The experimental site is located in the Caxiuanã National Forest, Pará State, north-eastern Brazil (1°43′3·5″S, 51°27′36″W). The forest is a lowland terra firme rain forest situated on a level plain 10–15 m above river water level, with a high annual rainfall (∼2500 mm) and a pronounced seasonality in leaf fall which peaks during the strong dry season (see Table 1 for additional plot details). Plant species diversity is high at around 100 species per hectare, of which over half are Sapotaceae, Fabaceae, Violaceae and Chrysobalanacae, and less than 1% are lianas. Mean annual air temperature is c. 25 °C and the diurnal variation is typically less than 3 °C. The most widespread soil type is a highly weathered yellow Oxisol (US Department of Agriculture soil taxonomy). In January 2002, a 1-ha area of forest was modified with the installation of plastic panels at 2 m height to exclude c. 50% of incident rainfall (TFR plot). This reduction in rainfall is similar to a key early long-term climate prediction for the region (Cox et al. 2000). The perimeter of the TFR plot was trenched to a mean depth of 1 m and lined with plastic sheeting to minimize lateral flow of water into the site. Intercepted water was channelled away to a run-off area 50 m away from the plot. An adjacent 1-ha Control plot with similar topography, soil type and vegetation structure (Fisher et al. 2007) was used to assess natural patterns of L, S and R, in the absence of any TFR treatment. Supplementary measurements during the first 3 years of the TFR treatment demonstrated that soil water potential, tree stem sapflow, stomatal conductance and photosynthesis were all substantially reduced in the TFR plot compared to the Control, particularly during the dry season (Fisher et al. 2007). At the beginning of the experiment in January 2001, 30 m tall canopy access towers were installed near the centre of both plots. All measurements were taken at least 20 m inside the perimeter of each plot to minimize edge effects.
Table 1. Key vegetation and soil features for each plot surveyed
| Tree number ha−1||434||421|
| Stem basal area (m2 ha−1)||23·9||24·0|
| Tree species ha−1||118||113|
|Soil 0–10 cm|
| Clay content (%)||18||13|
| Silt content (%)||5||4|
| Sand content (%)||77||83|
| Carbon content (g kg−1)||9||12|
| Nitrogen content (g kg−1)||0·4||0·3|
| Phosphorus content (mg dm−3)||3||3|
| Carbon : nitrogen ratio||23||35|
| Soil cation exchange (cmol dm−3)||0·8||0·7|
Measurement of leaf dark respiration and specific leaf area
R and S from trees on both plots were recorded on six occasions between November 2001 and January 2007: once before and five times after imposition of the TFR treatment. L data are also available from the same periods on both plots (Fisher et al. 2007). All measurement campaigns sampled fully expanded, non-senescent, un-diseased leaves, and recorded additional information about the height and tree species of the sampled leaves. Thus, R measurements from these leaves should primarily reflect ‘maintenance’ respiration rather than ‘growth’ respiration associated with metabolic costs of constructing new plant tissue (McCree 1970). All leaves were sampled during the daytime (08.30–15.00 h) and kept in the dark until CO2 gas exchange had stabilized (usually after 5–10 min) before R at ambient air CO2 concentration (360–380 p.p.m.) and humidity (60–80%) was recorded, thus minimizing biases potentially introduced by light-enhanced dark respiration and the photorespiratory post-illumination burst (Atkin, Evans & Siebke 1998).
The first five measurement campaigns (conducted between November 2001 and 2003) used the following methodology: 17–26 leaves from nine trees, and 18–26 leaves from eight trees were sampled around the canopy access towers on the Control and TFR plots respectively (Table 2). Measurements were taken from the same trees and from leaves at the same canopy heights in each measurement campaign. R was measured in situ from un-excised leaves with an infra-red gas analyser (IRGA) connected to a leaf measurement cuvette (LI-COR 6400 portable photosynthesis system with 6400-02B leaf cuvette; Lincoln, NE, USA). Leaf discs of a known area were cut from leaves on the same branch as leaves used for R measurement, dried at 70 °C until constant mass and weighed. S was calculated for each leaf disc sampled by dividing dry mass by one-sided area.
Table 2. Tree species sampled on the plots
|Duguetia echinophora||Duguetia echinophora|
|Hasseltia floribunda||Hirtela bicornis|
|Licania heteromorpha||Lecythis confertiflora|
|Manilkara bidentata||Licaria armeniaca|
|Mezilaurus lindawiana||Licania canescens|
|Pouteria lateriflora||Manilkara paraensis|
|Protium heptaphyllum||Mouriri duckeana|
|Quiina florida||Swartzia racemosa|
The final measurement campaign (in January 2007) sampled a total of 33 and 28 individual leaves from the Control and TFR plots, respectively, from 15 separate trees per plot. Of these trees sampled, 10 were randomly selected as the closest tree to every 10 m intersection point along two 40 m long transects in the centre of the plot. A branch from the outer canopy of each selected tree was excised at between one and three different canopy heights. No attempt was made to cut and re-cut branches under water because this would not have guaranteed that gas exchange remained unaltered (Santiago & Mulkey 2003). Instead, we designed an experiment to quantify and, if necessary, correct for any impacts of branch excision (see text in Methods section). To facilitate sampling of leaves higher up in the emergent canopy and to replicate measurements on individual trees made earlier, an additional five trees per plot were similarly sampled around the canopy access towers on each plot. R was measured for most leaves within 3 h of branch excision, using an IRGA connected to a leaf measurement cuvette (CIRAS-1 IRGA with PLC6 leaf cuvette; PP Systems, Hitchen, UK). The interval of time between branch excision and R measurement was noted for each leaf sampled. There was no significant difference in the mean time between excision and R measurement on the plots.
After measurement, the same leaves were photographed to calculate leaf area with digital image analysis, and then dried at 70 °C until constant mass and weighed. S was calculated for each entire leaf sampled (including petioles) by dividing dry mass by one-sided area.
Measurements made with the LI-COR 6400 IRGA maintained a flow rate of 500 μmol s−1, with a mean ± standard error (SE) difference between Cr and Cs of 0·52 ± 0·07 p.p.m. The CIRAS-1 IRGA was set to a lower flow rate of 200 μmol s−1, and consequently the observed mean ± SE CO2 difference was 0·91 ± 0·06 p.p.m. The inward diffusion of respired CO2 from leaf material clamped under the cuvette gasket (Pons & Welschen 2002) and the diluting effect of water vapour produced by the leaf was corrected for.
Leaf temperatures recorded automatically by the IRGA systems during R measurement varied between 22 and 31 °C. Species-specific R temperature response functions were not available for all of the trees sampled, so measurements were standardized to a reference temperature of 25 °C (R25) with the following formula that describes the average R temperature response across 116 terrestrial plant species (Atkin & Tjoelker 2003; Atkin, Bruhn & Tjoelker 2005):
where Ra is R recorded at ambient temperature (Ta).
To investigate the potential confounding influences of branch excision on the R values recorded in January 2007, the following experiment was devised. An un-excised leaf was placed within the IRGA cuvette and R was measured every minute for 1 h. After this period, the branch attached to the leaf within the cuvette was excised, but R measurement was continued at the same temporal frequency for 5 h, to observe whether there was any change in R with time since branch excision. Over this period, the sensor was regularly automatically calibrated with air passed through a molecular sieve to remove all CO2. Before and during measurements the molecular sieve was frequently checked to ensure that it was not exhausted. This procedure was repeated three times, on consecutive days from three individual leaves each on separate trees of different species. All leaves sampled showed no change in R over the hour prior to excision, but after excision R rose gradually over time, approximately doubling after 5 h compared to the pre-excision mean value (data not shown). A third-order polynomial model was fitted to the mean trend of R over time since branch excision (R2 = 0·77). This model was not chosen as a realistic mechanistic simulation of plant gas exchange, but purely for limited predictive purposes over the duration of the measurements because it provided the best fit to the data. This equation, together with data collected on the interval of time between branch excision and R measurements for each leaf, was used to correct for the confounding effect of excision and storage on January 2007 measurements by calculating R at time since excision = 0 for each leaf sampled. No immediate effect of excision itself on R was apparent.
Measurement of leaf area index and foliage mass
Mean plot L estimated during the first five measurement campaigns (conducted between November 2001 and 2003) is presented in Fisher et al. (2007). These data were derived from canopy images captured at 100 points per plot with LAI-2000 plant canopy analysers (LI-COR Inc.). For this study, additional L data were collected in January 2007 based upon canopy images per plot collected at 25 locations along a with a digital camera and fish-eye lens (Nikon Coolpix 900; Nikon Corporation, Melville,USA) and subsequently analysed with digital image analysis software (Hemiview 2.1 SR1; Delta-T Devices Ltd, Cambridge, UK). All images of the canopy were recorded in the early morning or late afternoon, during periods of fully diffuse incoming radiation along a regular grid within both plots (following the methodology of Aragão et al. (2005). The distribution of L with height above the ground on both plots was estimated once – in November 2001 – by recording L with the LAI-2000 plant canopy analysers (LI-COR Inc.) every 2 m up each of the plot canopy access towers. Plot-level M was estimated for each measurement campaign by multiplying mean plot leaf mass per unit leaf area (1/S) by L.
Estimating stand-scale night-time foliar carbon efflux
To illustrate how R, S and L interact, and to facilitate direct comparison of our leaf-level R measurements with other ecosystem C fluxes, we derived approximate estimates of stand-scale foliar C efflux. To do so, we calculated mean ± 95% confidence intervals of L and R per unit area separately for three canopy height layers (≤10, 11–20, ≥21 m). Night-time foliar C emissions per unit ground area were estimated for each canopy layer as the product of R per unit leaf area multiplied by L. Given the low temporal frequency of our direct measurements and the focus on between-plot (rather than seasonal or annual) differences we opted for the relatively simple, more transparent, up-scaling approach of assuming constant night-time air temperature of 25 °C and 12 h of dark conditions each day throughout the year. For the purposes of this analysis, we also assumed that dark-equilibrated R recorded during the day in this study was representative of night-time leaf respiration (Chambers et al. 2009; but see Hubbard, Ryan & Lukens 1995). Means from each canopy layer were summed to derive total plot estimates. Where necessary, 95% confidence intervals were propagated by quadrature of absolute errors for addition and subtraction, and quadrature of relative errors for multiplication and division (Mood, Graybill & Boes 1974; Cavaleri, Oberhauer & Ryan 2008). This assumes that errors are independent and normally distributed.
To assess the impact of the TFR treatment on S and R the following two statistical analyses were performed. (i) Within-plot change over time since the imposition of the TFR treatment was quantified with a repeated-measures analysis of variance (RM-ANOVA). Data from the final measurement campaign were not included in the RM-ANOVA because a different set of trees were sampled with a different methodology, whereas the previous five campaigns repeatedly sampled leaves from the same trees and same canopy heights. To examine specifically which time periods differed from each other in terms of S and R, pairwise comparisons between measurement campaigns were conducted within the RM-ANOVA analysis. (ii) Between-plot differences in R and S over all measurement campaigns were quantified with a Generalized Linear Model (GLM) with plot as a fixed-effects factor, and leaf height, tree family and sampling time specified as random-effects factors to control for the potentially confounding effect of sampling differences between plots. Using this method, plot differences were examined both for all data at each measurement campaign, and for all data in different canopy height categories (≤10, 11–20, ≥21 m). In addition, the links between leaf height, R and S were assessed with a Spearman’s Rank Correlation. Statistical analyses were carried out with SPSS 14·0 for Windows (SPSS Inc., Chicago, IL, USA). Key outputs of the analyses were an F-statistic (for the RM-ANOVA and GLM), a correlation coefficient (r, for the Spearman’s Rank Correlation analysis) and a significance P-value for all tests. Data were transformed with a natural logarithm, where necessary, to conform to the assumptions of parametric analysis.