Material and methods
Eutrophic, mesotrophic and oligotrophic sites in the northeast tropical Atlantic (see legend of Fig. 1) were occupied for 3, 4 and 6 days during the EUMELI #4 cruise (June 1992). Nutrients and pigments, optical properties, PB vs. E incubations and FRR fluorometry measurements were made daily. Sampling was generally performed at dawn, midday and dusk at 10 depths encompassing the whole algal vertical distribution. Samples collected at dawn using 10-l GoFlo bottles fixed on a Kevlar® wire were used for in situ primary production measurements (JGOFS protocols 1994). Midday and dusk samples were collected using a rosette sampler with 12-l Niskin bottles equipped with silicone washers, and analyzed for nutrient and pigment concentrations. Micromolar NO 2 +NO 3 and PO 4 concentrations were determined using a Technicon Autoanalyzer® using methods of Wood et al. (1967) and Murphy and Riley (1962), respectively. Nanomolar determinations of NO 2 and NO 3 concentrations were performed also using a Technicon Autoanalyzer®, following methods of Raimbault et al. (1990). Vertical profiles of temperature, salinity and chl a fluorescence were measured using a CTD (SBE911plus, SEA-BIRD Electronics Inc.) and an in situ fluorometer (FL3000, Sea Tech Inc.).
Discrete sample pigment concentrations were determined using fluorometry, spectrofluorometry, and HPLC. In all cases, filtration was performed using glass fiber filters (Whatman GF/F). Fluorometric determination of chl a + pheopigment concentration was performed as described by Parsons et al. (1984) using 100% methanol (rather than acetone) and extraction duration of 0.5 h. Spectrofluorometric determination of chlorophylls was carried out onboard using the method of Neveux and Lantoine (1993). Samples for liposoluble pigment analysis were stored in liquid nitrogen immediately following filtration and later measured by high pressure liquid chromatography (HPLC) after extraction in 100% methanol at a shore-base laboratory as described by Claustre and Marty (1995). The HPLC system allowed a separation of divinyl-chl a (DV-chl a) and chl a sufficiently well resolved to quantify both concentrations using the peak heights. The extinction coefficient used for DV-chl a was 105.6 l g-1 (Jeffrey and Humphrey 1975). All three pigment analysis methods provided similar vertical patterns for total chl a (chl a and DV-chl a; from hereafter denoted Chl), yet fluorometric and spectrofluorometric measurements always led to values clearly above those obtained by HPLC. As a rule, interpretation was based on HPLC results. When HPLC data were missing, spectrofluorometric (or fluorometric) measurements were used; they were "converted into HPLC data" by using conversion factors (calculated from samples for which all methods were simultaneously applied). A "non-photosynthetic pigment index" (NPP index) was computed from the HPLC analyses as the ratio (weight : weight) of non-photosynthetic pigments to which photoprotection is often ascribed (i. e. zeaxanthin plus diatoxanthin, diadinoxanthin and
-carotene) to total pigments (i.e. non-photosynthetic, chlorophylls and carotenoids; Bidigare et al. 1990).
Daily measurements of submarine photosynthetically available radiation (PAR: 400-700 nm) performed using a custom-built quantum meter, were used to calculate optical depth (
) at each station as:
where Kd is the vertical attenuation coefficient for downward PAR irradiance (m-1 ) and z is the depth (m);
In vivo absorption measurements of suspended particles were performed using 1 to 9 l of seawater filtered onto 25 mm Whatman GF/F glass-fiber filters under low vacuum pressure. Wet filters were placed between a light source (tungsten lamp) and the entrance to an integrating sphere connected by an optic fiber to a LI-1800UW Licor irradiance meter. Transmitted light was measured between 380 and 750 nm with 1-nm resolution. The baseline was measured prior to filtration using the same filter soaked for 1 h in filtered seawater. Absorption was also measured after pigment extraction, in order to determine the spectral absorption due to unpigmented detrital particles. Pigments were extracted by immersion of the filter in a small volume of methanol (Kishino et al. 1985, 1986). Care was taken not to wash off particles from the filter, even if glass-fiber filters retain rather efficiently filtered material. Both spectra (total particulate matter and non-algal matter) were corrected for the pathlength amplification ("
)] were normalized to the sum of Chl and pheopigments (when present) contents to derive algal Chl-specific spectral absorption coefficients [aph*(
values.
The PB vs. E curves were determined using a radial photosynthetron (described in Babin et al. 1994). Each seawater sample was dispensed into twelve 50-ml subsamples in polystyrene tissue culture flasks inoculated with 2 to 12 µCi of H14 CO3 - and stacked within an irradiance gradient in front of a 250W arc lamp (OSRAM, HQI-T 250 W/D). All samples were exposed to a PAR range going from ca. 15 to 550 µmol quanta m-2 s-1 , except those collected near surface at the OLIGO site which were exposed to ca. 25 to 900 µmol quanta m-2 s-1 . Samples from ten depths were simultaneously incubated for 60 to 120 min; each sample was maintained at a temperature reproducing the in situ temperature (at ± 1 °C).
The Chl-specific initial slope (
B ) and saturation level (PB max) of the PB vs. E curves were derived by fitting to experimental data points a hyperbolic tangent function (Jassby and Platt 1976) with a variable intercept, using the Quasi-Newton algorithm of Systat® statistical package. Photoinhibition was generally observed at high irradiances for samples collected below the euphotic zone at the OLIGO site. The corresponding points were excluded from the curve-fitting procedure.
For determinations of ƒ, 50-100 ml samples from discrete depths were dark adapted for 30 minutes. Fluorescence flash yields were measured in duplicate subsamples with a deck-based fast repetition rate (FRR) fluorometer (described in Greene et al. 1994, Vassielev et al. 1994, and Falkowski and Kolber 1995). Briefly, in the FRR method, PS2 reaction centers are rapidly closed by a train of 5-µs flashlits that cumulatively saturate the photochemical reaction. The rate of closure of reaction centers is used to derive the effective absorption cross section of PS2 by fitting the following expression on fluorescence and energy measurements:
(5)
where Fi is the fluorescence flash yield recorded following the i th flash, Fo is the fluorescence flash yield prior to the FRR protocol, Fv is the maximum variable fluorescence, i. e. (Fm - Fo), and Ij is the energy of the j th flash in the burst train of flashes. The average of 20 sets of measurements on each sample were used to calculate Fo, Fm and
PS2. The average coefficient of variation for the measurements was < 2% on an individual sample, and < 5% on replicate samples from the same station and depth.
The spectral output of the arc lamp used in the radial photosynthetron was determined using a spectroradiometer (LI-COR LI-1800UW) equipped with a cosine collector fixed at the end of a 2-m optic-fiber (see Babin et al. 1994). The spectral composition of the xenon flash tube used in the FRR fluorometer was derived from the manufacturers data (EG&G) and spectral absorption characteristics of the blocking filters (see Kolber and Falkowski 1993). The incubator source includes all PAR wavelengths whereas the FRR flash source has a restricted spectrum centered on 440 nm with half-maximum width of 70 nm. These differences must be accounted for before comparisons between
PS2can be made. For example, a change in the absorption maximum wavelength between different phytoplankton populations without a necessary change in the absorption amplitude, will alter
PS2.. To correct for spectral difference in experimental lamps, measured values of
PS2 were normalized for an ideal "white" spectrum [i. e., with E(
(6a) and
(6b)
where the subscript "white" refers to values for hypothetical white illumination and "exp" refers to experimental values measured on the actual sources (lamp or flash tube). Discussion below is based on
PS2(white), even if
PS2(exp) are used without normalization when calculating fC max (Eq. 1) or the ratio of
PS2 to
(Eq. 7 and Fig. 10 later on).
