Vertical partitioning of phosphate uptake among picoplankton groups in the P-depleted Mediterranean Sea

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Introduction
Understanding nutrient uptake strategies in microorganisms is a necessity to predict their biogeochemical response to environmental changes.Heterotrophic (Hprok) and phototrophic prokaryotes (cyanobacteria) dominate the planktonic biomass in oligotrophic areas of the surface ocean and account for most of the carbon fluxes through the microbial loop (Azam et al., 1983).The Sargasso and the Mediterranean Figures Sea seasonally undergo phosphate (P i ) deficiency relative to living organisms demand, with nanomolar concentrations of P i and P i turnover times as low as minutes or hours (McLaughlin et al., 2013;Moutin et al., 2002;Sebastián et al., 2012;Thingstad et al., 1998;Wu et al., 2000).Phosphate nutrition is preferentially achieved by harvesting orthophosphate (P i ) in most osmotrophs, but recent studies show that dissolved organic phosphorus (DOP) can be a significant source of P as well, particularly in its most labile forms like ATP (e.g.Björkman et al., 2012;Casey et al., 2009;Duhamel et al., 2012;Glibert et al., 2004;Lomas et al., 2010;Sebastián et al., 2012;Fu et al., 2006;Björkman and Karl, 1994).It is now accepted that P i uptake capacities of a microbial community are mostly influenced by the degree of P i limitation in the environment.Along with an increased stratification and oligotrophication of the surface ocean, a widely spread size-shift in the structure of phytoplankton communities is expected, from nano-and micro-eukaryotes to pico-sized cells, as observed in the North Pacific Subtropical Gyre by Church et al. (2002) and Karl et al. (2001).How phytoplankton and bacteria share P resources when they are poorly available has been debated for over 30 years.The existence of different P i acquisition systems in microorganisms was highlighted in studies where Hprok systems were found to be saturating at much lower P i concentrations than phytoeukaryotes (> 3 µm: Currie et al., 1986;Currie and Kalff, 1984).Some eukaryotes may develop mixotrophic capabilities and realize a significant fraction of their P from preying on P-richer prokaryotes (Christaki et al., 1999;Hartmann et al., 2011) and from DOP hydrolysis induced by ectoenzymes (ATPases, alkaline phosphatase, e.g.Webb, 1992).Eukaryotic phytoplankton may compensate their low affinity for the substrate at low concentrations (high Km) with high P i storage capacity (Cotner and Biddanda, 2002) compared to prokaryotes and a more efficient growth mechanism, i.e. a low half-saturation constant for growth (Ks, Rhee, 1973).P i uptake by microbes in natural environments has largely been assessed using size fractionation.Studies concur on the high contribution of the small size fractions (< 0.8, < 1, < 2 or < 3 µm) to P i uptake fluxes (Björkman and Karl, 1994;Currie et al., 1986;Moutin et al., 2002;Tanaka et al., 2003;Thingstad et al., 1998, Figures Back Close 1993).This contribution generally increases in aquatic systems with short P i turnover times and increases in low-P i systems after P amendments, emphasizing the idea that heterotrophic prokaryotes are high competitors in P-limited areas (e.g.Björkman et al., 2012;Currie et al., 1986;Drakare, 2002;Labry et al., 2002).However, size fractionation offers a limited resolution of microbial processes, especially in oligotrophic environments where osmotrophs are small and where taxonomic and functional types overlap in size.The development of combined radiolabeling techniques and cell sorting by flow cytometry has improved the level of resolution to study P i uptake strategies in heterotrophic and phototrophic microbes, as shown by at least 6 articles published since 2007 (Casey et al., 2009;Michelou et al., 2011;Talarmin et al., 2011;Zubkov et al., 2007;Björkman et al., 2012;Duhamel et al., 2012).
When looking into the contribution of picoplanktonic groups to total P i uptake, prokaryotes are better competitors than eukaryotes, and among prokaryotes, taxonspecific uptake rates of P i are significantly higher for cyanobacteria compared to Hprok.Nevertheless, the comparison among those studies is not always possible, due to methodological differences (fixation, inducing P i leakage) or to different targeted groups.Finally, only one study suggests the adaptation of microorganisms to P amendments (Björkman et al., 2012), with concentration kinetics experiments providing useful insights into their coexistence in P-deficient environments.
The present study investigates the contribution of sorted picoplankton groups to total P i uptake flux in the P i -depleted surface waters of the stratified Mediterranean Sea (down to 200 m).An estimation of kinetic constants (maximum P i uptake rate-V max , and the sum of the half-saturation constant plus P i natural concentration K + S n ) of 33 P i -radiolabeled orthophosphate uptake in sorted Hprok, cyanobacterial and picophytopanktonic cells is also provided to compare competitive abilities of the groups at different depths and under different ambient P i concentrations.In spite of the amount of phosphorus-related studies conducted in the Mediterranean Sea, such experiments have never been carried out in this system.Our results contribute to Introduction

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Full further understand how prokaryotic autotrophs and heterotrophs share P i resources when the phytoplankton biomass is dominated by prokaryotes.

Study sites and collection
An east-west transect across the Mediterranean Sea was realized during the BOUM cruise (Biogeochemistry from Oligotrophic to Ultraoligotrophic Mediterranean) on the French R/V L'Atalante from 16 June to 20 July 2008 (Fig. 1).Samples were collected using 12 L Niskin bottles mounted on a rosette equipped with a conductivity-temperature-density (CTD) profiler and sensors for pressure, oxygen, photosynthetically available radiation (PAR), and chlorophyll fluorescence.P i uptake and turnover time measurements reported in this study were fully processed onboard.Total chlorophyll a (TChl a) concentrations were determined as described in Crombet et al. (2011).Critical depths and integrated nutrient concentrations were calculated as described in Moutin and Prieur (2012).P i turnover times were measured at each of the 33 stations while taxon-specific processes were assessed at 3 long duration stations (St.A, B, C) and at 4 short duration stations (St.5, 9, 21, and 25) along the euphotic zone.

Enumeration of phototrophs and heterotrophs using flow cytometry
Triplicated aliquots of 1.8 mL were sampled and fixed with a 2 % (w/v) formaldehyde solution, stored for at least 30 min at room temperature, flash frozen in liquid nitrogen equipped with an air-cool argon laser (488 nm, 15 mW).The red fluorescence signal of the chlorophyll a was collected on a long-pass filter (> 650 nm) to identify phytoplankton groups including Prochlorococcus and pico-phytoeukaryotes, according to Marie et al. (2000).Synechococcus cells were discriminated from other phototrophs using their orange fluorescence signal (585/21 nm).
Samples for the enumeration of heterotrophic prokaryotes were stained with SYBRGreen I (Invitrogen-Molecular Probes) at 0.025 % (vol/vol) final concentration for 15 min at room temperature in the dark.Counts were performed using a FACSCalibur flow cytometer (BD Biosciences) equipped with an air-cooled argon laser (488 nm, 15 mW).Stained bacterial cells, were discriminated and enumerated according to their right-angle light scatter (SSC) and green fluorescence of the nucleic acid dye (530/30 nm).Autotrophic prokaryotes were discarded from this analysis based on their red fluorescence.

Sample preparation for P i uptake measurements
Acid-washed and sample-rinsed Nalgene bottles were filled with 10 mL of seawater samples.Preliminary measurements were conducted at the beginning of the cruise to adjust the incubation conditions so that P i uptake was linear with time.The methodology and calculations employed for bulk and cell-normalized, taxon-specific P i uptake rates were based on the protocol by Talarmin et al. (2011).Briefly, a radioactive working solution was freshly prepared on board by diluting a carrier-free [ 33 P]-H 3 PO 4 mother solution (Perkin Elmer, USA, 569.5 × 10 12 Bq mmol −1 ) in 0.2 µm-filtered milliQ water, and added to the samples. 33P i incorporation was stopped by adding an excess amount (0.1 mM final concentration) of a cold P i solution.Samples were split in 3 aliquots of 5 mL (for bulk-whole water-P i uptake measurements), 3.5 mL (for sorting of phototrophic groups) and 1.5 mL (for sorting of Hprok cells) mL and kept in the dark at +4 • C until further processing (< 1 h for the 5 mL subsamples, 10 min to 6 h for the others).Blank samples were added 0.1 mmol L −1 (final concentration) of cold P on average 5.0 to below 13 % of regular values estimated for bulk (n = 11) and sorted groups (n = 36), respectively.The radioactivity was counted within 5 h after addition of the scintillation cocktail using an embarked Packard LS 1600 liquid scintillation counter.

In situ P i uptake rates
At St. A, 9, 21 and 25, samples were collected at several depths in order to assess the vertical distribution of the taxon-specific P i uptake under ambient concentrations of P i .
Samples were spiked with a final concentration of 20 pmol L −1 of the 33 P-orthophoshate working solution and incubated for 15 min.

Concentration kinetics of P i uptake
Concentration kinetics experiments were conducted 15 m above the depth of the deep chlorophyll maximum (DCM) at St. A, B and C by adding increasing quantities of a cold KH 2 PO 4 solution (0-100 nmol L −1 added concentration, Sa).Samples were spiked with the radioactive working solution 15 min later, with an activity of 0.34 MBq per sample, i.e. a final radioactive P i concentration of 60 pmol L −1 (Sa*), and incubated for 45 min.
Bulk P i uptake rates, bulk P i turnover times, bulk and taxon-specific kinetic constants V max (maximum velocity of P i uptake) and K + S n (K being the half-saturation constant and S n the natural P i concentration, their sum being determined as the intersection of the plotted line with the x axis), were based on the work by Thingstad et al. (1993).Such estimations were obtained using the linear regression of P i turnover time vs. added P i concentrations (Sa, considering Sa* negligible).

Bulk P i uptake measurements
The 5 mL aliquots were gently filtered on a 0.2 µm polycarbonate membrane superimposing a GF/D filter soaked with a cold P i solution without rinsing.A 5 s increase of the vacuum pressure ended the filtration to remove non-incorporated P i .Introduction

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Full Filters were then placed in scintillation vials with 5 mL of Phase Combining System scintillation cocktail (PCS, GE Healthcare).

Flow sorting of labeled picoplankton groups
Other aliquots were processed by flow sorting on an embarked FACSAria cell sorter (BD Biosciences) equipped with two lasers: a 488 nm (13 mW, Coherent, Sapphire Solid State) and a 633 nm (11 mW, JDS Uniphase air-cooled HeNe), using the same detection strategy as for enumeration of phototrophic and heterotrophic cells.The sheath fluid was 0.2 µm filtered seawater.The instrument was controlled by a computer equipped with the FACSDiva software set on the four-way sorting 0/32/0 purity mode.Re-sorting of sorted samples was conducted randomly to check for the purity of the sorts, which reached 98 %.Sorted cells were collected in 2 mL microcentrifuge tubes where PCS scintillation cocktail was added up to a final volume of 2 mL.
Experiments were replicated on one occasion, and per cell P i uptake rates varied by less than 4 % in prokaryotic sorts and by 11 % in Pic (n = 4).

Soluble reactive phosphorus (SRP)
SRP was measured at the nanomolar level according to the Rimmelin and Moutin procedure (Rimmelin and Moutin, 2005), derived from the initial MAGIC method proposed by Karl and Tien (1992), using 250 mL triplicates per depth.The detection limit of this technique was around 5 nmol L −1 .

Statistical analyses
Averaged values are reported as mean ± one standard deviation (SD).The Michaelis-Menten model was fitted to some of the data points on a plot of the total P i concentration (S = S a + S n ) vs. P i uptake rate, once V max and K + S n constants had been determined and when a significant correlation was found (p < 0.05).Introduction

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Full

Environmental and biological conditions
A westward shoaling of the DCM (Table 1) and nutricline depths (120 to 50 m and 200 to 50 m, respectively) was observed along the transect.Total chlorophyll a concentrations integrated over 150 m were up to twice as high in the western basin (e.g.St. 25) compared to the Levantine basin (e.g.St. 9; Table 1), emphasizing the strong P ideficiency over the euphotic zone at most of the stations sampled.SRP concentrations ranged 2-379 nmol L −1 , varying with depth and location (Table 2).The highest maximum surface values were measured in the Western Basin (St.21 and 25), possibly due to mesoscale variability.However, short P i turnover times (0.8 to 10 h) were estimated (Fig. 2), revealing a high turnover of P i in the surface communities along the whole transect at surface depths.

Enumeration of microbial groups
Hprok cells were the most abundant group, and varied little over sampled stations and depths, with an average of 3.9 ± 0.3 × 10 5 cells mL −1 (n = 24), with extreme values of 2.7 × 10 5 cells mL −1 and 10.2 × 10 5 cells mL −1 (Fig. 3).Abundances of Proc cells varied between undetected levels to 1.4 × 10 5 cells mL −1 .Their pigment signature was too weak to be detected in surface waters without staining, therefore many experiments are missing data for Proc cells below 50 m.Syn cells were the most abundant at the coastal station 25 (7.7 × 10 4 cells mL −1 at 40 m) and the least abundant in the deep euphotic zone (130 m St. A, 1.4×10 5 cells mL −1 ; Fig. 3).Finally, Pic cells were the most abundant in the Western Basin, ranging 230 cells mL −1 up to 0.3 × 10 5 cells mL −1 .

Bulk P i uptake
P i uptake rates ranged between 0.03 and 21.6 nmol P L −1 h −1 and varied by a factor of 10 along the water column at stations with the lower turnover times (9 and 21), while Introduction

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Full rates could be multiplied by 200 from one depth to the other at St. 25 and A (Fig. 4).
No significant correlation was found between P i uptake rates and SRP concentrations.

Taxon-specific P i uptake
When all available, summed contributions of the 4 sorted groups to P i uptake under ambient P i concentrations reached 25 to 110 % of the bulk signal.At the 4 stations, a higher contribution (up to 82.5 % at St. A; Fig. 4) of Hprok cells was observed, with taxon-specific uptake rates between 0.74 and 27.75 amol P cell −1 h −1 (Table 1) between surface and 25 or 50 m.In the vicinity of the DCM, cyanobacterial cells were the major contributors to bulk signal (up to 72 % at St. A; Fig. 4), mostly Proc cells in open sea samples (Fig. 3a and c).Syn cells contributed the most to bulk signal (53 %) closer to the coast, where they reached the highest abundance measured during the cruise (7.7 × 10 5 cells mL −1 ; Fig. 3).At St. A, taxon-specific P i uptake rates were the highest for picoeukaryotes, and consistently high for all sorted groups (Table 1).Over all experiments, taxon-specific uptake rates ranged 2.6-402, 3.6-40.0,8.4-134.8,and 0.05-36.1×10−18 mol P cell −1 h −1 for Syn, Proc, Pic, and Hprok, respectively (Table 1).

Kinetics of the P i uptake
The bulk community exhibited a wide range of maximum P i uptake rates (V max , 0.2-4 nmol L −1 h −1 ), about 20 times lower at St. A compared to the other sites (Fig. 5; Table 2).Signals measured at St. B and C in sorted Pic cells during the isotope dilution experiments were not significant (less than twice the blank values).Unfortunately, technical problems prevented Hprok cells from being processed at St. A in a reasonable delay after the cold chase.At St. A, B and C, the highest V max value was obtained for cyanobacterial populations (1.8 nmol L −1 h −1 ; Table 2).Taxon-specific maximum uptake rates (V c max ) were higher for Syn (35 and 132 amol cell −1 h −1 ) compared to the other groups (0.2-21 amol cell −1 h −1 ) at the most P i -depleted stations (B and C), but V c max was higher in sorted Pic cells at St. A (Table 2).Estimations of the K +S n constant were Introduction

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Full 3 to 6.5 times higher for Syn cells than for other groups, while Proc and Hprok values at St. C were comparable.In most cases, the ascendant part of the fitted Michaelis-Memten curve did not coincide with our data because P i uptake rates were already approximating the V max value in the lowest added P i concentrations (Proc and Hprok at St. B and C; Fig. 5).Out of 9 successful sorts, 5 groups exhibited a subsaturated P i uptake under natural conditions (V exp /V max ): Syn at St. A, B and C (13, 58 and 37 %, respectively), Proc and Pic at St. A (44 and 86 %, respectively).

General considerations and methodological caveats
Consistently low P i turnover times (< 10 h) were estimated in surface samples from the eastern basin, which is congruent with previous studies (Flaten et al., 2005;Moutin et al., 2002).These conditions were favorable to use short-term incubations with trace concentrations of radiolabeled P i .The embarked equipment allowed to perform all experiments on live samples with a minimum of manipulations, within a time window where signal loss was minimal (Talarmin et al., 2011).
It is important to point out that the detection limit for SRP measurements was close to the ambient concentrations, hence a potential overestimation of the latter and of P i uptake rates calculated using Sn.This caveat would not affect the main results about the relative capabilities of sorted groups in a given sample to take up P i , as the same P i concentration was multiplied by the turnover rates (h −1 ) of each sorted group or bulk community for a given depth.Our inability to detect Prochlorococcus in most surface samples cut back any generalizations regarding this group, in spite of significant signals measured deeper in the euphotic zone.The added contributions of the 3 or 4 sorted groups did not necessarily add up to 100 %.Analytical error associated with per cell rates is about 10 % and error on cell abundances around 5 %.Furthermore, viruses and mixotrophic bacterivorous ciliates or nanoflagellates, potentially by-passing nutrients to Introduction

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Full the highest trophic levels of the microbial loop (Krom et al., 2005;Paffenhöfer et al., 2007), could also contribute to bulk P i uptake fluxes.Attached bacteria, excluded from our cell sorting experiments might also represent a significant contributor to the bulk P i uptake, if their specific activity is higher than that of free-living bacteria.

Taxon-specific P i uptake rates
In half of the samples for which Syn and Pic were sorted, taxon-specific P i uptake rates were higher in Syn cells than in Pic cells, and in all samples, Hprok-specific rates were consistently lower compared to cyanobacteria.Considering the importance of converting per cell rates to per volume rates (Casey et al., 2009), our results would still highlight the major role of cyanobacteria in P i uptake fluxes as outcompetitors of phytoeukaryotes under the lowest ambient SRP concentrations.

Contribution of the sorted groups to total P i uptake
Down to about 25 m above the DCM, at the 4 stations sampled for depth profiles, the main contributors to P i uptake fluxes were the heterotrophic prokaryotes.Michelou et al. (2011) found dominance of bulk P i uptake by Hprok in the Sargasso Sea, in surface and DCM layers.These authors determined that cyanobacteria (Proc + Syn) contributed to less than 10 % of the P i uptake fluxes.These contributions were a result of (1) low Syn abundances (on average 2 × 10 4 cells mL −1 ), although this group exhibited 2 to 5-fold higher taxon-specific uptake rates than Proc and Hprok and intermediate Proc abundances (on average 2.8 × 10 5 cells mL −1 ), combined with (2) lower cell specific rates for Proc (0.4 amol P cell −1 h −1 ).In contrast, we found in the Mediterranean Sea a higher contribution of cyanobacteria close to the DCM, mostly due to abundances higher than those observed in the North Atlantic at the time of the cruise (Michelou et al., 2011).
The companion paper of these experiments published earlier by Casey et al. (2009) found no difference in the contribution of Proc and Syn to P i utilization.Prochlorococcus Figures

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Full contributed to 45 % of the bulk P i uptake in the North Atlantic (Zubkov et al., 2007).
In the North Pacific Subtropical Gyre, Pro and Hprok cells did not reveal different contributions to total P i uptake fluxes, despite taxon-specific rates 3-fold higher for Proc than for Hprok (Björkman et al., 2012).At the SOLA station of the eastern Mediterranean Sea (Banyuls sur mer, France), Syn contributed the most to the total P i uptake (35 %) among 3 sorted groups, while Pic and Hprok showed low and nonsignificantly different contributions (Talarmin et al., 2011).For the first time, our results suggest that the uptake of P i at an offshore oligotrophic location was not dominated by a single group of microbes in the upper euphotic zone and that Prochlorococcus cells significantly contributed to microbial P i cycling in the Mediterranean Sea.Our findings are comparable to measurements carried out in the North Atlantic where Syn and Pro contributed up to 7 and 45 % to bulk P i uptake, respectively (Zubkov et al., 2007).

P i uptake kinetics
Maximum P i uptake rates of the bulk community were 2 to 40 times higher than any of the V max estimated for sorted groups, while bulk K + S n values were included in the range measured for sorted groups.This shows that the community is globally well adapted to low P i concentrations but has the potential to take up P i much faster.Unsorted groups (larger protists) may not have a significant biomass or contribute highly to bulk P i uptake fluxes, but they may have the ability to store large amounts of P i in case of upwelled or deposited inputs.
Among the sorted groups, a continuum of P i uptake capabilities was observed in the Mediterranean Sea.Per cell data suggested a higher maximum uptake rate for Pic (only 1 sample) than Syn, followed by Proc and Hprok cells, respectively.Also, observed Syn P i uptake was not saturated (i.e.V exp < V max , data not shown), which reinforces the potential of Synechococcus as an organism able to respond rapidly and significantly to pulsed P i events (Moutin et al., 2002).The lack of replication in the experiments did not allow statistical comparison of the kinetic constants.Our experiments indicated comparable uptake ability under low P i concentrations in Proc and Hprok cells.the cruise, cell volume was not estimated, in spite of its importance in the diffusion limitation model (Vadstein and Olsen, 1989).Per cell P i uptake rates converted in per volume P i uptake rates showed opposite trends, with cyanobacteria harvesting P i more efficiently than larger phytoplankton (Casey et al., 2009).This is consistent with a recent study showing that the theoretical surface-limited growth rate does not apply to cells with a volume below 40 µm 3 (Dao, 2013), due to a poor cellular machinery compared to the absorbing capability (surface).

Ecological and biogeochemical implications
The vertical partitioning of P i uptake detected in the present study may show that P i concentrations in low P i environments are a major factor explaining the distribution of osmotrophs, due to their different nutrient uptake strategies.The concept of competition for limited bioavailability of P i may actually be closer to a more common occupation of different ecological niches where one or more limiting resources regulate the biodiversity.In the surface where P i concentrations are the lowest, only organisms with the lowest K + S n can utilize P i efficiently, i.e.Proc and Hprok cells.It is likely that the cyanobacterial contribution to P i uptake decreased below the DCM because of light limitation (Duhamel et al., 2012), while Hprok in the subsurface layers might have been carbon limited.More details about such limitations could be provided by linking phylogenetic diversity of cyanobacteria to their P i uptake performances, as shown for different taxa of heterotrophic prokaryotes in mesocosms from the Mediterranean Sea (Sebastián et al., 2012).In most studies coupling flow sorting with radiolabeled techniques, the biomass of sorted groups was not evaluated.Recently, Casey et al. (2013) showed that carbon biomass of various taxa was the only variable linking changes in phytoplankton community structure and interannual trends in carbon export fluxes and elemental stoichiometry.
Additional data on the utilization of dissolved organic phosphorus across the Mediterranean Sea would be necessary to establish P i requirements for the most abundant microbes.It would also allow to estimate C : P uptake ratios in order to 14652 Introduction

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Full compare them to the eastward increasing C : P export ratios (from 40.8 : 1 at St. A to 212 : 1 at St. C, cf.Moutin and Prieur, 2012).N 2 fixation in the Mediterranean sustained up to 35 % of the new production in the Western Basins, while it was negligible in the Eastern Basin (Bonnet et al., 2011).This further supports the fact that P cycling is the key to establishing accurate biogeochemical budgets in the Mediterranean Sea.

Conclusions
P i uptake kinetic constants and P i uptake fluxes through the microbial communities of the stratified Mediterranean Sea were estimated along a gradient of oligotrophy.Our findings indicated that the spatial distribution of heterotrophic and photo-autotrophic picoplankters in this P i -deficient environment was partly attributable to their respective capabilities to take up P i .Heterotrophic prokaryotes dominated P i uptake fluxes in the surface layers, where the lowest P i turnover times were detected, which was consistent with their low V max and low half-saturation constant.Their abundance did not decrease drastically with depth, however P i uptake fluxes in the subsurface, above the DCM layer, were dominated by Prochlorococcus offshore and Synechococcus closer to the coast.Presumably, carbon limitation of bacterial activity reduced the capabilities of heterotrophic prokaryotes to take up P i , while cyanobacteria would be able to acquire carbon via photosynthesis.While a few taxon-specific P i uptake rates from various areas were published in the past 7 years, the vertical partitioning of P i uptake fluxes among sorted picoplankton groups was shown for the first time in our study.The   Table 2. P i uptake kinetic characteristics of the bulk community and sorted groups: V max is the theoretical volumetric maximum uptake rate of a group, V c max is the cell-normalized maximum uptake rate, and K + S n is the theoretical sum of the half saturation constant for uptake and the natural [P i ].LD: below the limit of detection (< 3 blank values).NA: data not available.
V c max (amol P cell −1 h −1 ) V max (nmol P L −1 h −1 ) K + S n (nmol P L Full .Bulk and taxon-specific P i uptake rates (V exp ) at increasing P i concentrations (in situ + added) in sorted Synechococcus (Syn), Prochlorococcus (Proc), picophytoeukaryotes (Pic) and heterotrophic prokaryotes (Hprok) at stations where concentration bioassays were conducted.Straight horizontal and vertical lines represent computed V max and K + S n , respectively.If a significant correlation (p < 0.05) between measured P i uptake rates and those calculated from the Michaelis-Menten model, the uptake curve was fitted to the data.
Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | and then stored at −80 • C until further processing onshore within 6 months.Samples were thawed an hour before analysis and 1 µm yellow-green beads (Fluoresbrite, Polysciences) were systematically added as a standard.The sheath fluid was filtered (< 0.2 µm) seawater and analyses were conducted with the software Cell Quest Pro.Microbes were enumerated using a FACScan flow cytometer (BD Biosciences) Discussion Paper | Discussion Paper | Discussion Paper | i 15 min prior to radiolabeling and processed like the other samples.Blank values represented Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | 3 Results Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | iteration of such experiments in other areas of the world ocean would contribute to tease apart the roles of nutrient limitation and biodiversity in P i uptake fluxes.Discussion Paper | Discussion Paper | Discussion Paper | time measurements and all the scientists who participated in the BOUM cruise.Finally, we acknowledge the crucial contribution of Claude Courties who sorted all the samples on board.This work is a contribution of the BOUM project (Biogeochemistry from the Oligotrophic to Ultraoligotrophic Mediterranean, http://www.com.univ-mrs.fr/BOUM).It was funded by the French national CNRS-INSU (LEFE-CYBER) program and the European IP SESAME (Southern European Seas: Assessing and Modelling Ecosystem Changes), EC Contract No. GOCE-036949.We acknowledge the French Research and Education Council for the funding of Agathe Talarmin's Ph.D. training.Discussion Paper | Discussion Paper | Discussion Paper | Wu, J., Sunda, W., Boyle, E. A., and Karl, D. M.: Phosphate depletion in the Western North Atlantic Ocean, Science, 289, 759-762, doi:10.1126/science.289.5480.759,2000.Zubkov, M. V., Mary, I., Woodward, E. M. S., Warwick, P. E., Fuchs, B. M., Scanlan, D. J., and Burkill, P. H.: Microbial control of phosphate in the nutrient-depleted North Atlantic subtropical gyre, Environ.Microbiol., 9, 2079-2089, 2007Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper |

Figure 1 .Figure 2 .Figure 3 .Figure 4 .
Figure 1.Map of the Mediterranean Sea with locations of sampling sites during the BOUM cruise transect.Numbered and lettered stations denote locations where our experiments were conducted.

Table 1 .
Environmental context of the experiments: sampled depth (m), depth of the deep chlorophyll maximum (DCM d ), 0-150 m integrated Chl a concentrations, range of SRP concentrations measured at those stations between 0 and 150 m, and per cell rates of P i uptake measured in sorted groups Synechococcus (Syn), Prochlorococcus (Proc), picoeukaryotes (Pic) and heterotrophic prokaryotes (Hprok) measured under ambient concentrations.