/ 16 O measured in leaf, stem, and soil water[END_REF].

High instrument precision at short detection intervals

Free of sample preparation and processing, new optical techniques can achieve much faster detection than IRMS. Insitu measurements of CO 2 and H 2 O isotope ratios in ambient air, especially if made on a long-term basis and calibrated precisely, can provide a powerful tool for atmospheric inverse analysis of the terrestrial carbon sink and tracking of water transport in the atmosphere. However, to measure the source/sink signature properly near the land surface, one should interface the isotopic analyser with plant ( [START_REF] Barbour | A new measurement technique reveals temporal variation in δ 18 O of leaf-respired CO 2[END_REF][START_REF] Barthel | The diel imprint of leaf metabolism on the δ 13 C signal of soil respiration under control and drought conditions[END_REF] and soil chambers (e.g. Wingate et al., 2010a, b) and deploy it in the gradientdiffusion mode either over the vegetation [START_REF] Griffis | Seasonal variation and partitioning of ecosystem respiration in a southern boreal aspen forest[END_REF] or over the soil surface inside the canopy [START_REF] Santos | 13 CO 2 and C 18 O 16 O temporal variation near the ground and above a temperate deciduous forest[END_REF], or combine it with a sonic anemometer for direct eddy covariance measurement of isotopic fluxes [START_REF] Lee | In-situ measurement of water vapor 18 O/ 16 O isotope ratio for atmospheric and ecological applications[END_REF][START_REF] Griffis | Direct measurement of biosphere-atmosphere isotopic CO 2 exchange using the eddy covariance technique[END_REF][START_REF] Griffis | Determining the oxygen isotope composition of evapotranspiration using eddy covariance[END_REF] or landscape scale measurements in high elevation or airborne conditions (e.g. Tuszon et al., 2010). In all these configurations, suitable interfaces between the analyser and the sample and calibration periphery are useful. The system as a whole must be robust and designed and tuned for minimal interference, memory effects or signal drifts. Fast detection is particularly critical for eddy covariance applications, which require an instrument response to be faster than 10 Hz and relies on continuous-flow sampling. However, fast detection is also desired for chamber based measurements in studies of short-term events, such as water vapour and CO 2 flux pulses after rain [START_REF] Santos | 13 CO 2 and C 18 O 16 O temporal variation near the ground and above a temperate deciduous forest[END_REF][START_REF] Unger | Interpreting post-drought rewetting effects on soil and ecosystem carbon dynamics in a Mediterranean oak savannah[END_REF]. Maximizing precision at short integration times and maintaining accuracy for long periods should be a high priority in future instrument development.

Instrument and infrastructure cost

High instrument and maintenance costs limit the broad adoption of new technologies in field research. It is highly desirable that the costs are brought down to a level comparable to that of a broadband infrared gas analyser, which is now an indispensible tool for ecosystem carbon and water flux monitoring worldwide [START_REF] Baldocchi | Fluxnet: A new tool to study the temporal and spatial variability of ecosystem-scale carbon dioxide, water vapor and energy flux densities[END_REF]. We envision the development of a network with real-time observations of isotopic fluxes of CO 2 and H 2 O to help diagnose changes in biospheric processes. This can become a realistic goal if instrumentation costs are lower.

Multi-isotopologue instruments

New instruments are becoming available and enable socalled "clumped isotope" measurements [START_REF] Eiler | Clumped-isotope" geochemistry-The study of naturally-occurring, multiply-substituted isotopologues[END_REF], wherein the occurrence of two heavy isotopes in the same molecule can serve as a unique stable isotope tracer itself.

Currently its applicability as a paleo-thermometer is being tested. The basic assumption is based on the observation that the heavier molecules and atoms are not randomly distributed within the same matrix, but rather form a clumped aggregate of substrates with the heavier isotope. For the distinction of such clumped isotopes highly sensitive instruments are needed.

Outlook

New research opportunities at all scales of isotope biogeochemistry of carbon and water are arising from deepened process-based understanding and improved analysis tools, together with the development of mechanistic models. Especially combinations of multiple isotope and non-isotope variables have the potential to stimulate our understanding across a wide range of scales, including leaves, plants, mesocosms, natural ecosystems, and the atmosphere. The scale-spanning assessment of carbon and water fluxes is, on the one hand, a great opportunity offered by stable isotope approaches. On the other hand, deeper insights into the multitude of processes affecting carbon and oxygen isotope discrimination during photosynthesis and transpiration, as well as during downstream metabolic processes, are challenging a generalisation of the information contained in the isotopic signature and a transfer to higher temporal and/or spatial scale. One example is the knowledge that plant phenology or growth patterns (Sect. 2.1.3) might complicate the comparison of the isotopic composition of bulk material between species. However, we can apply appropriate techniques such as the assessment of organic matter pools with a well defined turnover time and chemical composition to avoid misinterpretation. Moreover, experimental designs focussing on changes in environmental conditions or species interactions and on the effect of such changes on the isotopic composition can often overcome the problem. While the isotopic composition might not be directly comparable between species, the direction and magnitude of change can give quantitative information on physiological reactions within and between species, communities and ecosystems.

At the leaf-level (see Sect. 2.1), combined analyses of different isotopes might lead to a better understanding of mesophyll conductance and related components, including diffusion through intercellular airspaces and transport through barriers in cells such as the cell wall, membranes, or stroma. It might also help to assess the possible role of cooporins (membrane proteins acting as pores for CO 2 ) in facilitating and controlling transport of CO 2 . Combined measurements of the isotopologues of CO 2 and H 2 O will further allow quantifying the extent of equilibration between dissolved CO 2 and leaf water, and thus can provide a non-invasive reconstruction of leaf water dynamics. These are critical aspects for validation and further development of carbon and water isotope approaches and models. Information on different species and ecotypes will in turn enhance our understanding of the different morpho-physiological factors controlling carbon and water fluxes and, hence, water use efficiency of leaves.

Although the last ten years have seen a large increase in knowledge of post-carboxylation fractionation phenomena (see Sect. 2.1.2), we expect no slowdown in the development of this field. In part, empirical progress will be facilitated by improvements in NMR technology as well as in derivatisation techniques (see Sect. 3) which permit measurements of natural intramolecular isotope distribution patterns in intermediates of primary and secondary metabolism, and respiratory substrates. Dynamic labelling experiments with 13 C-enriched or depleted CO 2 or with (intra-molecular) position-labelled metabolites will permit better assessment of metabolic networks and turnover times of different carbon pools. Such work will also enhance our understanding for the metabolic causes of variations in post-carboxylation fractionation. Temporal dynamics of apparent fractionation during dark respiration may vary, depending on the identity of the different metabolic intermediates, their synthesis pathways and metabolic functions as well as on the demand for substrates in the respiratory pathways.

Investigations of natural intra-molecular 13 C and 18 O distribution patterns might also be key to quantify isotope fractionation phenomena during loading, phloem transport and unloading of different organic compounds (see Sect. 2.2). These include assessments of isotopic exchange reactions along the path from leaves to sites of assimilate use, and fractionation or isotopic exchange during biosynthetic processes such as cellulose synthesis. Such approaches may elucidate the mechanisms underlying spatio-temporal variation of δ 13 C and δ 18 O during transfer from the chloroplast to heterotrophic tissues, the rhizosphere/soil and atmosphere. The mechanistic understanding, on the other hand, will strengthen climatological and physiological interpretation of tree ring cellulose and similar isotope archives such as grass, sediments, hair, horn, or tooth enamel of herbivores (see Sect. 2.6). We are of the strong opinion that a deeper knowledge of fractionation during photosynthesis, transport and post-carboxylation metabolism is an important basis for understanding ecosystem-scale isotope discrimination and for linking the carbon balance with water relations at different scales. Whereas the mechanistic understanding of photosynthetic carbon isotope fractionation and evaporative 18 O enrichment of water in leaves is relatively advanced, equivalent understanding of fractionation phenomena in the downstream metabolism -as expressed in quantitative models -is still in its infancy.

It is, therefore, not surprising that the interpretation of ecosystem scale fractionation remains challenging (see Sect. 2.4). We expect that significant steps for resolving this complexity will include similar approaches as advocated for leaf-and plant-level studies: (i) joint flux measurements of the different isotopologues of CO 2 and H 2 O in natural systems -which will enable a better distinction of the CO 2 and H 2 O flux components and pools, (ii) tracing metabolite and intramolecular labelling patterns between and within system components in artificial setups as well as in field labelling experiments -shedding light on allocation, turnover of different carbon pools as well as plant-soil-atmosphere interaction, and (iii) hypothesis-testing mesocosm-scale experimentstesting our system-scale understanding. Insights from these approaches may then help to improve and test stable-isotopeenabled models of carbon and water fluxes at the ecosystem scale.

Regional-scale studies (see Sect. 2.5) of the water isotope cycle are becoming more important to our understanding of synoptic climates, ecosystem processes, the role of abiotic processes (e.g. temperature of condensation), moisture sources, and storm tracks on the ecohydrology of entire landscapes and continents. However, isotope fractionations are quite uncertain on global and continental scales and it is therefore important to identify robust features that can be constrained by large-scale isotope observations. C 4plant distribution is one such feature that might become well constrained by 13 C isotopes. But isotope studies will benefit greatly from the combination with other non-isotope tracers also on landscape, regional and continental scales. It might be the tapping into the above-mentioned multitude of information that will advance the usage of isotope signals on the global scale.

In conclusion, we are in the midst of a rapid growth in process-based understanding of the behaviour of carbon and oxygen stable isotopes in organisms and in the environment. On the one hand, we are increasingly recognising the complexity of 13 C and 18 O fractionation processes and their spatial and temporal variation. On the other hand, new technologies (see Sect. 3) can deliver high resolution records of shortand long-term variability in isotope signatures, overcoming the constraints of earlier laborious procedures. New analytical tools and process-based understanding will allow further development of isotope-enabled biogeochemical models for investigations of the complex interplay of soil, plant, ecosystem and atmosphere processes in the carbon and water cycles.

www.biogeosciences.net/9/3083/2012/ Biogeosciences, 9, 3083-3111, 2012

Biogeosciences, 9, 3083-3111, 2012www.biogeosciences.net/9/3083/2012/

Acknowledgements. We gratefully acknowledge helpful comment from anonymous referees and Nina Buchmann. This review resulted from a workshop during the SIBAE (Stable Isotopes and Biogeochemical Cycles in Terrestrial Ecosystems) meeting in Monte Verità, Switzerland. We gratefully acknowledge financial support from SIBAE COST action for workshop organisation and publication. Further, financial support to AG from DFG and the EU project FundivEurope; CM from Portuguese FCT; MJZ from Swiss NSF (SNSF; grant PBEZP2-127819), Marie Curie Excellence Grant (MEXT-CT-2006-042268) and BASIN; JW from US NSF (J Welker-AGS-0080952, DBI-0923571); and CW, MC and AG from German DFG (WE 2681/3-1, 5-1, 6-1, CU 173/2-1, GE 1090/7-1, 8-1, 9-1, 10-1, 11-1) is acknowledged.

Edited by: N. Buchmann