In a more detailed analysis, eight species in each of the two forest types and the tree species present in the agroforestry plantations were selected (19 species in total). The species were chosen because of their abundance and also with the aim of covering a broad morphological spectrum concerning leaf size and structure. Five of the pioneer species and two of the species of the natural forest belonged to the family Euphorbiaceae. The other families represented were Anacardiaceae, Annonaceae, Celastraceae, Lauraceae, Fabaceae, Meliaceae, Sabiaceae, Sterculiaceae, Tiliaceae, Ulmaceae and Urticaceae.
Wooden towers were built for access to the sun leaves of the trees in the natural and secondary forests. The towers were 10 - 15 m high in the natural forest, and 4 - 5 m high in the secondary forest. All trees in the agroforestry systems could be reached from the ground.
3.6.1 Photosynthetic rate
3.6.1.1 The Li-6400 system
Net rates of photosynthesis were measured with a portable gas exchange measurement system (Li-6400, LiCor Inc., Lincoln, NE, USA). This equipment allows in situ gas
exchange measurements in the field at intact leaves in natural position at the tree. The system measures concentration differences in CO2 and H2O between the air stream flowing through the leaf chamber and a reference air stream. CO2- and H2O-concentrations are measured by infrared gas analysis. The Li-6400 system consists of a console and a sensor
3 MATERIAL AND METHODS
head. The console contains a computer, a display, and a small keyboard for managing the system. The leaf chamber and the gas analysers are located in the sensor head. The temperature in the chamber can be controlled by Peltier devices, enabling adjustment of defined vapour pressure deficits (VPD). A red and blue light source enables controlled irradiation of the leaf.
Several times during the working period the analysers were additionally calibrated with a gas of a defined CO2-concentration and air of defined humidity generated by a dew-point generator (Li-610, Li-Cor Inc., Lincoln, NE, USA).
A zero-calibration of the sample and reference gas analysers (IRGA) of the Li-6400 was carried out every morning before starting the measurements.
It was regularly checked that the two IRGAs, matched each other internally. This check was done before logging of each data point.
3.6.1.2 Measurement protocol
Net rates of photosynthesis were measured at 10 – 15 fully sunlit, mature leaves of each species, on at least two different mature or premature trees (typically the fifth leaf, counted from the terminal bud of a twig). During the measurements leaf temperature was maintained at 28°C and VPD around 1.4 kPa. Ambient CO2-levels were used. Mean CO2-level was 369
± 6 ppm.
The central part of a leaf, close to the mid rib, was clamped into the 6 cm2 leaf chamber.
The photosynthetic light response at seven irradiation levels (photon flux densities, PPFD, 2000, 1500, 1000, 500, 200, 50 and 0 µmol s-1 m-2) was determined, starting at the highest irradiance. Values were logged after a time period of at least 12 minutes of irradiation, when steady state was reached, which could be controlled on the display of the Li-6400 system.
Maximum photosynthetic net rate at saturating light intensity and ambient CO2
-concentration, Amax, is assumed to be equivalent to the level of the plateau reached in the light response curve. From the Amax-area value (in µmol CO2 m-2 s-1) given by the Li-6400 system and specific leaf area data of the species (in cm2 g-1), Amax per mass unit (in µmol CO2 g-1 s-1) was calculated.
All measurements were conducted during the hours when leaves shoved maximum photosynthetic capacity (between 9 am and 3 pm). After rainfall the measurements were discontinued until the next day. The investigations were carried out between October 2001 and June 2002.
19
3.6.2 Stomatal conductance for water vapour
Stomatal conductance for water vapour at natural light intensity and humidity
conditions were measured. This was done with a portable steady state porometer (Li-1600, LiCor Inc., Lincoln, NE, USA). Ten to twenty fully expanded, mature, vigorous sun leaves per species (typically the fifth leaf, counted from the terminal bud of a twig) from at least two different mature or premature tree individuals were measured in their natural position.
Each leaf was repeatedly measured on 7 to 9 occasions during a day, in 45 minutes intervals, creating 10 - 20 daily courses of stomatal conductance per species. All measurements took place between 9 am and 2:30 pm.
Leaf and cuvette temperature, VPD and flux density of photosynthetically active radiation (PAR) were recorded simultaneously with all measurements. Transpiration of a leaf placed in the Li-1600 leaf chamber is determined by measuring the flow rate of dry air needed to maintain a constant relative humidity inside the cuvette against the transpiration of the leaf. This target humidity was set to the surrounding humidity at the site of measurement just before starting measuring each leaf. Stomatal conductance (gs) is calculated directly from the measured values of relative humidity, leaf and air temperature, and flow rate by the Li-1600 in mmol m-2 s-1. The VPD (kPa) was calculated from the relative humidity (RH %) and leaf temperature (T Cº) as follows:
VPD = 6.1078((17.08 * T) / (234.18 + T)) - RH/100 * 6.1078((17.08 * T) / (234.18 + T))
To determine maximum stomatal conductance (gsmax) of a leaf, only peak gs values recorded at humidity readings < 80% were considered. Readings at RH > 80% (mainly in early morning hours and immediately before afternoon rainfall) were often thought to be less reliable.
Conductance values reported are all stomatal conductance of the lower leaf side.
3.6.3 Water use efficiency
For calculating water use efficiency (WUE) as
WUE = Amax-area / gs [µmol CO2 (mol H2O)-1]
the gs values measured by the Li-6400-system, corresponding to each Amax value were used.
Thus, the two values used for calculating WUE were obtained simultaneously, in a given leaf, at the same RH, light intensity (2000 µmol s-1 m-2), leaf temperature (28°C), VPD (1.4
3 MATERIAL AND METHODS
kPa) and ambient CO2-concentrations (369 ± 6 ppm). The measurements were conducted in 10 – 15 leaves from at least two different tree individuals.