This interpretation of whole-plant carbon isotope contents provided the theoretical basis for the use of δ¹³C values to screen plants for CAM, particularly in the tropics (Rundel et al., 1979; Winter, 1979; Teeri et al., 1981; Griffiths and Smith, 1983; Winter et al., 1983; Earnshaw et al., 1987; Arroyo et al., 1990; Kluge et al., 1991; Zotz and Ziegler, 1997). In recent analyses on the evolution of CAM in the Bromeliaceae and the closely related, reportedly C₃ Rapateaceae, the δ¹³C values of about two-thirds of the 2,800 known species of bromeliads and 85 of the approximately 100 species of the Rapateaceae were determined (Crayn et al., 2001; D.M. Crayn, J.A.C. Smith, and K. Winter, unpublished data). The survey of Bromeliaceae yielded a bimodal distribution of δ¹³C values, with frequency peaks around −13‰ (indicating strong CAM) and −26‰ (indicating C₃ species). The rapateads exhibited a broad but unimodal distribution of δ¹³C values between −37.7‰ and −19.8‰. From such and other studies, it has been estimated that more than 6% of vascular plant species have some ability to exhibit CAM (Smith and Winter, 1996; Winter and Smith, 1996).

A shortcoming of using whole-tissue carbon isotope surveys to obtain an integrated value of the contributions of dark and light CO₂ fixation to total carbon gain is that, in the absence of measurements of acidity, CO₂ exchange, or instantaneous carbon isotope discrimination, it is unclear where C₃ ends and CAM begins. In general, surveys of plant ¹³C/¹²C composition indicate a least negative limit for C₃ species of around −23‰ to −20‰, values that can also indicate some CAM activity. Confirmed CAM species such as Tillandsia usneoides, Didierea madagascariensis H. Baill., and Microsorium punctatum (L.) Copel. have been reported to exhibit values of −19.8‰ (Griffiths and Smith, 1983), −21.2‰ (Winter, 1979), and −22.6‰ (Holtum and Winter, 1999), respectively. Moreover, diel acid fluctuations have been measured in Tillandsia elongata H.B.K. var subimbricata (Baker) L.B. Sm. and Guzmania monostachia (L.) Rusby ex Mez var monostachia, species for which δ¹³C values of −26.4‰ and −26.5‰, respectively, have been reported (Griffiths and Smith, 1983).

O'Leary (1988) predicted a linear relationship between δ¹³C values of CAM plants and the proportions of CO₂ fixed at night and during the day. Surprisingly, given the number of isotopic surveys of vegetation that have been published, O'Leary's prediction has never been tested experimentally. O'Leary cautioned that his qualitative model did not take into account environmental interactions. If one assumes that, for a given species subject to a given set of conditions, −28‰ is the isotope composition when 100% of the CO₂ is fixed by Rubisco in the light then, theoretically, any δ¹³C value less negative than −28‰ indicates that some dark fixation occurs. However, in the real world, chemical and diffusional processes contribute to the isotopic composition of plants, such that integrated tissue δ¹³C values are affected by plant biochemistry, plant-environment interactions and the ¹³C/¹²C composition of the source air, all of which exhibit variation (O'Leary, 1981; Farquhar et al., 1989; Griffiths, 1992). Osmond et al. (1976) concluded “nothing short of a complete balance sheet of CO₂ assimilation throughout the life of the plant is required to adequately predict these (dark versus light fixation) relationships.”

We aim to afford a better interpretation of intermediate δ¹³C values measured during vegetation surveys and to provide experimentally measured values for the relationship put forward by O'Leary (1988). To this end, a balance sheet was generated of light and dark CO₂ assimilation and δ¹³C compositions for well-watered, nonstressed CAM plants. Shoots exhibiting different proportions of daytime to nighttime CO₂ fixation were grown inside a gas-exchange cuvette for up to 51 d, during which time the proportions of CO₂ fixed in the light and dark were quantified. The relationships between the proportions of CO₂ fixed in the light and dark and the δ¹³C value of the biomass accumulated were then assessed.

Table I Dry mass, leaf area, and carbon gain during light and dark periods of shoots of CAM and C3 species developing in a gas-exchange cuvette under various temperature regimes for between 12 and 51 d

Figure 1 Twenty-four-hour net CO2 exchange by shoots of Kalanchoe pinnata exposed to four temperature regimes. A, 25°C, 190 μmol m−2 s−1 light/25°C dark, dew point 18°C; B, 25°C, 180 μmol m−2 (more ...)

Figure 4 Twenty-four-hour net CO2 exchange by shoots of two species of C3 plants. A, Teak: 28°C, 360 μmol m−2 s−1 light/22°C dark, dew point 20°C; and B, Clusia sp.: 28°C, 360 μmol m−2 s−1 (more ...)

Figure 2 Twenty-four-hour net CO2 exchange by shoots of three species of CAM plants. A, K. daigremontiana: 30°C, 185 μmol m−2 s−1 light/17°C dark, dew point 15°C; B, Kalanchoe beharensis: 30°C, 185 μmol (more ...)

Figure 3 Twenty-four-hour net CO2 exchange by shoots or cladodes of three species of CAM plants. A, Aloe vera: 28°C, 193 μmol m−2 s−1 light/22°C dark, dew point 15°C; B, H. monocanthus: 28°C, 350 μmol (more ...)

Some peculiarities in the CO₂ exchange patterns depicted probably reflect the heterogeneous nature of the tissues in the cuvette. These include the split phase-II CO₂ uptake burst in V. pauciflora and the rise and fall of CO₂ uptake by H. monocanthus during the early dark period (Fig. 3).

Teak (Tectona grandis), a C₃ species, and Clusia sp., exhibited net CO₂ uptake only during the light (Fig. 4). The Clusia sp. exhibited a phase II-like CO₂ uptake burst similar to that observed for K. pinnata growing in a 25°C/25°C day/night regime.

In general, the small amounts of tissues initially placed in the gas-exchange cuvette exhibited net CO₂ exchange patterns roughly similar to those expressed in the tissues harvested at the end of the experiments. However, in H. monocanthus the patterns of carbon exchange changed markedly (Fig. 5). The tissue initially exhibited net carbon loss during both the light and the dark. C₃-like net carbon gain in the light and carbon loss in the dark occurred after d 6, whereas CAM-like net carbon gain during the dark began after d 10. By d 18, dark CO₂ fixation accounted for 81.9% of the CO₂ fixed.

Figure 5 Net CO2 balances for 18 12-h-light () and dark (●) cycles during which the stem succulent H. monocanthus grew inside the gas-exchange cuvette.

Table II The δ13C compositions of carbon in the tissues initially placed in the gas-exchange cuvette, in the tissues harvested from the cuvette, and calculated to have been incorporated during the experimental periods (per Eq. 2) in shoots of CAM and C (more ...)

The δ¹³C values of the total and the new tissues were correlated in a linear manner with the cumulative proportions of carbon gained during the dark and the light (Fig. 6, A and B). The linear regressions, which exhibited r² values of 0.95 and 0.96, respectively, predicted δ¹³C values of −8.3‰ and −8.7‰ after CO₂ fixation exclusively in the dark by the total and new tissues, respectively. A value of −26.9‰ was predicted for 100% light fixation in both tissues.

Figure 6 Relationships between the cumulative net carbon gain in the light and the dark and the δ13C values of tissues harvested after between 12 and 51 d (A) or the δ13C values of tissues formed (calculated per Eq. 2) after between 12 and 51 d (more ...)

The δ¹³C values of the total and new tissues were also correlated in a linear manner with the proportions of carbon gained in the light and the dark during the final 24 h within the gas-exchange cuvette (Fig. 6C). The linear regressions predicted δ¹³C values of −26.9‰ and −27.0‰ after exclusively CO₂ fixation in the light by the total and new tissues, respectively. The predicted values for 100% dark fixation were −10.5‰ and −10.9‰, respectively. The δ¹³C values for H. monocanthus were excluded from the regressions because the assumptions of Equation 2 were not met.

The proportion of CO₂ fixed during the light and dark was the major contributor to variation in whole-tissue carbon isotope composition of the C₃ and CAM plants examined. A range of 18.6‰ was estimated for the difference between tissues that assimilated carbon exclusively during the dark or the light (Fig. 6). Because the relationship between the contributions of light and dark CO₂ uptake and whole-tissue δ¹³C was linear, each 10% contribution of dark CO₂ fixation to net CO₂ exchange should shift δ¹³C values by about +1.8‰.

The above observations have major implications for the interpretation of the bimodal distributions of δ¹³C values characteristic of isotope-based C₃-CAM vegetation surveys and define a “typical” CAM plant growing in the field (Rundel et al., 1979; Winter, 1979; Teeri et al., 1981; Griffiths and Smith, 1983; Winter et al., 1983; Earnshaw et al., 1987; Arroyo et al., 1990; Kluge et al., 1991; Zotz and Ziegler, 1997; Crayn et al., 2001). In surveys such as those summarized in Figure 7, the individuals in the more positive isotopic cluster, which usually has a mode of around −13‰ to −14‰ and ranges between about −9‰ and −19‰, are generally categorized as CAM. On the basis of the regression presented in Figure 6B, one could conclude that the typical CAM plant with a δ¹³C value of −13‰ to −14‰ obtained around 71% to 77% of its carbon from dark fixation. A plant at the more negative edge of the CAM cluster, at around −19‰, would gain 44% of its carbon from dark fixation. The more negative of the two clusters, which generally exhibits a mode around −27‰ and a range between about −21‰ and −32‰, is typically designated as a C₃ cluster but, according to our observations, may contain both C₃ species and plants that obtain up to 33% of their carbon via dark CO₂ fixation. That is, the carbon isotope method alone does not distinguish between field-grown C₃ species and plants that obtain up to one-third of their carbon during the dark, which include weak and many facultative CAM plants. Such plants can only be distinguished by careful measurements of diel titratable acidity changes or dark CO₂ uptake. Measurements of low levels of dark CO₂ uptake in the field may be at the limits of resolution of currently available portable gas-exchange systems.

Figure 7 δ13C values of 506 species from the Asclepiadaceae, Bromeliaceae, Crassulaceae, Cucurbitaceae, Didiereaceae, Euphorbiaceae, Orchidaceae, Polypodiaceae, and Vittariaceae plant families known to contain both C3 and CAM members. Values were selected (more ...)

It has been suggested that in some CAM species, PEP carboxylase may be active for parts of the photoperiod (Winter and Tenhunen, 1982). The isotopic compositions of the CAM species that exhibited 100% total carbon gain in the light were approximately 2‰ more positive than those of C₃ species, but because we were unable to distinguish between PEP carboxylase or diffusional effects in the light and PEP carboxylase-based recycling of CO₂ in the dark, we can neither rule out nor confirm the possibility that PEP carboxylase may be fixing external CO₂ during the light. However, if PEP carboxylase fixation was occurring during the light in the CAM species, the linearity of the relationship between isotopic signature and the proportions of light and dark CO₂ uptake is consistent with a constant proportional contribution of PEP carboxylase-fixed carbon to carbon gain during the light for the species in our study.

Although the proportion of CO₂ fixed during the light and dark was the major contributor to variation in carbon isotope composition of newly fixed carbon in our study of well-watered plants, the range in δ¹³C values measured in the field is greater than that observed in our study (compare Figs. 6B and 7). The extent to which diffusion or carboxylation limit CO₂ fixation in the light and in the dark contributes to this difference. The isotopic compositions for carbon fixed in the light or in the dark estimated in this study, −26.9‰ and −8.7‰, respectively, support the postulate that both light and dark CO₂ fixation in well-watered CAM plants are colimited by diffusion and carboxylation (O'Leary and Osmond, 1980; O'Leary, 1981, 1988). If one assumes an average source CO₂ isotopic composition of −8.7‰ (see “Materials and Methods”), then the expected Rubisco- and diffusion-limited isotopic compositions of carbon fixed in the light would be −35.7‰ and −13.1‰, respectively, and the corresponding values for dark-fixed carbon would be −3‰ and −13.1‰, respectively (Farquhar et al., 1989). Uncertainty in estimating the contributions of dark- and light-fixed CO₂ using the correlations developed in Figure 6 can arise when plants are collected from environments that affect the relative limitations of diffusion and carboxylation (Farquhar et al., 1989), e.g. from extreme habitats characterized by edaphic stress (Farquhar et al., 1982), along altitudinal gradients (Körner et al., 1988; Friend et al., 1989; Crayn et al., 2001), or from forest understoreys with different levels of canopy closure and CO₂ stratification (Holtum and Winter, 2001). It is good field practice to collect, as a reference, material from sympatric C₃ plants. When collecting in forests or close to the ground, air samples should also be taken if practicable.

Other processes that complicate the isotopic signal as an index of light and dark fixation include the translocation of carbon into developing photosynthetic organs, variations in the amounts of respiratory CO₂ that are refixed, loss of isotopically heavy CO₂ in the light during phase III, and short-term variations in the δ¹³C content of ambient CO₂. In our experiments, with the exception of the stem-succulent H. monocanthus, translocation of carbon into the growing tissues was small because the plants had little or no photosynthesizing tissue outside the cuvette. Net carbon gains were more than sufficient to account for the increases in the dry masses of the tissues inside the cuvette, indicating that the incubated tissues were carbon sources rather than sinks. For H. monocanthus, it was calculated, on the basis of measured net CO₂ exchange and a determination that 43% of the dry matter was carbon, that 86% of the 90-fold increase in dry mass observed during 18 d in the gas-exchange cuvette was due to the reallocation of resources from mature stem outside the cuvette to the enclosed developing stem. Because of the massive importation of carbon into the developing stem enclosed within the cuvette, the isotopic composition of the 17.3 cm of the stem inside the cuvette did not reflect the proportions of CO₂ incorporated by that tissue during the light and dark, suggesting that meaningful isotopic compositions of such stem-succulents in the field will only be obtained if sampling is restricted to mature regions well behind the growing tip. Moreover, the reasonably rapid developmental change from C₃ to CAM in H. monocanthus resulted in a gas-exchange pattern for the final 24 h inside the cuvette that, in contrast to the other species studied, correlated poorly with the cumulative proportions of dark and light carbon gain measured throughout the period that the tissue was enclosed (Table I; Figs. 3 and 5).

The observations for H. monocanthus are of relevance to interpreting the isotopic signatures of tissues undergoing developmental change. The shifts between C₃ and CAM in leaves of facultative CAM species like Mesembryanthemum crystallinum and Clusia spp. may be accompanied by changes in the proportions of ¹³C-enriched or -depleted metabolites that are exchanged with the rest of the plant. In such circumstances the interpretation of analyses of single leaves collected during vegetation surveys will be superficial until more extensive sampling is undertaken.

During phase III in the light, X. danguyi, A. vera, and V. pauciflora evolved 1.6%, 0.5%, and 1.4%, respectively of total carbon fixed. Although the proportion of the daily carbon gain lost during phase III tends to be small, it may be considerably enriched in ¹³C. In Tillandsia utriculata, a 1% loss of net carbon uptake during phase III was sufficient to change the plant δ¹³C value by −0.64‰ (Griffiths et al., 1990). In vegetation surveys, such a shift is unlikely to change the interpretation of strong CAM that would be attributed to plants such as A. vera (−16.1‰) and V. pauciflora (−14.0‰) and even to X. danguyi, which exhibited an intermediate δ¹³C value of −20.1‰. In well-watered plants, the net loss of CO₂ during phase III tends to be observed in strong CAM species, whereas in plants that exhibit less developed CAM, such as the K. pinnata plants depicted in Figure 1, CO₂ uptake tends to remain positive during phase III.

The δ¹³C values of the limits for the regressions depicted in Figure 6 will vary with the isotopic composition of atmospheric CO₂. However, the slope of the regression should remain similar. Variation due to the annual shift of about −0.05‰ generated by the combustion of fossil-fuels and the small annual seasonal fluctuation of about 0.2‰ (Mook et al., 1983), can be predicted. Correction of field data will be easier if one collects sympatric C₃ species. To our knowledge, few δ¹³C values reported from herbarium-based surveys have been corrected for the atmospheric δ¹³C composition, which has changed from about −6.4‰ to about −8.7‰ since 1750 (Mook et al., 1983; Friedli et al., 1986). The δ¹³C values of herbarium specimens of 12 C₃ species collected between 1750 and 1988 changed from −25.8‰ ± 0.25‰ to −26.4‰ ± 0.29‰ (Peñuelas and Azcón-Bieto, 1992). The shift is small probably because the associated increase in [CO₂] has reduced stomatal apertures, thereby increasing diffusive resistance to CO₂ and reducing the contribution of the Rubisco-based fractionation to the isotopic signal.

Diel variations, generally associated with the vertical stratification of CO₂ beneath forest canopies, can add significant uncertainty to CAM isotopic signals. δ¹³C values about 2‰ more negative during the night than during the day, as has been reported for a semi-evergreen seasonal forest in Trinidad (Broadmeadow et al., 1992), would result in tissues becoming 0.2‰ more negative for every 10% CO₂ fixed during the dark. Because the vertical gradients of up to 5‰ that have been reported for CO₂ under forest canopies (Vogel, 1978; Medina and Minchin, 1980; Francey et al., 1985; Medina et al., 1986; Sternberg et al., 1989; Broadmeadow et al., 1992) are usually associated with changes in CO₂ concentration that reflect soil, root, and leaf respiration, correction for the effects of the position of the plant under the canopy requires at least isotopic knowledge of the CO₂ concentration of ambient atmosphere and the δ¹³C value of leaf litter (Sternberg et al., 1989; Broadmeadow et al., 1992). In this study in which the gas-exchange cuvette was supplied with well-mixed air sampled from above a 5-storey building, the diel variation of atmospheric δ¹³C was 1.3‰. CO₂ sampled at dawn (7 am) was −9.4‰ ± 0.06‰ (n = 5), whereas CO₂ sampled at dusk (6 pm) was −8.1‰ ± 0.04‰ (n = 7).

In conclusion, we have provided experimental confirmation for the hypothesis of O'Leary (1988) that the relationship between carbon isotope ratio and the proportion of dark and light CO₂ fixation in plants that exhibit CAM is linear and have established the slope and limits of the relationship for well-watered plants exposed to an ambient CO₂ concentration. Extrapolation of our observations to plants surveyed under natural conditions suggests first, that the most commonly expressed version of CAM in the field, the typical CAM plant, involves plants that gain about 71% to 77% of their carbon by dark fixation, and second, that the isotopic signals of plants that obtain less than one-third of their carbon in the dark may be confused with C₃ plants when identified on the basis of carbon isotope content alone. This uncertainty in the interpretation of isotopic signals begs the question of whether the isotopic distribution of CAM plants in the field is unimodal or bimodal. Does the isotopic distribution of terrestrial CAM plants exhibit a mode around −13‰ to −14‰ with a skewed margin that tails out to values close to −27‰? Or does the so-called C₃ isotopic cluster conceal a second peak of abundance indicative of ecological niches for plants, such as some tropical epiphytic ferns (e.g. Holtum and Winter, 1999), with low capacities for dark CO₂ fixation? The striking observation that, in mature source tissues, the proportions of carbon incorporated during the light and dark in a single day correlates with the δ¹³C composition (Fig. 6C) should, in conjunction with measurements of titratable acidity, greatly assist in sorting out these relationships.

Net CO₂ exchange by intact shoots exposed to different light/dark temperature regimes under a 12-h-light/12-h-dark cycle was measured using a through-flow gas-exchange system (Walz, Effeltrich, Germany) operating at 1 or 2 L min⁻¹ air. The CO₂ concentration of the gas supply, ambient air sourced 16 m above ground level and passed through a 2 m³ buffer, varied between 360 and 430 μL L⁻¹.

Developing shoots were sealed inside a 1.2-L Plexiglas cuvette of the gas-exchange system in a controlled environment cabinet (GEC, Chagrin Falls, OH). Duplicates of similar shape and size were harvested at the beginning of each experiment. Root systems still attached to the plants were outside the cuvette but still in the cabinet. Lower leaves on the stem outside the cuvette were removed except for X. danguyi. For K. beharensis, K. daigremontiana, and K. pinnata, the apex including the top leaf pair about 4 cm high was used. For A. vera, an entire four-leaf 2.5-cm-high juvenile shoot was used. For H. monocanthus, approximately 0.5 cm of a developing stem was sealed in the cuvette. For X. danguyi, the cuvette initially contained four small leaves attached to about 3 cm of cane from which the apex had been removed. During the study, the four leaves increased in size. For V. pauciflora, the cuvette contained an initiating leaf and a developing leaf shoot attached to a 20-cm defoliated shoot that was outside the cuvette. For teak and Clusia sp., the upper leaf pair of about 4-cm-tall seedlings was inserted in the cuvette.

H. monocanthus, X. danguyi, teak, Clusia sp., and V. pauciflora were grown in potting mix during the gas-exchange measurements, whereas the roots of A. vera, K. beharensis, K. daigremontiana, and K. pinnata were maintained in one-half-strength Johnson's solution (Winter, 1973).

Tissue harvested at either d 0 or between 12 and 51 d growth in the gas-exchange cuvette was dried for 48 h at 65°C, weighed, and ground to a fine powder using a mortar and pestle. ¹³C/¹²C analyses were performed on three aliquots of the powdered extracts of tissue harvested on d 0, and on five to eight aliquots of the extracts of tissue grown in the gas-exchange system.

Following the appropriate corrections for other isotopes, the abundance of ¹³C in each sample was calculated relative to the abundance of ¹³C in standard CO₂ that had been calibrated against Pee Dee belemnite (Belemnitella americana). Relative abundance was determined using the relationship

1

2

Carbon isotope ratios of CO₂ from 12 0.5-L samples of air collected at dawn and dusk in August, 2001, from the inlet of the gas-exchange cuvette were determined at the Institute of Arctic and Alpine Research, University of Colorado (Boulder), using a Micromass Optima dual gas inlet spectrometer.

Larry Giles (Duke University), Wolfgang Wanek (University of Vienna, Vienna), and Bruce Vaughn (University of Colorado) performed the measurements of δ¹³C, and V. pauciflora was identified by Neal Smith (Smithsonian Tropical Research Institute, Panama) under conditions of stress.