Winter pelagic photosynthesis in the NW Mediterranean

ARTICLE IN PRESS

Deep-Sea Research I 52 (2005) 1806–1822 www.elsevier.com/locate/dsr

Winter pelagic photosynthesis in the NW Mediterranean Xose´ Anxelu G. Mora´na,, Marta Estradab a Instituto Espan˜ol de Oceanografı´a, Centro Oceanogra´fico de Xixo´n, Camı´n de L’Arbeyal s/n, E-33212 Xixo´n, Spain Departament de Biologia Marina i Oceanografia, Institut de Cie`ncies del Mar, CSIC, Pg. Marı´tim, 37-49, E-08003 Barcelona, Spain

b

Received 15 November 2004; received in revised form 30 March 2005; accepted 31 May 2005 Available online 10 August 2005

Abstract There is a large gap in our knowledge of winter pelagic primary production in offshore Mediterranean waters, despite the widespread observation of phytoplankton blooming during that season. On two cruises performed in the NW region in March 1999 and January–February 2000, primary production was estimated by means of concurrent in situ incubations and photosynthesis–irradiance (P– E) experiments with the 14C technique. Although the water column was generally well-mixed below the euphotic zone, photosynthetic parameters displayed vertical differences within the mixed layer, indicating that photoacclimation rates were faster than mixing. Their values were similarly variable during both cruises, except for the maximum chlorophyll a-normalized photosynthetic rate ðPBm Þ, which was significantly higher in March. A strong relationship was found between the two methods of estimating integrated primary production rates (log P– E PPint ¼ 0.36+0.87 log in situ PPint; r2 ¼ 0:88, po0:001, n ¼ 18), which ranged from 0.1 to 2 g C m2 d1. March 1999 values (1.070.5 g C m2 d1) were notably higher than in other more intensively sampled periods. Water column chl a-normalized rates were similar on both cruises (mean 8.6 mg C mg chl a1 d1) despite great differences in phytoplankton biomass and composition, but were also positively related to incident irradiance. This finding, together with the marked seasonality of surface photosynthetic parameters obtained in previous surveys in the western Mediterranean, stresses the fundamental role played by irradiance in determining the photosynthetic response during the annual cycle. r 2005 Elsevier Ltd. All rights reserved.

1. Introduction The seasonal variation of phytoplankton in temperate seas is typically characterized by the occurrence of the maximum annual biomass during the so-called spring bloom (e.g. Harris, Corresponding author. Fax: +34 985 308 672.

E-mail address: [email protected] (X.A.G. Mora´n).

1986), when the interaction between availability of light and nutrients and water-column stability promotes the exponential growth of large-celled microalgae. In the Mediterranean Sea, however, the largest annual bloom is frequently found as early as February (Margalef and Castellvı´ , 1967; Estrada et al., 1985; Mura et al., 1996; Ribera d’Alcala` et al., 2004), when the mixed-layer thickness notably exceeds the critical depth as

0967-0637/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.dsr.2005.05.009

ARTICLE IN PRESS X.A.G. Mora´n, M. Estrada / Deep-Sea Research I 52 (2005) 1806–1822

defined by Sverdrup (1953) and a thermocline is not well-established. The causes of the variability in the timing and extent of winter blooms are complex (Townsend et al., 1992, 1994; Waniek, 2003; Backhaus et al., 2003). Recent modelling work in the Adriatic suggests that in addition to the maximum convective depth, deep nutrient pools and mixing rates determine phytoplankton biomass production in a given year (Santoleri et al., 2003). However, most observations of winter blooms in the Mediterranean have been largely limited to biomass or taxonomic accounts (e.g. Estrada et al., 1999; Ribera d’Alcala` et al., 2004), hence hampering further investigations into this phenomenon, supposedly general to all its pelagic ecosystems (Estrada et al., 1985; Duarte et al., 1999). Since input of temporal variations in photobiology is critical for global primary production models (Behrenfeld and Falkowski, 1997), lack of data on the photosynthetic performance of phytoplankton assemblages for this time of the year (Estrada, 1996) probably explains some discrepancies between different satellite-based estimates of primary production for the whole basin (e.g. Bricaud et al., 2002; Morel and Andre´, 1991). Photosynthetic parameters obtained from photosynthesis–irradiance (P– E) relationships at representative depths are a less time-consuming alternative to in situ primary production incubations, and also provide relevant information on the photoacclimation response and physiological state of algal assemblages (Sakshaug et al., 1997; MacIntyre et al., 2002). In a well-mixed water column with high and uniform nutrient concentrations, the initial slope of the P– E curve (aB), the maximum chlorophyll a (chl a)-normalized photosynthetic rate (PBm ) and the saturation irradiance (Ek) should at most display small differences between the surface layer and the bottom of the photic zone (Maran˜o´n and Holligan, 1999; Tilstone et al., 2003). However, rapid physiological adjustments to a varying light regime can alter this expected uniformity in a different fashion for specific parameters (Lewis et al., 1984). For instance, in their work with a diatom culture subjected to irradiance shifts, Cullen and Lewis (1998) showed that aB changes more rapidly than PBm . Other authors have assumed that aB, being a

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function of basic photochemical reactions rather than enzymatic processes (Falkowski and Raven, 1997), tends to behave more conservatively (see refs. in Behrenfeld et al., 2004). P– E experiments have long been used to estimate water-column primary production (e.g. Harrison et al., 1985; Morel et al., 1996; Mora´n and Estrada, 2001). Although the use of artificial light sources may in principle result in systematic differences with the in situ or in situ-simulated methods because of spectral differences with the underwater light field (Kirk, 1994; Laws et al., 1990; Markager and Vincent, 2001), concurrent assessments of both types of approaches are not frequent in the literature (e.g. Harrison et al., 1985; Lohrenz et al., 1997; Kyewalyanga et al., 1992). In this paper, we report on primary production calculated from both in situ incubations and P– E measurements using artificial light during two winter cruises (late January to mid March) in the NW Mediterranean, spanning a wide range of phytoplankton biomass (23–250 mg chl a m2). The goals were to compare the performance of both types of short-term incubations in estimating primary production and to gain knowledge of the photosynthetic response of Mediterranean phytoplankton assemblages under the mixing conditions found in winter. We also attempt to describe the seasonal variation of photosynthetic parameters based on additional data from our previous work in different areas of the western sub-basin.

2. Material and methods The HIVERN cruises were conducted in the Catalano-Balearic Sea (NW Mediterranean) on board R.V. Garcı´a del Cid in March 1999 (HIVERN 99) and January–February 2000 (HIVERN 00). Primary production experiments were performed at six biological stations located in two transects perpendicular to the Catalan coast: stations C, F and D off Barcelona, and PC, PD and PF off Palamo´s (Fig. 1). Some stations were sampled twice during the cruise (Table 1). Vertical profiles of temperature, salinity and fluorescence were obtained at each station from CTD casts. Water samples for biological determi-

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nations were taken at selected depths down to 60 m with Niskin bottles mounted on a rosette attached to the CTD probe. The mixed-layer depth was established, where a density (st) change 42.0

X0.05 kg m3 over a 5 m interval was first attained (Mitchell and Holm-Hansen, 1991). A stratification index (SI) was calculated as per meter difference in st between the surface and 60 m depth. 2.1. Irradiance measurements

PC PF

41.5 Barcelona

C

PD

20 00

0

50

10 00

20 0

Latitude (°N)

F

41.0

D

40.5

40.0

Menorca

Mallorca

39.5 0.5

1.0

1.5

2.0 2.5 3.0 Longitude (°E)

3.5

4.0

4.5

Fig. 1. Map of the NW Mediterranean showing the positions of the sampled stations.

Surface irradiance (E0) was measured continuously by a net radiation sensor (Aanderaa Instruments). In addition, at each station, water-column downward PAR (400–700 nm) was measured 2–3 times per day with a spherical quantum sensor (LiCor LI-193SA), coincident with CTD casts for sampling. Vertical profiles of log2-PAR permitted the calculation of the vertical light extinction coefficients (Kd) for the whole upper water column or for discrete, adjoining layers when abrupt changes in the slope of the plots were observed. The bottom of the euphotic zone (Zph) was established at the 1% isolume and ranged from 33 to 68 m (Table 1). For better assessment of

Table 1 Sampling dates, upper mixed layer and euphotic zone depths (Zuml and Zph, respectively), stratification index (SI), dissolved inorganic  +  nitrogen (DIN ¼ NO 2 +NO3 +NH4 ) and PO4 concentrations at the surface, and chlorophyll a concentration, both at the surface (Chl a0) and integrated down to 60 m depth (Chl aint) of the stations sampled during the HIVERN cruises Station

Date

Zuml (m)

Zph (m)

SI (st m1)

DIN (mmol l1)

1 PO 4 (mmol l )

Chl a0 (mg m3)

Chl aint (mg m2)

C1 C2 F1 D1 PC1 PC2 PF1 PD1

1999 3-Mar 13-Mar 8-Mar 7-Mar 5-Mar 12-Mar 10-Mar 11-Mar

19 73a 4100 4100 80a 78a 4100 17

49 43 54 47 37 33 39 64

5.53  103 1.38  103 9.09  104 4.00  104 2.73  104 1.75  103 4.63  104 5.22  103

— — 5.28 3.78 — — 1.21 2.57

— — 0.08 0.07 — — 0.02 0.06

0.51 4.49 0.76 1.24 3.88 5.01 4.56 1.41

63 165 38 82 162 231 250 44

C1 C2 F1 F2 D1 D2 PC1 PC2 PF1 PD1

2000 31-Jan 9-Feb 30-Jan 8-Feb 29-Jan 7-Feb 6-Feb 10-Feb 2-Feb 1-Feb

81a 77a 4100 4100 16 53 104a 103a 4100 4100

57 67 64 67 68 45 50 56 48 55

3.99  104 3.95  104 1.70  104 7.00  104 8.45  103 4.54  103 4.64  104 2.56  104 8.68  104 1.08  103

3.73 2.50 2.97 4.67 0.60 0.69 3.33 4.33 3.27 4.22

0.07 0.37 0.10 0.22 0.01 0.09 0.15 0.18 0.04 0.09

0.38 2.27 0.45 0.89 1.33 0.72 1.14 0.99 1.28 1.70

23 119 23 25 46 39 62 60 61 67

Station codes as in Fig. 1, with the corresponding figure representing either the 1st or 2nd visit. —not determined. a Station depth. Water column mixed to the bottom.

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vertical differences in photosynthetic performance, optical depths were also calculated (z ¼ zK d ). Optical depth values of 2.3 and 4.6 correspond to 10% and 1% isolumes, respectively.

photosynthetic 14C fixation. No isotope discrimination factor was used. Conversion to carbon units was performed assuming an ambient inorganic C concentration of 25 000 mg C m3.

2.2. Chlorophyll a and nutrients

2.4. Photosynthesis– irradiance (P– E) relationships

Samples (100 ml) for estimating chl a concentration were filtered through Whatman GF/F filters of 25 mm diameter, which were immediately frozen until extraction. Filters were then placed in 90% acetone at 4 1C for 24 h and the fluorescence of the extract measured with a Turner Designs fluorometer (Yentsch and Menzel, 1963). Although measurements were performed before and after acidification, no phaeopigment correction was applied because of great uncertainties in the estimated values, especially in the presence of chl b (Welshmeyer, 1994). Samples for analyzing dissolved inorganic nitro + gen (DIN ¼ NO 2 +NO3 +NH4 ), phosphate and silicate were immediately frozen and their concentrations determined on land with a Technicon autoanalyzer. 2.3. In situ primary production Samples were taken between 07:30 and 09:30 GMT at eight depths from the surface to 60 m. Two light and one dark acid-cleaned polycarbonate bottles were filled with 160 ml water samples from each depth, inoculated with 3.7  105 Bq (10 mCi) of 14C-bicarbonate (VKI, Denmark) and deployed at the sampling depths, attached to a rope hanging from a buoy. The buoy was separated by enough distance from the ship so as to prevent shading. Incubations were started within 1 h upon sampling and lasted for 2 h. Samples were then filtered onto Whatman GF/F filters (25 mm diameter). Filters were placed in 6 ml vials and fumed overnight with 35% HCl. Finally, 4.5 ml of Ready Safe liquid scintillation cocktail were added before determination of disintegrations per minute (dpm) in the laboratory by means of a Beckman LS6500 liquid scintillation counter. Dark bottle dpm, which were consistently low on both cruises (2179% SD of light-bottle values on average), were subtracted for correction of non-

Additional CTD casts were performed between 14:00 and 15:30 GMT for collecting samples for the photosynthesis–irradiance experiments. Although changes in the vertical distribution of phytoplankton were sometimes detected between early morning and midday sampling, a high correlation was found between chl a concentrations in the in situ and P– E samples at coincident depths (r ¼ 0:81, po0:001, n ¼ 36). Samples were taken from the surface (5 m) and at the depth of maximum biomass (15–60 m) in 1999, while in 2000 all experiments were performed with water from 5 and 30 m depth. At each depth, 12 light bottles plus 1 dark bottle were filled with 70 ml of water, inoculated with 3.7  105 Bq 14C-bicarbonate, and incubated for 2 h in a linear incubator refrigerated with circulating surface seawater. Quartz halogen lamps were used as the light source. Irradiance levels at each position (range 2–2000 mmol photons m2 s1) were measured after each experiment with a cosine quantum sensor (Li-Cor LI-190SZ). Intercalibration with the LI-193SA underwater sensor used for in situ measurements was made in order to avoid biases due to the use of different sensors. Treatment of samples and conversion into carbon units was done in the same way as for the in situ incubations. The model of Platt et al. (1980) was fitted to hourly carbon production rates normalized by chl a from the experiments (most of them) in which photoinhibition was detected. The simpler model of Webb et al. (1974) was used in the absence of photoinhibition. The photosynthetic parameters considered hereafter are: the maximum or lightsaturated chl a-normalized photosynthetic rate PBm (mg C mg chl a1 h1), the initial slope of the P– E relationship aB [mg C mg chl a1 h1 (mmol photons m2 s1)1], the photoinhibition parameter bB (same units as aB) and the light

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saturation parameter or saturation irradiance Ek (mmol photons m2 s1). 2.5. Integrated primary production The euphotic zone was significantly shallower (ttest, p ¼ 0:02, n ¼ 18, Table 1) in March 1999 than in January–February 2000, partly because of the higher phytoplankton biomass during the former year (see further). Although on a few occasions the euphotic zone was deeper than 60 m, our deepest in situ measurements were systematically performed at that depth, so we estimated integrated or areal primary production rates by trapezoidal integration down to 60 m for both methods. In order to estimate daily rates, in situ hourly rates were multiplied by a factor that was the average of daylength in hours according to Straskraba and Gnauck (1985) and the ratio of daily irradiance to that received during each incubation. Mean7SD values of that factor were 9.070.2 h in 1999 and 8.070.3 h in 2002, similar to the empirical factor used (8.8 h) in previous work (e.g. Estrada, 1981).

Daily primary production was also estimated from P– E relationships as described in Mora´n and Estrada (2001), using the vertical profiles of chl a and mean hourly irradiance and linear interpolation between surface and deep values of photosynthetic parameters. No correction was applied for phytoplankton respiration at night.

3. Results 3.1. Hydrography and phytoplankton The water column was generally well-mixed. As an example, Fig. 2 shows that both temperature and salinity differed little (o4%) between the surface and 150 m in the different visits to station F, precluding significant density stratification. As a consequence, except for specific instances, the upper mixed layer either reached the bottom of the shelf stations C and PC or was deeper than 100 m (Table 1). The shallower mixed layers found at stations C1 and PD1 in 1999 were due to the presence of surface waters of low salinity (o38),

Salinity

Temperature

Chlorophyll a

12 0

13

In situ primary production

(mg m-3)

(°C) 37

38

0

1

(mg C m-3 h-1) 0

1

2

20

Depth (m)

40 60 80 100 120

08-Mar-99 20-Jan-00 08-Feb-00

140 Fig. 2. Vertical profiles of temperature, salinity, chl a concentration and in situ primary production rates during the three visits to station F.

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of integrated chl a (chl aint) were consistently mirrored at the surface (chl a0), as shown in Fig. 3 (r ¼ 0:96, po0:001, n ¼ 18). Chl a0 frequently exceeded 1 mg m3 on both cruises (Table 1). Differences between years in both chl a0 amd chl aint were significant (t-test, p ¼ 0:02, n ¼ 18), although the vertical distribution was similar during both periods. Chl a was generally detectable several 10s of m below the euphotic zone (Fig. 2). On average, 64713% (SD) in 1999 and 6078% of chl a in the 0–60 m depth range accumulated in the upper 30 m. This percentage was significantly correlated with SI (r ¼ 0:52, p ¼ 0:028, n ¼ 18). The increase in phytoplankton biomass in the water column decreased the depth of the euphotic zone from 70 to less than 40 m (Fig. 3). 3.2. Photosynthetic parameters Mean7SD values of surface and deep photosynthetic parameters for both periods are presented in Table 2. The experiment at station PC1 was not considered because of malfunctioning of the recirculating water system. Overall, maximum photosynthetic rates ðPBm Þ ranged from 0.8 to 3.9 mg C mg chl a1 h1 and, for each individual station, tended to be higher at the surface (13 out of 17 experiments). The initial slope of the P– E curve (aB) was slightly more variable, from 0.006 to 0.032 mg C mg chl a1 h1 (mmol photons m2 s1)1. Half of the experiments in 1999 displayed higher values of aB at the surface,

70 5 60

4 3

50

2

40

1 0

30 0

50

100 150 200 250 Integrated chl a (mg m-2)

Euphotic zone depth (m)

6 Surface chl a (mg m-3)

whereas in 2000 a marked thermohaline stratification persisted in the two visits to station D. In spite of marked differences in hydrography (see further) between years, SI was similarly low for both periods (t-test, p40:05, n ¼ 18). As a consequence of deep winter mixing, nutrient-sufficient conditions for phytoplankton growth were generally found at all sampling depths (surface values shown in Table 1). Average surface concentrations of DIN were 3.270.9 mmol l1 in 1999 and 3.070.5 mmol l1 in 2000, and either held constant or increased deeper in the water column. Surface PO4 concentrations were seldom below 0.05 mmol l1, with an average value of 0.0670.01 mmol l1 in 1999 and 0.1370.04 mmol l1 in 2000. At the beginning of the 1999 cruise, a large anticyclonic eddy (80 km in diameter) of Modified Atlantic Water occupied the southwestern part of the study area (Pascual et al. 2002). High phytoplankton biomass (4100 mg chl a m2), associated with the presence of diatoms (mainly Chaetoceros spp., Asterionellopsis glacialis and Pseudo-nitzschia spp.), developed near the coast and in the northern part of the study area. The anticyclonic eddy contained lower chl a and was dominated by haptophytes (including Emiliania huxleyi and other coccolithophores) and small autotrophic flagellates (Estrada et al. unpublished). When the biological stations (Table 1) were visited, the eddy had moved northeast, so that the sampling area was occupied by the diatom assemblage. In contrast, the hydrographic scenario in 2000 was typical for the winter period, with a well-marked salinity front close to the F station and a doming of isopycnals approximately midway between the Iberian coast and the Balearic Islands (data not shown). During most of the cruise, algal biomass, dominated by small dinoflagellates, coccolithophores such as E. huxleyi, other haptophytes and flagellates, was relatively low and integrated values did not exceed 70 mg chl a m2. High chl a values and a diatom-dominated community (with Chaetoceros spp., Pseudonitzschia spp. and Thalassiosira spp.) were found only on the second visit to station C (Table 1), presumably as a consequence of the development of the winter phytoplankton bloom. High values

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300

Fig. 3. Integrated chlorophyll a vs. surface values (filled symbols) and euphotic zone depth (open symbols) for all data pooled.

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Table 2 Mean (7SD) photosynthetic parameters in surface (5 m) and deep (15–60 m) samples of the two cruises PBm

aB

bB

Ek

Surface Deep Jan–Feb 2000

2.3270.76 1.7770.57

0.01370.005 0.01570.007

070.0001 0.000870.0012

2047114 127748

Surface Deep

1.6470.55 1.5670.48

0.01270.001 0.01770.006

0.000270.0003 0.001870.0016

154790 97735

Overall CV

35%

39%

167%

57%

Mar 1999

PBm: maximum chlorophyll a-normalized photosynthetic rate (mg C mg Chl a1 h1), aB: slope of the P– E relationship [mg C mg Chl a1 h1 (mmol photons m2 s1)1], bB: photoinhibition parameter (same units as aB), Ek: light saturation parameter (mmol photons m2 s1). Also shown the coefficients of variation (CV) of each parameter for all data pooled.

whereas it was always higher at 30 m than at the surface in 2000. The light saturation parameter (Ek) was quite variable, ranging from 52 to 442 mmol photons m2 s1. Ek was significantly correlated with the mean irradiance received during the day at the sampling depth (r ¼ 0:48, p ¼ 0:003, n ¼ 36). Photoinhibition was commonly observed in deep samples but only on a few occasions at the surface. bB was consistently higher at depth than at the surface of individual stations. This difference increased with water-column stability, as shown by the correlation (r ¼ 0:52, p ¼ 0:03, n ¼ 17) between the per meter change in bB (i.e. the difference between the surface and deep values at each station divided by the depth interval) and SI. For these reasons, the photoinhibition parameter bB had the highest coefficient of variation (167%, Table 2). In contrast with other studies (e.g. Basterretxea and Arı´ stegui, 1999; Mora´n and Estrada, 2001; Behrenfeld et al., 2004), no correlation was found between PBm and aB either for the whole data-set or the 1-year subsets. Nutrient concentrations were not related to changes in any of the parameters. Both the sampling period, characterized by different means of total incident irradiance, and sampling depth could affect photosynthetic parameters. Two-way ANOVAs were conducted with the pooled data-set with year and depth (surface vs. deep) as factors. PBm was slightly but significantly higher in March 1999 (F ¼ 4:77, p ¼ 0:037, df ¼ 30) whereas both Ek and bB were significantly different in surface and deep samples, higher

(F ¼ 6:73, p ¼ 0:015) and lower (F ¼ 10:98, p ¼ 0:002), respectively, at the surface. Interaction effects were not significant for any of the variables. Since the maximum sampling depth differed among cruises (see Material and Methods), photosynthetic parameters were also plotted against optical depth for between-year comparisons (Fig. 4). Optical depth affected the parameters in a different fashion. In 1999, only PBm was significantly and negatively correlated with optical depth (r ¼ 0:53, p ¼ 0:03, n ¼ 16) while bB significantly increased with optical depth in 2000 (r ¼ 0:71, p ¼ 0:001, n ¼ 18). Paired t-tests between values at 5 and 30 m in 2000 showed significant differences for b (p ¼ 0:009), aB (p ¼ 0:007) and Ek (p ¼ 0:04). There were no systematic changes in watercolumn stability at the stations that were sampled twice (see Table 1); hence no pattern was observed in absolute values or vertical relationships of photosynthetic parameters between visits. 3.3. In situ primary production Volumetric hourly rates of primary production obtained from in situ incubations (Fig. 2) usually peaked at the surface on both cruises (maxima of 11.8 and 7.6 mg C m3 h1, respectively), with the exception of stations D1 and C1 in 1999, which showed distinct maxima of biomass and production at 20–25 m. In 2000, maxima were found at 5

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0

0.01

αB 0.02 0.03

P Bm 0.04

0

1

2

3

0

0.002

1813

βB 0.004

0.006

0

Optical depth

2

4

6

8 Mar '99 Jan-Feb '00 10 Fig. 4. Optical depth distribution of the photosynthetic parameters aB [mg C mg chl a1 h1 (mmol photons m2 s1)1], PBm (mg C mg chl a1 h1 (mmol photons m2 s1)1]) and bB (same units as aB) for the two periods. Dashed lines represent significant correlations for March PBm data and January–February bB data. More details are given in the text.

or 10 m at all stations. Overall, 9379% and 8379% of primary production occurred in the first 30 m of the water column in 1999 and 2000, respectively. A marked variability was observed on the successive visits to the stations that were sampled twice. In 1999, an increase in biomass was accompanied by an increase in productivity at station C but not at PC. In contrast, during 2000, changes in biomass were always reflected in productivity with the highest difference observed at station C, in agreement with a 5-fold increase in algal biomass (Table 1). 3.4. In situ vs. P– E-derived estimates of daily primary production The two approaches for estimating watercolumn primary production yielded notably similar results. First, we compared chl a-normalized in situ hourly rates with the corresponding rates obtained with photosynthetic parameters and irradiance levels at the sampling depths of the

P– E experiments. Values ranged from 0.7 to 3.9 mg C mg chl a h1 near the surface and were generally o1 mg C mg chl a h1 deeper in the water column. In situ and P– E-derived estimates were significantly correlated (r ¼ 0:68, po0:001, n ¼ 36; r ¼ 0:82 with one outlier excluded). Despite the difference in sampling times for the two types of experiments, the slope of the corresponding model II linear regression was not significantly different from 1 (t-test, p ¼ 0:28). A paired t-test did not show significant differences between the two estimates of assimilation numbers (p ¼ 0:16, n ¼ 36). Estimates of integrated daily rates of primary production (PPint) using photosynthetic parameters and diel variations of irradiance were also highly correlated with those derived from in situ experiments (r ¼ 0:94, po0:001, n ¼ 18, Fig. 5). Moreover, differences between the two estimates were not significant (paired t-test, p ¼ 0:85, n ¼ 18) and the slope of the model II linear regression (log P– E PPint ¼ 0.36+0.87 log in situ

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1000

Mar '99 Jan-Feb '00 100 100

Integrated PP (mg C m-2 d-1)

(mg C m-2 d-1)

P-E primary production

2000

1500

1000

500

0

1000 In situ primary production

Mar '99 Jan-Feb '00 0

50

(mg C m-2 d-1)

Fig. 5. Relationship between in situ and P– E-derived daily rates of integrated primary production. Dashed line represents the model II linear regression given in the text. Solid line corresponds to the 1:1 relationship.

Daily primary production usually exceeded 1 g C m2 d1 during March 1999 (Fig. 5), with a maximum of 1.7 g C m2 d1 at station PF1. In the earlier sampling of 2000, values were considerably lower, except at station C2, where 1 g C m2 d1 was also reached. Mean7SD values for the two cruises were 10007471 and 4047 248 mg C m2 d1, respectively. A high correlation was obtained between integrated values of chl a and PP (Fig. 6, r ¼ 0:89, po0:001, n ¼ 18). A common areal assimilation number for both cruises could be estimated either as the slope of the model II linear regression (6.67 mg C mg chl a d1, Fig. 6) or by averaging data (8.627 0.85 SE mg C mg chl a1 d1). The latter values were significantly correlated with SI (r ¼ 0:60, p ¼ 0:008, n ¼ 18) indicating that, although stratification of the euphotic layer was seldom achieved, a decrease in the mixing rate increased photosynthetic efficiency. Mean daily water-column-integrated production to biomass (P:B) ratios

150

200

250

300

Fig. 6. Relationship between integrated chl a and P– E-derived integrated primary production. Dashed line represents the model II linear regression: PPint ¼ 91.17+6.67 chl aint; r2 ¼ 0:79, po0:0001, n ¼ 18.

PPint) was not significantly different from 1 (95% confidence limits 0.73–1.04, Ricker, 1975). Therefore, only P– E-derived estimates of PPint will be considered hereafter.

0.4

Mar '99 Jan-Feb '00

0.3 P:B ratio

3.5. Between-year comparisons and relationships with biomass

100

Integrated chl a (mg m-2)

0.2

0.1 0

0

10

20

30

40

Surface irradiance (mol photons m-2 d-1) Fig. 7. Relationship between the production to biomass (P:B) ratio using a C:chl a ratio of 50 and surface incident irradiance.

estimated with a C:chl a ratio of 50 were 0.1970.10 and 0.1670.05 in 1999 and 2000, respectively. P:B ratios and daily incident irradiance at the surface were significantly correlated (r ¼ 0:56, po0:016, n ¼ 18, Fig. 7).

4. Discussion In spite of our improved capability of remotedetection of chlorophyll a, field measurements of phytoplankton photosynthetic properties are still

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needed in many areas of the world ocean in order to refine existing bio-optical models. A critical aspect is the temporal and vertical variation of those properties in water columns characterized by substantially different physico-chemical conditions over the year, alternating between periods of mixing and stratification. We are aware of only a few papers reporting on photosynthetic parameters in the Mediterranean Sea (Videau et al., 1994; Mora´n et al., 2001; Mora´n and Estrada, 2001; Ignatiades et al., 2002; Marty and Chiave´rini, 2002), and none of them has specifically addressed winter values. Results reported here should be useful in future revision of current models attempting to resolve the seasonal cycle of primary production in the basin (e.g. Bosc et al., 2004). 4.1. Photosynthetic parameters Given the relative uniformity of the thermohaline distributions, with a water column generally well-mixed to a depth greater than that of the euphotic zone (Fig. 2, Table 1), photosynthetic parameters were not expected to show either high variability or vertical patterns (e.g. Dower et al., 1996; Videau et al., 1994). However, noticeable variability was shown by all parameters in this study (Table 2), and strikingly, the 5-fold change in both aB and PBm equalled the variation measured in strongly stratified waters of the subtropical North Atlantic (Bouman et al., 2000). aB and PBm are functions of light and dark biochemical reactions, respectively (Falkowski and Raven, 1997), and the former is still frequently regarded as more constant based on photochemistry considerations (Behrenfeld et al., 2004), in spite of its demonstrated plasticity (Cullen and Lewis, 1988). The slightly higher coefficient of variation for the initial slope than for the maximum photosynthetic rate (Table 2) confirms previous findings (Coˆte´ and Platt, 1983; Kyewalyanga et al., 1998) and indicates that algae may respond to environmental constraints with changes in both parameters alike. The above-mentioned variability of the photosynthetic parameters was not random, but related to photoacclimation processes. The strong photo-

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inhibition in deep samples during January–February 2000 (Fig. 4) should be interpreted with caution because of the possibility that exposure of the samples during 2 h of incubation to irradiances that exceeded Ek values by an order of magnitude (Table 2) could have induced an artefactual response (Macedo et al., 2002). However, surface populations subjected to the same incubation conditions showed few photoinhibition effects, indicating a vertical differentiation in physiological properties. Moreover, vertical patterns of aB and PBm (Fig. 4), showing no clear trend or decreasing with depth, respectively, caused a significant decrease of Ek between the surface and greater depths in the whole data-set, indicating that deeper phytoplankton responded to in situ PAR levels. Variability of P– E relationships of Mediterranean phytoplankton during winter would thus fall into the ‘‘Ek-dependent’’ category recently coined by Behrenfeld et al. (2004). With a vertical turbulent diffusion coefficient (Kz) of 105 m2 s1, typical for the Mediterranean mixing layer in winter in the absence of deep convection (Mikhail Emelianov, pers. comm.), according to Denman and Gargett (1983) several days would be needed for a phytoplankton vertical displacement of the order of 10 m. While significant photoacclimation would certainly occur, thermohaline stratification is not necessarily achieved at this time scale. Although the water column was fairly well-mixed during both surveys, as expressed by the low SI values (Table 1), the enhanced shade acclimation during March 1999, detected as a significant decrease in PBm with depth (Falkowski, 1981), could be linked to relatively slower mixing rates. All in all, high mixing and low absolute irradiances could increase the relevance of the light-limited portion of the P– E relationship during winter. Our results showed overall higher values of aB when compared with studies performed from spring to autumn in other Mediterranean sites (see below). Physiological adjustments undertaken by mixed populations in response to a varying light regime do not necessarily imply equal rates of change for all parameters (Lewis et al., 1984; Cullen and Lewis, 1988). We hypothesize that PBm is the first parameter to respond vertically in a coherent fashion as thermal stratification

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progresses, causing physical separation of deep populations from the upper, well-illuminated layers. In two surveys performed later in the year in the SW Mediterranean, both aB and PBm showed distinct vertical patterns. Interestingly, although PBm decreased significantly with depth in both periods, aB values were higher at the surface in May (Mora´n and Estrada, 2001) while the opposite situation was found in October (Mora´n et al., 2001). Only with a strong physical stability of the water column promoting constant and different growth conditions for upper and deeper layers, can aB be significantly greater at depth (e.g. Maran˜o´n and Holligan, 1999; Bouman et al., 2000; Lorenzo et al., 2004).

winter populations to capture the comparatively low PAR levels experienced in a mixed water column. Conversely, winter PBm values were 0.020 (A) 0.015


B

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0.010

0.005

0.000 (B)

7

4.2. Seasonal variability of photosynthesis

P Bm

5

Fig. 8. Seasonal variability of surface chl a-normalized maximum photosynthetic rate ðPBm Þ, initial slope of the P– E relationship (aB), saturation irradiance (Ek) and incident irradiance (E0) on different cruises performed in the W Mediterranean from 1996 to 2000. Units are given in the text. Additional data from cruises MATER 1 (May 1998, Mora´n and Estrada 2001), MATER 3 (May 1998), ARO (July 2000), MATER 2 (September 1999) and ALGERS (October 1999, Mora´n et al. 2001). Dashed lines represent smooth fits to data.

4 3 2 1 0 1200

(C)

1000

Ek

800 600 400 200 0 (D)

60 50 E0

With the exception of PBm , the photosynthetic response was similar during the two surveys (Table 2). However, winter photosynthetic parameters largely differed from results obtained later in the year. Mean7SE values of photosynthetic parameters measured by us at the surface of several western Mediterranean sites during recent years (1996–2000) were plotted together for comparison (Fig. 8). Despite very large differences in hydrographic conditions and nutrient fluxes (e.g. Mora´n and Estrada, 2001; Mora´n et al., 2001), a notable seasonal pattern emerged. Thus, aB was highest in autumn and winter and lowest close to the summer solstice (Fig. 8A). Temporal variations have sometimes been suggested (e.g. Finenko et al., 2002) but in general no seasonality has been reported for this parameter (Tillmann et al., 2000; Shaw and Purdie, 2001). It is likely that the 42-fold variation of aB found in our results reflects an adaptation of autumn and

6

40 30 20

0

50

100

150 200 250 Day of year

300

350

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significantly lower than those found later in the year (Fig. 8B), as shown for other regions (Karl et al., 2002; Tillmann et al., 2000; Finenko et al., 2002). The variability of PBm during spring and summer agrees with the pattern reported by Estrada et al. (1993) for the water-column maximum productivity index in the Catalano-Balearic Sea, which increased from mid-to-late spring and decreased again in summer. Although temperature, which in principle should be associated with higher growth rates, explained up to 74% of the variance of PBm during short periods for Black Sea samples (Finenko et al., 2002), the variability shown in Fig. 8B appears to be larger than expected considering the typical temperature change from winter to late spring (13–20 1C) and the Q10 relationships proposed by Eppley (1972) and Raven and Geider (1988). There are also marked seasonal changes in size and taxonomic composition of the phytoplankton assemblages. For instance, the microalgal (420 mm) fraction represented 6% of integrated biomass in the Catalano-Balearic Sea in May 1989 and 55% in February 1990, whereas the o5 size-fraction contributed 67% and 27%, respectively (Delgado et al., 1992). Seasonal changes of the nano- and micro-phytoplankton (hereafter referred to as phytoplankton) composition in the study area were studied by Estrada (1999). The winter bloom was characterized by a diatom assemblage with strong contribution of Chaetoceros spp., as in the present case. After the winter bloom, diatoms declined and the phytoplankton community became dominated by small dinoflagellates, haptophytes (including coccolithophorids and Phaeocystis sp.) and other flagellates. From spring to summer, with increasing stratification, there was a shift towards a higher contribution of dinoflagellates with respect to that of haptophytes. In general, smaller size and larger surface to volume ratio have been associated with higher growth rates, although the degree of reported dependence varies depending on author (Banse, 1982; Tang, 1995). In addition, diatoms tend to present higher growth rates than dinoflagellates (Brand and Guillard, 1981; Chan, 1980). Changes in species composition from relatively large to small cells could have contributed to the spring peak shown

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in Fig. 8B, but other factors should be taken into consideration. Low PBm under high nutrient and low irradiance conditions could simply be a consequence of a higher chl a content per cell. Seasonal variability of the C:chl a ratio has long been recognized (e.g. Taylor et al., 1997) with maxima in summer and minima in winter, while for groups such as cyanobacteria, an increase in cell-specific chl a fluorescence with almost no change in size has been found during autumn and winter (Worden et al., 2004). As a derived parameter, the seasonal trend was also evident for Ek (Fig. 8C). As stated above, many potentially explanatory environmental factors such as irradiance, temperature or water-column stability present marked seasonal trends. However, neither nutrient nor species composition were able to explain the variability in the mean surface values of photosynthetic parameters shown in Fig. 8. Although nutrient status is known to affect C:chl a ratios (Geider et al. 1997) and hence indirectly PBm and aB, its proxies, seawater DIN or PO4 concentrations, did not show significant relationships with any parameter (p40:25), while similar phytoplankton assemblages were characterized by largely different parameters on the different cruises. In contrast, total daily irradiance at the surface (E0, Fig. 8D) strongly covaried with the initial slope aB and the saturation irradiance Ek with respective correlation coefficients of 0.89 and 0.90 (po0:008, n ¼ 7). When we considered individual data rather than mean values, in addition to the above-mentioned correlations, PBm was significantly correlated with temperature (r ¼ 0:51, po0:001, n ¼ 46) but strikingly showed a negative correlation with DIN (r ¼ 0:41, p ¼ 0:012, n ¼ 37), which points to a complex environmental determination of maximum photosynthetic rates. In conclusion, when within-cruise measurements are averaged, surface photosynthetic parameters seem to be greatly and coherently determined by incident PAR, in agreement with recent studies (Tilstone et al., 2003; Smyth et al., 2004), contradicting previous claims of the smaller effect of irradiance vs. other factors such as nutrient concentrations or phytoplankton community composition (Geider, 1993).

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4.3. In situ vs. P– E-derived primary production The validity of estimating primary production from P– E experiments has sometimes been questioned because of the failure of the light sources employed in the incubations to simulate ambient spectral characteristics (Laws et al., 1990; Markager and Vincent, 2001), although comparisons between in situ and P– E-derived estimates are not frequent in the literature. Together with studies performing such comparisons (Lohrenz et al., 1997; Riegman and Colijn, 1991; Platt and Sathyendranath, 1988; Harrison et al., 1985), our results show that the constant light spectrum provided by halogen lamps was not critical for estimating daily rates. The absence of significant differences between the two estimates, either for volumetric or integrated measurements (Fig. 5), do not support earlier statements of marked underestimations by the P– E method compared to in situ incubations (Laws et al. 1990), especially at low irradiance depths (Harrison et al., 1985). We believe that the absence of a winter deep chlorophyll maximum (DCM) rendered errors of the broad-band (PAR) approach used here of minor importance in comparison to spectrally resolved assessments (Tilstone et al., 2003; Kyewalyanga et al., 1992), although Lorenzo et al. (2004) recently questioned the need for spectral resolution even with a well-developed DCM. Other possible errors, due to bottle confinement, incubation time (Macedo et al., 2002), diel changes in physiological conditions or vertical distribution of algal biomass (Mora´n and Estrada, 2001), are common to both 14 C uptake methods and at present can be circumvented only by instantaneous approaches such as those based on in vivo fluorescence (Kolber and Falkowski, 1993; Smyth et al., 2004). According to the present results, P– E experiments performed with samples from two selected depths appear as a reliable and logistically convenient way of estimating primary production with the 14C method, in addition to providing insight into the physiological state of algal assemblages and their response to environmental fluctuations. Since vertical differentiation of photosynthetic parameters became apparent even under mixing conditions (Fig. 4), we should no

longer consider measurements of photosynthesis at a single depth (e.g. Dower and Lucas, 1993) as representative of the whole upper mixed layer. 4.4. Winter primary production in the NW mediterranean The observations of high primary productivity presented here (up to 2 g C m2 d1) coincide with the period of maximum algal biomass during the annual cycle (Estrada et al. 1999). Winter phytoplankton blooms were first reported for the Catalano-Balearic Sea by Margalef and Castellvı´ (1967) and are a common occurrence in the whole Mediterranean basin (Ribera d’Alcala` et al., 2004). This feature contrasts with the ‘‘spring bloom’’ of many temperate regions and may be related to the climatic conditions in the Mediterranean, which resemble those of the subtropical belt. High levels of phytoplankton biomass and production in the absence of thermal stratification, also observed in other regions (e.g. Townsend et al., 1992; Keller et al., 2001), could be explained by sustained periods of calm weather limiting vertical mixing in isopycnal water columns (Townsend et al., 1994), associated in the Mediterranean with a westward migration of the Siberian high-pressure system (Duarte et al., 1999). In this connection, although a stratified water column was observed only on three occasions (Table 1), enhanced areal assimilation numbers were consistently observed at increasing SI values. The generally assumed oligotrophy of the Mediterranean Sea, although more marked in its eastern sub-basin (Gotsis-Skretas et al., 1999; Ignatiades et al., 2002), corresponds to modelled estimates of primary production usually well below 500 mg C m2 d1 (Morel and Andre´, 1991; Bricaud et al., 2002; Bosc et al., 2004), similar to open-ocean subtropical waters. The high primary production values reported here, characteristic of meso- to eutrophic conditions (e.g. Morel et al., 1996; Keller et al., 2001; Tilstone et al., 2003), probably contribute to the relatively high fisheries yield of the NW Mediterranean, not fully explained by the primary production data-set available to date (Estrada, 1996). Prior to this work, no value greater than 900 mg C m2 d1 had been

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reported for the repeatedly sampled transect between the Catalan coast and the Balearic Islands (stations C, F and D). Some researchers have expressed their concern with the emphasis usually on chl a rather than on photobiology determinations in satellite-based models of primary production (Behrenfeld and Falkowski, 1997; Sakshaug et al., 1997; Maran˜o´n and Holligan, 1999). Underrepresented winter measurements in available data-sets may also help explain the failure of some of these models to reproduce the peaks of primary production in winter (Bricaud et al., 2002), in clear disagreement with the scarce in situ measurements reporting coincident maxima of production and biomass (Conan et al., 1998; Marty and Chiave´rini, 2002). Total phytoplankton biomass was tightly correlated with primary production for all data pooled (Fig. 6), indicating the utility of chl a as a productivity proxy over narrow temporal ranges (Coˆte´ and Platt 1983, Malone et al. 1986, Keller et al. 2001). In spite of large differences in chl a (11fold) and community composition during the sampled periods, algal assemblages appeared to be able to integrate environmental fluctuations by showing a remarkably constant areal assimilation number (8.62 mg C mg chl a d1, cf. the slope of the regression in Fig. 6). However, this overall constancy was further modulated by incident irradiance. Estimated P:B ratios increased from 0.1 to 0.4 following a 4-fold increase in incident irradiance due to variations in cloud cover and sampling periods (Fig. 7). At daily to weekly scales, variability of incident irradiance has been demonstrated to strongly affect primary production measurements in coastal waters (Falkowski, 1981; Kelly and Doering, 1997). A stepwise multiple regression with integrated primary production rate (PPint) as the dependent variable included phytoplankton biomass (chl aint), daily irradiance (E0) and stratification index (SI) as significant variables, altogether explaining 91% of its variability: PPint ¼  388:2 þ 6:7chl aint þ 15:6E 0 þ 43676:7SI; r2 ¼ 0:91; po0:0001; n ¼ 18. The moderate increase in mean incident irradiance observed from January–February to March

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(Fig. 8D) was not followed by any significant change in nutrient concentrations, which remained high throughout the sampled period; therefore, neither DIN nor PO4 were included in the regression model. These results illustrate the major role played by irradiance in determining the photosynthetic response of Mediterranean phytoplankton, and specifically in the development of winter blooms once the standing stock begins to accumulate in the upper layers as a consequence of vertical mixing relaxation.

Acknowledgments We thank the crew and others on board the R.V. Garcı´a del Cid for their help during the cruises. Comments by A´ngel Lo´pez-Urrutia and Emilio Maran˜o´n on an earlier version of the manuscript, as well as those of two anonymous referees, are greatly appreciated. This research was supported by the Spanish CICYT Grant MAR980932.

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