Three adjacent sites (around 10 000 m2 each), situated in the subtidal region at Cap Martin (French Riviera) from 9 to 11 m, presenting similar environmental conditions (substrate, exposure, depth), were sampled every two months, from May 1999 to July 2000 using SCUBA diving. Three levels of interaction between C. taxifolia and P. oceanica were identified and replicate sampling was performed in each interaction category (Additional file 1). Formation of the P. oceanica meadow primarily resulted from the growth of orthotropic (erect) shoots. No seedling recruitment was observed during the course of the study. Shoot density differences were not significant between monospecific stands L0 (435 ± 64 shoots.m-2) and locations with co-occurring populations of P. oceanica and C. taxifolia fronds (565 ± 158). The density of C. taxifolia fronds was also similar. Within each site we collected randomly 30 individual sterile adult shoots of P. oceanica with intact rhizomes and 40 fronds of C. taxifolia connected to different stolons.
Sample Processing of individual shoots and fronds
Leaf lengths were measured and the number of adult (oldest external leaves with a sheath) and intermediate leaves (younger internal leaves without sheath) was recorded according to Giraud' method . Dry weights for leaf blades and leaf sheaths were computed separately. Lepidochronological analysis was also carried out to establish the average cycle of leaf renewal and estimate the annual production of leaves and rhizomes . In this method, the mean leaf primary production corresponds to the mean number of leaves produced per shoot and per year multiplied by sheath and blade biomass. The length of C. taxifolia fronds was measured to the nearest millimeter.
Tannin cells analysis
In September 1999, a sub-sample of three shoots of P. oceanica was preserved in ethanol (ethanol – 95%), to observe tannin cells. Once rinsed with fresh water, transverse sections (50 μm thick) were performed along the adult leaves at 2 cm intervals (sheath) and 5 cm intervals (blade). Tannin cells were then counted using optical transmission microscopy, enlargement ×100 and density expressed as a number of cells cm2.
Preparation and chromatographic analysis of phenolic compounds
P. oceanica shoots were kept at low temperatures (1–5°C) during transport. In the laboratory, leaf epiphytes were removed using a razor blade and leaves were freeze dried for 72 hours (HETO®, Lab Equipment-FD4). Extraction of phenolic compounds was then initiated [52, 38]. 1 to 3 g dry weight of leaf tissues were infused for 3 h in 200 ml of aqueous ethanol (1:1), in darkness (40°C). Extraction was carried out with ethyl acetate after vacuum evaporation of the ethanol, at 45°C. The organic phase was thus separated in the separatory funnel, dried using anhydrous sodium sulphate, and then evaporated until a dry residue was obtained. The liposoluble phenolic compounds extracted were stabilized by conversion of the hydroxyl groups into trimethylsilyl groups and the dry extract was added to 50 μl of the mixture trimethylchlorosilane: hexamethyldisilazane: pyridine (1:3:9), 100 μl of bis(trimethylsilyl)trifluoroacetamide and 1.5 μl of trimethylchlorosilane and heated at 70°C for 30 minutes in an inert atmosphere.
For each interaction level, three stabilized samples were analysed by GC and by GC-MS. The GC analyses were carried out using a Perkin-Elmer Autosystem GC apparatus equipped with FID detectors and fused-silica capillary column (30 m × 0.25 mm i.d., film thickness 0.25 μm), Rtx-1 (dimethyl polysiloxane). Oven temperature was programmed to increase the temperature environment by 2°C/m increments between 60°C to 230°C and then hold temperature at 230°C for 35 min. Injector and detector temperatures were maintained at 280°C. Samples were injected in the split mode (1:80), using helium as a carrier gas (1 ml/min).
The GC-MS analyses were performed on a Perkin-Elmer quadrupole MS system (model Q-mass 910). MS conditions occurred as follows: ionisation voltage of 70 eV, scan rate 1 scan/s, mass range 35–350 Da, ion source temperature 200°C. The spectrometer was directly coupled to a Perkin-Elmer Autosystem GC. A fused-silica capillary column (30 m × 0.25 mm i.d., film thickness 0.25 μm), Rtx-1 (dimethyl polysiloxane) was employed. The temperature conditions and the carrier gas were the same as above.
Compound identification was based on: (i) comparison between retention times on an apolar column, and those of standards injected beforehand, and (ii) computer matching with commercial mass spectra libraries . A standard curve derived from pure products enabled the concentrations of phenolic compounds in the samples to be quantified.
Preparation and chromatographic analysis of Caulerpenyne (CYN)
Five samples of C. taxifolia were taken from each experimental site. Algal fronds were processed by rinsing in fresh water, storing at -20°C in MeOH at a concentration of 5 g wet weight of each frond in 50 ml of MeOH (MeOH, 95–98%, Chromanorm; HPLC quality), in order to avoid any degradation of the CYN. Extraction of CYN, the major terpene compound produced by C. taxifolia , was performed directly in the MeOH. To ensure the total diffusion of the CYN present within each frond, samples were sonicated for five minutes. CYN measurements were performed using High Performance Liquid Chromatography. Thus, 10 μl of each sample was injected into the glass column of 5 μm silica (100 × 3 mm, Chrompack) and eluted with a MeOH – water solvent mixture (8:2) at a speed of 0.5 ml min-1. As the retention time of CYN is of the order of 2.8 min, the injection time for each sample was set at 6 min. Measurements were performed at a UV wavelength of 254 nm, sensitivity = 0.32. The HPLC pump (Waters 600), equipped with an automatic injector (Waters 717), was monitored using specially-designed software (Millennium Waters software), that also controlled the PAD data acquisition (Photodiode Array Detector Waters 996). This system enables CYN peaks to be identified during elution both in real time and under the measurement conditions. Three replicates were performed for each sample to assess the analytical dispersion. The standard curve, established on the basis of purified CYN, allowed determination of CYN levels in the samples. A direct relationship between HPLC peaks and CYN levels was obtained.
After checking for normality (Shapiro-Wilks test) and homogeneity of the variances (Bartlett's test), analysis of variance (ANOVA) was carried out using Statgraphic v.3.0 software. The factors were represented by season, the station and tissue type (adult sheaths and blades and intermediate leaves) and in the case of tannin cells, by the position of the section on the leaf. These ANOVA were then completed by a Tukey's multiple range test, in order to locate the differences. It should be noted that because of the small sample size for the study of the phenolic compounds (n = 3), the normality of the data could not be determined. However, ANOVA is a robust test under the conditions of application . For each test, the null hypothesis was rejected with a probability of 95%.