**3.3. Chemical analyses of the samples**

stand (<5 m tall) of the species *Rhizophora mangle* and *Avicennia germinans* growing in a hypersaline lagoon (salinity >1000 mmol kg−1 ≈ 35 ppt) located south of the village Tacuato on the Paraguaná peninsula (11°41′40″N, 69°49′52″W). The climate is dry (<400 mm rainfall) with one rainy season from September to December. The lagoon has access to the gulf of Tacuato with an average salinity of 45 ppt (1600 mmol kg−1). The water depth of the lagoon in the sampling area varied between 0 and 20 cm, depending on rainfall events and tides. Diurnal air humidity was about 70–80%, whereas day air temperatures ranged from 27 to 37°C. The tallest trees of both species in the middle part of the lagoon reached a height of 5 m. Trees used for measurements were smaller, but mature, as they were flowering and fruiting. The site was divided into two sub-sites: the fringe-region and an inner site nearly 20 m apart from the fringe, differing in their average osmolality of interstitial soil water (1600 and 1800 mmol kg−1, respectively).

72 Photosynthesis - From Its Evolution to Future Improvements in Photosynthetic Efficiency Using Nanomaterials

The second study site, further on called Ricoa, is located at the fringes of the estuary of the Ricoa River west to the village of Tocópero (11°30′21″N, 69°12′19″W). Annual precipitation is about twice that of Tacuato (970 mm in average) with peaks in May–July and November– December. The soil water salinity averaged 127 mol kg−1 (2–3 ppt), the diurnal air humidity was about 70–80%, like that at Tacuato; day air temperatures were in general lower with highest values around 33°C. Reduced soil salinity was a consequence of higher rainfall and the contribution of the river water run-off. At this site, the trees used for measurements were located at the estuary flood plain, and had approximately the same height as the plants used in Tacuato. Measurements and sample collection of *R. mangle* and *A. germinans* (from now on designated by their genus names) were carried out during seven field trips distributed over

9 months (from October to June), thus including dry and rainy seasons at both sites.

Interstitial water was sampled by digging 10–20 cm into the mud with a perforated plastic tube. Water salinity was determined *in situ* with a refractometer (ATAGO) calibrated with distilled water just before the measurement. Osmolality of the sampled water was calculated

Gas exchange measurements were carried out with an open IRGA system of the type LCA 3 (ADC3, Analytical Development Co.) combined with a Parkinson leaf chamber of 6.25 cm<sup>2</sup>

photometer and a thermocouple attached to the chamber allowed the measurement of incoming light intensity and leaf temperature. Photosynthetic rates used for correlations with leaf

conditions at saturating intensities of photosynthetic active radiation (PAR) ≥ 1000 μmol m−2 s−1 (Asat). Leaves were oriented at 90° to the incoming radiation during measurements. To obtain a range of quantum fluxes, leaves were shaded in the field by a set of fine wire nets. The wire

), and the concentrations of N and chlorophyll, were measured under natural

. A

**3. Materials and methods**

from salinity (in ppt) as in [22].

**3.2. Gas exchange measurements**

**3.1. Interstitial water**

conductance (gl

After gas exchange measurements, leaves were detached and gently cleaned with a wet tissue to remove salt from their surfaces. About 7–10 leaves were used to obtain one sample. Petioles and midribs were removed. Every leaf was cut into halves of which one was put into a plastic syringe (for leaf sap extraction) and the other was put into a plastic bag (for the determination of chlorophyll, N, and P). The samples prepared in that way were immediately frozen on dry ice. Upon returning to the laboratory, they were stored in a freezer at −5°C.

Fresh mass was determined in the field by a battery powered balance (precision ±0.01 g). Samples were dried at approximately 70°C in a ventilated oven until constant weight. Total chlorophyll (a + b) (Chlortot) concentration of leaf disks was measured by spectrophotometry of acetone extracts [25]. Syringes containing the samples were thawed, and leaf sap was squeezed out with a pressure device [20]. Osmolality of the leaf sap was determined with a dew point osmometer (WESCOR 5500). Total P concentration was measured in acid digested dry leaf material following the procedure of Murphy and Riley [26]. Nitrogen concentration was measured using a standard microKjeldahl procedure [27]. These measurements were contrasted with the parallel analysis of calibrated leaf material (peach leaf or citrus leaf, National Institute of Standards and Technology, USA). Sample preparation for organic compounds analyses and the chromatographic determinations of cyclitols in *Rhizophora mangle* and glycinebetain in *Avicennia germinans* have been described in detail elsewhere [14, 28, 29]. Measurement of carbon isotope ratios (δ13C values) of leaf material was performed at the Institute of Botany and Microbiology, University of Munich following standard procedures described elsewhere [30].
