Mar Biol

DOI 10.1007/s00227-009-1185-2

ORIGINAL PAPER

Picophytoplankton responses to changing nutrient and light regimes during a bloom

Katherine R. M. Mackey · Tanya Rivlin · Arthur R. Grossman · Anton F. Post · Adina Paytan

Received: 17 November 2008 / Accepted: 16 March 2009© The Author(s) 2009. This article is published with open access at Springerlink.com

Abstract The spring bloom in seasonally stratiWed seas isoften characterized by a rapid increase in photosyntheticbiomass. To clarify how the combined eVects of nutrientand light availability inXuence phytoplankton compositionin the oligotrophic Gulf of Aqaba, Red Sea, phytoplanktongrowth and acclimation responses to various nutrient andlight regimes were recorded in three independent bioassaysand during a naturally-occurring bloom. We show thatpicoeukaryotes and Synechococcus maintained a “bloomer”growth strategy, which allowed them to grow quickly whennutrient and light limitation were reversed. During thebloom picoeukaryotes and Synechococcus appeared tohave higher P requirements relative to N, and were respon-sible for the majority of photosynthetic biomass accumula-tion. Following stratiWcation events, populations limited bylight showed rapid photoacclimation (based on analysis of

cellular Xuorescence levels and photosystem II photosyn-thetic eYciency) and community composition shifts with-out substantial changes in photosynthetic biomass. Thetraditional interpretation of “bloom” dynamics (i.e., as anincrease in photosynthetic biomass) may therefore be con-Wned to the upper euphotic zone where light is not limiting,while other acclimation processes are more ecologicallyrelevant at depth. Characterizing acclimation processes andgrowth strategies is important if we are to clarify mecha-nisms that underlie productivity in oligotrophic regions,which account for approximately half of the global primaryproduction in the ocean. This information is also importantfor predicting how phytoplankton may respond to globalwarming-induced oligotrophic ocean expansion.

Introduction

Phytoplankton blooms occur when the rates of cell growthand division signiWcantly exceed the rates of cell loss,resulting in rapid biomass accumulation. SuYcient nutri-ents and light, as well as an absence of grazers, are condi-tions that stimulate phytoplankton growth. A transientoccurrence of any of these favorable growth factors can bethe causal event in bloom initiation. For example, mixingperiods in permanently and seasonally stratiWed oligo-trophic seas can result in episodic inputs of growth-limitingnutrients that trigger rapid, transient increases in photosyn-thetic biomass (i.e., blooms). Blooms are also initiated fol-lowing delivery of exogenous nutrients via processes suchas nitrogen Wxation and atmospheric deposition. The natureand magnitude of a bloom is linked to composition of thephytoplankton community, which determines growth rates,acclimation strategies, and competitive interactions thatensue during the bloom. However, understanding how

Communicated by U. Sommer.

K. R. M. Mackey (&)Department of Civil and Environmental Engineering, Stanford University, Stanford, CA 94305, USAe-mail: kmackey@stanford.edu

K. R. M. Mackey · A. PaytanInstitute of Marine Science, University of California Santa Cruz, Santa Cruz, CA 95064, USA

T. Rivlin · A. F. PostH. Steinitz Marine Biology Laboratory, The Interuniversity Institute of Marine Sciences, POB 469, 88103 Eilat, Israel

K. R. M. Mackey · A. R. GrossmanDepartment of Plant Biology, The Carnegie Institution of Washington, Stanford, CA 94305, USA

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nutrient levels and physiological factors contribute tobloom dynamics is confounded by the ambient physicalcharacteristics of the water column, where light may super-sede nutrient availability as the growth limiting variable.

The Gulf of Aqaba, an oligotrophic water body fed bynutrient-depleted surface waters from the Red Sea, has sea-sonal cycles of stratiWcation and mixing similar to othersubtropical oligotrophic seas. Its water column is stratiWedduring summer, and surface water nutrient levels are nearthe limits of detection (Levanon-Spanier et al. 1979; Reissand Hottinger 1984; Mackey et al. 2007). During the sum-mer months atmospheric dry deposition is a signiWcantsource of nutrients to the euphotic zone, supporting tran-sient phytoplankton blooms (Chen et al. 2007; Paytan et al.2009). Beginning in the fall, cooling of surface waters initi-ates convective mixing, and a deeply mixed (300 m ormore) water body is observed by winter (Wolf-Vetch et al.1992). Nutrients entrained from depth as a consequence ofthis mixing result in mesotrophic conditions (Lindell andPost 1995), with productivity likely limited by light (Labi-osa et al. 2003). The water column begins to re-stratify inthe spring as surface waters warm, trapping nutrients andphytoplankton in the euphotic zone along a steep lightgradient. Phytoplankton take advantage of the favorablenutrient and light conditions within the newly stratiWedeuphotic zone forming a surface bloom over a period ofdays (Labiosa 2007), while the growth of phytoplanktontrapped at depth is limited by low light. Picophytoplankton(cells < 2 �m) are the dominant cell type in the Gulf ofAqaba; however, ultraplankton (cells < 8 �m) and somelarger diatoms and dinoXagellates (cells 5–100 �m) alsooccur in phytoplankton assemblages, particularly during themesotrophic winter season (Lindell and Post 1995; Sommer2000; Mackey et al. 2007).

Permanently and seasonally stratiWed waters similar tothat of the Gulf of Aqaba account for a signiWcant portion(>50%) of the surface area of the ocean and therefore repre-sent a signiWcant fraction of the area available for carbonsequestration. Identifying the major factors that controlphytoplankton growth, species abundance, and acclimationprocesses in these habitats is an important step in under-standing their role in the global carbon cycle. Indeed, manystudies have sought to address these issues, and recent Wnd-ings suggest that the factors controlling Chl a levels andprimary production may be diVerent from those controllingcell division rate (Davey et al. 2008). Ecotypic variationamong phytoplankton genera adds another level of com-plexity by helping to shape local and global phytoplanktondistributions (Moore et al. 1998; Partensky et al. 1999;Rocap et al. 2003; Fuller et al. 2005; Johnson et al. 2006;Litchman and Klausmeier, 2008). Lindell et al. (2005) sug-gest that the dominance of certain phytoplankton taxo-nomic groups may be due to the ability of diVerent ecotypes

within those groups to thrive under speciWc environmentalconditions, thereby allowing the entire population to accli-mate to environmental stimuli. Clearly the factors inXuenc-ing phytoplankton abundance in oligotrophic waters, and asa consequence the marine carbon cycle, are complex andmore work is needed to fully elucidate phytoplanktongrowth dynamics in these regions. Moreover, observationsof prolonged stratiWcation (Karl et al. 2001) and projectionsof oligotrophic ocean expansion in response to globalwarming (Sarmiento et al. 1998; Matear and Hirst 1999;Sarmiento et al. 2004) highlight the importance of charac-terizing phytoplankton dynamics in these regions.

This study investigates survival strategies of naturallyoccurring phytoplankton communities in the oligotrophic,stratiWed Gulf of Aqaba in response to nutrient availabilityunder various light regimes. We use Weld measurementsfrom the euphotic zone of the Gulf of Aqaba, Red Seabefore and during stratiWcation at the onset of a springbloom, as well as nutrient addition incubation experiments(at diVerent light regimes) performed during the ultraoligo-trophic summer season to: (1) assess shifts in phytoplank-ton community composition and photophysiology inresponse to the combined eVects of nutrient and light avail-ability; (2) identify key competition, acclimation, and nutri-ent uptake strategies of picoeukaryotes, Synechococcus,and Prochlorococcus during a bloom; and (3) estimatethe contribution of picoeukaryotes, Synechococcus, andProchlorococcus to total photosynthetic biomass duringblooms as well as periods of sustained oligotrophy.

Materials and methods

Spring bloom monitoring

Water samples were collected from Station A (29°28�N,34°55�E) in the Northern Gulf of Aqaba during the springseason when the water column transitions from mixed tostratiWed. Station A is located oV shore in a deep (>700 m)open water region where the water column extends wellbelow the euphotic zone; this permits analysis of an entirelypelagic euphotic zone. Depth proWles were taken on 12March 2007 and 16 March 2007 using a sampling CTD-Rosette (SeaBird). Dissolved nutrient concentrations, Chla, and Xow cytometry measurements were taken asdescribed below with the following modiWcations. For Chla samples, Wlters were transferred immediately to 10 mL90% acetone aboard the ship, and the pigment wasextracted for 24 h at 4°C. Flow cytometry measurementswere taken with a Becton Dickenson FACScan Xow cytom-eter. On the basis of autoXuorescence characteristics, cellswere classiWed as picoeukaryotes or Synechococcus. Due totheir relatively low autoXuorescence compared to other

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phytoplankton, Prochlorococcus cannot be reproduciblydiVerentiated from other non-photosynthetic bacteria usingthe FACScan Xow cytometer. Therefore, Prochlorococcusconcentrations are not available from the Weld data set dueto the Xuorescence detection limit of the machine.

Simulated stratiWcation experiment

A simulated stratiWcation experiment was conducted tomeasure photoacclimation with higher time resolution thanwas possible for the Weld measurements described above.The experiment was designed to simulate the reversal oflight limitation experienced by cells during the natural tran-sition from mixing to stratiWcation that occurs during thespring bloom. Surface water (1 m) was collected from apier 20 m oVshore on 12 March 2007 (prior to the springbloom), and placed into 10 L sample-rinsed translucentpolyethylene bottles (3 replicate bottles per treatment) 1 hbefore sunrise at the InterUniversity Institute for MarineScience (IUI) in Eilat, Israel. Water was pre-Wlteredthrough 20 �m mesh to remove grazers. Because nutrientswere naturally abundant in sample waters as a consequenceof deep mixing that preceded the experiment, it was notnecessary to amend the sample water with additional nutri-ents to elicit a bloom response.

Sample bottles were incubated under light conditionsdesigned to simulate either “shallow” (high light, HL) or“deep” (low light, LL) locations in the stratiWed water col-umn. Bottles were incubated in an outdoor tank throughwhich water from the Gulf circulated, and screening mate-rial was used to attenuate the sunlight intensity to which thebottles were exposed. In HL treatment, 50% light attenua-tion yielded maximum midday irradiance of »1,000 �molquanta m¡2 s¡1 and was equivalent to the upper 10 m of theeuphotic zone of the Gulf during summer months. In theLL treatment, »95% light attenuation yielded maximummidday irradiance of »100 �mol quanta m¡2 s¡1 and wasequivalent to the light intensity at approximately 80 m inthe summer months [similar to the upper boundary of thedeep chlorophyll maximum (D. Iluz, personal communica-tion)]. No alteration of the natural light spectrum was donein the HL treatment. The light spectrum in the LL treatmentwas enriched in blue-green wavelengths using a tintedshade cloth to more closely resemble the natural light spec-trum at 70–80 m depth. Comparison of spectra for unatten-uated sunlight and light transmitted through the tinted cloth(each normalized to irradiance at 694 nm) showed that thecloth enriched the proportions of blue wavelengths at 443and 490 nm by 280 and 210%, respectively, and greenwavelengths at 510 and 555 nm by 190 and 150%, respec-tively, relative to the unWltered visible spectrum.

The bottles were incubated for 2 days and aliquots wereremoved approximately every 3–4 h during the day for

sampling. Flow cytometry samples were removed and pro-cessed as described below at each time point. PhotosystemII (PSII) chlorophyll Xuorescence measurements weretaken by concentrating cells in the dark onto GF/F Wlters(Whatman) using a low pressure peristaltic pump. Oneliter of seawater was collected onto each sample Wlter.Chlorophyll Xuorescence measurements were made witha WATER-PAM Xuorometer and WinControl software(Heinz Walz GmbH). An automated program was run todetermine the photochemical eYciency of PSII under dark-adapted (Fv/Fm) and light-adapted (�PSII) states. (Fordetailed discussion of Xuorescence parameter calculationsand measurement techniques used in this study see Mackeyet al. 2008). The program delivered the following lighttreatments and measuring pulses: 10 min dark adaptationand delivery of a saturating pulse to determine Fv/Fm, fol-lowed by 3 min exposure to actinic light at 100 �molquanta m¡2 s¡1 and delivery of a saturating pulse to deter-mine �PSII at this light intensity (hereafter �PSII¡100), fol-lowed by 3 min exposure to actinic light at 1,000 �molquanta m¡2 s¡1 and delivery of a saturating pulse to deter-mine �PSII at this light intensity (hereafter �PSII–1,000).These actinic light exposure durations were determinedempirically as suYcient for the cells to reach steady state.

Nutrient enrichment experiments

Nutrient enrichment experiments were conducted to ascer-tain phytoplankton responses to fertilization with exoge-nous inorganic nutrients (N and P) under incubation withHL and LL intensities simulating “shallow” and “deep”depths, respectively, within the water column. These exper-iments were designed to simulate bloom conditions result-ing from delivery of new nutrients (e.g., via nitrogenWxation or atmospheric deposition) to a stratiWed, oligo-trophic water column. Surface water was collected at 1 mdepth from an oVshore site in the Northern Gulf of Aqabaduring two Weld excursions in September 2005 and October2006 [note that community structure during these months isquite consistent from year to year (Lindell and Post 1995;Mackey et al. 2007)] Surface water was pre-Wltered with20 �m nylon mesh to remove grazers larger than 20 �m,collected into sample-rinsed translucent polyethylene bot-tles, and kept in the dark during transport (less than twohours) to the IUI facility. All incubations were performedwith three replicate bottles per treatment.

The LL nutrient enrichment experiment was conductedin September 2005 and the HL nutrient enrichment experi-ment in October 2006. Light attenuation and incubationsetup were performed exactly as described above for thesimulated stratiWcation experiment, using the same screeningmaterials and incubation conditions. Phosphorus was added as0.4 �mol L¡1 sodium phosphate monobasic (hereafter PO4),

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and N was added as 7 �mol L¡1 sodium nitrate (hereafterNO3) or ammonium chloride (hereafter NH4). Sample bot-tles contained 6 L water and were incubated for 4 days. Thenutrient concentrations used in the enrichments were basedon typical PO4 and NO3 concentrations for deep water fromthe Gulf (Mackey et al. 2007) because phytoplankton arenaturally exposed to N and P at these levels following deepmixing and upwelling events. Nutrients were thereforeadded at an N:P ratio of 17.5:1, rather than the canonicalRedWeld ratio of 16:1, to better simulate natural bloomforming conditions.

Nutrient analyses

Total oxidized nitrogen (nitrate and nitrite, N + N) and sol-uble reactive phosphorus (SRP) concentrations were deter-mined using colorimetric methods described by Hansen andKoroleV (1999), modiWed for a Flow Injection Autoana-lyzer (FIA, Lachat Instruments Model QuickChem 8000).In all treatments not receiving sodium phosphate, SRP waspre-concentrated before analysis by a factor of about 20using the magnesium co-precipitation (MAGIC) method(Karl and Tien 1992). Using standard additions, the recov-ery of inorganic P in this procedure was determined to be100% and the blank was always below detection limits. TheFIA was calibrated using standards prepared in low nutrientWltered seawater (summer surface water from the Gulf)over a range of 0–10 �mol L¡1. The precision of the meth-ods used in this work is 0.05 �mol L¡1 for N + N, and0.02 �mol L¡1 for SRP. The detection limit for these nutri-ents was 0.02 �mol L¡1.

Chlorophyll a

Daily samples for chlorophyll a (Chl a) determinationwere taken during both nutrient addition bioassays byWltering 200 mL aliquots onto Whatman GF/F glass WberWlters. In the HL nutrient enrichment experiment, sampleswere analyzed daily following extraction at 4°C in 90%acetone for 24 h in the dark. In the LL nutrient enrichmentexperiment, aliquots were removed from each bottlebeginning at 3 pm each day, refrigerated overnight in thedark and Wltered beginning at 10 am the following morn-ing. These Wlters were placed within sterile 2 mL cryovi-als, stored at ¡80°C, and extracted for 24 h in 90%acetone (7 mL per sample) within 1 week. No signiWcanteVect of storage was observed based on comparisonsbetween sample sets analyzed with and without storage;Chl a concentrations determined by the two methods werestatistically indistinguishable. Fluorescence was measuredwith a Turner Fluorometer (Turner Designs 10-AU-005-CE) before and after acidiWcation with 3.7% HCl and wasconverted to a Chl a concentration using a standard

conversion method (JGOFS Protocols 1994). The standarderror determined from triplicate samples was below 0.03and 0.06 mg m¡3 in the HL and LL nutrient enrichmentexperiments, respectively.

Flow cytometry

Aliquots were removed daily during the nutrient additionbioassays for Xow cytometry, Wxed with glutaraldehyde(Wnal concentrations 0.1%) and stored at ¡80°C untilanalyzed on a FACSAria Xow cytometer. Data analysiswas performed using FlowJo software (TreeStar, Inc.).Picophytoplankton (photosynthetic cells <2 �m diameter)were classiWed as picoeukaryotes, Prochlorococcus, orSynechococcus on the basis of autoXuorescence character-istics. Growth curves generated from the cell concentrationdata represent net growth for each group, i.e., growth minusmortality. Cell concentrations were determined by spikingsamples with a known volume and concentration of 1 �mXuorescent yellow-green beads (Polysciences). The coeY-cient of variation for cell concentrations determined fromtriplicate samples was below 0.15 in both experiments.Mean and median cellular red Xuorescence levels for pic-oeukaryotes, Synechococcus, and Prochlorococcus popula-tions were estimated from the logarithmic signals collectedon the Cy55-PE channel (488 nm excitation, 665 nm emis-sion) of the Xow cytometer.

Estimation of photosynthetic biomass

The contribution of each cell type to the overall picophyto-plankton photosynthetic carbon biomass was estimatedusing the equation of Verity et al. (1992), which relates cellvolume to carbon (C) biomass. Estimates of cell dimen-sions for Prochlorococcus (»0.7 �m) and picoeukaryotes(»1.5 �m) were based on light microscopy using a NikonepiXuorescent microscope at 400£ magniWcation and fromside-scatter measurements (a proxy for cell size) taken dur-ing Xow cytometry of Wxed cells collected in the Weld. Mea-surements fell within the typical range observed in otherstudies (Campbell et al. 1994). Both Prochlorococcus andpicoeukaryotes were spherical, giving conversion factors of98 and 708 fg C cell¡1, respectively. The conversion factorfor Prochlorococcus is within the range of calculated esti-mates (29 and 124 fg C cell¡1) reported in other Weld stud-ies (Partensky et al. 1999 and references therein; Zubkovet al. 2000; Grob et al. 2007), but is higher than an estimateof 49 fg C cell¡1 measured from laboratory cultures (Cail-liau et al. 1996). Conversion factors for picoeukaryotesvary substantially due to the broader range of possible celldiameters; however, our estimate is within the range of cal-culated estimates (530–863 fg C cell¡1) for picoeukaryotesfrom the PaciWc Ocean (Worden et al. 2004) with diameters

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similar to those determined for this study. Microscopyrevealed rod-shaped Synechococcus cells (dimensions »0.8to »1.2 �m), with a composition of 279 fg C cell¡1, whichis similar to previous estimates of »250 fg C cell¡1 (Kanaand Glibert 1987; Campbell et al. 1994). Conversion fac-tors were multiplied by cell concentration (determined fromXow cytometry) to estimate photosynthetic biomass. Whilenatural variability of phytoplankton cell size within thepopulation introduces some error into our estimates,approximations of cellular C content from cell biovolumecan inform productivity models if the potential for error isadequately considered when interpreting the results (Camp-bell et al. 1994; Worden et al. 2004).

Results

Spring bloom monitoring

To investigate phytoplankton responses to nutrient andlight availability during the spring bloom, in situ monitor-ing of Chl a, Xow cytometry, and nutrient draw down werecarried out before and during the 2007 spring bloom.Figure 1 shows depth proWle measurements of SRP, NO3,Chl a, and Synechococcus and picoeukaryotes cell concen-trations from the euphotic zone before (12 March 2007) andafter (16 March 2007) stratiWcation had begun to occur.Prior to stratiWcation, 60 m is the typical depth at whichlight is attenuated to 1% of surface intensity (»20 �molquanta m¡2 s¡1) in the mixed Gulf water column (D. Iluz,personal communication). Because the water column washomogenously mixed down to >600 m on March 12 and thesalinity proWles did not change over the course of the sam-pling (data not shown), we conclude that the observedchanges in the water column during this period are due to

stratiWcation rather than introduction of diVerent watermasses to the euphotic zone via other physical processes inthe water column. This interpretation is consistent withthe current understanding of the circulation in the Gulf(Wolf-Vetch et al. 1992). NO3 and SRP levels were rela-tively homogenous (1.7 and 0.11 �mol L¡1, respectively)throughout the mixed water column (on March 12) (Fig. 1a,b), whereas drawdown was evident in the upper 50 m of thestratiWed water column (March 16). Based on temperatureproWles, the surface mixed layer was approximately 25 mon March 16 during stratiWcation. In these proWles, NO3

levels were 0.8 �mol L¡1 at the surface and increased to1.8 �mol L¡1 at depth, and SRP levels were 0.05 �mol L¡1

at the surface and increased to 0.08 �mol L¡1 at depth(Fig. 1a, b).

A Chl a maximum (1.26 mg m¡3) was observed in thestratiWed water column at 20 m depth, whereas the Chl adistribution was homogenous throughout the mixed watercolumn on 12 March (0.2 mg m¡3; Fig. 1c). In the mixedproWle, picoeukaryotes were present at approximately3,000–4,000 cells mL¡1, and Synechococcus were present atapproximately 2,000–3,000 cells mL¡1 throughout theeuphotic zone (Fig. 1d, e). The maximum picoeukaryote cellconcentration in the stratiWed proWle on 16 March occurredat 60 m (17,000 cells mL¡1), and another smaller peakwas apparent near 120 m (11,000 cells mL¡1). In contrast,Synechococcus cell concentration proWles showed twosmall peaks at 20 m (7,000 cells mL¡1) and 100 m (8,000cells mL¡1) that were oVset from the picoeukaryote peaks(Fig. 1d, e). Cell abundances of Prochlorococcus and largerphytoplankton were not available for these casts, so it is notpossible to know their vertical distributions. However, intypical years in the Gulf, Prochlorococcus numbers remainrelatively constant throughout the spring bloom (Lindell andPost 1995; A. Paytan unpublished data). The proWles of Chl

Fig. 1 Depth proWles of a SRP, b NO3, c Chl a, d Synechococcus cell concentration, and e picoeukaryote cell concentration for the mixed (closedcircles) and stratiWed (open circles) water columns

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a would include input from both Prochlorococcus andlarger cells. In particular, inputs from larger cells in surfacewaters (Mackey et al. 2007) may explain the Chl a peak at20 m that is oVset from Synechococcus and picoeukaryotepeaks.

On 16 March, picoeukaryote cellular Xuorescenceshowed no systematic trends with depth (Fig. 2a), whereasSynechococcus cellular Xuorescence was higher for cells at100 m, the depth of the deep cell maximum, than at 20 m,the depth of the surface maximum (Fig. 2b).

Figure 3a shows the ratio of NO3 to PO4 (NO3:PO4) cal-culated from NO3 and SRP measurements at each depth forthe mixed and stratiWed casts. A dashed line at 16 indicates

the value of the RedWeld ratio (RedWeld et al. 1963). In themixed proWle the NO3:PO4 ratio was approximately 16throughout the water column except at the surface where itwas approximately 14. In the stratiWed cast the NO3:PO4

ratio was also near 14 in surface waters, but increased toapproximately 20 at greater depths. The drawdown of NO3

and SRP determined from changes in water nutrient concen-trations over the period of sampling are shown in Fig. 3band c, respectively, and show that larger decreases in theconcentrations of both nutrients (i.e., greater drawdown)occurred in the upper 50 m of the euphotic zone. The ratiosof NO3 and PO4 drawdown (Fig. 3d) are close to the Red-Weld ratio of 16 in the surface, but decrease drastically at60 m (NO3:PO4 of approximately 1) before increasing againwith depth but still remaining below RedWeld ratios(NO3:PO4 between 5 and 10). These uptake ratios are indic-ative of the ratios at which these nutrients are removed fromthe water during the sampling period, e.g., through biologi-cal immobilization or export (Eppley and Peterson 1979)assuming (1) the inventory of nutrients (i.e., dissolved plusparticulate) in the euphotic zone was constant over the timeof our sampling, and (2) that there was no vertical or hori-zontal Xux of nutrients due to lateral diVusion or isopycnalmixing. These assumptions are reasonable for the northernGulf, given that the north-south gradient in surface watersnutrients is low throughout the year (Klinker et al. 1977; A.Paytan, unpublished data), the short time scale of our calcu-lation (four days), and given that a one dimensional mixedlayer model accurately describes the convective/advectivewater balance at the northern end of the Gulf where oursampling was conducted (Wolf-Vetch et al. 1992).

Simulated stratiWcation experiment

The picophytoplankton community responded similarly toincubation under HL and LL conditions in the simulated

Fig. 2 Cellular Xuorescence (and corresponding cell densities) fora picoeukaryotes, and b Synechococcus on 16 March

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Fig. 3 Depth proWles of a seawater NO3:PO4 ratios for the mixed (closed circles) and stratiWed (open circles) water columns; and the changes in b NO3 concentration; c PO4 concentration; and d NO3:PO4 uptake ratios during the transition from mixing to stratiWcation

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stratiWcation experiment. The initial population was com-posed of picoeukaryotes (14,500 cells mL¡1), Synechococ-cus (19,600 cells mL¡1), and Prochlorococcus (12,900cells mL¡1). Picoeukaryotes and Synechococcus increasedin abundance, and Prochlorococcus decreased in abun-dance, over 2 days in both the HL (Fig. 4a) and LL(Fig. 4b) treatments.

Midday (noon) PSII Xuorescence measurements for thesimulated stratiWcation experiment are shown in Fig. 5.Fv/Fm and �PSII measurements increased slightly comparedto initial midday levels within one day of incubation in theHL treatment (Fig. 5a). In contrast, Fv/Fm measurementsremained relatively stable in the LL treatment (Fig. 5b),while both �PSII¡100 and �PSII¡1,000 decreased by over 75%by midday on the second day of the experiment (i.e., hour36) compared to initial midday levels (hour 12).

Nutrients uptake in nutrient enrichment experiments

Ambient seawater nutrient concentrations measured priorto the addition of experimental amendments in the HL andLL nutrient enrichment experiments are shown in Table 1,and reXect the oligotrophic character of surface waters inthe Gulf during the stratiWed season in which the experi-ments were conducted in both years. The nutrient data fromthe HL and LL nutrient enrichment experiments are dis-cussed semi-quantitatively because nutrient levels in sev-eral of the treatments were close to detection limits anddiVerences between treatments were, in some cases, close

to the analytical precision. However, while statistical state-ments are not made, the conclusions herein are neverthelessbased on comparison of averages and standard deviationsof triplicate samples. In the HL nutrient enrichment experi-ment, SRP decreased in treatments receiving PO4 togetherwith added sources of N (i.e., NO3 or NH4, Table 1). Thechanges in SRP concentration for all other treatments werenot substantially diVerent from the control. Decreases ininorganic N (Ni) were observed in treatments with NO3 andPO4 together, and NO3 alone. In the LL nutrient enrichmentexperiment, SRP concentrations similarly decreased only intreatments with PO4 when NH4 or NO3 were also added.All other treatments showed slight decreases in SRPconcentration but were not substantially diVerent from thecontrol. Ni decreased following treatment with NO3 andPO4 together but not with NO3 alone, while all otherchanges were similar to the control (Table 1).

Chlorophyll a in nutrient enrichment experiments

Chl a levels were monitored daily throughout the HL andLL nutrient enrichment experiments as described above.Figure 6a shows the Chl a time series in the HL nutrientenrichment experiment. The initial Chl a level was0.15 mg m¡3. The Chl a concentration in the controldecreased to 0.02 mg m¡3 by the Wnal day of the experiment.

Fig. 4 Cell concentration time series for the a high light treatmentand b low light treatment in the simulated stratiWcation experiment.Error bars represent SE of the mean of triplicate measurements andare contained within the symbol when not visible

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SII¡100 is the photochemical eYciency of PSII during 100 �mol quantam¡2 s¡1 actinic light, and �PSII¡1,000 is the photochemical eYciency ofPSII during 1,000 �mol quanta m¡2 s¡1 actinic light. Error bars repre-sent SE of the mean of triplicate measurements

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The largest increase in Chl a was observed in the treatmentreceiving NH4 and PO4 together (0.41 mg m¡3), whichshowed a greater than 3-fold increase over the initial leveland a 17-fold increase relative to the control on the Wnalday (Fig. 6a). Treatment with PO4 and NO3 togetheryielded a smaller but measurable increase (0.18 mg m¡3).By the Wnal day of the experiment, treatments of PO4, NO3,or NH4 alone (i.e., single nutrient amendments) showeddecreased Chl a levels relative to the initial level (0.06,0.05, and 0.05 mg m¡3, respectively) that were similar orslightly higher than that of the control (Fig. 6a). The stan-dard error determined from triplicate samples in the HLnutrient enrichment experiment was below 0.03 mg m¡3

for all treatments.In the LL nutrient enrichment experiment all treatments

(including the control) resulted in elevated Chl a levels rel-ative to the initial level (0.08 mg m¡3); however, none ofthe nutrient addition treatments resulted in Chl a levels thatwere signiWcantly higher than the control (0.24 mg m¡3)by day 4 of the experiment (Fig. 6b). Treatment withPO4, NH4, or NO3 alone resulted in twofold increases inChl a compared to the initial values (0.17, 0.13, and0.21 mg m¡3, respectively). Treatments of PO4 given together

with either NH4 or NO3 showed Chl a levels above initiallevels but similar to the control (0.27 and 0.19 mg m¡3,respectively). The standard error determined from triplicatesamples in the LL nutrient enrichment experiment wasbelow 0.06 mg m¡3 for all treatments.

Flow cytometry cell counts in nutrient enrichment experiments

To determine how picophytoplankton community composi-tion changes in response to nutrients and light availability,we measured the changes in cell densities of picoeukary-otes, Synechococcus, and Prochlorococcus in the HL andLL nutrient enrichment experiments using Xow cytometry.Figure 7 shows picoeukaryotes, Synechococcus, andProchlorococcus cell concentrations throughout the course

Table 1 Nutrient data

Initial inorganic N (Ni) and SRP levels following nutrient additionswere estimated as the sum of the mean background concentrations ofNi and SRP at day zero plus the calculated amount of Ni or SRP addedfrom the nutrient addition treatments. Standard errors are given follow-ing the means. Changes in NO3 and NO2 (�N) and P (�P) concentra-tions between these initial levels and levels at day 4 of the LL and HLnutrient enrichment experiments are also shown. Negative values indi-cate consumption and positive values indicate production of nutrientsthroughout the experiments (�mol L¡1). Values are averages of tripli-cate samples

Treatment Initial nutrient levels after nutrient addition treatment

Changes in nutrient levels

Ni SRP �N �P

HL experiment

NH4,PO4 7.08 § 0.01 0.43 § 0.01 0.07 ¡0.14

NO3,PO4 7.08 § 0.12 0.43 § 0.01 ¡1.41 ¡0.08

NO3 7.08 § 0.11 0.03 § 0.00 ¡0.22 0.01

NH4 7.08 § 0.03 0.03 § 0.00 0.00 0.00

PO4 0.08 § 0.00 0.43 § 0.00 ¡0.02 ¡0.01

Control 0.08 § 0.00 0.03 § 0.00 0.02 0.00

LL experiment

NH4,PO4 7.13 § 0.03 0.44 § 0.02 0.07 ¡0.16

NO3,PO4 7.13 § 0.05 0.44 § 0.01 ¡1.18 ¡0.16

NO3 7.13 § 0.10 0.04 § 0.01 0.05 ¡0.02

NH4 7.13 § 0.01 0.04 § 0.01 0.01 ¡0.01

PO4 0.13 § 0.03 0.44 § 0.01 ¡0.05 ¡0.07

Control 0.13 § 0.02 0.04 § 0.01 0.15 ¡0.02

Fig. 6 Chlorophyll a concentrations for the a high light and b lowlight nutrient enrichment experiments. Error bars represent SE of themean of triplicate measurements

0.3

0.2

0.0

0.4

0.1

NO3, PO4

NH4, PO4

NO3

NH 4

PO4

Control

0.3

0.2

0.0

0.4

0.5

0.1

Chl

a(m

g m

-3)

(a)

532

Chl

a(m

g m

-3)

(b)

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0.5

51 4 1 432

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of the HL and LL nutrient enrichment experiments, andTable 2 shows the Wnal concentrations and the relativeincreases for each treatment compared to the control on thelast day of the experiment. (The coeYcient of variation forcell concentrations determined from triplicate samples wasbelow 0.15 for both the HL and LL nutrient enrichmentexperiments.) Prochlorococcus (Table 2, Fig. 7a.3) wasthe dominant cell type at the start of the HL experiment,followed by Synechococcus (Table 2, Fig. 7a.2) and pic-oeukaryotes (Table 2, Fig. 7a.1), whereas Synechococcus(Fig. 7b.2) was the dominant cell type at the start of the LLnutrient enrichment experiment, followed by Prochloro-coccus (Fig. 7b.3) and picoeukaryotes (Fig. 7b.1). In theHL nutrient enrichment experiment, the greatest increasesin picoeukaryote and Synechococcus cell numbers (1–2orders of magnitude increase compared to control) occurredin treatments receiving N and P together. Prochlorococcuscell concentrations also increased relative to the control fortreatments receiving PO4 together with either NO3 or NH4,but were less than an order of magnitude greater than thecontrol and remained similar to initial levels (Table 2,Fig. 7a). In contrast, cell concentration measurements forall three organisms were typically less than or comparableto that of the control for treatments in the LL nutrientenrichment experiment (Table 2, Fig. 7b).

Photosynthetic biomass in nutrient enrichment experiments

To estimate the relative contributions of picoeukaryotes,Synechococcus, and Prochlorococcus to primary productionduring a bloom, we estimated changes in the photosyntheticbiomass of each of these groups during the HL and LL nutri-

ent enrichment experiments. Figure 8 shows the changefrom initial picophytoplankton photosynthetic biomass lev-els to those on day 4 of each experiment for picoeukaryotes,Synechococcus, and Prochlorococcus. There were no sub-stantial net changes in picophytoplankton photosyntheticbiomass for any treatments relative to initial levels in the LLnutrient enrichment experiment, where the photosyntheticbiomasses for all treatments were similar to initial levels(6.8 mg C m¡3). In these low light samples, the contributionfrom Prochlorococcus and Synechococcus decreased in alltreatments except for the control, while the contribution ofpicoeukaryotes increased for all treatments (Fig. 8b). TheHL nutrient enrichment experiment (Fig. 8a), in contrast,showed net increases in picophytoplankton photosyntheticbiomass relative to initial levels (2.4 mg C m¡3) when N andP were added together (16.3 and 19.3 mg C m¡3 for treat-ments NH4 + PO4 and NO3 + PO4, respectively), similarlevels following additions of PO4 (4.2 mg C m¡3) or NH4

(4.6 mg C m¡3), and decreased levels for the control andonly NO3 treatments (1.1 mg C m¡3).

Flow cytometry cellular Xuorescence in nutrient enrichment experiments

To assess photoacclimation in the picoeukaryote, Synecho-coccus, and Prochlorococcus populations HL and LLnutrient enrichment experiments we estimated cellularXuorescence, a proxy for cellular pigment content (Sosiket al. 1989; Li et al. 1993), from Xow cytometry. Figure 9shows the mean and median red Xuorescence of cells onday 0 and 4 of the LL nutrient enrichment experiment. Byday 4 of the experiment, red Xuorescence increased twofold

Fig. 7 Cell concentration time series for the a high light and b low light nutrient enrichment experiments for (a.1) picoeuk-aryotes; (a.2) Synechococcus; and (a.3) Prochlorococcus. The coeYcient of variation for triplicate samples was below 0.15; error bars (SE) are contained within symbols where not visible

b.3

Prochlorococcus

a.3

Day

432 321

b.2

Synechococcus

a.2

Day

432 1 41 4321

LL

exp

erim

ent

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103

102

101

HL

exp

erim

ent

a.1

Picoeukaryotes

NO3

NH4

PO4

NO3, PO4

NH4, PO4

Control

104

103

102

101

Cel

l den

sity

(ce

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L-1

)

Day

4321 4321

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in the picoeukaryote population (Fig. 9a) and Wvefold in theSynechococcus population (Fig. 9b) regardless of nutrienttreatment. Red Xuorescence in the Prochlorococcus popu-lation was similar on day 4 and day 0 in all nutrient treat-ments (Fig. 9c). Red Xuorescence levels in cell populationson day 4 of the HL nutrient enrichment experiment did notshow any systematic trends relative to day 0 levels (data notshown).

Discussion

Nutrient limitation refers to the slowing or cessation ofphotosynthetic biomass production in response to lowavailability of an essential nutrient. Together, the RedWeldratio and Liebig’s law of the minimum have been used inoceanography to identify nutrient limitation at the phyto-plankton community level (von Liebig 1840; RedWeld et al.1963). These interpretations are based on the assumptionthat phytoplankton communities assimilate macronutrientsat a constant ratio, and therefore deviations from such ratiosindicate limitation by one nutrient over another. However,nutrient co-limitation can occur in oligotrophic waters(Seppala et al. 1999; Mills et al. 2004) where addition ofone nutrient may rapidly induce limitation for anothernutrient as soon as limitation by the former is relieved. Inthese cases phytoplankton communities are eVectively co-limited because addition of both nutrients is required toelicit substantial increases in biomass. Therefore, while

only one nutrient can be physiologically limiting at a givenpoint in time, more than one nutrient may eVectively co-limit phytoplankton on ecologically relevant time scales. Inthis study, the term “co-limitation” refers to inhibition ofphotosynthetic biomass production, rather than to the phys-iological nutrient status of a cell or group of cells per se.

In the stratiWed Gulf of Aqaba, N and P co-limit the phy-toplankton community. Under irradiances similar to levelsthat would be seen in the upper euphotic zone (HL nutrientenrichment experiment), additions of inorganic N and Ptogether resulted in elevated Chl a levels, while addition ofN or P independently did not (Fig. 6a) (Fe, Si, and othertrace metal concentrations are elevated in the waters of theGulf (Longhurst 1998; Chase et al. 2004; Chen et al. 2007),thus limitation by these elements was not considered here).Indeed, estimates of picophytoplankton photosynthetic bio-mass for the HL nutrient enrichment experiment showedthat the largest increases occurred when N and P wereadded simultaneously (Fig. 8a). We therefore conclude thatduring the time of our experiments, oligotrophic conditionsin surface waters (Table 1) caused co-limitation of the phy-toplankton community by N and P when light was not lim-iting (Figs. 6a, 8a; see also Labiosa 2007). This type oflimitation is referred to as “multi-nutrient co-limitation,”and results when the levels of two or more nutrients aredepleted beyond the levels required for cellular uptake(Arrigo 2005 and references therein).

The HL nutrient enrichment experiment showed that thephytoplankton community did not respond homogenously

Table 2 Absolute cell concentrations (cells mL¡1) of each picophytoplankton cell type on day 4 of the HL and LL nutrient enrichment experi-ments as determined by Xow cytometry

The total concentration of all picophytoplankton cells is shown in the last column, and concentrations at day zero are also shown. Percentage ofcells in each experimental treatment relative to time zero and control are given in parentheses

Treatment Picoeukaryotes (cells mL¡1)

Synechococcus (cells mL¡1)

Prochlorococcus (cells mL¡1)

All picophytoplankton (cells mL¡1)

HL experiment

NH4,PO4 4,300 (1,433, 2,150%) 33,300 (3,700, 16,650%) 40,400 (205, 878%) 78,000 (373, 1560%)

NO3,PO4 8,700 (2,900, 4,350%) 29,600 (3,289, 14,800%) 49,800 (253, 1083%) 88,100 (422, 1762%)

PO4 600 (200, 300%) 8,500 (944, 4,250%) 14,500 (74, 315%) 23,600 (113, 472%)

NH4 700 (233, 350%) 11,800 (1,311, 5,900%) 8,200 (42, 178%) 20,700 (99, 414%)

NO3 300 (100, 150%) 20 (2, 10%) 9,200 (47, 200%) 9,500 (45, 190%)

Control 200 (67, 100%) 200 (22, 100%) 4,600 (23, 100%) 5,000 (24, 100%)

Day zero 300 (100, 150%) 900 (100, 450%) 19,700 (100, 428%) 20,900 (100, 418%)

LL experiment

NH4,PO4 7,200 (514, 120%) 4,700 (29, 62%) 8,600 (64, 54%) 20,500 (66, 69%)

NO3,PO4 8,600 (614, 143%) 700 (4, 9%) 3,500 (26, 22%) 12,800 (41, 43%)

PO4 3,000 (214, 50%) 8,300 (52, 111%) 10,400 (78, 66%) 21,700 (70, 74%)

NO3 4,500 (321, 75%) 5,000 (31, 66%) 8,400 (63, 53%) 17,900 (58, 61%)

NH4 2,400 (171, 40%) 5,900 (37, 79%) 5,600 (42, 35%) 13,900 (45, 47%)

Control 6,000 (429, 100%) 7,500 (47, 100%) 15,900 (119, 100%) 29,400 (95, 100%)

Day zero 1,400 (100, 23%) 16,100 (100, 213%) 13,400 (100, 84%) 30,900 (100, 105%)

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to nutrient additions under suYcient irradiances. Cell con-centration measurements indicated that picoeukaryotes andSynechococcus responded to nutrient additions during the4-day incubation period, whereas Prochlorococcus (thedominant organism at the start of our sampling) did not(Fig. 7a). Therefore, the majority of the photosyntheticbiomass produced following addition of N and P was attrib-utable to picoeukaryotes and Synechococcus (Fig. 8a).Similar results were observed in the simulated stratiWcationexperiment, where picoeukaryote and Synechococcus popu-lations bloomed over a 2 day period when light limitationwas reversed (Fig. 4). In this study, we focused onpicophytoplankton bloom dynamics; we note, however,that larger cells also contribute to blooms components inthe Gulf of Aqaba (Lindell and Post 1995; Mackey et al.2007), contributing additional photosynthetic biomass. Ourresults indicate that when growth limiting nutrients becomeavailable and light is not limiting, blooms of speciWc sub-populations within the overall phytoplankton communitycan yield substantial increases in photosynthetic biomass asa result of their rapid growth responses.

The relatively rapid increase in the cell densities ofpicoeukaryotes and Synechococcus when N and P were

provided together in the HL nutrient enrichment (Fig. 7a.1,a.2), and the simulated stratiWcation (Fig. 4a, b) experi-ments suggested that these phytoplankton have a “bloomer”growth strategy (Margalef 1978; Smayda and Reynolds2001; Klausmeier et al. 2004; Labiosa 2007; see Arrigo

Fig. 8 Estimates of picophytoplankton photosynthetic biomass on theWnal day of the a high light and b low light nutrient enrichment exper-iments contributed by picoeukaryotes (black bars), Synechococcus(hatched bars), and Prochlorococcus (white bars)

- 6 0 6 12 18

(a)

(b)

Change in photosynthetic biomass(mg C m-3)

euk

Syn

Pro

NH4,PO4

NO3,PO4

NH4

NO3

PO4

control

NH4,PO4

NO3,PO4

NH4

NO3

PO4

control

Fig. 9 Cellular Xuorescence during the LL nutrient enrichment exper-iment for a picoeukaryotes, b Synechococcus, and c Prochlorococcus.Plots show Xuorescence per cell on day 0 for the initial populations,and on day 4 for each treatment and the control. Error bars show thestandard deviations of the cellular Xuorescence distributions

101

102

103

(a)

(b)

(c)

Tim

e ze

ro

Con

trol

PO4

NO

3

NH

4

NO

3,PO

4

NH

4,PO

4

103

104

105

103

104

105

Red

flu

ores

cenc

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mean

median

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2005 for discussion of terminology). This growth responseis similar to the in situ changes that occurred as the watercolumn started to stratify and nutrient and light limitationare reversed, which show that the cell concentrations ofpicoeukaryotes (and to a lesser extent Synechococcus)increase two to threefold in the upper euphotic zone over aperiod of days (Fig. 1d, e). Moreover, picoeukaryotes werenot numerically dominant in day zero samples, indicatingthat they were unable to remain abundant during prolongedoligotrophic summer conditions (Fig. 7a.1, b.1; Lindell andPost 1995; Labiosa 2007; Mackey et al. 2007), as would beexpected for a bloomer strategist.

By contrast, Prochlorococcus was abundant in day zerosamples and showed no rapid, substantial growth responsesregardless of nutrient additions when light was not limitingin the HL nutrient enrichment experiment (Fig. 7a.3, b.3;see also Labiosa 2007). Similar results were observed in thesimulated stratiWcation experiment, where the Prochloro-coccus population declined slightly over a 2 day periodwhen light limitation was reversed but nutrients were stillavailable from deep winter mixing. These data may suggestthat Prochlorococcus adopts a “survivalist” growth strat-egy, enabling them to sustain growth during periods of lownutrient levels, possibly owing to lower growth rates, lowernutrient requirements (Bertilsson et al. 2003; Fuller et al.2005; Van Mooy et al. 2006) and high aYnity nutrientacquisition systems (Scanlan and Wilson 1999) comparedto other picophytoplankton. Indeed, Lindell and Post(1995) observed that Prochlorococcus became the domi-nant phytoplankter in the Gulf only after several months ofstratiWcation and prolonged nutrient depletion had causedmarked decreases in picoeukaryote and Synechococcusnumbers.

However, while this pattern of succession is typicalduring most years in the Gulf (Israel National MonitoringProject, Eilat, http://www.iui-eilat.ac.il/NMP/, unpublisheddata), Prochlorococcus was a signiWcant contributor to thespring bloom in 1999 (Fuller et al. 2005), suggesting that itmay share characteristics of bloom strategists as well.SpeciWcally the hydrographic and chemical conditions (sta-bility and duration of the stratiWcation, amount of nutrientsinjected, temperature and light intensities etc.) duringbloom events and the related variable responses of Prochlo-rococcus should be evaluated in more detail. In the simu-lated stratiWcation experiment, the lack of Prochlorococcusgrowth could be a function of the antecedent conditions ofthe sample water, rather than a function of a survivalistgrowth strategy. In contrast to the in situ monitoring at sta-tion A (a location in open water away from the coastalshelf), this experiment used water collected closer to shore.The higher concentrations of Synechococcus and picoeuk-aryotes in the time zero samples (Fig. 4) compared to thesurface water from station A from the same day (12 March,

Fig. 1) suggests that growth conditions varied slightlybetween the locations; coastal stratiWcation may have pre-ceded stratiWcation at station A due to faster warming inshallower water, thereby accelerating the bloom. Therefore,it is possible that the relatively high Prochlorococcus con-centrations in time zero samples could indicate that thegrowth response of these cells had already occurred andreached steady state before our sample water was collected,even though Synechococcus and picoeukaryotes continuedto bloom for the duration of the experiment. Alternately,the size of the seed population of competent high light eco-types of Prochlorococcus (rather than growth strategy)could explain the growth response (Fuller et al. 2005).Therefore, while Prochlorococcus has optimized its growthat lower nutrient levels and is abundant in nutrient poorwaters (Partensky et al. 1999and references therein), morework is needed to accurately characterize Prochlorococcusgrowth strategies, particularly with respect to ecotypicdiversity.

In addition to identifying multi-nutrient co-limitation inthe phytoplankton community and the species speciWcresponse to alleviation of this co-limitation, our results alsosuggested that the RedWeld ratio represents an averagevalue of species-speciWc N:P ratios in the Gulf of Aqaba, asobserved in other areas of the ocean (Elser et al. 1996). Amodel by Klausmeier et al. (2004) reports a range of phyto-plankton elemental stoichiometries (N:P) of 8.2 to 45depending on environmental conditions, and notes that var-iability occurs between and within species due to geneticdiversity and physiological Xexibility. The range of N:Pratios reported for Synechococcus (13.3-33.2) and Prochlo-rococcus (15.9-22.4) (Bertilsson et al. 2003; Heldal et al.2003) demonstrate the Xexibility in phytoplankton elemen-tal stoichiometries in oligotrophic marine environmentswhen faced with variable nutrient availability.

Our study demonstrates that the physiological Xexibilitywith which nutrients are assimilated into photosyntheticbiomass is important for phytoplankton dynamics duringthe spring bloom, where phytoplankton regulate and main-tain their elemental stoichiometries via preferential uptakeof certain nutrients. Phytoplankton with a “bloomer”growth strategy tend toward lower N:P ratios (Elser et al.1996). Nutrient stoichiometries in the water column wereclose to RedWeldian prior to stratiWcation (Fig. 3a). Duringthe transition from mixing to stratiWcation, the net removalof NO3 and PO4 from the water continued at near-RedWeldratios in the surface (likely reXecting a contribution fromlarger phytoplankton cells not counted in our analysis),whereas bulk uptake ratios were lower (<10) in the middleand lower euphotic zone (Fig. 3d) where Synechococcusand picoeukaryotes dominated (Fig. 1d, e). Moreover, theratio reached a minimum value of 1 at 60 m, coincidingwith the picoeukaryote maximum identiWed in Fig. 1e.

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These results showed that as stratiWcation progressed, moreP was taken up overall relative to N than would be pre-dicted by the RedWeld ratio, possibly indicating luxury Puptake. These trends were consistent with the phytoplank-ton growth observed during the bloom (Fig. 1d, e), whichfavor resource partitioning toward P-rich cellular assemblymachinery, including ribosomes, high energy adenylates,and nucleic acids (Elser et al. 1996, 2000; Geider and LaRoche 2002). While this preferential uptake has been sug-gested based on theory and laboratory experiments, ourWeld results also support it by relating changes in phyto-plankton community composition (i.e., bloomers vs. sur-vivalists) to nutrient uptake ratios.

The term “photoacclimation” encompasses the pheno-typic changes that occur in an organism in response tochanges in environmental factors aVecting photosynthesis(MacIntyre et al. 2002). The photochemical yield of PSII isa measure of photoacclimation because it shows howeYciently PSII is able to use light to drive photochemistry.Fv/Fm, which is the maximum photochemical eYciency ofPSII in dark adapted cells, is a measure of the number offunctional PSII reaction centers, and tends to reach its low-est values at midday due to photoinhibition (Mackey et al.2008). �PSII is a measure of the photochemical eYciency ofPSII under a given background (actinic) light intensity. ItdiVers from Fv/Fm in that some of the functional PSII reac-tion centers are in the reduced state due to exposure to lightat the time of measurement. In the HL treatment, anincrease in �PSII under both 100 and 1,000 �mol quantam¡2 s¡1 shows that after only one and a half days of incu-bation under relatively high light (incubation irradiance of1,000 �mol quanta m¡2 s¡1), the cells in these sampleswere more eYcient at keeping PSII oxidized during expo-sure to light compared to initial measurements when cellswere acclimated to deep mixing (Fig. 5a). In contrast, cellsincubated under lower irradiance (i.e., maximum middayintensity of 100 �mol quanta m¡2 s¡1 in the LL treatment)showed a decreased capacity to cope with light and use it todrive photochemistry on the second day of incubation. Inthese samples a larger number of PSII reaction centersbecame reduced (i.e., �PSII decreased, Fig. 5b) followingexposure to the actinic light, suggesting that the cells wereless eYcient at coping with exposure to light [e.g., by draw-ing electrons away from PSII, possibly due to a lack oflight-inducible photoprotective strategies (Cardol et al.2008)] compared to the Wrst day of incubation.

This response demonstrates that photoacclimationoccurs rapidly at the onset of stratiWcation, and likely inXu-ences which taxonomic groups become dominant during abloom. For example, photoacclimation typically leads to anincrease in photosynthetic pigment content under decreasedirradiance, such that more light is harvested by thephotosynthetic apparatus. For both picoeukaryotes and

Synechococcus populations in the LL nutrient enrichmentexperiment, similar cellular Xuorescence increases wereobserved in all treatments and the control (relative to initiallevels, Fig. 9). The incubation of these cells under low lightintensities was therefore either associated with a concurrentcellular synthesis of pigments, or a community shift inwhich cells with higher pigment content became moreabundant, replacing their low-pigment counterparts.

When nutrient limitation was reversed but the incubationirradiance was similar to levels that would be experiencedat greater depths in the euphotic zone, i.e., in the LL nutri-ent enrichment experiment, nutrient additions had a smallereVect on photosynthetic biomass than in the HL nutrientenrichment experiment. SpeciWcally, the increase in Chl a(Fig. 6b) observed under low light conditions did not corre-spond to increased photosynthetic biomass in the LL nutri-ent enrichment experiment (Fig. 8b) even though shifts inthe phytoplankton community were observed relative toinitial samples (Fig. 7b). For example, picoeukaryote cellconcentrations in the LL nutrient enrichment experimentincreased over an order of magnitude regardless of nutrientaddition treatment (Fig. 7b.1). (Picoeukaryote cell numbersin the deep euphotic zone during the spring bloom showsimilar trends, increasing at 120 m (Fig. 1e)). In contrast tothe picoeukaryotes, Synechococcus and Prochlorococcusdeclined slightly in abundance for all treatments andthe control in the LL nutrient enrichment experiment(Fig. 7b.2, b.3), possibly due to the presence of small(<20 �m) grazers that were able to pass through the mesh atthe start of the experiment (Sommer 2000). The results ofthis LL nutrient enrichment experiment show that whenlight (rather then nutrients) limits growth, both photoaccli-mation and considerable shifts in community compositioncan occur within the phytoplankton community without anet gain or loss of photosynthetic biomass. Therefore, thetraditional interpretation of “bloom” dynamics (i.e., as anincrease in photosynthetic biomass) may be conWned to theupper euphotic zone where light is not limiting, while otheracclimation processes are more ecologically relevant atdepth.

While Synechococcus declined in abundance during theLL nutrient enrichment experiment, samples from the deepeuphotic zone during the spring bloom show a slightincrease in Synechococcus abundance at 100 m (Fig. 1d)along with a concurrent increase in cellular Xuorescence(Fig. 2), suggesting that Synechococcus cells are able togrow and acclimate within the light limited regions of thedeep euphotic zone. Low-light adapted Prochlorococcusecotypes have also been shown to be dominant and ubiqui-tous members of deep euphotic zone phytoplankton com-munities (West and Scanlan 1999; Rocap et al. 2003;Mackey et al. 2008) but did not grow in the LL experi-ments. One explanation for the diVerence in Synechococcus

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and Prochlorococcus growth responses between cells in theLL nutrient enrichment experiment and cells from the deepeuphotic zone during the spring bloom could be the diVer-ent antecedent conditions to which these populations wereacclimated prior to sampling. The LL nutrient enrichmentexperiment was conducted with water and phytoplanktoncollected from stratiWed surface waters; therefore, it islikely that these cells were acclimated to high light and mayhave required more than 4 days to adjust to lower incuba-tion irradiances before growing. Moreover, low-lightadapted ecotypes were likely less abundant in the samplewater used in the nutrient addition experiments than high-light ecotypes (Fuller et al. 2005; Lindell et al. 2005), andwould have required more time to become numericallydominant following a light shift. In contrast, cells sampledfrom the deeply mixed water column (>300 m) wouldlikely be acclimated to a very diVerent light regime, asbefore the bloom the water column was mixed from the sur-face (high light) to below the euphotic depth (very lowlight). Following stratiWcation of the water column, cellstrapped at depth would be primed to acclimate to low irra-diances, allowing them to bloom immediately (Fig. 1d, e).In addition, larger seed populations of competent low lightadapted ecotypes would have been present in the mixedwater column than in the surface waters used during the LLnutrient enrichment experiment.

Our data suggest that in the Gulf of Aqaba, physiologicalacclimation to diVerent nutrient and light regimes help shapethe phytoplankton community. However, it is importantto note that this physiological Xexibility is likely only onefactor inXuencing phytoplankton survival and communitysuccession: multiple taxonomic groups of picoeukaryotes(Moon-van der Staay et al. 2001; Worden 2006), Synecho-coccus (Fuller et al. 2005; Penno et al. 2006), and Prochlo-rococcus (Moore et al. 1998; Rocap et al. 2003) coexist inthis and other open ocean regions, and this extra layer ofdiversity may further enable picophytoplankton communi-ties to adjust rapidly to changes in nutrient and light avail-ability. The dominance of one group of picoeukaryotesduring a bloom may therefore be at least partially attribut-able to the ability of diVerent ecotypes within that group tothrive under diVerent environmental nutrient and lightregimes, rather than entirely through sustained expression ofnutrient and light stress-response genes (Lindell et al. 2005).

Clarifying the combined inXuence of nutrient and lightlimitation on phytoplankton strategies and bloom dynamicsin oligotrophic regions such as the Gulf of Aqaba is impor-tant for understanding how oceanic productivity could beaVected by global change. Estimates suggest that by theyear 2050 climate warming will lead to longer periods ofstratiWcation with fewer deep mixing events in seasonallystratiWed seas, causing expansion of low productivity,permanently stratiWed subtropical gyre biomes (i.e., ultraol-

igotrophic waters) by 4.0% in the Northern Hemisphere and9.4% in the Southern Hemisphere (Sarmiento et al. 2004).Following oligotrophic ocean expansion, the eVect of non-deepwater nutrient sources (atmospheric dry deposition,precipitation, nitrogen Wxation, etc.) on oceanic net carbonsequestration, export production, and climate could becomemore signiWcant as the relative contribution of deepwaternutrients decreases. This shift in nutrient availability andsource, which would be overlaid upon a more static lightenvironment, could substantially alter phytoplankton com-munity structure and bloom dynamics in these regions. Ourresults from the oligotrophic Gulf of Aqaba suggest thatunder present-day conditions the introduction of exogenousnutrients into stratiWed surface waters supports blooms ofpicoeukaryotes that, when present, increase in numberquickly by exploiting the new nutrients (Figs. 7a.1, 8; Labi-osa 2007). However, because survivalists like Prochloro-coccus are more likely to be abundant following prolongedultraoligotrophic periods (Lindell and Post 1995; thiswork), the eVect of exogenous nutrient sources (e.g., dust,nitrogen Wxation, precipitation, etc.) on carbon sequestra-tion within future permanently stratiWed oligotrophic seasremains diYcult to predict. SpeciWc investigations on theeVects of exogenous new nutrients are still needed to fullyunderstand if and how they will inXuence primary produc-tion and shape phytoplankton community structure in theoligotrophic ocean at present and in the future.

Acknowledgments We thank our colleagues at the InteruniversityInstitute for Marine Science in Eilat, Israel for assisting in data collec-tion and providing laboratory space and equipment during the study.We also thank the anonymous reviewers who provided comments onthe manuscript. M. Chernichovsky, Y. Chen, R. Foster, E. Grey, andJ. Street assisted with sampling. D. Parks assisted with Xow cytometrymeasurements at Stanford and I. Ayalon provided Xow cytometry dataanalyzed at IUI. R. Labiosa helped develop experimental methods.This research was supported under the National Aeronautics and SpaceAdministration (NASA) New Investigator Program NAG5-12663 toAP, the North Atlantic Treaty Organization (NATO) Science for PeaceGrant SfP 982161 to AP and AFP, a grant from the Koret Foundationto AP, National Science Foundation (NSF) Oceanography grant OCE-0450874 to ARG., and Israel Science Foundation grant 135/05 to AFP.KRMM was supported through the National Science Foundation(NSF) Graduate Research Fellowship Program and the Department ofEnergy (DOE) Global Change Education Program. All experimentscomply with the current laws of the countries in which the experimentswere performed.

Open Access This article is distributed under the terms of the Crea-tive Commons Attribution Noncommercial License which permits anynoncommercial use, distribution, and reproduction in any medium,provided the original author(s) and source are credited.

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