Seasonal dynamics and punctuated carbon sink reduction suggest photosynthetic capacity of boreal silver birch is reduced by the accumulation of hexose.

The 'assimilates inhibition hypothesis' posits that accumulation of nonstructural carbohydrates (NSCs) in leaves reduces leaf net photosynthetic rate, thus internally regulating photosynthesis. Experimental work provides equivocal support mostly under controlled conditions without identifying a particular NSC as involved in the regulation. We combined 3-yr in situ leaf gas exchange observations (natural dynamics) in the upper crown of mature Betula pendula simultaneously with measurements of concentrations of sucrose, hexoses (glucose and fructose), and starch, and similar measurements during several one-day shoot girdling (perturbation dynamics). Leaf water potential and water and nitrogen content were measured to account for their possible contribution to photosynthesis regulation. Leaf photosynthetic capacity (A/Ci) was temporally negatively correlated with NSC accumulation under both natural and perturbation states. For developed leaves, leaf hexose concentration explained A/Ci variation better than environmental variables (temperature history and daylength); the opposite was observed for developing leaves. The weaker correlations between NSCs and A/Ci in developing leaves may reflect their strong internal sink strength for carbohydrates. By contrast, the strong decline in photosynthetic capacity with NSCs accumulation in mature leaves, observed most clearly with hexose, and even more tightly with its constituents, provides support for the role of assimilates in regulating photosynthesis under natural conditions.

Aerobic methane production in Scots pine shoots is independent of drought or photosynthesis.

Shoot-level emissions of aerobically produced methane (CH4) may be an overlooked source of tree-derived CH4, but insufficient understanding of the interactions between their environmental and physiological drivers still prevents the reliable upscaling of canopy CH4 fluxes. We utilised a novel automated chamber system to continuously measure CH4 fluxes from the shoots of Pinus sylvestris (Scots pine) saplings under drought to investigate how canopy CH4 fluxes respond to the drought-induced alterations in their physiological processes and to isolate the shoot-level production of CH4 from soil-derived transport and photosynthesis. We found that aerobic CH4 emissions are not affected by the drought-induced stress, changes in physiological processes, or decrease in photosynthesis. Instead, these emissions vary on short temporal scales with environmental drivers such as temperature, suggesting that they result from abiotic degradation of plant compounds. Our study shows that aerobic CH4 emissions from foliage are distinct from photosynthesis-related processes. Thus, instead of photosynthesis rates, it is more reliable to construct regional and global estimates for the aerobic CH4 emission based on regional differences in foliage biomass and climate, also accounting for short-term variations of weather variables such as air temperature and solar radiation.

Partitioning seasonal stem carbon dioxide efflux into stem respiration, bark photosynthesis and transport-related flux.

Stem CO2 efflux is an important component of the carbon balance in forests. The efflux is considered to principally reflect the net result of two dominating and opposing processes: stem respiration and stem photosynthesis. In addition, transport of CO2 in xylem sap is thought to play an appreciable role in affecting the net flux. This work presents an approach to partition stem CO2 efflux among these processes using sap-flux data and CO2-exchange measurements from dark and transparent chambers placed on mature Scots pine (Pinus sylvestris) trees. Seasonal changes and monthly parameters describing the studied processes were determined. Respiration contributed most to stem net CO2 flux, reaching up to 79% (considering the sum of the absolute values of stem respiration, stem photosynthesis and flux from CO2 transported in xylem sap to be 100%) in June, when stem growth was greatest. Photosynthesis contribution accounted for up to 13 % of the stem net CO2 flux, increasing over the monitoring period. CO2 transported axially with sap flow, decreased towards the end of the growing season. At a reference temperature, respiration decreased starting around midsummer, while its temperature sensitivity increased during the summer. A decline was observed for photosynthetic quantum yield around midsummer together with decreasing light-saturation point. The proposed approach facilitates modeling net stem CO2 flux at a range of time scales.

Drivers of intra-seasonal δ13 C signal in tree-rings of Pinus sylvestris as indicated by compound-specific and laser ablation isotope analysis.

Carbon isotope composition of tree-ring (δ13 CRing ) is a commonly used proxy for environmental change and ecophysiology. δ13 CRing reconstructions are based on a solid knowledge of isotope fractionations during formation of primary photosynthates (δ13 CP ), such as sucrose. However, δ13 CRing is not merely a record of δ13 CP . Isotope fractionation processes, which are not yet fully understood, modify δ13 CP during sucrose transport. We traced, how the environmental intra-seasonal δ13 CP signal changes from leaves to phloem, tree-ring and roots, for 7 year old Pinus sylvestris, using δ13 C analysis of individual carbohydrates, δ13 CRing laser ablation, leaf gas exchange and enzyme activity measurements. The intra-seasonal δ13 CP dynamics was clearly reflected by δ13 CRing , suggesting negligible impact of reserve use on δ13 CRing . However, δ13 CP became increasingly 13 C-enriched during down-stem transport, probably due to post-photosynthetic fractionations such as sink organ catabolism. In contrast, δ13 C of water-soluble carbohydrates, analysed for the same extracts, did not reflect the same isotope dynamics and fractionations as δ13 CP , but recorded intra-seasonal δ13 CP variability. The impact of environmental signals on δ13 CRing , and the 0.5 and 1.7‰ depletion in photosynthates compared ring organic matter and tree-ring cellulose, respectively, are useful pieces of information for studies exploiting δ13 CRing .

Xylem embolism and bubble formation during freezing suggest complex dynamics of pressure in Betula pendula stems.

Freeze-thaw-induced embolism, a key limiting factor for perennial plants results from the formation of gas bubbles during freezing and their expansion during thawing. However, the ice volumetric increase generates local pressures, which can affect the formation of bubbles. To characterize local dynamics of pressure tension and the physical state of the sap during freeze-thaw cycles, we simultaneously used ultrasonic acoustic emission analysis and synchrotron-based high-resolution computed tomography on the diffuse-porous species Betula pendula. Visualization of individual air-filled vessels and the distribution of gas bubbles in frozen xylem were performed.. Ultrasonic emissions occurred after ice formation, together with bubble formation, whereas the development of embolism took place after thawing. The pictures of frozen tissues indicated that the positive pressure induced by the volumetric increase of ice can provoke inward flow from the cell wall toward the lumen of the vessels. We found no evidence that wider vessels within a tissue were more prone to embolism, although the occurrence of gas bubbles in larger conduits would make them prone to earlier embolism. These results highlight the need to monitor local pressure as well as ice and air distribution during xylem freezing to understand the mechanism leading to frost-induced embolism.

Exploring optimal stomatal control under alternative hypotheses for the regulation of plant sources and sinks.

Experimental evidence that nonstomatal limitations to photosynthesis (NSLs) correlate with leaf sugar and/or leaf water status suggests the possibility that stomata adjust to maximise photosynthesis through a trade-off between leaf CO2 supply and NSLs, potentially involving source-sink interactions. However, the mechanisms regulating NSLs and sink strength, as well as their implications for stomatal control, remain uncertain. We used an analytically solvable model to explore optimal stomatal control under alternative hypotheses for source and sink regulation. We assumed that either leaf sugar concentration or leaf water potential regulates NSLs, and that either phloem turgor pressure or phloem sugar concentration regulates sink phloem unloading. All hypotheses led to realistic stomatal responses to light, CO2 and air humidity, including conservative behaviour for the intercellular-to-atmospheric CO2 concentration ratio. Sugar-regulated and water-regulated NSLs are distinguished by the presence/absence of a stomatal closure response to changing sink strength. Turgor-regulated and sugar-regulated phloem unloading are distinguished by the presence/absence of stomatal closure under drought and avoidance/occurrence of negative phloem turgor. Results from girdling and drought experiments on Pinus sylvestris, Betula pendula, Populus tremula and Picea abies saplings are consistent with optimal stomatal control under sugar-regulated NSLs and turgor-regulated unloading. Our analytical results provide a simple representation of stomatal responses to above-ground and below-ground environmental factors and sink activity.

Sunflecks in the upper canopy: dynamics of light-use efficiency in sun and shade leaves of Fagus sylvatica.

Sunflecks are transient patches of direct radiation that provide a substantial proportion of the daily irradiance to leaves in the lower canopy. In this position, faster photosynthetic induction would allow for higher sunfleck-use efficiency, as is commonly reported in the literature. Yet, when sunflecks are too few and far between, it may be more beneficial for shade leaves to prioritize efficient photosynthesis under shade. We investigated the temporal dynamics of photosynthetic induction, recovery under shade, and stomatal movement during a sunfleck, in sun and shade leaves of Fagus sylvatica from three provenances of contrasting origin. We found that shade leaves complete full induction in a shorter time than sun leaves, but that sun leaves respond faster than shade leaves due to their much larger amplitude of induction. The core-range provenance achieved faster stomatal opening in shade leaves, which may allow for better sunfleck-use efficiency in denser canopies and lower canopy positions. Our findings represent a paradigm shift for future research into light fluctuations in canopies, drawing attention to the ubiquitous importance of sunflecks for photosynthesis, not only in lower-canopy leaves where shade is prevalent, but particularly in the upper canopy where longer sunflecks are more common due to canopy openness.

Photosynthetic acclimation and sensitivity to short- and long-term environmental changes in a drought-prone forest.

Future climate will be characterized by an increase in frequency and duration of drought and warming that exacerbates atmospheric evaporative demand. How trees acclimate to long-term soil moisture changes and whether these long-term changes alter trees' sensitivity to short-term (day to months) variations of vapor pressure deficit (VPD) and soil moisture is largely unknown. Leaf gas exchange measurements were performed within a long-term (17 years) irrigation experiment in a drought-prone Scots pine-dominated forest in one of Switzerland's driest areas on trees in naturally dry (control), irrigated, and 'irrigation-stop' (after 11 years of irrigation) conditions. Seventeen years of irrigation increased photosynthesis (A) and stomatal conductance (gs) and reduced gs sensitivity to increasing VPD and soil drying. Following irrigation-stop, gas exchange decreased only after 3 years. After 5 years, maximum carboxylation (Vcmax) and electron transport (Jmax) rates in irrigation-stop recovered to similar levels as to before the irrigation-stop. These results suggest that long-term release from soil drought reduces the sensitivity to VPD and that atmospheric constraints may play an increasingly important role in combination with soil drought. Moreover, our study indicates that structural adjustments lead to an attenuation of initially strong leaf-level acclimation to strong multiple-year drought.

Assessing model performance via the most limiting environmental driver in two differently stressed pine stands.

Climate change will impact forest productivity worldwide. Forecasting the magnitude of such impact, with multiple environmental stressors changing simultaneously, is only possible with the help of process-based models. In order to assess their performance, such models require careful evaluation against measurements. However, direct comparison of model outputs against observational data is often not reliable, as models may provide the right answers due to the wrong reasons. This would severely hinder forecasting abilities under unprecedented climate conditions. Here, we present a methodology for model assessment, which supplements the traditional output-to-observation model validation. It evaluates model performance through its ability to reproduce observed seasonal changes of the most limiting environmental driver (MLED) for a given process, here daily gross primary productivity (GPP). We analyzed seasonal changes of the MLED for GPP in two contrasting pine forests, the Mediterranean Pinus halepensis Mill. Yatir (Israel) and the boreal Pinus sylvestris L. Hyytiälä (Finland) from three years of eddy-covariance flux data. Then, we simulated the same period with a state-of-the-art process-based simulation model (LandscapeDNDC). Finally, we assessed if the model was able to reproduce both GPP observations and MLED seasonality. We found that the model reproduced the seasonality of GPP in both stands, but it was slightly overestimated without site-specific fine-tuning. Interestingly, although LandscapeDNDC properly captured the main MLED in Hyytiälä (temperature) and in Yatir (soil water availability), it failed to reproduce high-temperature and high-vapor pressure limitations of GPP in Yatir during spring and summer. We deduced that the most likely reason for this divergence is an incomplete description of stomatal behavior. In summary, this study validates the MLED approach as a model evaluation tool, and opens up new possibilities for model improvement.

Leaf carbon and water status control stomatal and nonstomatal limitations of photosynthesis in trees.

Photosynthetic rate is concurrently limited by stomatal limitations and nonstomatal limitations (NSLs). However, the controls on NSLs to photosynthesis and their coordination with stomatal control on different timescales remain poorly understood. According to a recent optimization hypothesis, NSLs depend on leaf osmotic or water status and are coordinated with stomatal control so as to maximize leaf photosynthesis. Drought and notching experiments were conducted on Pinus sylvestris, Picea abies, Betula Pendula and Populus tremula seedlings in glasshouse conditions to study the dependence of NSLs on leaf osmotic and water status, and their coordination with stomatal control, on timescales of minutes and weeks, to test the assumptions and predictions of the optimization hypothesis. Both NSLs and stomatal conductance followed power-law functions of leaf osmotic concentration and leaf water potential. Moreover, stomatal conductance was proportional to the square root of soil-to-leaf hydraulic conductance, as predicted by the optimization hypothesis. Though the detailed mechanisms underlying the dependence of NSLs on leaf osmotic or water status lie outside the scope of this study, our results support the hypothesis that NSLs and stomatal control are coordinated to maximize leaf photosynthesis and allow the effect of NSLs to be included in models of tree gas-exchange.

Determinants of legacy effects in pine trees - implications from an irrigation-stop experiment.

Tree responses to altered water availability range from immediate (e.g. stomatal regulation) to delayed (e.g. crown size adjustment). The interplay of the different response times and processes, and their effects on long-term whole-tree performance, however, is hardly understood. Here we investigated legacy effects on structures and functions of mature Scots pine in a dry inner-Alpine Swiss valley after stopping an 11-yr lasting irrigation treatment. Measured ecophysiological time series were analysed and interpreted with a system-analytic tree model. We found that the irrigation stop led to a cascade of downregulations of physiological and morphological processes with different response times. Biophysical processes responded within days, whereas needle and shoot lengths, crown transparency, and radial stem growth reached control levels after up to 4 yr only. Modelling suggested that organ and carbon reserve turnover rates play a key role for a tree's responsiveness to environmental changes. Needle turnover rate was found to be most important to accurately model stem growth dynamics. We conclude that leaf area and its adjustment time to new conditions is the main determinant for radial stem growth of pine trees as the transpiring area needs to be supported by a proportional amount of sapwood, despite the growth-inhibiting environmental conditions.

Stem emissions of monoterpenes, acetaldehyde and methanol from Scots pine (Pinus sylvestris L.) affected by tree-water relations and cambial growth.

Tree stems are an overlooked source of volatile organic compounds (VOCs). Their contribution to ecosystem processes and total VOC fluxes is not well studied, and assessing it requires better understanding of stem emission dynamics and their driving processes. To gain more mechanistic insight into stem emission patterns, we measured monoterpene, methanol and acetaldehyde emissions from the stems of mature Scots pines (Pinus sylvestris L.) in a boreal forest over three summers. We analysed the effects of temperature, soil water content, tree water status, transpiration and growth on the VOC emissions and used generalized linear models to test their relative importance in explaining the emissions. We show that Scots pine stems are considerable sources of monoterpenes, methanol and acetaldehyde, and their emissions are strongly regulated by temperature. However, even small changes in water availability affected the emission potentials: increased soil water content increased the monoterpene emissions within a day, whereas acetaldehyde and methanol emissions responded within 2-4 days. This lag corresponded to their transport time in the xylem sap from the roots to the stem. Moreover, the emissions of monoterpenes, methanol and acetaldehyde were influenced by the cambial growth rate of the stem with 6-10-day lags.

The influence of soil temperature and water content on belowground hydraulic conductance and leaf gas exchange in mature trees of three boreal species.

Understanding stomatal regulation is fundamental to predicting the impact of changing environmental conditions on vegetation. However, the influence of soil temperature (ST) and soil water content (SWC) on canopy conductance (gs ) through changes in belowground hydraulic conductance (kbg ) remains poorly understood, because kbg has seldom been measured in field conditions. Our aim was to (a) examine the dependence of kbg on ST and SWC, (b) examine the dependence of gs on kbg and (c) test a recent stomatal optimization model according to which gs and soil-to-leaf hydraulic conductance are strongly coupled. We estimated kbg from continuous sap flow and xylem diameter measurements in three boreal species. kbg increased strongly with increasing ST when ST was below +8°C, and typically increased with increasing SWC when ST was not limiting. gs was correlated with kbg in all three species, and modelled and measured gs were well correlated in Pinus sylvestris (a model comparison was only possible for this species). These results imply an important role for kbg in mediating linkages between the soil environment and leaf gas exchange. In particular, our finding that ST strongly influences kbg in mature trees may help us to better understand tree behaviour in cold environments.

Research frontiers for improving our understanding of drought-induced tree and forest mortality.

Accumulating evidence highlights increased mortality risks for trees during severe drought, particularly under warmer temperatures and increasing vapour pressure deficit (VPD). Resulting forest die-off events have severe consequences for ecosystem services, biophysical and biogeochemical land-atmosphere processes. Despite advances in monitoring, modelling and experimental studies of the causes and consequences of tree death from individual tree to ecosystem and global scale, a general mechanistic understanding and realistic predictions of drought mortality under future climate conditions are still lacking. We update a global tree mortality map and present a roadmap to a more holistic understanding of forest mortality across scales. We highlight priority research frontiers that promote: (1) new avenues for research on key tree ecophysiological responses to drought; (2) scaling from the tree/plot level to the ecosystem and region; (3) improvements of mortality risk predictions based on both empirical and mechanistic insights; and (4) a global monitoring network of forest mortality. In light of recent and anticipated large forest die-off events such a research agenda is timely and needed to achieve scientific understanding for realistic predictions of drought-induced tree mortality. The implementation of a sustainable network will require support by stakeholders and political authorities at the international level.

Stand dynamics modulate water cycling and mortality risk in droughted tropical forest.

Transpiration from the Amazon rainforest generates an essential water source at a global and local scale. However, changes in rainforest function with climate change can disrupt this process, causing significant reductions in precipitation across Amazonia, and potentially at a global scale. We report the only study of forest transpiration following a long-term (>10 year) experimental drought treatment in Amazonian forest. After 15 years of receiving half the normal rainfall, drought-related tree mortality caused total forest transpiration to decrease by 30%. However, the surviving droughted trees maintained or increased transpiration because of reduced competition for water and increased light availability, which is consistent with increased growth rates. Consequently, the amount of water supplied as rainfall reaching the soil and directly recycled as transpiration increased to 100%. This value was 25% greater than for adjacent nondroughted forest. If these drought conditions were accompanied by a modest increase in temperature (e.g., 1.5°C), water demand would exceed supply, making the forest more prone to increased tree mortality.

An empirical method that separates irreversible stem radial growth from bark water content changes in trees: theory and case studies.

Substantial uncertainty surrounds our knowledge of tree stem growth, with some of the most basic questions, such as when stem radial growth occurs through the daily cycle, still unanswered. We employed high-resolution point dendrometers, sap flow sensors, and developed theory and statistical approaches, to devise a novel method separating irreversible radial growth from elastic tension-driven and elastic osmotically driven changes in bark water content. We tested this method using data from five case study species. Experimental manipulations, namely a field irrigation experiment on Scots pine and a stem girdling experiment on red forest gum trees, were used to validate the theory. Time courses of stem radial growth following irrigation and stem girdling were consistent with a-priori predictions. Patterns of stem radial growth varied across case studies, with growth occurring during the day and/or night, consistent with the available literature. Importantly, our approach provides a valuable alternative to existing methods, as it can be approximated by a simple empirical interpolation routine that derives irreversible radial growth using standard regression techniques. Our novel method provides an improved understanding of the relative source-sink carbon dynamics of tree stems at a sub-daily time scale.

Coordination of physiological traits involved in drought-induced mortality of woody plants.

Accurate modelling of drought-induced mortality is challenging. A steady-state model is presented integrating xylem and phloem transport, leaf-level gas exchange and plant carbohydrate consumption during drought development. A Bayesian analysis of parameter uncertainty based on expert knowledge and a literature review is carried out. The model is tested by combining six data compilations covering 170 species using information on sensitivities of xylem conductivity, stomatal conductance and leaf turgor to water potential. The possible modes of plant failure at steady state are identified (i.e. carbon (C) starvation, hydraulic failure and phloem transport failure). Carbon starvation occurs primarily in the parameter space of isohydric stomatal control, whereas hydraulic failure is prevalent in the space of xylem susceptibility to embolism. Relative to C starvation, phloem transport failure occurs under conditions of low sensitivity of photosynthesis and high sensitivity of growth to plant water status. These three failure modes are possible extremes along two axes of physiological vulnerabilities, one characterized by the balance of water supply and demand and the other by the balance between carbohydrate sources and sinks. Because the expression of physiological vulnerabilities is coordinated, we argue that different failure modes should occur with roughly equal likelihood, consistent with predictions using optimality theory.

Balancing the risks of hydraulic failure and carbon starvation: a twig scale analysis in declining Scots pine.

Understanding physiological processes involved in drought-induced mortality is important for predicting the future of forests and for modelling the carbon and water cycles. Recent research has highlighted the variable risks of carbon starvation and hydraulic failure in drought-exposed trees. However, little is known about the specific responses of leaves and supporting twigs, despite their critical role in balancing carbon acquisition and water loss. Comparing healthy (non-defoliated) and unhealthy (defoliated) Scots pine at the same site, we measured the physiological variables involved in regulating carbon and water resources. Defoliated trees showed different responses to summer drought compared with non-defoliated trees. Defoliated trees maintained gas exchange while non-defoliated trees reduced photosynthesis and transpiration during the drought period. At the branch scale, very few differences were observed in non-structural carbohydrate concentrations between health classes. However, defoliated trees tended to have lower water potentials and smaller hydraulic safety margins. While non-defoliated trees showed a typical response to drought for an isohydric species, the physiology appears to be driven in defoliated trees by the need to maintain carbon resources in twigs. These responses put defoliated trees at higher risk of branch hydraulic failure and help explain the interaction between carbon starvation and hydraulic failure in dying trees.

The effect of ectomycorrhizal fungal exposure on nursery-raised Pinus sylvestris seedlings: plant transpiration under short-term drought, root morphology and plant biomass.

Drought is a major environmental stressor that limits seedling growth. Several studies have found that some ectomycorrhizal fungi may increase the drought tolerance of nursery-raised seedlings. However, the precise role that different ectomycorrhizal fungi species play in drought tolerance remains unclear. We evaluated the transpiration rate of Pinus sylvestris seedlings under drought stress in greenhouse conditions by exposing seedlings to 10 ectomycorrhizal fungi species, with different functional traits (exploration type and hydrophobicity), and to 3 natural soil inoculums. We measured the transpiration and water potential of the seedlings during a 10-day drought period and a 14-day recovery period. We then analyzed their root morphology, stem, needle, root biomass and needle chlorophyll fluorescence. We showed that exposing seedlings to ectomycorrhizal fungi or soil inoculum had a positive effect on their transpiration rate during the driest period and through the recovery phase, leading to 2- to 3-fold higher transpiration rates compared with the nonexposed control seedlings. Seedlings exposed to medium-distance ectomycorrhizal fungi performed better than other exploration types under drought conditions, but ectomycorrhizal fungi hydrophobicity did not seem to affect the seedlings response to drought. No significant differences were observed in biomass accumulation and root morphology between the seedlings exposed to different ectomycorrhizal fungi species and the control. Our results highlight the positive and species-specific effect of ectomycorrhizal fungi exposure on drought tolerance in nursery-raised Scots pine seedlings. The studied ectomycorrhizal fungi functional traits may not be sufficient to predict the seedling response to drought stress, thus physiological studies across multiple species are needed to draw the correct conclusion. Our findings have potential practical implications for enhancing seedling drought tolerance in nursery plant production.

Tree organ growth and carbon allocation dynamics impact the magnitude and δ13C signal of stem and soil CO2 fluxes.

Incomplete knowledge of carbon (C) allocation dynamics in trees hinders accurate modeling and future predictions of tree growth. We studied C allocation dynamics in a mature Pinus sylvestris L. dominated forest with a novel analytical approach, allowing the first comparison of: (i) magnitude and δ13C of shoot, stem and soil CO2 fluxes (Ashoot, Rstem and Rsoil), (ii) concentration and δ13C of compound-specific and/or bulk non-structural carbohydrates (NSCs) in phloem and roots and (iii) growth of stem and fine roots. Results showed a significant effect of phloem NSC concentrations on tracheid growth, and both variables significantly impacted Rstem. Also, concentrations of root NSCs, especially starch, had a significant effect on fine root growth, although no effect of root NSC concentrations or root growth was detected on Rsoil. Time series analysis between δ13C of Ashoot and δ13C of Rstem or δ13C of Rsoil revealed strengthened C allocation to stem or roots under high C demands. Furthermore, we detected a significant correlation between δ13C of Rstem and δ13C of phloem sucrose and glucose, but not for starch or water-soluble carbohydrates. Our results indicate the need to include C allocation dynamics into tree growth models. We recommend using compound-specific concentration and δ13C analysis to reveal C allocation processes that may not be detected by the conventional approach that utilizes bulk organic matter.