Controlling for anthropogenically induced atmospheric variation in stable carbon isotope studies

Increased use of stable isotope analysis to examine food-web dynamics, migration, transfer of nutrients, and behavior will likely result in expansion of stable isotope studies investigating human-induced global changes. Recent elevation of atmospheric CO₂ concentration, related primarily to fossil fuel combustion, has reduced atmospheric CO₂ δ¹³C (¹³C/¹²C), and this change in isotopic baseline has, in turn, reduced plant and animal tissue δ¹³C of terrestrial and aquatic organisms. Such depletion in CO₂ δ¹³C and its effects on tissue δ¹³C may introduce bias into δ¹³C investigations, and if this variation is not controlled, may confound interpretation of results obtained from tissue samples collected over a temporal span. To control for this source of variation, we used a high-precision record of atmospheric CO₂ δ¹³C from ice cores and direct atmospheric measurements to model modern change in CO₂ δ¹³C. From this model, we estimated a correction factor that controls for atmospheric change; this correction reduces bias associated with changes in atmospheric isotopic baseline and facilitates comparison of tissue δ¹³C collected over multiple years. To exemplify the importance of accounting for atmospheric CO₂ δ¹³C depletion, we applied the correction to a dataset of collagen δ¹³C obtained from mountain lion (Puma concolor) bone samples collected in California between 1893 and 1995. Before correction, in three of four ecoregions collagen δ¹³C decreased significantly concurrent with depletion of atmospheric CO₂ δ¹³C (n ≥ 32, P ≤ 0.01). Application of the correction to collagen δ¹³C data removed trends from regions demonstrating significant declines, and measurement error associated with the correction did not add substantial variation to adjusted estimates. Controlling for long-term atmospheric variation and correcting tissue samples for changes in isotopic baseline facilitate analysis of samples that span a large temporal range.     

Inorganic carbon acquisition by red seaweeds grown under dynamic light regimes

Palmaria palmata, which is able to use HCO _inf3 ^sup– as a carbon source for photosynthesis, and Lomentaria articulata, which is dependent on diffusive uptake of dissolved CO₂, were grown under constant light and light with sunflecks designed to model wave-induced fluctuations of near-shore underwater light. Both species exhibited significantly increased stable carbon isotope discrimination (more negative values of ¹³C relative to PDB) when grown with sunflecks. More negative ¹³C values were associated with decreased growth rate of P. palmata but not of L. articulata. The contrasting effects of sunflecks on the carbon-use characteristics of the two species are discussed in terms of the energetic cost of HCO _inf3 ^sup– use and the susceptibility of CO₂ diffusion-dependent species to photoinhibition.     

The effects of nitrogen fertilisation and elevated CO2 on the lipid biosynthesis and carbon isotopic discrimination in birch seedlings (Betula pendula)

The effects of nitrogen (N) fertilisation and elevated [CO₂] on lipid biosynthesis and carbon isotope discrimination in birch (Betula pendula Roth.) transplants were evaluated using seedlings grown with and without N fertiliser, and under two concentrations of atmospheric CO₂ (ambient and ambient+250 μmol mol^-1) in solar dome systems. N fertilisation decreased n-fatty acid chain length (18:0/16:0) and the ratios of α-linolenate (18:2)/linoleate (18:1), whereas elevated [CO₂] showed little effect on n-fatty acid chain length, but decreased the unsaturation (18:2+18:1)/18:0. Both N fertilisation and elevated [CO₂] increased the quantity of leaf wax n-alkanes, whilst reducing that of n-alkanols by 20–50%, but had no simple response in fatty acid concentrations. ¹³C enrichment by 1–2.5‰ under N fertilisation was observed, and can be attributed to both reduced leaf conductance and increased photosynthetic consumption of CO₂. Individual n-alkyl lipids of different chain length show consistent pattern of δ¹³C values within each homologue, but are in general 5–8‰ more depleted in ¹³C than the bulk tissues. Niether nitrogen fertilisation and elevated CO₂ influenced the relationship between carbon isotope discrimination of the bulk tissue and the individual lipids. This revised version was published online in June 2006 with corrections to the Cover Date.     

Carbon isotope discrimination and stomatal responses of mature Pinus sylvestris L. trees exposed in situ for three years to elevated CO2 and temperature

The Climate Change Experiment (CLIMEX) is a unique large scale facility in which an entire undisturbed catchment of boreal vegetation has been exposed to elevated CO₂ (560 ppm) and temperature (+3°C summer, +5°C winter) for the past three years with all the soil-plant-atmosphere linkages intact. Here, carbon isotope composition and stomatal density have been analysed from sequential year classes of needles of mature Scots pine trees (Pinus sylvestris L.) to investigate the response of time-integrated water-use efficiency (UWE) and stomatal density to CO₂ enrichment and climate change. Carbon isotope discrimination decreased and WUE increased in cohorts of needles developing under increased CO₂ and temperature, compared to needles on the same trees developing in pretreatment years. Mid-season instantaneous gas exchange, measured on the same trees for the past four years, indicated that these responses resulted from higher needle photosynthetic rates and reduced stomatal conductance. Needles of P. sylvestris developing under increased CO₂ and temperature had consistently lower stomatal densities than their ambient grown counterparts on the same trees. The stomatal density of P. sylvestris needles was inversely correlated with δ¹³C-derived WUE, implying some effect of this morphological response on leaf gas exchange. Future atmospheric CO₂ and temperature increases are therefore likely to improve the water economy of P. sylvestris, at least at the scale of individual needles, by affecting stomatal density and gas exchange processes.     

Species-dependent responses of juniper and spruce to increasing CO<Subscript>2</Subscript> concentration and to climate in semi-arid and arid areas of northwestern China

Qilian juniper (Sabina przewalskii Kom.) and Qinghai spruce (Picea crassifolia Kom.) represent different tree functional types, which can be found extensively in northwestern China. The former is drought-tolerant, whereas the latter is hygrophilous and shade-tolerant. We compared their intrinsic water-use efficiency (iWUE, inferred from carbon isotopic discrimination, δ¹³C, in their wood) as a function of atmospheric CO₂ concentration, [CO₂], and climate. δ¹³C of spruce was consistently about higher than that of juniper in semi-arid areas but was lower in arid areas. This difference was stable over time and demonstrated strong cross-correlations between species, although some subtle high-frequency (2 or 3 years) variations existed in both species, suggesting that regional climate may control carbon isotope discrimination. The ratio (the [CO₂] values in leaf intercellular and the atmosphere, respectively) of the juniper increased steadily over time, whereas that of the spruce showed a long-term downward trend. IWUE increased at all sites over the 150-year study period, mainly caused by increasing [CO₂]. The relationship between iWUE and [CO₂] reveals that the spruce was more sensitive than the juniper to increasing [CO₂], suggesting a species-specific adaptation to long-term environmental changes. Correlations between the high-frequency variations in stable carbon discrimination (Δ) and climate indicate similar intra-site responses to climate in both species, but different response strengths. Overall, complex interactions of temperature and moisture on stable carbon discrimination during current growth seasons of both species were environmental-determined. Regulation of gas exchange and reduced transpiration may influence water and energy budgets directly; therefore species-dependent responses of trees to elevated CO₂ should be considered in future research on global plant physiological ecology.     

A global survey of carbon isotope discrimination in plants from high altitude

Summary Carbon 13/12 isotope ratios have been determined from leaves of a hundred C₃ plant species (or ecotypes) from all major mountain ranges of the globe, avoiding drought stressed areas. A general increase in ¹³C content was found with increasing altitude, i.e. overall discrimination against the heavy isotope is reduced at high elevation. The steepest decline of discrimination is observed in taxa typically ranging to highest elevations (e.g. the genus Ranunculus). Mean ¹³C for all samples collected between 2500 and 5600 m altitude is-26.15 compared to the lowland average of-28.80 (P<0.001). Forbs from highest elevations reach-24. According to theory of ¹³C discrimination this indicates decreasing relative limitation of carbon uptake by carboxylation. In other words, we estimate that the ratio of internal to external partial pressure of CO₂ (p _i /p _a )in leaves of high elevation plants is lower than in leaves of low altitude. These results confirm recent gas exchange analyses in high and low elevation plants.     

Fossil plant evidence for Early and Middle Jurassic paleoenvironmental changes in Lanzhou area, Northwest China

Estimating the paleoclimate changes through CO₂ levels has become a promising area of geological research. This paper focuses on analysis of fossil Ginkgo in continuous sedimentary series in northwestern China using plant anatomy and organic geochemistry approaches. The CO₂ variation curve during Early and Middle Jurassic is reconstructed based on the stomatal ratio method, which is consistent with the estimated results of GEOCARB III. In comparison with the carbon isotopic composition measured from the fossil leaves of Ginkgo, we suggest that the stomata-based CO₂ concentrations for the Jurassic are generally consistent with the predictions of the geochemical model, ranging from 1000 to 1600 ppmv. Measurements of carbon isotope values demonstrate that the water use efficiency of the fossil Ginkgo in a “green house” world is higher than that of the living Ginkgo. Investigations of physiological responses of plants to the increasing CO₂ level at the present and in the future should help demonstrate this effect. The cause of the carbon isotopic shift at the boundary between Aalenian and Bajocian for the Yaojie Basin is unclear and therefore needs further investigation.     

Carbon autonomy of reproductive shoots of Siberian alder (<Emphasis Type="Italic">Alnus hirsuta</Emphasis> var.<Emphasis Type="Italic"> sibirica</Emphasis>)

Carbon autonomy of current-year shoots in flowering, and of current-year shoots plus 1-year-old shoots (1-year-old shoot system) in fruiting of Siberian alder (Alnus hirsuta var. sibirica) was investigated using a stable isotope of carbon, ¹³C. The current-year shoot and 1-year-old shoot systems were fed ¹³CO₂ and the atom% excess of ¹³C in flowers and fruits was determined. The majority of photosynthate allocated to flower buds was originally assimilated in the leaves of the flowering current-year shoots. Of all the current-year shoots on fruiting 1-year-old shoots, only those nearest to the fruits allocated the assimilated photosynthate to fruit maturation. These results indicate that the current-year shoots and 1-year-old shoot systems are carbon-autonomous units for producing flowers and maturing fruits, respectively.     

Mechanisms of CO2 fixation in bacterial photosynthesis studied by the carbon isotope fractionation technique

1. The carbon isotope discrimination properties of a representative of each of the three types of photosynthetic bacteria Chlorobium thiosulfatophilum, Rhodospirillum rubrum and Chromatium and of the C₃-alga Chlamydomonas reinhardii were determined by measuring the ratio of ¹³CO₂ to ¹²CO₂ incorporated during photoautotrophic growth. 2. Chromatium and R. rubrum had isotope selection properties similar to those of C₃-plants, whereas Chlorobium was significantly different. 3. The results suggest that Chromatium and R. rubrum assimilate CO₂ mainly via ribulose 1,5-diphosphate carboxylase and the associated reactions of the reductive pentose phosphate cycle, whereas Chlorobium utilizes other mechanisms. Such mechanisms would include the ferredoxin-linked carboxylation enzymes and associated reactions of the reductive carboxylic acid cycle.Abbreviations RuDP ribulose 1,5-disphosphate - PEP phosphoenolpyruvate     

Discrimination Against 13CO2 in Leaves,Pod Walls,and Seeds of Water-stressed Chickpea

The rate of photosynthesis (P _N) in leaves and pods as well as carbon isotope content in leaves, pod walls, and seeds was measured in well-watered (WW) and water-stressed (WS) chickpea plants. The P _N, on an area basis, was negligible in pods compared to leaves and was reduced by water stress (by 26%) only in leaves. WS pod walls and seeds discriminated less against ¹³CO₂ than did the controls. This response was not observed for leaves as is usually the case. Pod walls and seeds discriminated less against ¹³CO₂ than did leaves in both WW and WS plants. Measurement of carbon isotope composition in pods may be a more sensitive tool for assessing the impact of water stress on long-term assimilation than is the instantaneous measurement of gas exchange rates.     

Decomposition of leaf and root tissue of three perennial grass species grown at two levels of atmospheric CO2 and N supply

Leaf and root tissue of Lolium perenne L., Agrostis capillaris L. and Festuca ovina L. grown under ambient (350 μl l^-1 CO₂) and elevated (700 μl l^-1) CO₂ in a continuously ¹⁴C-labelled atmosphere and at two soil N levels, were incubated at 14°C for 222 days. Decomposition of leaf and root tissue grown in the low N treatment was not affected by elevated [CO₂], whereas decomposition in the high N treatment was significantly reduced by 7% after 222 days. Despite the increased C/N ratio (g g^-1) of tissue cultivated at elevated [CO₂] when compared with the corresponding ambient tissue, there was no significant correlation between initial C/N ratio and ¹⁴C respired. This finding suggests that the CO₂-induced changes in decomposition rates do not occur via CO₂-induced changes in C/N ratios of plant materials. We combined the decomposition data with data on ¹⁴C uptake and allocation for the same plants, and give evidence that elevated [CO₂] has the potential to increase soil C stores in grassland via increasing C uptake and shifting C allocation towards the roots, with an inherent slower decomposition rate than the leaves. An overall increase of 15% in ¹⁴C remaining after 222 days was estimated for the combined tissues, i.e., the whole plants; the leaves made a much smaller contribution to the C remaining (+6%) than the roots (+26%). This shows the importance of clarifying the contribution of roots and leaves with respect to the question whether grassland soils act as a sink or source for atmospheric CO₂. This revised version was published online in June 2006 with corrections to the Cover Date.     

Seasonal and geographic climate variabilities during the Last Glacial Maximum in North America: Applying isotopic analysis and macrophysical climate models

Climate models provide estimates of climatic change over periods of time in the ancient past. Macrophysical climate models (MCM) differ from the more widely used general circulation models (GCM), in that MCMs provide temporally high-resolution (~ 100 years) and site-specific estimations of monthly values of climate variables such as temperature and precipitation. In this paper, seasonal changes in climate variables are modeled for six ¹⁴C-dated fossil localities in North America. Five of these localities represent the time of maximum extent of ice during the most recent glacial episode, the Full Glacial (25 + –15 ka), including one at the peak of the Last Glacial Maximum (17–15 ka). The other locality represents the time as the ice began to recede, the Late Glacial (15–11 ka). Seasonal variations in temperature and precipitation modeled by MCM are herein compared with interpretations of seasonal variation based upon oxygen isotopes from serially sampled hypsodont teeth (mostly Equus and Bison) collected from each of these localities. Additionally, the MCM-modeled seasonal variations are used to predict the expected abundances of different plant functional groups (PFG) during those times, especially C3 and C4 functional groups, using modern relationships. These predictions are compared with carbon isotopic values from the same teeth. The importance of atmospheric pCO² for the relative abundance of plants utilizing the C4 metabolic pathway is discussed, given that glacial episodes are known to have been times of lower atmospheric pCO². Interpretations of seasonal variability and the relative abundance of C3 versus C4 vegetation based upon isotopes from tooth enamel are in broad agreement with predictions using the MCM and the modern distribution of PFGs with climate variables. The influence of pCO² on the distribution of C4 vegetation during glacial times appears to be negligible.     

Nitrogen and Carbon Concentrations, and Stable Isotope Ratios in Mediterranean Shrubs Growing in the Proximity of a CO2 spring

Seasonal changes in foliage nitrogen (N) and carbon (C) concentrations and δ¹⁵N and δ¹³C ratios were monitored during a year in Erica arborea, Myrtus communis and Juniperus communis co-occurring at a natural CO₂ spring (elevated [CO₂], about 700 μmol mol⁻¹) and at a nearby control site (ambient [CO₂], 360 μmol mol⁻¹) in a Mediterranean environment. Leaf N concentration was lower in elevated [CO₂] than in ambient [CO₂] for M. communis, higher for J. communis, and dependent on the season for E. arborea. Leaf C concentration was negatively affected by atmospheric CO₂ enrichment, regardless of the species. C/N ratio varied concomitantly to N. Leaves in elevated [CO₂] showed lower δ¹³C, and therefore likely lower water use efficiencies than leaves at the control site, regardless of the species, suggesting substantial photosynthetic acclimation under long-term CO₂-enriched atmosphere. Leaves of E. arborea showed lower values of δ¹⁵N under elevated [CO₂], but this was not the case of M. communis and J. communis foliage. The use of the resources and leaf chemical composition are affected by elevated [CO₂], but such an effect varies during the year, and is species-dependent. The seasonal dependency and species specificity suggest that plants are able to exploit different available water and N resources within Mediterranean sites. This revised version was published online in July 2006 with corrections to the Cover Date.     

Screening of certain mangroves for photosynthetic carbon metabolic pathway

The mangroves Rhizophora lamarkii, Ceriops roxburghiana, Bruguiera gymnorrhiza, Aegiceras corniculatum, and Lumnitzera racemosa were screened for their carbon metabolic pathways by measuring net photosynthetic rate (P _N), ¹³C discrimination rate, leaf anatomy, titratable acidity, and activities of phosphoenolpyruvate carboxylase, NADH-malate dehydrogenase, alanine aminotransferase, aspartate aminotransferase, and pyruvate phosphate dikinase. The tested mangroves had a well developed succulence, opening of stomata during day time and closure in the night hours, and absence of diurnal fluctuation of organic acids in their leaves which excludes the possibility of these species being CAM plants. Moreover, the leaf anatomy had not exhibited Kranz syndrome. The high values of discrimination against ¹³C, low P _N, high CO₂ compensation concentration, and the activities of aminotransferases in the direction of alanine formation suggest that the species may follow C₃ mode of carbon metabolic pathway.     

Two distinct plant respiratory physiotypes might exist which correspond to fast-growing and slow-growing species

The origin of the carbon atoms in CO₂ respired by leaves in the dark of several plant species has been studied using ¹³C/¹²C stable isotopes. This study was conducted using an open gas exchange system for isotope labeling that was coupled to an elemental analyzer and further linked to an isotope ratio mass spectrometer (EA–IRMS) or coupled to a gas chromatography–combustion-isotope ratio mass spectrometer (GC–C-IRMS). We demonstrate here that the carbon, which is recently assimilated during photosynthesis, accounts for nearly ca. 50% of the carbon in the CO₂ lost through dark respiration (R_d) after illumination in fast-growing and cultivated plants and trees and, accounts for only ca. 10% in slow-growing plants. Moreover, our study shows that fast-growing plants, which had the largest percentages of newly fixed carbon of leaf-respired CO₂, were also those with the largest shoot/root ratios, whereas slow-growing plants showed the lowest shoot/root values.     

Does carbon isotope discrimination correlate with biological nitrogen fixation?

It has recently been reported that N₂ fixation and carbon isotope discrimination (Δ) are negatively correlated. To further test this hypothesis, a greenhouse experiment was conducted to investigate if Δ is correlated with the efficiency of lentil (Lens culinaris cv Laird) in fixing atmospheric nitrogen. Lentil seed was inoculated with one of 10 Rhizobium leguminosarum strains that varied in their effectiveness in symbiotic N₂ fixation. Carbon-13 discrimination was positively correlated with N₂ fixation (r²=0.60^*). Although the amount of N₂ fixed ranged from 1.5 mg N to 13.5 mg N shoot⁻¹, the range of Δ values was only 25.8 to 26.6%.. It is unlikely that variability of such small magnitude could be of any practical use in selecting for N₂-fixing efficiency.     

Carbon allocation in shoots of alpine treeline conifers in a CO<Subscript>2</Subscript> enriched environment

With a new approach we assessed the relative contribution of stored and current carbon compounds to new shoot growth in alpine treeline conifers. Within a free air CO₂ enrichment experiment at the alpine treeline in Switzerland, ¹³C-depleted fossil CO₂ was used to trace new carbon in the two tree species Larix decidua L. and Pinus uncinata Ramond over two subsequent years. The deciduous L. decidua was found to supply new shoot growth (structural woody part) by 46% from storage. Surprisingly, the evergreen P. uncinata, assumed to use current-year photosynthates, also utilized a considerable fraction of storage (42%) for new wood growth. In contrast, the needles of P. uncinata were built up almost completely from current-year photosynthates. The isotopic composition of different wood carbon fractions revealed a similar relative allocation of current and stored assimilates to various carbon fractions. Elevated CO₂ influenced the composition of woody tissue in a species-specific way, e.g. the water soluble fraction decreased in pine in 2001 but increased in larch in 2002 compared to ambient CO₂. Heavy defoliation applied as an additional treatment factor in the second year of the experiment decreased the lipophilic fraction in current-year wood in both species compared to undefoliated trees. We conclude that storage may play an important role for new shoot growth in these treeline conifers and that altered carbon availability (elevated CO₂, defoliation) results in significant changes in the relative amount of mobile carbon fractions in woody tissue. In particular, stored carbon seems to be of greater importance in the evergreen P. uncinata than has been previously thought.     

Seasonal, daily and diurnal variations in the stable carbon isotope composition of carbon dioxide respired by tree trunks in a deciduous oak forest

The stable C isotope composition (δ¹³C) of CO₂ respired by trunks was examined in a mature temperate deciduous oak forest (Quercus petraea). Month-to-month, day-to-day and diurnal, measurements were made to determine the range of variations at different temporal scales. Trunk growth and respiration rates were assessed. Phloem tissue was sampled and was analysed for total organic matter and soluble sugar ¹³C composition. The CO₂ respired by trunk was always enriched in ¹³C relative to the total organic matter, sometimes by as much as 5‰. The δ¹³C of respired CO₂ exhibited a large seasonal variation (3.3‰), with a relative maximum at the beginning of the growth period. The lowest values occurred in summer when the respiration rates were maximal. After the cessation of radial trunk growth, the respired CO₂ δ¹³C values showed a progressive increase, which was linked to a parallel increase in soluble sugar content in the phloem tissue (R = 0.95; P < 0.01). At the same time, the respiration rates declined. This limited use of the substrate pool might allow the discrimination during respiration to be more strongly expressed. The late-season increase in CO₂ δ¹³C might also be linked to a shift from recently assimilated C to reserves. At the seasonal scale, CO₂ δ¹³C was negatively correlated with air temperature (R = −0.80; P < 0.01). The diurnal variation sometimes reached 3‰, but the range and the pattern depended on the period within the growing season. Contrary to expectations, diurnal variations were maximal in winter and spring when the leaves were missing or not totally functional. By contrast to the seasonal scale, these diurnal variations were not related to air temperature or sugar content. Our study shows that seasonal and diurnal variations of respired ¹³C exhibited a similar large range but were probably explained by different mechanisms.     

The effects of salinity,crassulacean acid metabolism and plant age on the carbon isotope composition of <Emphasis Type="Italic">Mesembryanthemum crystallinum</Emphasis> L., a halophytic C<Subscript>3</Subscript>-CAM species

The carbon isotope composition of the halophyte Mesembryanthemum crystallinum L. (Aizoaceae) changes when plants are exposed to environmental stress and when they shift from C₃ to crassulacean acid metabolism (CAM). We examined the coupling between carbon isotope composition and photosynthetic pathway by subjecting plants of different ages to salinity and humidity treatments. Whole shoot ¹³C values became less negative in plants that were exposed to 400 mM NaCl in the hydroponic solution. The isotopic change had two components: a direct NaCl effect that was greatest in plants still operating in the C₃ mode and decreased proportionally with increasing levels of dark fixation, and a second component related to the degree of CAM expression. Ignoring the presumably diffusion-related NaCl effect on carbon isotope ratios results in an overestimation of nocturnal CO₂ gain in comparison to an isotope versus nocturnal CO₂ gain calibration established previously for C₃ and CAM species grown under well-watered conditions. It is widely taken for granted that the shift to CAM in M. crystallinum is partially under developmental control and that CAM is inevitably expressed in mature plants. Plants, cultivated under non-saline conditions and high relative humidity (RH) for up to 63 days, maintained diel CO₂ gas-exchange patterns and ¹³C values typical of C₃ plants. However, a weak CAM gas-exchange pattern and an increase in ¹³C value were observed in non-salt-treated plants grown at reduced RH. These observations are consistent with environmental control rather than developmental control of the induction of CAM in mature M. crystallinum under non-saline conditions.     

Precession-forced changes in South West African vegetation during Marine Isotope Stages 101–100 (2.56–2.51 Ma)

The intensification of Northern Hemisphere Glaciation (iNHG) is one of the critical climate thresholds in the Cenozoic. This study focuses on marine sediments recovered from Marine Isotope Stages 101/100 at the Ocean Drilling Program Site 1083 to assesses the impact of the iNHG on continental southern African vegetation through n-alkane (straight-chain hydrocarbon) abundance and δ¹³C values. The n-alkane abundance data yield a convoluted signal due to the number of controlling factors such as the source area, transportation routes and vegetation type. The C₃₁ n-alkane δ¹³C values, however, exhibit a cyclic pattern with a periodicity of c. 20 ka, and are not correlated to the abundance data. It is inferred that the signal does not represent a change in the geographical source of n-alkanes. Instead, we suggest that the variations are caused by water-stress-induced changes in either carbon isotope fractionation during C₃ photosynthesis or subtle changes in the proportion of C₃ and C₄ plants. These changes, unlike variations in oceanographic proxies, closely track precessional forcing factors and are independent of the prevailing obliquity-forced glacial/interglacial cycles. We conclude that the varying monsoon strength, rather than pCO₂ or temperature change, forced changes in southern African vegetation during this period.