Evidence of Substantial Carbon Isotope Fractionation among Substrate, Inorganic Carbon, and Biomass during Aerobic Mineralization of 1,2-Dichloroethane by Xanthobacter autotrophicus

Carbon isotope fractionation during aerobic mineralization of 1,2-dichloroethane (1,2-DCA) by Xanthobacter autotrophicus GJ10 was investigated. A strong enrichment of ¹³C in residual 1,2-DCA was observed, with a mean fractionation factor α ± standard deviation of 0.968 ± 0.0013 to 0.973 ± 0.0015. In addition, a large carbon isotope fractionation between biomass and inorganic carbon occurred. A mechanistic model that links the fractionation factor α to the rate constants of the first catabolic enzyme was developed. Based on the model, it was concluded that the strong enrichment of ¹³C in 1,2-DCA arises because the first irreversible step of the initial enzymatic transformation of 1,2-DCA consists of an S_N2 nucleophilic substitution. S_N2 reactions are accompanied by a large kinetic isotope effect. The substantial carbon isotope fractionation between biomass and inorganic carbon could be explained by the kinetic isotope effect associated with the initial 1,2-DCA transformation and by the metabolic pathway of 1,2-DCA degradation. Carbon isotope fractionation during 1,2-DCA mineralization leads to 1,2-DCA, inorganic carbon, and biomass with characteristic carbon isotope compositions, which may be used to trace the process in contaminated environments.     

A skew in poo: Biases in primate fecal isotope analysis and recommendations for standardized sample preparation

Feces are a treasure trove in the study of animal behavior and ecology. Stable carbon and nitrogen isotope analysis allows to assess the dietary niches of elusive primate species and primate breastfeeding behavior. However, some fecal isotope data may unwillingly be biased toward the isotope ratios of undigested plant matter, requiring more consistent sample preparation protocols. We assess the impact of this potential data skew in 114 fecal samples of wild bonobos (Pan paniscus) by measuring the isotope differences (Δ¹³C, Δ¹⁵N) between bulk fecal samples containing larger particles (>1 mm) and filtered samples containing only small particles (<1 mm). We assess the influence of fecal carbon and nitrogen content (ΔC:N) and sample donor age (subadult, adult) on the resulting Δ¹³C, Δ¹⁵N values (n = 228). Additionally, we measure the isotope ratios in three systematically sieved fecal samples of chimpanzees (Pan troglodytes verus), with particle sizes ranging from 20 μm to 8 mm (n = 30). We found differences in fecal carbon and nitrogen content, with the smaller fecal fraction containing more nitrogen on average. While the Δ¹³C values were small and not affected by age or ΔC:N, the Δ¹⁵N values were significantly influenced by fecal ΔC:N, possibly resulting from the differing proportions of undigested plant macroparticles. Significant relationships between carbon stable isotope ratios (δ¹³C) values and %C in large fecal fractions of both age groups corroborated this assessment. Δ¹⁵N values were significantly larger in adults than subadults, which should be of concern in isotope studies comparing adult females with infants to assess breastfeeding. We found a random variation of up to 3.0‰ in δ¹³C and 2.0‰ in nitrogen stable isotope ratios within the chimpanzee fecal samples separated by particle sizes. We show that particle size influences isotope ratios and propose a simple, cost-effective filtration method for primate feces to exclude larger undigested food particles from the analysis, which can easily be adopted by labs worldwide.     

Geochemically distinct carbon isotope distributions in Allochromatium vinosum DSM 180T grown photoautotrophically and photoheterotrophically

Anoxygenic, photosynthetic bacteria are common at redox boundaries. They are of interest in microbial ecology and geosciences through their role in linking the carbon, sulfur, and iron cycles, yet much remains unknown about how their flexible carbon metabolism—permitting either autotrophic or heterotrophic growth—is recorded in the bulk sedimentary and lipid biomarker records. Here, we investigated patterns of carbon isotope fractionation in a model photosynthetic sulfur‐oxidizing bacterium, Allochromatium vinosum DSM180^T. In one treatment, A. vinosum was grown with CO₂ as the sole carbon source, while in a second treatment, it was grown on acetate. Different intracellular isotope patterns were observed for fatty acids, phytol, individual amino acids, intact proteins, and total RNA between the two experiments. Photoautotrophic CO₂ fixation yielded typical isotopic ordering for the lipid biomarkers: δ¹³C values of phytol > n‐alkyl lipids. In contrast, growth on acetate greatly suppressed intracellular isotopic heterogeneity across all molecular classes, except for a marked ¹³C‐depletion in phytol. This caused isotopic “inversion” in the lipids (δ¹³C values of phytol < n‐alkyl lipids). The finding suggests that inverse δ¹³C patterns of n‐alkanes and pristane/phytane in the geologic record may be at least in part a signal for photoheterotrophy. In both experimental scenarios, the relative isotope distributions could be predicted from an isotope flux‐balance model, demonstrating that microbial carbon metabolisms can be interrogated by combining compound‐specific stable isotope analysis with metabolic modeling. Isotopic differences among molecular classes may be a means of fingerprinting microbial carbon metabolism, both in the modern environment and the geologic record.     

Combining meteorology, eddy fluxes, isotope measurements, and modeling to understand environmental controls of carbon isotope discrimination at the canopy scale

Estimates of terrestrial carbon isotope discrimination are useful to quantify the terrestrial carbon sink. Carbon isotope discrimination by terrestrial ecosystems may vary on seasonal and interannual time frames, because it is affected by processes (e.g. photosynthesis, stomatal conductance, and respiration) that respond to variable environmental conditions (e.g. air humidity, temperature, light). In this study, we report simulations of the temporal variability of canopy‐scale C₃ photosynthetic carbon isotope discrimination obtained with an ecophysiologically based model (ISOLSM) designed for inclusion in global models. ISOLSM was driven by half‐hourly meteorology, and parameterized with eddy covariance measurements of carbon and energy fluxes and foliar carbon isotope ratios from a pine forest in Metolius (OR). Comparing simulated carbon and energy fluxes with observations provided a range of parameter values that optimized the simulated fluxes. We found that the sensitivity of photosynthetic carbon isotope discrimination to the slope of the stomatal conductance equation (m, Ball–Berry constant) provided an additional constraint to the model, reducing the wide parameter space obtained from the fluxes alone. We selected values of m that resulted in similar simulated long‐term discrimination as foliar isotope ratios measured at the site. The model was tested with ¹³C measurements of ecosystem (δ_R) and foliar (δ_f) respiration. The daily variability of simulated ¹³C values of assimilated carbon (δ_A) was similar to that of observed δ_f, and higher than that of observed and simulated δ_R. We also found similar relationships between environmental factors (i.e. vapor pressure deficit) and simulated δ_R as measured in ecosystem surveys of δ_R. Therefore, ISOLSM reasonably simulated the short‐term variability of δ_A controlled by atmospheric conditions at the canopy scale, which can be useful to estimate the variability of terrestrial isotope discrimination. Our study also shows that including the capacity to simulate carbon isotope discrimination, together with simple ecosystem isotope measurements, can provide a useful constraint to land surface and carbon balance models.     

Depth variation in isotopic composition of benthic resources and assessment of sculpin feeding patterns in an oligotrophic Alaskan lake

Stable isotopes of carbon (C) and nitrogen (N) are commonly used to track resource flows through lake food webs. However, there remains a weak understanding of the spatial variation in isotopic composition of benthic resources and how this variation affects inference about energy flows among species. Boundary layers at the interface between benthic substrates and the overlying water column restrict diffusion of nutrients and carbon from the water column to benthic algae and may affect the isotopic composition of benthic algae as nutrient and CO₂ concentrations can become locally depleted in the benthic boundary layer. We quantified the variation in C and N stable isotope composition of benthic resources along a depth gradient in a large oligotrophic lake to assess the magnitude of change in stable isotope composition. Snails were increasingly depleted in ¹³C with depth, by about 10 ‰ from 0 to 20 m, while ¹⁵N in snails showed only subtle enrichment over this depth range. Sculpin (Cottas aleuticus) δ ¹³C and δ ¹⁵N signatures did not significantly change with depth and were more enriched in ¹⁵N than would be expected from consumption of snails alone. A comparison of δ ¹³C and δ ¹⁵N values from sculpins relative to shallow and deep snails, and alternative prey (marine-derived salmon resources), within a mixing model suggested sculpins feed selectively on deep grazers in this system in addition to marine-derived resources provided by migrating sockeye salmon. This study illustrates the importance of accounting for depth-related variation in isotope patterns when assessing benthic resource contributions to food webs using stable isotope data.     

Decarboxylation of glycine contributes to carbon isotope fractionation in photosynthetic organisms

Carbon isotope effects were investigated for the reaction catalyzed by the glycine decarboxylase complex (GDC; EC 2.1.2.10). Mitochondria isolated from leaves of pea (Pisum sativum L.) and spinach (Spinacia oleracea L.) were incubated with glycine, and the CO₂ evolved was analyzed for the carbon isotope ratio (δ¹³C). Within the range of parameters tested (temperature, pH, combination of cofactors NAD⁺, ADP, pyridoxal 5-phosphate), carbon isotope shifts of CO₂ relative to the C₁-carboxyl carbon of glycine varied from +14‰ to −7‰. The maximum effect of cofactors was observed for NAD⁺, the removal of which resulted in a strong ¹²C enrichment of the CO₂ evolved. This indicates the possibility of isotope effects with both positive and negative signs in the GDC reaction. The measurement of δ¹³C in the leaves of the GDC-deficient barley (Hordeum vulgare L.) mutant (LaPr 87/30) plants indicated that photorespiratory carbon isotope fractionation, opposite in sign when compared to the carbon isotope effect during CO₂ photoassimilation, takes place in vivo. Thus the key reaction of photorespiration catalyzed by GDC, together with the key reaction of CO₂ fixation catalyzed by ribulose-1,5-bisphosphate carboxylase, both contribute to carbon isotope fractionation in photosynthesis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Scope for use of stable carbon isotopes in discerning the incorporation of forest detritus into aquatic foodwebs

Stable isotope analysis of carbon has been proposed as a means for discerning the incorporation of terrestrial forest detritus into aquatic foodwebs, and as such, has the potential to be used as a biomonitor of the aquatic effects of riparian deforestation. A synthesis of ¹³C/¹²C data from the literature indicates, however, that the scope for successful use of carbon isotope analysis in separating allochthonous and autochthonous food provenance is much more limited than was once thought. This occurs due the overlap in carbon isotope ratios between terrestrial forest detritus and those of both lotic attached algae and lentic filamentous attached algae. Only within rockyshored, oligotrophic lakes without macrophytes, and forest-fringed estuaries and lagoons, where the carbon isotope ratios for attached algae and forest detritus are significantly different, is there any likelihood of discerning the incorporation of allochthonous carbon into aquatic foodwebs using ¹³C/¹²C values alone.     

Experimental Evidence for the Isotope Effect in Photorespiration

Recent data on carbon isotope fractionation in photosynthesis are reviewed. Analysis of the carbon isotope composition in photosynthates and derivative products supports the hypothesis of the isotope effect in photorespiration. This hypothesis envisages the existence in a photosynthesizing cell of two carbon flows differing in isotope composition. One of these flows is enriched in ¹²C and associated with the assimilation pathway of photosynthesis. The other flow enriched in ¹³C circulates in the photorespiratory pathway. The relation between stimulated photorespiration and the carbon isotope composition of biomass supports this view. Our hypothesis of two interrelated isotope effects in photosynthesis leads to the conclusion that photosynthesis and photorespiration are coupled processes subject to periodical oscillations, where Rubisco acts as a main switch regulating these two pathways.     

Fractionation of Carbon Isotopes during Biogenesis of Atmospheric Isoprene

The stable carbon isotope composition of isoprene emitted from leaves of red oak (Quercus rubra L.) was measured. Isoprene was depleted in ¹³C relative to carbon recently fixed by photosynthesis. The difference in isotope composition between recently fixed carbon and emitted isoprene was independent of the isotopic composition of the source CO₂. β-Carotene, an isoprenoid plant constituent, was depleted in ¹³C relative to whole leaf carbon to the same degree as isoprene, but fatty acids were more depleted. Isoprene emitted from leaves fed abscisic acid was much less depleted in ¹³C than was isoprene emitted from unstressed leaves. We conclude that isoprene is made from an isoprenoid precursor that is derived from acetyl-CoA made from recent photosynthate. The carbon isotope composition of isoprene in the atmosphere is likely to be slightly more negative (less ¹³C) than C₃ plant material but when plants are stressed the isotopic composition could vary.     

Biological fractionation of stable carbon isotopes at the aerobic/anaerobic water interface of meromictic water bodies

Mass-spectrometric investigation of carbon isotope composition (δ¹³C) was carried out for suspended organic matter and dissolved mineral compounds for the water column of some meromictic water bodies differing in salinity and trophic state. As a rule, a more pronounced carbon isotope fractionation (resulting from the metabolism of phytoplankton and anoxygenic phototrophic bacteria) was revealed in the zones of enhanced oxygenic and anoxygenic photosynthesis. Carbon isotope fractionation at the border between oxidized and reduced waters depends both on the activity of microbial communities and on the dominant species of phototrophic microorganisms. Analysis of the distribution profiles of the isotopic composition of suspended organic matter and dissolved mineral carbon revealed active mineralization of the organic matter newly formed via anoxygenic photosynthesis in the monimolimnion by microbial communities, resulting in the release of isotopically light carbon dioxide. Mineral carbon in the anaerobic zones of highly productive meromictic water bodies is therefore enriched with the light ¹²C isotope.     

Fractionation of Stable Carbon Isotopes during Chemoautotrophic Growth of Sulfur-Oxidizing Bacteria

Laboratory-grown strains of chemoautotrophic Thiomicrospira sp. strain L-12 and Thiobacillus neapolitanus produced cell carbon that was 24.6 to 25.1 ppt (24.6 to 25.1 mg/g) lower in ¹³C isotope abundance than the ambient source of carbon dioxide and bicarbonate. This degree of ¹³C isotope depletion was comparable to that found in organic material produced in deep-sea hydrothermal-vent communities.     

Isotope alteration caused by changes in biochemical composition of sedimentary organic matter

Stable carbon (C) and nitrogen (N) isotope ratios of sedimentary organic matter (OM) can reflect the biogeochemical history of aquatic ecosystems. However, diagenetic processes in sediments may alter isotope records of OM via microbial activity and preferential degradation of isotopically distinct organic components. This study investigated the isotope alteration caused by preferential degradation in surface sediments sampled from a eutrophic reservoir in Germany. Sediments were treated sequentially with hot water extraction, hydrochloric acid hydrolysis, hydrogen peroxide oxidation and di-sodium peroxodisulfate oxidation to chemically simulate preferential degradation pathways of sedimentary OM. Residue and extracts from each extraction step were analyzed using elemental analyzer-isotope ratio mass spectrometry and solid-state ¹³C nuclear magnetic resonance spectroscopy. Our results show that stable C and N isotope ratios reacted differently to changes in the biochemical composition of sedimentary OM. Preferential degradation of proteins and carbohydrates resulted in a 1.2‰ depletion of ¹³C, while the isotope composition of ¹⁵N remained nearly the same. Sedimentary δ¹⁵N values were notably altered when lignins and lipids were oxidized from residual sediments. Throughout the sequential fractionation procedure, δ¹³C was linearly correlated with the C:N of residual sediments. This finding demonstrates that changes in biochemical composition caused by preferential degradation altered δ¹³C values of sedimentary OM, while this trend was not observed for δ¹⁵N values. Our study identifies the influence of preferential degradation on stable C isotope ratios and provide additional insight into the isotope alteration caused by post-depositional processes.

     

Limitations of stable carbon isotope analysis for determining natal host origins of tobacco budworm, Heliothis virescens

Differences in the stable carbon isotope ratios of plants utilizing the C3 vs. C4 photosynthetic pathway have been used to broadly identify the natal host origins of herbivorous insects. This study explored whether adequate variation exists between the carbon isotope ratios of different C3 plants in the host range of Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae) to enable accurate identification of natal host‐plant species. Isotope ratio mass spectrometry (IRMS) analysis of ¹³C/¹²C ratios of moths reared on four crop plant species [Gossypium hirsutum (L.), Nicotiana tabacum L., Glycine max (L.) Merrill, and Arachis hypogaea L.] and two common weed species [Geranium carolinianum L. and Linaria canadensis (L.) Chaz.] revealed a range of δ¹³C values within that expected for plants utilizing the C3 photosynthetic pathway. Analysis of vegetative and reproductive tissues from the plants utilized in the study resulted in statistically different δ¹³C values for some plant species; nevertheless, the range of δ¹³C values observed for many plant species overlapped. Significant differences in mean δ¹³C values were detected between groups of moths reared on different host‐plant species, but there was no significant correlation between the δ¹³C values of moths vs. the δ¹³C value of plant tissue on which they were reared. Feral tobacco budworm moths collected over 3 years were found to have carbon isotope ratios consistent with those having fed on C3 plants, confirming little utilization of C4 plant species by the insect. Results demonstrate that within the range of C3 host plants tested, carbon isotope signatures are not sufficiently unique to enable a reliable determination of natal origin of feral tobacco budworm with current IRMS technology.     

Evidence for a nonstatistical carbon isotope distribution in natural glucose

The relative carbon isotope content (δ¹³C value) in each position of glucose from a C₄ plant (maize starch) and a C₃ plant (sugar beet sucrose) has been determined by stepwise chemical and biochemical degradation of the molecule and stable isotope ratio measurement of the fragments. The suitability of the degradation methods has been tested through their chemical yield and isotope balance. The results from both methods agreed perfectly, revealing a defined and reproducible ¹³C distribution in glucose from both origins. Most prominent was a relative ¹³C enrichment by 5 to 6 δ-units in position 4 and a depletion by about 5 δ-units in carbon 6. As possible reasons for these nonstatistical isotope distributions, isotope effects of the aldolase, the triose phosphate isomerase, and the transketolase reactions during carbohydrate biosynthesis are discussed. The practical importance of the results in regard to isotope distributions in secondary plant products as a means for food authenticity control is outlined.     

Annual cyclicity in high-resolution stable carbon and oxygen isotope ratios in the wood of the mangrove tree Rhizophora mucronata

In the present study, the high‐resolution stable carbon (¹³C/¹²C) and oxygen (¹⁸O/¹⁶O) isotope ratio profiles in the wood of the mangrove Rhizophora mucronata Lam., a tropical tree species lacking distinct growth rings, were investigated. Variations of both isotope ratios revealed a remarkable annual cyclicity with lowest values occurring at the latewood/earlywood boundary (April–May) and highest values during the transition from earlywood to latewood (October–November). Based on the current knowledge of the physiology of this mangrove species, as well as on the current literature available on high‐resolution profiles of stable isotope ratios in tree rings, possible driving forces responsible for this seasonal pattern are discussed. The annual cyclicity, together with a conspicuous isotope pattern appearing in the El‐Niño year 1997, promises great potential for tropical dendrochronology.     

Stable nitrogen and carbon isotope ratios in wild native honeybees: the influence of land use and climate

The eastern hive bee Apis cerana is a major honeybee species in Asia providing numerous ecosystem services. Understanding how much the honeybees depend on natural and human-influenced plants and landscapes in different climates is important could contribute to evaluate how wild honeybees use food resources and to measure the ecosystem services. We investigated the effects of land use and climate changes on stable nitrogen and carbon isotope ratios in wild populations of A. cerana. In populations from 139 individual sites throughout Japan, we measured nitrogen (δ¹⁵N) and carbon (δ¹³C) stable isotope ratios and analyzed the effects of land use and climate. Our results showed that forested areas and annual precipitation had significant effects on δ¹⁵N, and that paddy fields and urban areas had significant effects on δ¹³C. These results suggest that A. cerana sensibly uses available food resources in the various environments and that stable nitrogen and carbon isotope ratios clearly reflect the effects of land use and climate changes on the populations of A. cerana. Thus, stable nitrogen and carbon isotope ratios in A. cerana, which widely distributes in Asia, can be used as indicators of the environments, such as land use and climate, of an area within its foraging range.     

Comparison of the exploitation of methane-derived carbon by tubicolous and non-tubicolous chironomid larvae in a temperate eutrophic lake

Carbon and nitrogen stable isotope ratios (δ¹³C and δ¹⁵N) in three sympatric species of larval chironomids were analyzed in a temperate eutrophic shallow lake in Japan. Markedly lower δ¹³C values were reported in Chironomus plumosus (?51.2 ‰) and Tanypus sp. (?43.5 ‰) than those in photoautotrophic carbon sources [particulate organic matter (POM) and sediment]. There were positive correlations between δ¹³C and δ¹⁵N in the two chironomid species. These results indicated that they assimilated carbon derived from biogenic methane by exploiting methane-oxidizing bacteria (MOB). In contrast, Propsilocerus akamusi exhibited similar δ¹³C values to those of POM or sediment. A δ¹³C-based isotope mixing model was used to estimate the dietary contributions of MOB to each chironomid species. The mean contributions ranged from 11 to 15 % in C. plumosus, 13 to 19 % in Tanypus sp., but only up to 5 % in P. akamusi. In an aquarium, we observed that individuals of C. plumosus and Tanypus sp., which exhibited low δ¹³C values, built U-shaped larval tubes in the sediment, and an oxidized layer developed around these tubes. Propsilocerus akamusi did not exhibit this behavior. These results suggest that tube building may provide larval chironomids with greater access to methane-derived carbon through increased opportunities to feed on MOB.     

Kinetic isotope effects significantly influence intracellular metabolite 13C labeling patterns and flux determination

Rigorous mathematical modeling of carbon-labeling experiments allows estimation of fluxes through the pathways of central carbon metabolism, yielding powerful information for basic scientific studies as well as for a wide range of applications. However, the mathematical models that have been developed for flux determination from ¹³C labeling data have commonly neglected the influence of kinetic isotope effects on the distribution of ¹³C label in intracellular metabolites, as these effects have often been assumed to be inconsequential. We have used measurements of the ¹³C isotope effects on the pyruvate dehydrogenase enzyme from the literature to model isotopic fractionation at the pyruvate node and quantify the modeling errors expected to result from the assumption that isotope effects are negligible. We show that under some conditions kinetic isotope effects have a significant impact on the ¹³C labeling patterns of intracellular metabolites, and the errors associated with neglecting isotope effects in ¹³C-metabolic flux analysis models can be comparable in size to measurement errors associated with GC–MS. Thus, kinetic isotope effects must be considered in any rigorous assessment of errors in ¹³C labeling data, goodness-of-fit between model and data, confidence intervals of estimated metabolic fluxes, and statistical significance of differences between estimated metabolic flux distributions.     

Kangaroo tooth enamel oxygen and carbon isotope variation on a latitudinal transect in southern Australia: implications for palaeoenvironmental reconstruction

Tooth enamel apatite carbonate carbon and oxygen isotope ratios of modern kangaroos (Macropus spp.) collected on a 900-km latitudinal transect spanning a C₃–C₄ transition zone were analysed to create a reference set for palaeoenvironmental reconstruction in southern Australia. The carbon isotope composition of enamel carbonate reflects the proportional intake of C₃ and C₄ vegetation, and its oxygen isotope composition reflects that of ingested water. Tooth enamel forms incrementally, recording dietary and environmental changes during mineralisation. Analyses show only weak correlations between climate records and latitudinal changes in δ¹³C and δ¹⁸O. No species achieved the δ¹³C values (~?1.0 ‰) expected for 100 % C₄ grazing diets; kangaroos at low latitudes that are classified as feeding primarily on C₄ grasses (grazers) have δ¹³C of up to ?3.5 ‰. In these areas, δ¹³C below ?12 ‰ suggests a 100 % C₃ grass and/or leafy plant (browse) diet while animals from higher latitude have lower δ¹³C. Animals from semi-arid areas have δ¹⁸O of 34–40 ‰, while grazers from temperate areas have lower values (~28–30 ‰). Three patterns with implications for palaeoenvironmental reconstruction emerge: (1) all species in semi-arid areas regularly browse to supplement limited grass resources; (2) all species within an environmental zone have similar carbon and oxygen isotope compositions, meaning data from different kangaroo species can be pooled for palaeoenvironmental investigations; (3) relatively small regional environmental differences can be distinguished when δ¹³C and δ¹⁸O data are used together. These data demonstrate that diet–isotope and climate–isotope relationships should be evaluated in modern ecosystems before application to the regional fossil record.     

Correlating δ13C and δ18O in cellulose of trees

We measured the carbon and oxygen isotopic composition of stem cellulose of Pinus sylvestris, Picea abies, Fagus sylvatica and Fraxinus excelsior. Several sites along a transect of a small valley in Switzerland were selected which differ in soil moisture conditions. At every site, six trees per species were sampled, and a sample representing a mean value for the period from 1940 to 1990 was analysed. For all species, the mean site δ¹³C and δ¹⁸O of stem cellulose are related to the soil moisture availability, whereby higher isotope ratios are found at drier sites. This result is consistent with isotope fractionation models when assuming enhanced stomatal resistance (thus higher δ¹³C of incorporated carbon) and increased oxygen isotope enrichment in the leaf water (thus higher δ¹⁸O) at the dry sites. δ¹⁸ O-δ¹³C plots reveal a linear relationship between the carbon and oxygen isotopes in cellulose. To interpret this relationship we developed an equation which combines the above-mentioned fractionation models. An important new parameter is the degree to which the leaf water enrichment is reflected in the stem cellulose. In the combined model the slope of the δ¹⁸O-δ¹³C plot is related to the sensitivity of the p_i/p_a of a plant to changing relative humidity.