The hydrogen isotopic signature (δ2H) of water in trees contains information on plant functional responses to climatic changes and on the origin of the water. This is also true for the non-exchangeable hydrogen isotopic signature (δ2HNE) of plant organic matter, which contains additional physiological and biochemical information that can be dated to specific years if extracted from annual rings of trees. Despite this potential for gaining unique insights from δ2HNE of tree-ring cellulose (δ2HTRC), it has not been widely used compared to other isotope signals, likely due to challenging methodological constraints and interpretations of these isotopic signals. In this chapter, we first summarize hydrogen isotope (2H-) fractionation that occurs between source water and tree rings and review methods (e.g. nitration, equilibration, position-specific applications) and calculations to determine δ2HNE in tree material. Building upon a summary of the current state of knowledge, this chapter also provides an exhaustive synthesis of δ2HTRC papers, applications, and associated data from approximately 180 sites across the globe (paired with modelled precipitation δ2H values and climate data). The data allow us to investigate the hydrological-climatic effects driving δ2HTRC pattern on a global scale, the relationship of hydrogen with oxygen isotopes in the same tree-ring material, as well as the influence of physiological-biochemical effects (e.g., species differences, tree growth) that appear to be more important on local or temporal scales than on a large spatial scales. Thus, when local hydro-climatic influences on source water δ2H can be isolated, δ2HTRC gives novel insights on tree physiological responses to abiotic and biotic stresses. We conclude that the growing constellation of tree-ring metrics, including advancements in 2H-processing (i.e., equilibration techniques allowing rapid determinations of δ2HNE) and further refinements to the understanding of post-photosynthetic 2H-fractionations will together provide many new opportunities to understand past climates and ecophysiology by using δ2H in tree rings.
1 General Introduction
Hydrogen isotopes in various biomarkers have been widely used by geochemists and paleo-climatologists to reconstruct past hydrological and climatic changes (Feng and Epstein 1994; Sachse et al. 2012). The underlying assumption of these studies has been that the hydrogen isotope ratio (δ2H) in a compound reflects the δ2H of water used during photosynthesis and during biosynthesis of these compounds in various plant organs. Given that δ2H of precipitation water is well known to be under the control of air humidity during evaporation and temperature during condensation, δ2H of plant compounds have primarily been used to reconstruct those two climatic variables (Libby et al. 1976; Voelker et al. 2014a). The more abundant protium (99.98%) is the lighter of the two stable hydrogen isotopes, consisting of one proton and one electron (mass 1). The less abundant deuterium (0.02%) is the heavier of the two, holding an additional neutron (mass 2). The large difference in mass between the two hydrogen isotopes is physically expressed as strong kinetic and equilibrium isotope fractionation that can cause δ2H variation of up to 550‰ in water and organic (plant) samples (Schmidt et al. 2007). Moreover, there are strong differences in 2H-fractionation among plant metabolic pathways leading to δ2H variation between carbohydrates, lipids, and proteins. In recent decades, measurements of δ2H have been increasingly applied on plant-derived lipids such as n-alkanes, that are highly recalcitrant to environmental degradation over timescales of thousands of years. These compounds can be extracted from soil or sediment cores, or other sources that are often dated using radiocarbon or other dating methods (Sachse et al. 2012). Yet, we are just beginning to understand the physiological and hydrological driving factors causing isotopic variations of plant-derived lipids (Kahmen et al. 2013; Newberry et al. 2015). In contrast, analyses of non-exchangeable δ2H of tree-ring cellulose (δ2HTRC) is still rarely applied, which is mainly caused by what has appeared to be a low sensitivity to local climatic changes and associated difficulties interpreting δ2H signals (Boettger et al. 2014; Lipp et al. 1991), as well as due to methods that hinder the rate of sample processing (Epstein and Yapp 1976; Schimmelmann 1991). However, further developments during the last two decades led to high-throughput methods for δ2HNE analyses of plant organic material (including δ2HTRC) and important new interpretations of metabolic controls over δ2HNE in plant material (Cormier et al. 2018; Sanchez-Bragado et al. 2019) and tree-ring cellulose (Kimak 2015; Lehmann et al. 2021; Mayr 2003). The coming decade promises more rapid developments in this field that should lead to more studies employing δ2HTRC to understand past climates and plant ecophysiology.
2 The Hydrogen Isotopic Signature of Water in Trees
The hydrogen isotopic compositions of precipitation and atmospheric water represent the primary determinants of δ2H in terrestrial plant tissues including tree rings. Establishment of the temporal and spatial dynamics of the hydrogen isotopic composition of source water (δ2HS, but see also Chapter 18), via measurements and modelling, is therefore an important prerequisite for optimal interpretation of δ2H values in tree rings (Allen et al. 2019; Dawson et al. 2002; Saurer et al. 2012). As δ2H values in precipitation and water progressively change due to various factors including temperature, elevation, latitude, distance to coast, amount of a rain event, weather events, and atmospheric circulation pattern, the variability of water isotopes across land surfaces are substantial. Ocean water is used as international reference point for isotope measurements (Vienna Standard Mean Ocean Water, VSMOW), with a δ2H value of 0‰. After evaporation from a water surface or from vegetation, vapor is depleted in 2H (lower δ2H) compared to its origin due to fractionation during phase transition from liquid to gas form; that transition favors the preferential evaporation of lighter isotopologues (e.g., 1H16O) over heavier ones (e.g., 2H18O). In contrast, when water condenses in clouds, precipitation is enriched in 2H (higher δ2H) than the water vapor remaining in the air (Fig. 11.1). Therefore, precipitation will become more depleted in 2H with each rain event as an air mass undergoes successive condensation and precipitation while moving inland or to higher elevations. This process, commonly termed “rainout”, depends strongly on temperature conditions during condensation, partially explaining the strong temperature dependency of precipitation δ2H values (Dansgaard 1964; Libby et al. 1976). Given that the isotopic composition of precipitation is not always available, global maps of average water isotope values have been modelled and can be used to infer δ2H values of precipitation in the absence of measurements (Bowen and Revenaugh 2003). The isotopic signals of precipitation can be modified by evaporation effects prior to when trees take up water in the soil, which has often been characterized by an enrichment in 2H from deep to more shallow soil layers (Allison et al. 1983; Lin and Sternberg 1993). On the other hand, δ2H of soil water that is taken up by trees is an admixture of recent and older precipitation that can depend on the size, depth, and geological properties of water catchments and groundwater aquifers, as well as on water turn-over rates in concert with soil characteristics, vegetation rooting depths and other processes modifying the mixing and uptake of water by tree roots (Brooks et al. 2010; Lin and Sternberg 1993). Overall, the isotopic composition of precipitation and source water taken up by trees can lag precipitation inputs by weeks to years, and can vary markedly among individuals and across regions (Allen et al. 2019; Voelker et al. 2019; Volkmann et al. 2016).
Source water (δ2HS) becomes enriched in heavy isotopes after reaching leaves due to preferential evaporation loss of lighter isotopologues from the leaf water pool during transpiration. The fractionation from δ2HS to δ2H of leaf water (δ2HL) has been closely predicted by the Craig-Gordon model (Dongmann et al. 1974), describing the isotopic enrichment at the evaporative site of a water pool as (Craig and Gordon 1965):
where εe is the temperature-dependent equilibrium isotope fractionation between vapor and liquid water (Horita and Wesolowski 1994), εk is the kinetic isotope fractionation occurring during the diffusion of water molecules through the stomata cavity and the boundary layer (Merlivat 1978), δ2HV is the hydrogen isotope ratio of atmospheric water vapor, and ea/ei is the ratio between the partial pressures of the atmosphere and intracellular water vapor. As implied by Eq. 11.1, δ2HL values are mainly driven by the leaf-to-air vapor pressure ratio, similar to relative humidity (i.e., which is often used as an estimate for ea/ei) or by the vapor pressure deficit (VPD, difference between saturated and ambient pressure) and have found widespread application for reconstruction of environmental and hydrological conditions from plant organic compounds (Sachse et al. 2012; Voelker et al. 2014a; Zech et al. 2013). Importantly, at high relative humidity (ca. > 60%), the isotopic value of water vapor dominates the isotopic signal of leaf water (Gerlein-Safdi et al. 2018; Lehmann et al. 2018; Roden et al. 2000). The basic leaf water model (Eq. 11.1) tends to overestimate δ2HL values; corrections include representing leaves as two pools as well as Péclet effects, to account for unenriched water pools (e.g., lignified leaf veins) or progressive leaf water enrichment (e.g., from the bottom to the tip or from the main vein to the margin in leaves) (Roden et al. 2015). Variation in δ2HL strongly influences δ2H of assimilates (Cormier et al. 2018) and thus δ2HTRC (Roden et al. 2000). The following section will therefore focus on the manifold isotope fractionations influencing δ2HTRC chronologies.
3 The Hydrogen Isotopic Signature of Tree-Ring Cellulose
2H-fractionations occurring in trees during the biosynthesis of organic compounds from leaf water and CO2 to sugars and subsequent molecules result from several biochemical processes (Luo and Sternberg 1992; Schmidt et al. 2003; Sternberg et al. 1984; Yakir 1992). The biosynthetic hydrogen isotope fractionation (εbio) between leaf water and organic compounds was (and still is) largely treated as a constant in practice in paleo-biogeochemical studies (Sachse et al. 2012). However, εbio may vary among species and compounds, as related to plant enzymatic reactions and the specific biochemical origin of H atoms during their biosynthesis (Cormier et al. 2019, 2018; Estep and Hoering 1981; Ziegler et al. 1976). For biosynthesis of compounds in plants, three origins of H are important in this respect (Fig. 11.1):
The organic precursor molecules in a biosynthetic pathway; for example, the H atoms of ribulose-1,5-bisphosphate that are transferred to the two triose phosphates synthesized in the Calvin cycle.
Redox cofactors (e.g. the biological reducing agent NADPH) that provide an important part of the H atoms in the biosynthesis of organic compounds.
The cellular water.
Yakir and DeNiro (1990) suggested that the overall 2H-fractionation factor occurring between leaf water (δ2HL) and carbohydrates (δ2HCarbo) can be divided into photosynthetic (F1) and post-photosynthetic 2H-fractionations (F2), and that the relative fraction (f) of these two processes drives the εbio according to the following equations:
The photosynthetic 2H-fractionation (F1) occurs in the chloroplast during the light reaction of photosynthesis where ferredoxin-NADP + reductase produces NADPH with reduced H that is strongly 2H-depleted compared with leaf water (Luo et al. 1991). This 2H-depleted H pool in NADPH is subsequently introduced into organic compounds in the Calvin cycle, causing the first building blocks of sugar pools to be 2H-depleted compared with leaf water. Cormier et al. (2018) suggests that this initial process is independent of the rate of photosynthesis within a species and possibly stable for any given species. In turn, this suggests variation in εbio of carbohydrates are driven by post-photosynthetic 2H-fractionations.
Post-photosynthetic 2H-fractionation (F2) processes are assumed to have strong 2H-enrichment effects, commencing in sugars produced by the Calvin cycle and reflecting several overlapping processes and enzymatic reactions. Triose phosphates (TP) synthesis in the Calvin cycle allows (partial) exchange of C-bound H atoms with (2H-enriched) cellular water in CH2 groups adjacent to CO groups via an enolic structure (Knowles and Albery 1977). Moreover, only one out of four C-bound H atoms in TPs is derived from 2H-depleted NADPH from the light reaction of photosynthesis; alternatively, the others derive from precursor molecules that become 2H-enriched after exchange with cellular water, as described earlier. During the fructose1,6-bisphosphate aldolase reaction, two TPs are used to build one hexose phosphate and one out of four C-bound H atoms is lost to the surrounding water (Schmidt et al. 2015). Phosphoglucose isomerase, used to inter-convert glucose 6-phosphate and fructose 6-phosphate, might further 2H-enrich the sugar pool by allowing partial exchange of specific H atoms with the surrounding cellular water (Schleucher et al. 1999). In non-autotrophic plants and plant tissues, NADPH is likely strongly 2H-enriched as it derives from the oxidative pentose phosphate pathway reactions. Overall, δ2HNE values of carbohydrates typically do not deviate as strongly from those in leaf water as we would expect from the primary 2H-depletion of the NADPH pool that is generated in the light reaction of photosynthesis because post-photosynthetic 2H-enrichment of the sugar pool provides nearly a net balance of isotopic fractionation events affecting tree rings and other plant tissues.
The 2H-fractionations in sugar pools of trees do not only occur at the leaf level. Roden and Ehleringer (1999) experimentally showed that about 36% of the leaf isotopic signature in a given tree-ring was replaced by the source water isotopic signal during cellulose biosynthesis. From their findings, the authors developed a mechanistic model for δ2HTRC:
The estimates of 2H-fractionation factors were derived from various experimental systems (Luo and Sternberg 1992; Yakir and DeNiro 1990), with F1 and F2 as −171‰ and + 158‰, respectively. The Roden et al. (2000) model provided a 1:1 relationship between observed and modeled δ2HTRC under two different relative humidity conditions. Similarly, the Roden model has been applied to paired tree-ring oxygen and hydrogen isotopes for reconstruction of relative humidity over space and time (Voelker et al. 2014a). Although the Roden model has found practical applications for tree-ring studies it is known to neglect that the compensating effects of F1 and F2 simultaneously determine δ2HCarbo (Eqs. 11.2 and 11.3) and that δ2HNE of leaf sugars are lower than δ2HTRC. Moreover, as discussed toward the end of this chapter, the use of heterotrophic 2H-enriched starch might play an important role for δ2HTRC values of mature trees (Kimak et al. 2015; Lehmann et al. 2021; Mayr 2003). Thus, further improvements to the Roden et al. (2000) model are needed to more accurately understand drivers of plant tissue δ2H and, consequently, better realize the research potential that δ2HTRC can provide.
4 Methods and Calculations for δ2H Analysis of Tree Carbohydrates
There are two kinds of hydrogen in carbohydrates or hydrocarbons such as sugars and cellulose. Hydrogen linked to carbon atoms (C-H) is called non-exchangeable hydrogen and cannot be easily exchanged without enzymatic catalysis in biochemical pathways. The δ2H ratio of such carbon-bound non-exchangeable hydrogen (often termed δ2HNE) in carbohydrates holds meaningful information on hydrological, climatic, or biochemical conditions during their synthesis and is therefore of interest in dendrochronological studies. In contrast, hydrogen that is linked to oxygen atoms in hydroxyl (O–H) groups of carbohydrate molecules is called exchangeable hydrogen. Exchangeable hydrogen is in equilibrium with the hydrogen of the surrounding water such as recent sap water or environmental water and thus does not reflect the hydrological, climatic, or biochemical conditions during cellulose synthesis. Moreover, during extraction of carbohydrates such as sugars or cellulose, the exchangeable hydrogen will be exchanged with extraction water or solutions and therefore its original isotopic composition will be again overwritten. Therefore, it is crucial to exclude the exchangeable hydrogen for an accurate determination of δ2HNE values in carbohydrates. Several methods have been developed to overcome this problem over the last decades to isolate the δ2HNE values of sugars and cellulose in plants. In Table 11.1, we summarized the different available δ2HNE methods and in the following section we explain the main principles.
4.1 Nitration Methods
Nitration is the most commonly employed method (Table 11.1) for determination of δ2HNE values in cellulose and particularly helpful to establish δ2HNE reference material (Boettger et al. 2007). The nitration of cellulose is a reaction where the hydroxyl groups are replaced by nitro groups (NO2) via an electrophilic substitution. The reaction can be performed using a mixture of HNO3 (90%) and H2SO4 (~98%) with a final concentration of HNO3 of about 25% in a 150 times weight excess at room temperature for ≥ 12 h. Alternatively, cellulose can also be nitrated with a 40 times weight excess of a solution of 28.7 m% P2O5 in HNO3 (90%) at room temperature (RT) for 4 h (modified after Alexander and Mitchell 1949). For both methods, the mixing of the two components is highly exergonic and the solution needs to be cooled down to 4 °C, e.g., in an ice-water bath. The complete dissolving of P2O5 can take several hours or overnight, where a slow temperature increase to RT is uncritical. For achieving highly nitrated cellulose, it is recommended to constantly stir the reaction solution and to keep the water content as low as possible. After the nitration, the product can be washed with distilled water and stabilised with methanol. Incompletely nitrated side products can be removed via the dissolution of the highly nitrated product in acetone and centrifugation. Cellulose nitrate with a nitrogen content of > 12.7% is assumed to reflect a nearly complete nitration, but more than 13.5% N are not achievable (de la Ossa et al. 2011). To complete these steps noted in the methods above and produce cellulose nitrate with an acceptably high nitrogen content, there is probably an upper limit of about 50 samples per week that can be processed by one person, which has limited its application in attempts to understand inter-annual variation in δ2H of tree rings. Note that cellulose nitrate is an explosive material and should be stored in a refrigerator to reduce degradation. In addition, nitration methods for sugars have been developed (Dunbar and Schmidt 1984) and applied in food authenticity studies (Doner et al. 1987). However, sugar nitration should be performed very carefully under constant cooling as temperature above 20 °C can cause an extremely vigorous reaction and explosion. Finally, δ2H analysis of cellulose nitrate with a TC/EA-IRMS system might be best performed in a reactor consisting of a mix of chromium and quartz chips to avoid potential isotope fractionation during pyrolysis of nitrogen-rich compounds (Gehre et al. 2015).
4.2 Equilibration Methods
In contrast to the cellulose nitration method, where the exchangeable H on hydroxyl (OH) groups is replaced by a nitro group (NO2), the equilibration method is based on a physico-chemical principle of exchanging the OH groups with those of standard water. This process of H isotope exchange happens naturally with ambient water or vapor. In order to guarantee stable and robust δ2HNE values, which makes up 70% of the hydrogen atoms in a cellulose molecule, it is accepted to follow an equilibration with in-house-standard water of known isotopic composition before sample analysis with a TC/EA-IRMS system. Samples are decomposed by pyrolysis at ca. 1400 °C to hydrogen (H2) and carbon monoxide (CO) gases, with the former being used to determine δ2H values. Given strong methodological improvements over the last decades, the equilibration method has become more popular for determining δ2HNE in cellulose.
First, offline methods were used to perform the equilibration (Feng et al. 1993; Grinsted and Wilson 1979; Schimmelmann 1991; Wassenaar and Hobson 2000). They followed different temperature regimes but all of them suffered from disadvantages: (1) slow equilibration process at low temperatures, (2) difficult and time-consuming removal of the excess water through sublimation for the method of Feng et al. (1993), (3) isotope fractionation potential between water and exchangeable hydrogen during the different process steps and (4) a rather large amount of sample (3–12 mg) that is required. Wassenaar and Hobson (2000) described a similar method with static offline equilibration that can be applied for cellulose, as well as for complex organic materials, for example, feathers or keratin. More recent developments in offline methods allow equilibration of a batch of cellulose, starch, or sugar samples in a metal chamber under a continuous flow of vapor with a known isotopic composition at high temperature conditions (ca. 70 to 130 °C; “Hot offline equilibration”, Table 11.1) in an oven and to dry them before TC/EA-IRMS analysis (Sauer et al. 2009; Schuler et al. 2022). This system was further optimized, enabling sample equilibration and drying to occur directly in a heated and helium purged autosampler to prevent contamination with humid laboratory air. The autosampler is placed on an TC/EA-IRMS system, so that multiple samples can be equilibrated with injected water vapor, dried, and directly measured (Wassenaar et al. 2015). Equilibrations are mostly performed at high temperatures to vaporize a water source and to accelerate the equilibration process. However, other studies showed that sufficient equilibration of plant material with a water source can also be achieved at room temperature in an evacuated glass desiccator over a longer time period (“Cold offline equilibration”, Table 11.1), followed by drying of the samples in a vacuum oven over several days (Qi and Coplen 2011; Sanchez-Bragado et al. 2019).
In addition to offline equilibration systems, Filot et al. (2006) developed an online equilibration method. An equilibration chamber temperature controlled at 110 °C with a moving piston selecting between three positions to load (sample derived from an autosampler), equilibrate and inject a sample for subsequent isotope ratio analysis. The equilibration chamber is flushed with helium and in-house-standard water that is immediately vaporized when entering the heated equilibration chamber. An analytical batch consists of 94 measurements (i.e. 55 samples and 39 standard materials), allowing the analysis of about 165 samples per week. Samples and standard materials are equilibrated sequentially within 600 s and then pyrolyzed by a TC/EA-IRMS to determine δ2H values. A GC-column held at 70 °C was used to separate H2 and CO gases to the degree that sequential isotope ratio measurements are feasible on IRMS, enabling to do triple isotope analysis (Loader et al. 2015). The “hot online equilibration” method (see Table 11.1) can actually be used for the quadruple isotope ratio determination by a corresponding adjustment of the GC-column that allows the separation of N2 from CO.
4.3 Position-Specific Methods to Determine δ2HNE in Wood Material
Deuterium in plant carbohydrates can also be position-specifically determined by nuclear magnetic resonance (NMR, Table 11.1) spectroscopy. δ2HNE methods have been developed for starch and sugars from different herb and tree species (Schleucher et al. 1999; Zhang et al. 2002) and for plant and tree-ring cellulose (Betson et al. 2006). The intra-molecular analysis reveals strong δ2H differences for carbon-bound hydrogen atoms within carbohydrate molecules. Thus for interpretation of δ2HTRC values the underlying δ2HNE variations on individual positions should be considered (but see Chapter 7 for more information). Moreover, a novel and simple preparation method was developed to measure the δ2H value of lignin methoxyl groups (δ2Hmeth, Table 11.1) by gas chromatograph/pyrolysis-IRMS (Greule et al. 2008). δ2Hmeth values of wood samples were strongly correlated with the δ2HP on a global scale. δ2Hmeth might therefore reflect a new climate proxy for temperature changes over time if extracted from annual rings of trees (Gori et al. 2013). However, the biosynthetic isotope fractionation between δ2Hmeth and δ2HP of ca. -217 ‰ can vary among different tree species and therefore care should be taken if used for climate reconstructions (Anhauser et al. 2017). δ2Hmeth might be of particular interest for paleo-wood samples, which have often been buried under anoxic conditions and have undergone substantial or nearly complete losses in cellulose due to degradation, but may still contain lignin.
4.4 Calculation of Non-exchangeable Hydrogen Isotopic Composition, International Standards, and Referencing
During our review for this chapter, we found that equations regarding δ2HNE can vary among studies using equilibration methods. To facilitate comparisons among past and future δ2HTRC values, we propose the following systematics and syntax. The total δ2H values (δ2HT), which is the result of an isotopic analysis of equilibrated samples, can be calculated as follows:
where ƒe is the fraction of total exchangeable hydrogen in the total H pool and δ2HE the δ2H value of the exchangeable hydrogen atoms. For a precise estimation of δ2HNE, a comprehensive evaluation of different potential cases for ƒe are needed. Theoretically, 30% of the hydrogen in cellulose should be exchanged, but in most cases less were exchanged, most probably due to difficulties in accessing all exchangeable hydrogen and to inhomogeneities of the cellulose preparation steps. Therefore, the range for ƒe of cellulose varies between 0 and 0.3 (Schimmelmann 1991; Wassenaar and Hobson 2000). The higher ƒe values of a measurement, the better the equilibration processes and the precision and accuracy of the δ2HNE analysis:
where δ2HT-A and δ2HT-B are δ2HT values of cellulose that have been equilibrated with in-house-standard water (A or B) and thus with water vapor of a known isotope ratio (δ2HW-A, δ2HW-B), respectively. The isotopic differences between the two in-house-standard waters A and B is mostly several 100‰ and allow for the determination of ƒe (approximately the slope of relationship between δ2HT and δ2HW of both waters). The 2H-fractionation (εe) occurring between δ2HE and δ2H of water vapor has been found to vary between 60‰ and 100‰ for most plant derived compounds. Most methods set εe to ca. 80‰, as it has been found to be an average isotope fractionation factor for most cellulose samples (Schimmelmann 1991; Wassenaar and Hobson 2000). Both, εe and ƒe are temperature dependent, so temperature needs to be tightly controlled during equilibration and also held constant among batches to provide consistent results.
A mass balance allows the determination of δ2HNE from the δ2HTA or δ2HTB where δ2HT values are normalized to the international VSMOW scale using an international standard, which has no exchangeable hydrogen (e.g., polyethylene foil, IAEA-CH-7) following Filot et al. (2006):
δ2HNE values of equilibrated cellulose samples should be checked against cellulose standards with known δ2HNE values to verify method accuracy. Cellulose nitrate derived from different wood material or chronologies might be ideal for verification of these methods (Filot et al. 2006; Schuler et al. 2022). Recently published international wood standards (USGS 54–56) might also be used for δ2H calibration (Qi et al. 2016). There is also an international cellulose standard available (IAEA-CH-3), however, there is no agreement yet on its actual δ2HNE value (Loader et al. 2015; Sauer et al. 2009). Equilibration can also be performed on other organic matter, however, the degree of exchangeable hydrogen and the exchangeability of hydrogen atoms itself can differ among substances.
5 Synthesis of δ2HTRC Data, Applications, and Interpretations
For this book chapter, we reviewed ca. 50 studies and unpublished records showing information on δ2HNE of tree-ring cellulose (δ2HTRC; mainly derived via equilibration or nitration methods) from ca. 180 sites. We approximated the mean δ2HTRC values for each site chronology and plotted the distribution on a global map (Fig. 11.2). Table S1 holds all information on publications, sample preparation and material, tree species, temporal resolutions, location, and tree-ring isotope data. For each site, we estimated the mean annual temperature (MAT) and mean annual sum of precipitation (MAP) using a gridded data product at 5-arc-minute resolution (Fick and Hijmans 2017). We also modelled long-term mean annual δ2H and δ18O of precipitation (δ2HP and δ18OP) for each site using grids available on Bowen and Revenaugh (2003). For studies that reported site elevation, δ2HP values were adjusted by a value calculated from multiplying the isotopic lapse rate, assumed to be −2.24‰ δ2H per 100 m (Poage and Chamberlain 2001) for the difference between site elevation and pixel-mean elevation; for studies that did not report site elevation, the gridded values were used without adjustment.
5.1 Global δ2HTRC Patterns and Hydro-Climatic Effects
For more than four decades, δ2HNE of wood or tree-ring cellulose has been recognized to closely correlate with MAT and that δ2HNE is generally sensitive to continental-scale climatic trends (Epstein and Yapp 1976; Gray and Song 1984; Libby et al. 1976; Schiegl 1974). In retrospect, the temperature dependence of δ2HTRC at these large scales is partially due to the isotopic composition of meteoric water, which also correlates with temperature variations (Dansgaard 1964; Yapp and Epstein 1982). Here we investigated the global relationship of δ2HTRC with δ2HP and MAT across various tree species and study sites (Fig. 11.3). Including δ2HTRC values from paleo study sites (< 1000 years before 2000 AD) only slightly affected the relationship. However, to be conservative, we excluded paleo studies (ca. 40 sites) from this analysis due to large uncertainties related to time-transgressive estimates of paleoclimate temperatures and associated estimates of δ2HP.
As expected, δ2HP is indeed significantly related to δ2HTRC (r2 = 0.69), demonstrating that spatial variability in δ2HP of precipitation determines more than two thirds of the spatial variation in tree-ring isotope ratios. Interestingly, the relationship between δ2HP and δ2HTRC largely envelopes the 1:1 line, albeit with a slightly shallower slope of 0.9 ± 0.05. The overlap of the 1:1 line is somewhat surprising, given that δ2HTRC is modified by 2H-enrichment of leaf water and by various 2H-fractionation in carbohydrates in leaf and sink tissues. Roden et al. (2000) showed that it is rather unlikely that the leaf water signal is fully lost during tree-ring cellulose synthesis for both oxygen and hydrogen isotopes. We therefore assume that the sum of the 2H-enrichment during leaf water evaporation and 2H-fractionations of carbohydrates (Eqs. 11.2 and 11.3) cancel each other out, so that the mean δ2HTRC of a chronology approximately reflects the average δ2HP of a site. While MAP showed no clear relationship with δ2HTRC (not shown), we found a significant relationship between MAT and δ2HTRC (r2 = 0.47), with a slope-derived change of 5.2 ± 0.5‰/℃ and an intercept of −21.5 ± 4.0‰. The linear relationship was very similar between MAT and δ2HP (r2 = 0.67, not shown), with a slope-derived change of 5.8 ± 0.3‰/℃ and an intercept of −122.2 ± 3.0‰. Our results agree with 5.5‰/℃ that was observed by Gray and Song (1984), demonstrating that the temperature-dependent equilibrium isotope effect in precipitation water is the dominant factor influencing δ2HTRC on a global scale (however, not on a local scale as discussed later).
Moreover, we additionally investigated the global δ2H-δ18O relationship for tree-ring cellulose of about 110 sites (70 modern/40 paleo) (Fig. 11.4). Previously, these two types of data had been used to model leaf water isotope across continental climate gradients (Voelker et al. 2014a). However, to our knowledge, a global δ2H-δ18O relationship for tree-ring cellulose across diverse tree species has not previously been presented. We found that the global δ2H-δ18O of tree-ring cellulose is shifted by an average δ18O offset of + 38.6‰ between precipitation and the signal incorporated in tree-ring cellulose. This offset approximates the difference between measured δ18O values of xylem water and tree-ring cellulose (Roden and Ehleringer 2000). The changes in δ2HTRC values are low (Fig. 11.4), which can be explained by the close relationship to δ2HP (Fig. 11.3). The slope of the δ2H-δ18O relationship for tree-ring cellulose is 8.1 and thus closely mirrors that of the global meteoric water line (the GMWL is here derived from the modelled precipitation isotope data), reflecting the dominance of isotope fractionations driving spatial variation in the signals of δ2HP and δ18OP taken up by trees. Global δ2H-δ18O relationships of tree-ring cellulose were not clearly influenced by broadleaf or coniferous tree species (Fig. 11.3 & 11.4) and species-effects might not play a significant role at this scale (but see species effects on the local scale below). Perhaps more importantly, variability of how δ2H vs δ18O diverge from this global relationship spatiotemporally and among and within species certainly has the potential to yield unique insights on climatic variability, ecohydrology and tree ecophysiology that needs further exploration.
5.2 Paleo-Climatic δ2HTRC Applications
Due to the strong temperature response, δ2HTRC analyses were undertaken to provide early paleoclimate or “paleo-thermometer” proxies that could be applied to ancient wood samples (Feng and Epstein 1994; Yapp and Epstein 1977). Other studies on ancient wood material combined δ2HNE and δ18O for the reconstruction of relative humidity (Edwards and Fritz 1986; Voelker et al. 2014a) and atmospheric circulations patterns (Feng et al. 2007). Some studies used the δ2HTRC difference between ancient and modern wood to infer climatic changes or monsoon intensity (Boettger et al. 2003; Feng et al. 1999). Moreover, δ2HTRC can function as a supplementary information, helping to date tree rings in wood of unknown sources (Becker et al. 1991). This is particularly important for dating of ancient wood samples, which are rarer and thus can be more difficult to cross date using conventional dendrochronological methods. The longest published δ2HTRC record so far (over 8000 years before 1950, at 50-year resolution) was established by Feng and Epstein (1994) from bristlecone pine trees. However, δ2HTRC was also analyzed from much older individual wood samples from the Late Glacial/Holocene (14 to 8 Ka) (Boettger et al. 2003; Edwards and Fritz 1986; Epstein 1995; Feng et al. 2007), Late Pleistosence (60 to 30 Ka) (Stojakowits et al. 2020), Early Pliocene (~4.5 Ma) (Csank et al. 2011), and Eocene (~45 Ma) (Jahren and Sternberg 2008). Despite this past work, hydrogen isotope analysis of tree rings is rarely conducted compared to the use of hydrogen isotopes from other paleo isotope archives (e.g., ice or sediment cores). Hence, novel approaches that combine δ2HTRC with carbon and oxygen isotope analyses and other tree-ring properties at inter-annual resolution may provide promising new multi-proxy tools for understanding past climates and their impact on plant ecophysiology.
5.3 Local δ2HTRC Pattern and Physio-Biochemical Effects
Despite known correlations between climate variables and δ2HTRC across large spatial scales, temporal variations in local δ2HTRC chronologies often do not strongly correlate with local climatic changes or hydrological influences (Boettger et al. 2014; Lipp et al. 1991; Loader et al. 2008; Pendall 2000). Oxygen and hydrogen isotopes in an individual tree should theoretically be derived from the same water sources and thus be correlated (as shown in Fig. 11.4), however, only weak relationships between δ2H and δ18O chronologies of tree-ring cellulose have been observed on local scales (Fig. 11.5). Early research indicates that climatic information in water-derived hydrogen isotopes is potentially overwritten by physiological factors driving changes in δ2HTRC, including differences in 2H-fractionation between autotrophic and heterotrophic tissues and metabolic pathways (Luo and Sternberg 1992; Yakir and DeNiro 1990). The following text reviews a growing body of evidence documenting strong physio-biochemical isotope effects on δ2H of plant-derived carbohydrates that are eventually incorporated into δ2HTRC and may explain the difference between global versus local isotopic patterns.
(1) Maturation effect: In Germany, δ2HTRC of oaks was shown to increase by about 20‰ between approximately age 0 and 150, with little change thereafter (Lipp et al. 1993; Mayr 2003). We term this trend over 100 + years a “maturation effect” because the juvenile stage for many oaks, biologically, usually only lasts up to about 40–50 years and sometimes much less if trees are from sprout rather than seed origin. This increase in δ2HTRC with age is the opposite of that which would be expected if rooting depths increased with development, leading to them tapping deeper soil water sources with more negative δ2HS values. Changes in δ2HTRC values with tree age might be related to an age-related increase in the availability or use of non-structural carbohydrates for growth (i.e. fresh assimilates vs. carbon storage) and/or increased phloem transport distance between canopy leaf area and the stem base (i.e. tree height) where wood samples are typically extracted for isotopic analyses. The aforementioned ideas should be tested with species that differ in growth rate and maturation as only a few δ2HTRC studies have investigated the maturation effect so far (Arosio et al. 2020).
(2) Intra-annual effects: An early δ2HTRC study investigated intra-annual variation (Epstein and Yapp 1976) and documented that earlywood was 2H-enriched by 7 to 52‰ compared to latewood within the same tree and year, with some exceptions; 2H-enriched earlywood was confirmed by other studies (Kimak 2015; Mayr 2003). Higher-resolution intra-annual measurements in fossil (Metasequoia sp.) or in modern (Quercus crispula, Pinus radiata) wood demonstrated that cellulose is most 2H-depleted in the center of an annual tree-ring, while the start and end are more 2H-enriched (Jahren and Sternberg 2008; Nabeshima et al. 2018; Wilson and Grinsted 1978). Leaf cellulose of two deciduous trees in Switzerland were also observed to be more 2H-enriched in the early than in the late growing season, while the opposite pattern was observed for an evergreen conifer (Kimak et al. 2015). The drivers of these intra-annual effects are not simply explained by changes in source water or environmental conditions. Winter/spring source water from temperate and boreal zones is 2H-depleted, which is in contrast to higher δ2HTRC values in wood formed at the growing season, assuming much of the water used by trees during this time are from dormant season precipitation recharge of soil moisture. The VPD-driven leaf water 2H-enrichment would also be lesser during the early growing season than in summer, which is again in contrast to the observed intra-annual δ2HNE pattern in leaf and tree-ring cellulose. It is likely, the intra-annual effects are caused by a higher relative use of 2H-enriched storage pools for the formation of earlywood and consequently 2H-depleted photosynthetic assimilates for the formation of late wood (Lehmann et al. 2021; Vitali et al. 2022). Cleary, the drivers of intra-annual isotope variation needs further exploration to provide better insights on seasonal changes in plant-water relations and physiology.
(3) Growth effects: Change in biomass productivity, as indicated by various metrics of tree-ring growth, have often been used as primary indicator of plant physiological performance under changing climatic conditions. Here we combined growth and δ2HTRC data of bur oaks (Quercus macrocarpa Michx.) at three North American sites with a range in environmental conditions (Voelker et al. 2014a, b). Using the Roden model (Eq. 11.4), we calculated the expected δ2HTRC for at least fifteen years at each of those three sites and then subtracted these modeled values from the observed δ2HTRC values and plotted these residuals versus growth rates as calculated by Voelker et al. (2014b), which yielded a significant negative relationship (r2 = 0.30; Fig. 11.6). For the model, we assumed constant δ2HS values for each year, as the species is known to be very deeply rooted, and δ2H values of vapor to be in equilibrium with those of source water. If the Roden model would have accounted for all δ2HTRC variation, then there should be no offset and no trend in data shown. There are two possibilities explaining the trend. The first is that the modelled δ2HS used in the Roden model were increasingly underestimated across regions with slower growth rates, which is unlikely, because the slower growth rates arise from a complex combination of climatic phenomena that would not have provided such shifts in modeled δ2HS values. The second explanation for this trend is that low growth rates are inherently associated with greater 2H-fractionation, which correspond to how slow-growing oak trees used a greater proportion of stored carbohydrates that were 2H-enriched. To some extent this would be expected, particularly in oak trees, because their earlywood vessels are largely formed early in the spring (i.e. before leaf expansion is complete) from stored carbohydrates and as oaks grow more slowly, a larger proportion of the tree-ring tissue in any given year is earlywood vessels (Voelker et al. 2012). Figure 11.6 demonstrates that correlations were weak within sites, which would be expected given a lack of year-specific knowledge of water δ2H values to plug into the Roden model. Importantly, the significant relationship across sites provides the first tree-ring based support for the contention of Cormier et al. (2018) that constraints on metabolic rates can increase δ2H in plant compounds. Besides, a recent publication suggest the use of carbon storage under stress conditions by observing a negative relationship between δ2HTRC values and tree-ring width chronologies on four sites in Europe and Asia, where growth is limited by precipitation or light (Lehmann et al. 2021). However, on a site where temperature was the growth-limiting factor, the relationship between δ2HTRC and TRW was slightly positive. Thus, further studies are needed to determine the driving factors of 2H-fractionaions related to annual growth (Vitali et al. 2022).
(4) Herbaceous tissue effects: Tissue-specific δ2HNE effects represent important physiological processes, but have rarely been analyzed in trees (e.g. stem vs. root cellulose, Roden and Ehleringer 1999). We thus focused on δ2HNE reports working with herbaceous plant tissues and assume that this knowledge is helpful to understand tree physiological responses. Sanchez-Bragado et al. (2019) documented the lowest δ2HNE values in plant assimilates of autotrophic flag leaf tissues of wheat, intermediate δ2HNE values in semi-autotrophic ears, and highest δ2HNE values in heterotrophic roots and grain tissues. These results indicate the importance of the photosynthetic reactions causing 2H-depleted NADPH pools in autotrophic tissues. The findings are supported by δ2HNE responses of leaf cellulose to light intensity and CO2 concentrations (Cormier et al. 2018), δ2HNE differences in leaves of different morphology between parasitic plants and their hosts (Cormier et al. 2019), leaf blade and vein tissues (Kimak et al. 2015), and photosynthetic pathways (Sternberg and DeNiro 1983). These findings collectively indicate that autotrophic leaf tissues and their compounds are likely more 2H-depleted compared to all other plant tissues of trees (Ruppenthal et al. 2015). It will remain a future challenge to determine δ2HNE values of compounds in different tree tissues and to better understand the most relevant processes leading to 2H-fractionations that occur during photosynthesis, transport of freshly assimilated sugars, and during cellulose biosynthesis in woody tissues.
(5) Species effects: We analyze the previously described synthesized dataset for species effects because they are under-described in the literature (but see Ziehmer et al. (2018)). Using sub-fossil wood remains from alpine locations in the European Alps, we show that δ2HTRC is consistently 40‰ lower in the deciduous Larix decidua than in evergreen Pinus cembra across a 9000-year chronology (Fig. 11.7). Such differences might be partly driven by δ2HS difference, or, on the other hand, they might indicate divergent metabolic 2H-fractionations associated with leaf formation, translocation of assimilates, and tree-ring formation (Arosio et al. 2020). To investigate this further, we corrected the δ2HTRC value from each site in the literature data with modelled δ2H values of precipitation (Δ2HTRC). As global-scale isotope variations in precipitation water are taken into account by this correction (see Fig. 11.3), Δ2HTRC can be compared between tree species from different sites. However, no clear Δ2HTRC pattern were observed on a global scale (Fig. 11.8), suggesting that Δ2HTRC variations must be related to some combination of species/genus and regional climatic differences (i.e. MAT, MAP). We further investigated the Δ2HTRC values across genus and species, with mean values ranging strongly between −57 and 30‰ and −19 and 34.5‰, respectively (Fig. 11.9). Δ2HTRC values were significantly different for some genera (P < 0.001) and species (P ≤ 0.02), showing that taxon-specific information is likely imprinted on the 2H-fractionations in mature trees. Interestingly, we found that Δ2HTRC values at the genus level were co-influenced by MAP (P ≤ 0.02, ANCOVA), indicating that regional climate conditions at a given site, likely a combination of precipitation and strongly co-varying relative humidity, also contributes to variation in 2H-fractionations. The influence of water availability on Δ2HTRC would make sense, since growth sometimes depends on carbon storage and thus on 2H-enriched heterotrophic starch rather than on 2H-depleted sugars from recent photoassimilates, particularly in extreme dry years or during defoliation events (Vitali et al. 2022). Thus, it is likely that the highly negative and positive Δ2HTRC variations at the species and genus levels mainly reflect plastic 2H-fractionations related to the carbohydrate metabolism in trees. In this sense, broad surveys of δ2HNE values of non-structural carbohydrates and cellulose in different tree species might be helpful to disentangle the drivers of 2H-fractionations and potential phylogenetic effects.
Our chapter reveals that we are at the beginning of a new phase of understanding the hydrogen isotopic signals in trees. Overall, deriving robust inter-annual climate signals from δ2HTRC is currently challenging, but δ2HTRC may retain important low-frequency climate variation and allow for additional and complementary inferences on tree physiology and carbon metabolism when paired with other isotopic signals and tree-ring growth. To maximize this potential the δ2HTRC signals need further study and calibration. Toward this end, the foremost need is to investigate the 2H-fractionation processes between water, photosynthetic assimilates, carbon storage, and cellulose at the leaf and stem level. More specifically, studies should be carefully designed to address how this varies with abiotic or biotic stressors, among species and genus, and across diel and seasonal cycles. Second, we need to develop new isotope modeling approaches that can better isolate hydrological 2H-fractionations (leaf water, source water) from the proportionally larger biochemical 2H-fractionations. Third, the new knowledge on 2H-fractionation processes need to be applied to δ2HTRC chronologies to assess the potential for obtaining improved multi-proxy climate reconstructions or improved understanding of changes in ecophysiology in response to climate variability or other environmental influences. Great progress has been made in understanding and calibrating the δ2HTRC signal. Continuing advancements in 2H-processing that allow for rapid determinations of δ2HNE, a growing constellation of ancillary tree-ring metrics, and advances in knowledge on post-photosynthetic 2H-fractionations together foretell that applications of δ2HTRC will be increasingly used in dendroclimatology and forest ecophysiology.
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We thank Neil Loader (Swansea University) and Christoph Mayr (Friedrich Alexander University Erlangen-Nuremberg) for reviewing this book chapter. We also thank Kurt Nicolussi for the organization and cross-dating of the EACC wood material. Steve Voelker was supported by US-NSF Award #1903721. Marco M. Lehmann was supported by the SNSF Ambizione grant (No. 179978).
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Lehmann, M.M., Schuler, P., Cormier, MA., Allen, S.T., Leuenberger, M., Voelker, S. (2022). The Stable Hydrogen Isotopic Signature: From Source Water to Tree Rings. In: Siegwolf, R.T.W., Brooks, J.R., Roden, J., Saurer, M. (eds) Stable Isotopes in Tree Rings. Tree Physiology, vol 8. Springer, Cham. https://doi.org/10.1007/978-3-030-92698-4_11
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