Introduction

The δ13C value of leaves is widely used to identify the pathway of photosynthesis and to estimate the water-use efficiency (WUE) of plants in natural vegetation assemblages. Such analyses can be successful because the principal carboxylases of the C3, C4 and CAM pathways of photosynthesis discriminate to different extents against 13C, and because H2O loss and CO2 uptake in leaves are related to the intercellular partial pressure of CO2/ambient partial pressure of CO2, p i/p a, which is in turn related to δ13C (Farquhar et al. 1989). Because of their size, trees are hydraulically constrained, which affects their ability to acquire carbon (Meinzer 2003; Tyree 2003). Canopy leaves from trees of tropical forests often show pronounced “midday depression” in stomatal conductance and CO2 uptake when subjected to high solar radiation, as is known from desert plants, a manifestation of the fact that tight control of H2O and CO2 fluxes is of paramount importance in these exposed habitats (Zotz et al. 1995).

This study was undertaken to explore whether there is interspecific and seasonal variation in the carbon isotope signatures of canopy vegetation within a seasonally dry tropical forest composed of species with diverse life forms and leaf phenologies. To this end, and aided by the availability of a canopy crane, we measured the δ13C values of mature and juvenile leaves from trees in an upper canopy, a mid-canopy, a gap, and in associated epiphytes and vines growing on the trees.

Materials and methods

Study site

Plant material was collected in March, April, July and November 1994 from trees growing in a 75- to 150-year-old secondary growth semi-deciduous tropical lowland forest in the 265 ha Parque Natural Metropolitano (PNM; 8°58′ N, 79°23′ W), near Panama City, Republic of Panama. Access to the canopy was afforded by a 42-m canopy crane operated by the Smithsonian Tropical Research Institute (STRI; Parker et al. 1992).

The study site receives an average rainfall of ca. 1,800 mm a−1, 92% of which falls from May to December (for 9 years of climate data at the site, see the STRI Web site at http://striweb.si.edu/esp/physical_monitoring/download_pnm.htm). Further climate data for PNM are provided in Winter et al. (2001).

Species studied

Leaves were sampled from the following species:

  • Upper canopy: Acrocomia aculeata (Jacq.) Lodd. ex Mart. (Arecaceae), Albizia adinocephala (Donn. Sm.) Britton & Rose (Fabaceae), Anacardium excelsum (Bertero & Balb. ex Kunth) Skeels (Anacardiaceae), Antirrhoea trichantha Hemsl. (Rubiaceae), Astronium graveolens Jacq. (Anacardiaceae), Castilla elastica Sessé ex Cerv. (Moraceae), Cecropia longipes Pittier (Cecropiaceae), Cecropia peltata L. (Cecropiaceae), Chrysophyllum cainito L. (Sapotaceae), Cordia alliodora (Ruiz & Pav.) Oken (Boraginaceae), Didymopanax morototoni (Aubl.) Decne. & Planch. (Araliaceae), Enterolobium cyclocarpum (Jacq.) Griseb. (Fabaceae), Ficus insipida Willd. (Moraceae), Ficus maxima Mill. (Moraceae), Luehea seemannii Triana & Planch. (Tiliaceae), Pseudobombax septenatum (Jacq.) Dugand (Bombacaceae) and Spondias mombin L. (Anacardiaceae).

  • Mid-canopy: Annona spraguei Saff. (Annonaceae), Nectandra gentlei Lundell (Lauraceae), Phoebe cinnamomifolia (Kunth) Nees (Lauraceae), Xylopia frutescens Aubl. (Annonaceae) and Zuelania guidonia (Sw.) Britton & Millsp. (Flacourtiaceae).

  • Gap: Carica papaya L. (Caricaceae), Castilla elastica Sessé ex Cerv. (Moraceae), Ficus insipida Willd. (Moraceae) and Piper sp. (Piperaceae).

  • Epiphytes: Aechmea tillandsioides (Mart. ex Schult. & Schult. f.) Baker (Bromeliaceae), Codonanthe uleana Fritsch (Gesneriaceae), Epidendrum imatophyllum Lindl. (Orchidaceae), Epiphyllum phyllanthus (L.) Haw. (Cactaceae) and Peperomia macrostachya (Vahl) A. Dietr. (Piperaceae).

  • Vines: Arrabidaea patellifera (Schltdl.) Sandwith (Bignoniaceae), Bonamia trichantha Hallier f. (Convolvulaceae), Gouania lupuloides (L.) Urb. (Rhamnaceae), Mikania leiostachya Benth. (Asteraceae) and an unidentified species.

From each plant, between 3 and 12 leaves were collected and oven-dried at 65°C prior to carbon isotope analyses.

Excision of intercostal tissue from Anacardium excelsum

A mature Anacardium excelsum leaf (13.5 cm long) was harvested from the upper canopy (34 m) and oven-dried at 65°C. Intercostal tissue between the mid-rib and the leaf periphery was sampled at 3–5 mm intervals between ribs 2 and 6 and between ribs 9 and 13 of the first-order ribs numbered from the apex.

Carbon isotope ratio determinations

Carbon isotope ratios were determined for CO2 derived from the leaf samples at the Duke University Phytotron (Durham, NC) using isotope ratio mass spectrometry (Crayn et al. 2001; Pierce et al. 2002). 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

$$ \delta ^{{13}} {\text{C}}{\left(\permille \right)} = {\left[ {{\left( {{}^{{13}}{\text{C/}}{}^{{{\text{12}}}}{\text{C}}\;{\text{of}}\;{\text{sample}}} \right)}/{\left( {{}^{{13}}{\text{C/}}{}^{{{\text{12}}}}{\text{C}}\;{\text{of}}\;{\text{standard}}} \right)} - 1} \right]} \times 1000. $$

Results and discussion

The influence of canopy position on leaf δ13C value

The interspecific variation in δ13C values of mature sun-exposed leaves harvested over an annual season from 17 C3 tree species in the upper canopy of a seasonally dry lowland tropical forest in Panama ranged between −29.6 and −25.4‰ (Table 1). The range in isotopic composition is small considering the pronounced seasonality and the phenotypic diversity at the site, which contains deciduous and semi-deciduous species, as well as some evergreens such as Ficus spp. and Acrocomia aculeata, a palm (Kitajima et al. 1997). The trees included pioneers with relatively short-lived leaves, such as Cecropia spp., early succession species such as Castilla elastica and Antirrhoea trichantha with leaf lifespans of about 6 months, early to late succession species such as Anacardium excelsum, Enterolobium cyclocarpum and Luehea seemannii with longer-lasting leaves, the deciduous emergent Pseudobombax septenatum, and the rainy season deciduous species Cordia alliodora.

Table 1 δ13C values of young (italic font) and mature (normal font) leaves collected during an annual seasonal cycle from trees and epiphytes growing in the upper- and mid- canopies of a seasonally dry tropical lowland forest beneath the Smithsonian Tropical Research Institute crane at Parque Natural Metropolitano, Panama. δ13C values are means ± standard deviation. The number of leaves measured are in parentheses
Full size table

Despite the small range of isotopic values in the upper canopy, a vertical gradient in leaf isotopic composition was detected (Fig. 1). The isotopic composition, averaged across the seasons, of the mature exposed mid-canopy leaves was significantly more negative than that of the upper canopy (P<0.01; \( \bar{x}_{{{\text{top}}}} = - 27.4\permille \pm 1.1\;{\text{SD}} \), \( \bar{x}_{{{\text{mid}}}} = - 29.9\permille \pm 1.0\,{\text{SD}} \)).

Fig. 1
figure 1

Relationship between δ13C value and height above the forest floor for mature sun-exposed leaves from upper-canopy (○) and mid-canopy (shaded circle) trees, from trees in a gap (▵) and from epiphytes (•) growing in a seasonally dry tropical lowland forest at Parque Natural Metropolitano, Panama. Samples were harvested in July 1994. A regression line (y=a+bx, where a=90.79053, b=2.46928 and r 2=0.339) is fitted to the upper and mid-canopy values

Full size image

The principal contributors to more negative leaf isotopic values in the mid-canopy are probably the reductions in light intensity and vapour pressure deficits (VPDs) that are associated with the more sheltered canopy layers. This can result in a higher ratio of stomatal conductance to net photosynthesis, and consequently greater p i/p a ratios, i.e. a reduction in the diffusion component and a greater expression of the Rubisco component of isotopic fractionation (Farquhar et al. 1989). This effect was particularly pronounced in shade leaves of Anacardium excelsum growing at about the height of the mid-canopy trees, but sampled from deep within the crown of a 34 m upper-canopy tree (Table 1). The shade leaves had δ13C values that were 2.9–3.8‰ more negative than sun-exposed leaves. Low isotope values may also be exhibited when trees assimilate more isotopically depleted CO2, which is produced during soil or plant respiration (Medina and Minchin 1980; Sternberg et al. 1989). The contribution of the respiratory δ13C signal to the isotopic signature of mid- to upper-canopy leaves at the PNM crane site is probably minor. During daylight hours, when plants are photosynthesizing, the [CO2] measured within 10 cm of the soil surface during wet and dry seasons did not diverge extensively from the above-canopy concentration, rarely exceeding 450 ppm (Holtum and Winter 2001). At the heights and conditions at which leaves were sampled during this study, the dilution of ambient CO2 by respiratory CO2 should thus be small (Broadmeadow et al. 1992; Berry et al. 1997; Buchmann et al. 1997; Ometto et al. 2002).

Leaves from the upper surfaces of trees growing in a gap exhibited mean δ13C values that were similar to the upper canopy throughout the season, which suggests that the combined effects of VPD, light intensity and respiratory CO2 on isotopic composition were similar at the two forest sites (Table 1).

For a mature sun-exposed leaf from the upper canopy of A. excelsum sampled in March 1994, the mean ± SD δ13C value of 29 intercostal segments excised from the mid-rib to the leaf margin between the 2nd and 6th first-order ribs was −26.4±0.1, and the value for 22 segments sampled from between the 9th and 13th first-order ribs was −26.3±0.2.The similarity of isotopic composition, which was at the resolution limits of the mass spectrometer, of the 51 intercostal samples from a single mature leaf demonstrates that although stomatal aperture may often be patchy across the intercostal regions such that p i/p a may vary locally across pneumatically isolated patches (Beyschlag et al. 1992), any variation in δ13C values must be extremely local, at least in Anacardium. The observation validates the widely used procedure of taking samples for isotope determination from the centre or top-centre of the leaf blade in order to maximize the sample of the intercostal tissue and to minimize the contributions of juvenile cells, non-photosynthetic mid-rib or petiolar material that may contain an atypical δ13C composition.

δ13C value is influenced by leaf age

Juvenile leaves in the upper canopy exhibited mean δ13C values that were 1.5‰ less negative than for mature leaves (P<0.01, paired two-tailed t-test). In the nine species in which isotopic composition was measured during a temporal transition from young to old leaves (Albizia adinocephala, Antirrhoea trichantha, Castilla elastica, Cecropia longipes, Enterolobium cyclocarpum, Pseudobombax septenatum, Spondias mombin, Annona spraguei and Zuelania guidonia in Table 1), mature leaves exhibited more negative δ13C values in every instance. In Anacardium excelsum and Ficus insipida, for which juvenile and mature leaves were sampled concurrently, the mature leaves also had more negative δ13C values. Similar differences have been measured between juvenile and mature leaves in temperate and tropical species (Lowden and Dyck 1974; Sobrado and Ehleringer 1997; Terwilliger et al. 2001), though differences may sometimes be small (Ometto et al. 2002). Although it has been suggested that juvenile leaf isotopic values are less negative because the leaves are formed from carbon captured during drier periods when plants conserve water by operating at lower p i/p a, less negative δ13C values were also observed in plants that form leaves using carbon gained during the wet season (Terwilliger 1997). Terwilliger et al. (2001) speculated that the mechanistic basis for less negative δ13C values in juvenile leaves is that a significant proportion of the carbon in their structural carbohydrates is captured from the atmosphere by PEPC. It still remains to be seen to what extent fractionation processes involved in carbon storage in old leaves and stems and carbon remobilization from old leaves and/or stems contribute to less negative δ13C values in juvenile leaves.

δ13C values of vines

The δ13C signatures of sun-exposed leaves from five vine species sampled in March 1994 from the upper canopy were remarkably uniform, varying by less than 1‰ (Table 2), and did not significantly differ from the adjacent sun-exposed leaves of the upper-canopy trees that were sampled concurrently. It is not known whether this relative constancy of carbon isotope ratio is maintained throughout the year. Compared to trees, vines tend to allocate more biomass to photosynthetic tissues than to supporting structures (Castellanos 1991) and, in general, leaf production and turnover rates are greater in vines than in trees (Hegarty 1990). As a consequence, one might expect the δ13C of vine leaves to respond more rapidly to changing environmental conditions. The relative area of the canopy that is covered by vine and tree leaves certainly differs seasonally at the PNM site; during the dry season vine and tree leaves occupy 14% and 51% of the canopy area respectively whilst 35% is bare, whereas during the wet season vine and tree leaves cover 31% and 44% respectively whilst 25% is bare (Avalos and Mulkey 1999).

Table 2 The δ13C values of leaves of five species of vine growing on top of the upper canopy at heights between 25 and 30 m. Samples were taken in March 1994. Values are the mean ± SD of measurements on six leaves per species
Full size table

δ13C values of epiphytes

The δ13C values between −12.3 and −22.0‰ for canopy epiphytes were consistent with the operation of crassulacean acid metabolism (CAM) in all five species examined (Table 1; Silvera et al. 2005). The δ13C ratios of Aechmea tillandsioides, Epidendrum imatophyllum and Epiphyllum phyllanthus indicate that these species from three different families, the Bromeliaceae, Orchidaceae and Cactaceae, are strong CAM plants that gain about 74–89% of their carbon via dark CO2 fixation (Winter and Holtum 2002). A. tillandsioides exhibited strong CAM yet grew under extreme shade conditions, an ability also exhibited by some other bromeliads such as the terrestrial species Aechmea magdalenae (Skillman and Winter 1997; Skillman et al. 1999; Crayn et al. 2004). E. phyllanthus sampled in this study, growing well-shaded or in patchy shade, did not exhibit seasonal variation in δ13C values. In contrast, shadehouse-grown plants exposed to 9.2 mol m−2 day−1 exhibited little CAM when well-watered but predominately dark fixation when water-stressed (Andrade and Nobel 1997). Analogy between the shade-house and canopy-grown plants suggests that the plants in the canopy either experienced similar levels of water stress the year round, which is unlikely considering the wet-dry seasonality of the site, or that the extent of the expression of CAM in this species is also affected by other factors, such as light intensity.

Codonanthe uleana is only the second species within the Gesneriaceae known to express CAM. Guralnick et al. (1986) reported that under well-watered conditions C. crassifolia (Focke) can refix respiratory CO2 and accumulate titratable acidity, but does not exhibit net CO2 uptake in the dark (CAM cycling; Holtum and Winter 1999). When water-stressed, C. crassifolia closes its stomata in the dark, but still exhibits diurnal acid fluctuations (CAM idling). The δ13C value of −24.6‰ published for C. crassifolia is consistent with dark CO2 fixation contributing about 14% to the leaf carbon gain (Winter and Holtum 2002, 2005). In contrast, it can be calculated that C. uleana, with δ13C values of between −19.9 and −22.1‰, gains between 29% and 43% of its carbon from dark CO2 uptake. It is possible that more CAM species will be detected in the genus Codonanthe, which lies within the Episcieae, a tribe that contains the majority of epiphytic species within the subfamily Gesnerioideae (Smith 2000).

Peperomia macrostachya has been reported as one of many species in the genus that exhibits CAM cycling (Ting et al. 1985; Holthe et al. 1992). However, the isotopic range of −14.6 to −22.0‰ observed by us is equivalent to a contribution of dark CO2 fixation to net carbon gain of between 30% and 75%, suggesting that dark CO2 uptake makes a substantial contribution to growth, rather than merely reducing carbon loss by refixing respiratory CO2. The range of δ13C values measured for P. monostachya indicates that the species can respond to environmental changes by expressing CAM to variable degrees in the field, as is known for members of the neotropical genus Clusia (Holtum et al. 2004).

δ13C and WUE

The use of δ13C to estimate intrinsic WUE, integrated over the lives of leaves, is insensitive to variations in VPD because the relationship between WUE and δ13C is not causal; rather, both are independently linked with p i/p a (Farquhar et al. 1988). Therefore, the importance given to WUE of factors that independently influence leaf-to-air VPD may be unrecognized in studies of WUE based solely on δ13C. Although we observed little variation in δ13C values amongst exposed canopy leaves from a variety of tropical tree species, there could be substantial differences in the actual amount of H2O transpired per CO2 fixed due to differences in leaf morphology and orientation (Winter et al. 2005). The nature of the canopy itself can also influence VPD. Depending upon the extent of canopy closure and mixing of the air, evapotranspiration can increase the local humidity and reduce leaf temperatures, thereby lowering H2O loss without overly affecting p i/p a.

The most obvious changes in δ13C observed in the upper- and mid-canopy leaves were those associated with leaf development (Table 1). However, the sampling design of the isotopic analyses reported here was of insufficient frequency to enable us to comment upon the intrinsic WUE of seasonal leaf phenotypes (Kitajima et al. 1997). An isotope-based analysis of WUE of leaves of such phenotypes requires more frequent monitoring of leaf cohorts of known time of leaf initiation. It would be of particular interest to examine the water-use strategies of leaves of species that produce a second flush around the end of rainy season, which supports the production of reproductive structures.