Deep-Sea Research II

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1 Deep-Sea Research II 58 (2011) Contents lists available at ScienceDirect Deep-Sea Research II journal homepage: Co-limitation of diatoms by iron and silicic acid in the equatorial Pacific Mark A. Brzezinski c,e,n, Stephen B. Baines b, William M. Balch a, Charlotte P. Beucher e, Fei Chai j, Richard C. Dugdale i, Jeffrey W. Krause f,e, Michael R. Landry h, Albert Marchi i, Chris I. Measures d, David M. Nelson f,k, Alexander E. Parker i, Alex J. Poulton g, Karen E. Selph d, Peter G. Strutton f, Andrew G. Taylor h, Benjamin S. Twining a a Bigelow Laboratory for Ocean Sciences, West Boothbay Harbor, ME 04575, USA b Department of Ecology and Evolution, State University of New York, Stony Brook, NY 11794, USA c Department of Ecology Evolution and Marine Biology, University of California, Santa Barbara, CA 93106, USA d Department of Oceanography, University of Hawai i at Manoa, Honolulu, HI 96822, USA e Marine Science Institute, University of California, Santa Barbara, CA 93106, USA f College of Oceanic and Atmospheric Sciences, Oregon State University, Corvallis, OR 97331, USA g National Oceanography Centre, University of Southampton, Southampton, Hampshire, S014 3ZH, UK h Scripps Institution of Oceanography, University of California San Diego, La Jolla, CA 92093, USA i Romberg Tiburon Center, San Francisco State University, 3152 Paradise Drive, Tiburon, CA , USA j School of Marine Sciences, University of Maine, Orono, ME 04469, USA k Institut Universitaire Européen de la Mer, CNRS, Universite! Europe!enne de Bretagne Occidentale Brest, France article info Article history: Received 9 August 2010 Accepted 9 August 2010 Available online 17 August 2010 Keywords: Co-limitation Nutrient limitation Silicon Iron Equatorial upwelling Diatoms abstract The relative roles of silicon (Si) and iron (Fe) as limiting nutrients in the eastern equatorial Pacific (EEP) were examined in a series of nine microcosm experiments conducted over two years between 1101W and 1401W longitude. Si and Fe additions had consistently different but synergistic effects on macronutrient use, phytoplankton biomass and phytoplankton community structure. Silicon addition increased silicic acid use and biogenic silica production, but had no significant effect on the use of inorganic nitrogen or orthophosphate, chlorophyll accumulation, particulate inorganic (PIC) carbon accumulation, or plankton community composition relative to controls. That result, together with observations that Si addition increased the cellular Si content of the numerically dominant diatom by 50%, indicates that the main effect of Si was to regulate diatom silicification. Like the effect of Si, Fe addition increased the rate of silicic acid use and biogenic silica production and had no effect on PIC production. Unlike the effect of Si, Fe addition also enhanced rates of organic matter production, had no effect on cellular Si content of diatoms, and resulted in the growth of initially rare, large (440 mm) diatoms relative to controls, indicating that Fe limitation acts mainly through its effects on growth rate and phytoplankton community composition. A pennate diatom of the genus Pseudo-nitzschia dominated the diatom assemblage in situ, grew readily in the controls and did not show a strong growth response to either Fe or Si addition suggesting that its growth was regulated by other factors such as grazing or light. Addition of germanium, an inhibitor of diatom cell division, eliminated the effects of Fe on macronutrient use, biogenic silica production and chlorophyll accumulation and phytoplankton community composition, consistent with a predominantly diatom response to Fe addition. The lack of a response of PIC production to Fe suggests that coccolithophores were not Fe limited. Addition of Fe and Si together resulted in the greatest levels of nutrient drawdown and biomass accumulation through the effect of Fe in promoting the growth of large diatoms. The results suggest a form of co-limitation with Si regulating diatom silicification and the rate of biogenic silica production while Fe regulates the production of organic matter through limitation of phytoplankton growth rates, in particular those of large diatoms. The results argue against Si regulation of new production in the EEP under average upwelling conditions. Iron addition was necessary and sufficient to stimulate complete removal of nitrate within the equatorial upwelling zone suggesting that new production was restricted by low ambient dissolved Fe consistent with results from in situ Fe fertilization experiments conducted to the south of the equator outside of the equatorial upwelling zone. & 2010 Elsevier Ltd. All rights reserved. n Corresponding author at: Marine Science Institute, University of California, Santa Barbara, CA 93106, USA. Tel.: ; fax: address: brzezins@lifesci.ucsb.edu (M.A. Brzezinski) /$ - see front matter & 2010 Elsevier Ltd. All rights reserved. doi: /j.dsr

2 494 M.A. Brzezinski et al. / Deep-Sea Research II 58 (2011) Introduction Inefficient consumption of upwelled CO 2 and macronutrients by phytoplankton is a major factor causing the equatorial Pacific to be the largest single oceanic source of CO 2 to the atmosphere (Takahashi et al., 2009). The cause of low phytoplankton productivity, despite abundant macronutrients, is generally attributed to iron (Fe) limitation (Coale et al., 1996a, b; Martin et al., 1994; de Baar et al., 2005; Boyd et al., 2007) leading to a High-Nutrient Low- Chlorophyll (HNLC) condition in the eastern equatorial Pacific (EEP) east of 1701W (Levitus, 2001). Inthe1990s, theironexexperi- ments provided direct experimental evidence for Fe limitation of phytoplankton growth rate in the EEP to the south of the main upwelling zone (Martin et al., 1994; Coale et al., 1996b; Landry et al., 2000a, b). Subsequent studies between 1601E and901w longitude have shown an influence of low [Fe] on phytoplankton photophysiology across most of the upwelling cold tongue in the equatorial Pacific (Behrenfeld et al., 1996, 2006). At approximately the same time as the IronEx experiments it was recognized that while nitrate is abundant in the surface waters of the EEP, the concentration of silicic acid is consistently much lower. Ku et al. (1995) were the first to suggest that new production in the region may be limited by the supply of dissolved silicon based on a low vertical supply rate of silicic acid, Si(OH) 4, relative to that of nitrate, NO 3. Dugdale and Wilkerson (1998) also argued for Si regulation of new and export production in the equatorial Pacific at 1401W longitude based on a silicic acid:nitrate drawdown ratio in surface waters that matched the elemental composition of nutrient-replete diatoms. Estimates of new production by diatoms in other regions of the equatorial Pacific are also significant, with the fraction of new production by diatoms estimated to be between 20 and 85% (Dunne et al., 1999). Direct experimental evidence for the Si limitation of the rate of diatom Si uptake has been obtained in the EEP between 1101W and 1801W longitude (Leynaert et al., 2001; Brzezinski et al., 2008). Those studies clearly demonstrate that low silicic acid concentrations restrict the rate of diatom silicification over a broad region of the equatorial Pacific. However, they do not necessarily indicate limitation of diatom growth rate by Si as diatoms can maintain nearmaximum division rates despite limitation of the rate of Si acquisition by producing thinner frustules (see Martin-Jézéquel et al., 2000). In evaluating the degree of limitation of Si uptake in the equatorial Pacific both Brzezinski et al. (2008) and Leynaert et al. (2001) concluded that the degree of substrate limitation of Si uptake in the EEP was insufficient to be interpreted as indicating Si limitation of diatom growth rates. A few studies have explicitly examined the interactive effects of Si and Fe on phytoplankton rate processes in the HNLC waters of the EEP. Shipboard microcosm experiments conducted during IronEx II showed that the addition of Si alone did not stimulate diatom growth over that observed in controls, but that the addition of Fe led to the proliferation of pennate diatoms (Coale et al., 1996b). However, the addition of both Si and Fe together produced a greater response than did the addition of Fe alone (Coale et al., 1996b). Brzezinski et al. (2008), working in the EEP between 1101W and 1401W longitude, showed that either Fe or Si addition stimulated rates of silica production with each nutrient increasing Si production rates to a similar degree. Similar to the effects of Si and Fe on diatom growth rate observed during IronEx II, the individual effects of Si and Fe on rates of Si uptake were synergistic with the greatest increase in Si uptake rates observed when both Si and Fe were added together. Synergies between the effects of added Si and Fe also have been observed in two other HNLC regions. Working off the coast of California, Hutchins et al. (1998) showed that more chlorophyll was produced in microcosms receiving both Fe and Si compared to those receiving only Fe. Important roles for both Fe and Si have also been observed in the subantarctic where there is a seasonal switch in the factors limiting silica production with Fe limitation in early spring, when Si is relatively abundant, transitioning to clear Si limitation late in the growing season, following silicic acid depletion in surface waters (e.g. Boyd et al., 1999; Franck et al., 2000; Hutchins et al., 2001). A similar switch from Fe to Si limitation has recently been documented on the Kerguelen plateau in the Indian sector of the Southern Ocean where natural iron inputs relieve Fe limitation resulting in diatom blooms that ultimately become Si-limited (Mosseri et al., 2008). Results such as these have led to the idea that Si and Fe may co-limit diatoms in some HNLC waters (e.g. Hutchins et al., 2001; Brzezinski et al., 2008). Modeling studies have produced conflicting results regarding the roles of Si and Fe as limiting resources in HNLC waters. Models that designate diatoms as the sole members of the larger net phytoplankton show a strong effect of Si limitation in controlling new production and carbon export in the EEP (Chai et al., 2002). Other models that include both diatoms and non-siliceous eukaryotic net phytoplankton result in new production and export being driven mainly by the larger non-diatoms with little support for Si regulation of new production or for Si limitation of diatom growth (Salihoglu and Hofmann, 2007a, b). The relative influence of Si and Fe in these models is highly sensitive to the choice of physiological parameters. The diatom-based models can be made to simulate the effects of in situ mesoscale iron fertilization if the sensitivity of photosynthetic performance to Fe is increased to favor Fe rather than Si limitation (Chai et al., 2007). Given the empirical evidence that both Fe and Si influence the relative contribution of diatoms to new and export production in the EEP and the conflicting results of model simulations of these processes, we designed a series of microcosm experiments to investigate the relative roles of Fe and Si as limiting resources for phytoplankton in this region. The results support the idea that Si and Fe co-limit diatom rate processes in the equatorial Pacific with the mechanism of co-limitation being that low dissolved Fe limits the rate of growth and organic matter production, especially of large rare diatoms, while low [Si(OH) 4 ] regulates diatom Si content in the more abundant small diatoms and the overall production of biogenic silica. 2. Methods 2.1. Study area and sampling Sampling was conducted in the EEP aboard the R/V Roger Revelle from 9-24 December, 2004 and from 8-24 September, 2005 (Fig. 1). Six microcosm experiments were performed during the 2004 cruise with three additional experiments conducted during Three of the experiments in 2004 were performed using water collected between 31N and 31S latitude along 1101W longitude with the other three performed using water collected along the equator between 1101W and 1401W. During 2005 water for experiments was collected at 11N and then at 2.51S latitude, along 1401W longitude with a third experiment conducted using water from 1.751N, 1251W near the leading edge of a tropical instability wave (TIW). In addition, profiles of water column properties in the upper 300m were obtained at 17 stations extending from 1101W to 1401W along the equator in December 2004 (Fig. 1) to examine zonal gradients in nutrient concentrations within the study region.

3 M.A. Brzezinski et al. / Deep-Sea Research II 58 (2011) Fig. 1. Cruise track and locations of stations where water for microcosms experiments was collected in 2004 (white circles, top panel) and 2005 (white circles, bottom panel). Additional stations used to access zonal gradients in nutrient concentrations along the equator (black circles, top panel). Cruise track and station locations are superimposed on composite images of ocean color covering the period of each cruise Microcosm experiments Microcosm experiments were conducted in 20 L polycarbonate carboys that were washed with 10% aqueous trace-metal grade hydrochloric acid and high purity (418 MO) deionized water between uses. Microcosms were incubated in deckboard incubators that were screened to 50% of ambient sunlight using neutral density screens and cooled with flowing surface seawater. Light levels in the incubators were higher than the average for the mixed layer and about 2-3 times higher than present over the depth range where water for the experiments was collected. Thus, to the extent that cells in situ were being actively redistributed within the mixed layer the light environment in the incubators was less variable and of a higher intensity than experienced by phytoplankton in situ. Because the water level in the rigid carboys fell with repeated sampling they became increasingly buoyant over time so the carboys were individually strapped to the bottom of the incubator with nylon ropes that kept each container vertical and completely submerged to maintain effective cooling. Seawater to fill the microcosms was collected as described by Brzezinski et al. (2008) using the trace-metal clean collection system described by Measures et al. (2008). Briefly, the system consists of 12-L GO-FLO bottles and a SeaBird CTD hung on an epoxy-coated rosette weighted with resin-coated lead weights and deployed on Kevlar conducting cable. Sample collection involved lowering the rosette to ca. 100 m and raising the package at a slow speed. All 12 GO-FLO bottles were tripped within the mixed-layer between approximately 30 m and 15 m depth, without stopping the CTD/rosette package. The constant upward motion of the rosette meant that the GO-FLO bottles were continuously exposed to water that had minimal contact with the CTD/rosette package reducing the potential for trace metal contamination. Upon recovery the GO-FLO bottles were immediately transferred to a trace metal clean van for sub-sampling in air that was constantly filtered through a Mac 10 HEPA filter with a turnover time of 2 min (Measures et al., 2008). Two casts were required to fill each of the 12 carboys with 20 L of water from the mixed layer. In order to minimize differences between the water introduced into each carboy from the two casts, each carboy was half filled from a single GO-FLO from the first cast and then completely filled from the second cast. The order of the GO-FLOs used to fill the carboys on the second cast was reversed from that of the first, to produce the same average depth of water in each carboy. Transfers between GO-FLOs and carboys were made by gravity draining through acid cleaned C-Flex tubing. Each experiment consisted of five treatments and a control, with each run in duplicate. Experimental treatments were: +Si (20 mm as aqueous sodium metasilicate, cleaned of trace metals using Chelex resin), +Fe (2 nm as acidified ferric chloride), +Ge (3 mm, as aqueous sodium germanate cleaned using Chelex resin), +Fe+Si (2 nm Fe, 20 mm Si), +Fe+Ge (2 nm Fe, 3 mm Ge). Shipboard analyses of total dissolved [Fe] in the nutrient stocks used to augment nutrient concentrations in the microcosm experiments revealed that the Si stock was significantly contaminated with Fe in the first three experiments in 2004 causing the addition of Si to increase total dissolved [Fe] in the microcosms by 0.4 nm. As a result the responses in the +Si treatments from the first three experiments from 2004 were very similar to those in the +Fe+Si treatments. A new Si stock solution was made at sea and was found to contain insignificant amounts of Fe by direct measurement such that the addition of the nutrient to the carboys would increase total dissolved [Fe] by o0.005 nm. The new Si stock was used in all subsequent experiments, dramatically changing the responses observed in the +Si treatments. Experiments in which Si and/or Fe were added have been performed successfully in several past microcosm studies (e.g. Coale et al., 1996b; Franck et al., 2000; Kudela and Dugdale, 2000; Hutchins et al., 2001), but Ge additions have rarely been employed (but see Scarratt et al., 2006). Germanium is taken up by the Si uptake system of diatoms and incorporated into their frustules (e.g. Azam et al., 1973; Azam and Chisholm, 1976).

4 496 M.A. Brzezinski et al. / Deep-Sea Research II 58 (2011) Germanium has no effect on diatom growth when the dissolved Ge/Si concentration mole ratio is o0.01, but at higher ratios Ge inhibits diatom cell division by preventing deposition of the siliceous cell wall (Azam et al., 1973). Germanium is not known to be taken up by nonsiliceous phytoplankton or to affect their growth. Our laboratory tests showed that at a silicic acid concentration of 8.7 mm (higher than is found in the eastern equatorial Pacific under even the strongest upwelling conditions) growth of the diatom Thalassiosira weissflogii is completely arrested at a Ge concentration of 1.0 mm. Dissolved Ge concentrations of up to 30 mm (the highest tested) had no effect on growth of nonsiliceous species, including the coccolithophore Emiliania huxleyi, the cyanobacterium Synechococcus bacillaris, the chlorophyte Dunaliella teriolecta, the prymnesiophyte Isochrysis galbana or the dinoflagellate Gymnodinium simplex. Given these results we view the treatments with Ge-addition to represent the response by phytoplankton other than diatoms. Each experiment was sampled over five days for total dissolved iron concentration, macronutrient concentrations and several measures of phytoplankton biomass. At each sampling the carboys were transferred to the sub-sampling section of the trace metal clean van (Measures et al., 2008) where samples were drawn using a peristaltic pump and acid cleaned C-Flex tubing. Sub-samples for Fe analysis were pumped directly into cleaned Teflon bottles (Measures et al., 2008) and analyzed at sea using the flow injection method of Measures et al. (1995). Water for all other measurements was pumped into acid-washed 6-L polyethylene narrow mouth bottles. Then the bottles were transferred from the clean van to the main laboratory of the ship and subsampled immediately for the following parameters: Nitrate+ nitrite (N+N), orthophosphate and silicic acid concentrations were measured at sea on a Bran and Luebee Autoanalyzer II (Dugdale et al., 2007). Samples for ammonium concentration were analyzed at sea using the manual spectrophotometer method of Solorzano (1969) with a 10 cm cuvette as described by Dugdale et al. (2007). Chlorophyll biomass in the 43-mm and mm size fractions were measured using standard fluorometric methods (JGOFS, 1996). Replicate water samples for chlorophyll concentration analysis were size fractionated in parallel using 0.45 and 3 mm nitrocellulose filters (Millipore Corporation) and extracted in 90% acetone at 20 1C for at least 12 hours before measurement on a Turner Designs 10AU fluorometer (see Balch et al., 2011). Biogenic silica concentrations were measured by sodium hydroxide digestion (Nelson et al., 2001). Particulate inorganic carbon (PIC or calcium carbonate) was measured at 0, 48 and 120 h using inductively-coupled plasma optical emission spectroscopy (Poulton et al., 2006) to estimate the contribution of coccolithophorids. This is a conservative estimate as some cells of this taxa may not have been calcifying during the experiments. Picophytoplankton abundance was determined in each experiment by shipboard flow cytometry (Selph et al., 2001). Subsamples for estimates of eukaryotic phytoplankton abundance as well as the abundance of non-pigmented single-cell heterotrophs (grazers) by epifluorescence microscopy were taken at time zero and at the end of selected experiments. FlowCAM analysis of plankton abundances was performed ashore on sub-samples from all microcosm experiments (except for the first experiment which was inadvertently lost) that were preserved with buffered formalin (2% final concentration) and stored in brown glass bottles. Particle size calibration for the FlowCAM was performed using a combination of latex beads and phytoplankton cultures (sized with an ocular micrometer). Calibration for particle concentration was done using serial dilutions of cultures of known cell concentration. Equivalent spherical diameters of particles were calculated based on the pixels subtended by each Table 1 Initial conditions in microcosm experiments. [bsio 2 ] [Chl-a] mm (mg L 1 ) [Chl-a] 43 mm (mgl 1 ) [Fe]:[NO 3 ] nm:mm [Fe]:[Si(OH) 4 ] nm:mm [Si(OH) 4 ] NH þ 4 [H 3 PO 4 ] ½NO 3 Šþ NO 2 Š [Fe] (nm) Exp. Lat. Long. T (1C) Salinity (psu) Cruise date December N 1101W N 1101W S 1101W W W W September N 1401W * S 1401W N 1251W n From separate cast at same location.

5 M.A. Brzezinski et al. / Deep-Sea Research II 58 (2011) particle. Cell carbon calculated using the equations of Verity et al. (1992) and converted to cell nitrogen using a C:N mole ratio of 6.6 for comparison with N+N use in the microcosms. Particles were then identified to broad class, down to particles of 4 mm diameter. Plankton were enumerated using standard microscopy on a subset of experiments. Because of the time involved in the manual processing of microscopy samples, only samples from experiment 5 in 2004 and experiment 3 in 2005 were analyzed (Table 1). Cells o10 mm were analyzed at a magnification of 630 on slides prepared with paraformaldehyde-preserved samples (50 ml) filtered onto 0.8-mm black polycarbonate filters (Brown et al., 2003). Cells 410 mm were analyzed at 200 magnification on 300-mL samples preserved according to Sherr and Sherr (1993) and filtered onto 8.0 mm polycarbonate filters. Both preparations were stained with proflavin (0.33%) and the DNA-specific fluorochrome DAPI (50 mgml 1 ). Prepared slides were imaged, digitized and analyzed by size class and for major taxa with a Zeiss Axiovert 200 microscope equipped with a motorized stage for automated processing. Cellular carbon estimates were calculated from biovolumes according to Eppley et al. (1970) and were converted to cellular nitrogen as described above for the FlowCAM analyses. In experiment 5 from 2004, samples for single cell elemental analysis by synchrotron x-ray fluorescence (SXRF; Twining et al., 2003) were taken to examine changes in silicification of the diatoms in response to added Si and Fe. Due to logistical constraints and to avoid differences in cellular stoichiometry between treatments that result from changing species composition, only cells of the dominant morphotype, a diatom belonging to the genus Pseudo-nitzschia, were analyzed. (Table 1, and see Kaupp et al., 2011; and Selph et al., 2011, for detailed distributions). Initial chlorophyll concentrations in each experiment showed a dominance of smaller phytoplankton on both cruises. The 43-mm chlorophyll size fraction accounted for an average of 22% of total chlorophyll a in 2004 and an average of 7% in 2005 (Table 1) in agreement with the pattern reported for the more extensive chlorophyll data set gathered on these cruises by Balch et al. (2011). Biogenic silica concentrations were between 87 and 166 nmol Si L 1 with upper water column maxima showing considerable coherence with TIW induced upwelling (Krause et al., 2011; Strutton et al., 2011). Analysis of the nutrient profiles taken along the equator reveals zonal gradients in the nutrient concentrations in the source waters upwelling along the equator. Nutrient concentrations from the profile stations were interpolated to the 24.4 s y isopycnal. This isopycnal was chosen as it was located near the top of the pycnocline along the equator between 1101W and 1401W. Both [Si(OH) 4 ] and ½NO 3 þno 2Š increased slightly from west to east (Fig. 2A) with silicic acid concentrations showing significant zonal variability possibly related to the influence of TIWs (Strutton et al., 2011). In contrast total dissolved [Fe] showed a concentration gradient that was stronger and in the opposite direction to that of the macronutrients, increasing from o0.1 nm in the east with values reaching 0.58 nm in the west (Fig. 2A), possibly due to 3. Results 3.1. Physical, biological and chemical setting A thorough description of nutrient distributions relative to circulation and TIW activity between 1101W and 1401W on the two cruises is presented elsewhere (Strutton et al., 2011). For context a brief analysis of physical properties and nutrient distributions is presented here. Mixed-layer temperatures at the locations where microcosm experiments were performed were between 23.9 and C with salinities between and psu. Slightly cooler and less saline waters were found to the north of the equator along 1101W than along the equator or along 1401W (Table 1). Mixed-layer macronutrient concentrations showed some variability along the equator in 2004 due to the influence of TIWs (Strutton et al., 2011), and at both 1101W and 1401W elevated concentrations were observed at stations on or south of the equator compared to those from the north of the equator. Those distributions are consistent with the known meridional asymmetry in macronutrient distributions across the equator in the EEP (Dugdale et al., 2002). N+N concentrations in the euphotic zone were between 3.6 and 8.3 mm. Silicic acid concentrations were generally less than half those of N+N, ranging between 1.4 and 3.8 mm. Ammonium concentrations were usually near the limit of detection with the highest being 0.39 mm. Orthophosphate concentrations were between 0.30 and 0.78 mm. The average N:Si:P mole ratio in the inorganic nutrient pool was 15.1: 5.9:1, indicating near Redfield N:P ratios, but a deficit of Si relative to inorganic N where a N:Si ratio of 1.0 is characteristic of nutrient-replete diatoms (Brzezinski, 1985). Concentrations of total dissolved Fe in the water used for the microcosm experiments ranged from o0.08 to 0.54 nm with generally higher values observed towards the west Fig. 2. Longitudinal gradients in nutrient concentrations and ratios on the 24.4 s y isopycnal between 110 and 1401W longitude along the equator in A) ½NO 3 þno 2Š, open circles, [Si(OH) 4 ], filled circles, and [Fe], open triangles. B) [Fe]/ [Si(OH) 4 ] ratios, filled circles; and [Fe]/[½NO 3 þno 2Š, open circles. Lines and equations are those determined by linear regression of each parameter as a function of longitude.

6 498 M.A. Brzezinski et al. / Deep-Sea Research II 58 (2011) continuous removal from its major source in the eastward-flowing EUC. Total dissolved [Fe] also showed zonal variability with low values between 125 and 1301W longitude in the region of TIW influence (Strutton et al., 2011). As a result of the opposing gradients in macronutrient and total dissolved Fe concentrations Fe/Si and Fe/N mole ratios both increased from the east to the west (Fig. 2B). Linear regression of the nutrient gradients as a function of longitude indicate that Fe/Si and Fe/N mole ratios in source waters upwelling at 1401W are each four-fold higher than the corresponding ratios in waters upwelling at 1101W (Fig. 2B). When compared to the Fe/Si and Fe/N stoichiometry of nutrient-replete diatoms of (based on a mole ratio of C/Fe of 10 5 ; Geider and Roche, 1994; Twining et al., 2011, and a Si:N mole ratio of 1:1; Brzezinski, 1985), the Fe/Si and Fe/N stoichiometry of the source water suggest possible Fe limitation in the east and possible Si limitation in the west. Nutrient ratios at stations where water for microcosm experiments was collected reflect this trend with Fe/Si values between and at stations along 1101W increasing to values between and near 1401W (Table 1) Microcosms Experimental constraints: macronutrient exhaustion There are a variety of approaches to evaluating differences in nutrient use among treatments in batch experiments. Our initial attempts to calculate macronutrient depletion rates from the time courses of nutrient concentration resulted in large uncertainty because the rates were variable through time, with most of the nutrient use in the treatments with added Fe occurring during the 24 h prior to macronutrient exhaustion in many experiments. The actual time of exhaustion in the +Fe and +Fe+Si treatments was not well constrained as depletion was only known to have occurred sometime in the 24 h preceding the sampling time when macronutrient exhaustion was first detected. Thus rates calculated for this period were subject to the largest uncertainty and we chose to compare treatment effects using the measured levels of net nutrient depletion. Using nutrient consumption to compare treatments effects is not without artifacts. In the majority of experiments macronutrient depletion in the treatments receiving Fe was less than it would have been at higher initial macronutrient concentrations, because essentially all macronutrients that were initially available were taken up in many experiments. A summary of the pattern of nutrient depletion in all nine experiments is given in Table 2. Macronutrient exhaustion occurred with added Fe in six of the nine experiments with multiple macronutrients going to nearzero concentrations simultaneously (Table 2). Macronutrient exhaustion in the +Fe+Si treatment paralleled that in the +Fe treatment in all cases except that silicic acid was not exhausted in microcosms augmented with Si. Thus, in six of the nine experiments the consumption of one or more macronutrients in the +Fe and +Fe+Si treatments (Table 2) underestimates actual uptake potential. Nutrient exhaustion also affected the statistical analysis of the data. ANOVA of the differences in the level of nutrient use or biomass production (defined as the change between time zero and the time when one or more macronutrient was depleted, see Section 3.2.3) among treatments (not including the +Si treatments that received Fe-contaminated Si stock) and locations across all experiments indicated significant differences among treatments, among locations and a significant interaction between the treatment response and location for all response variables (two-way ANOVA with interaction; Po0.001 on all interactions). Because one or more macronutrients were completely exhausted in over half of the treatments with added Fe and with the combined addition of both Fe and Si (Table 2), differences in nutrient use among locations were mainly due to variation in initial macronutrient concentrations and not to differences in uptake potential of the phytoplankton. For example, the amount of N+N removed by biota growing in response to added Fe at each location was largely a function of the initial N+N concentration, which varied among experiments conducted at different locations (Table 1) and was completely consumed at six locations, leading to statistically significant, but ecologically irrelevant, differences in N+N use among locations. The presence of a significant interaction between location and all response variables eliminates the power of an ANOVA to detect differences among treatments. As an alternative, the mean effects in each experimental treatment were compared independent of location using False Discovery Rate procedures (q-fdr; e.g. Benjamini and Hochberg, 1995; Verhoeven et al., 2005). q-fdr controls the expected proportion of type I errors rather than the probability that such errors will occur which can increase statistical power compared to family-wise error rate techniques for handling multiple comparisons (Benjamini and Hochberg, 1995) Example time courses A summary of the results from a single microcosm experiment is presented in detail in Fig. 3 for the fourth microcosm experiment conducted in 2004 using water collected on the equator at 1201W longitude. This experiment was chosen as an example over the others as it reveals the general response observed across all experiments and it also illustrates the issues associated with random Fe contamination and nutrient exhaustion. These issues did not obscure the main treatment effects in the overall data set, but we illustrate them here to bring out the caveats necessary when interpreting the results. Table 2 Nutrient depletion patterns in microcosms receiving Fe or both Fe and Si. Instances of nutrient depletion were always simultaneous in the +Fe and +Fe+Si treatments. Cruise date Exp. Lat. Long. Depleted Nutrients Time when nutrient depletion observed (h) December N 1101W Si(OH) 4,NO N 1101W Si(OH) 4,NO 3, HPO ¼ S 1101W none W Si(OH) 4,NO 3, HPO ¼ W none W Si(OH) 4,NO 3, HPO ¼ September N 1401W none N 1401W Si(OH) N 1251W HPO ¼ 4 96

7 M.A. Brzezinski et al. / Deep-Sea Research II 58 (2011) Fig. 3. Example of the temporal changes in nutrient concentrations and phytoplankton biomass in the microcosm experiments. Data are for the fourth experiment conducted in A) total dissolved Fe, B) (nitrate+nitrite), C) orthophosphate, D) ammonium, E) silicic acid), F) biogenic silica, G) mm chlorophyll a,h)43.0 mm chlorophyll a, I) particulate inorganic carbon (PIC). In this particular experiment, total dissolved [Fe] increased from 0.2 nm to ca. 0.4 nm over time in all treatments not receiving supplemental Fe (Fig. 3A). Total dissolved Fe declined dramatically over the first 48 h in all treatments receiving supplemental Fe and then remained between 0.5 and 0.8 nm except in the +Fe+Ge treatment where [Fe] remained near 1 nm. Significant iron contamination events that increased total dissolved [Fe] by 40.5 nm are apparent in Fig. 3 in the +Fe+Si and +Ge treatments at 120 h. Contamination of this kind was rare, occurring in only four out of a total of 108 microcosms used in the nine experiments. In 2004, Fe contamination occurred in one of the +Ge and in one of the +Fe+Si treatment microcosms during experiment 4 (Fig. 3) and in one of the +Ge replicates in experiment 5 (not shown). In 2005, contamination of this magnitude was observed in just one of the +Fe+Si replicates in experiment 2 (not shown). These events did not significantly alter

8 500 M.A. Brzezinski et al. / Deep-Sea Research II 58 (2011) the results obtained in these microcosms compared to their corresponding replicates. This was due mainly to the fact that three of the contaminated microcosms had already received purposeful additions of Fe, minimizing the effect of contaminating Fe; the fourth contained added Ge, which consistently eliminated the effect of added Fe (see section below). Thus, the results from these treatments were included in the calculation of the average responses to the treatments. There was significant macronutrient consumption in the controls (Fig. 3B-E). More than half of the N+N, orthophosphate and ammonium was consumed in the controls over the 120-h experiment and silicic acid was depleted in the controls by 96 h (Fig. 3B-E). The exhaustion of silicic acid in the control was unique to this single experiment and reflects the low initial [Si(OH) 4 ]. The pattern of N+N, ammonium, orthophosphate and Fe use in the +Si treatments in this experiment was similar to that in the controls (Fig. 3A-E). Silicic acid depletion accelerated after 72 hours in both the controls and in the +Si treatments leading to silicic acid exhaustion in the control while [Si(OH) 4 ] remained high (415 mm) in the +Si treatments (Fig. 3E). Silicic acid in the +Si treatments continued to be consumed until the experiment was terminated at 120 h. The results of Fe addition were dramatically different from those for added Si. Iron addition resulted in greater depletion of N+N and orthophosphate compared to controls and compared to the +Si treatment (Fig. 3). Added Fe did not affect the pattern of ammonium use relative to controls for the first 48 hours, but ammonium depletion was slightly higher at later time points with added Fe (Fig. 3D). Silicic acid was exhausted by 96 h in the +Fe treatments, as was observed in the controls (Fig. 3E). When Ge was added to inhibit diatom activity, either alone or in combination with Fe, less N+N, orthophosphate, ammonium and silicic acid was consumed than in the controls (Fig. 3B-E). Drawdown of these macronutrients was nearly the same in the +Ge and +Fe+Ge treatments. Thus, inhibition of the diatoms by Ge caused lower macronutrient use than in the controls and eliminated the stimulation of macronutrient drawdown that was observed with added Fe. Macronutrients were consumed at a higher rate when both Fe and Si were added together than when either was added separately. However, interpretation of the amount of nutrient depletion in the +Fe and +Fe+Si treatments is confounded by the depletion of nutrients to nearly undetectable levels. In the experiment shown in Fig. 3, silicic acid, orthophosphate and N+N were all depleted to 0 mm by 96 h in the +Fe treatments. Exhaustion (depletion to 0 mm) of most of these nutrients also occurred over this same timeframe in the +Fe+Si treatment, but Si exhaustion was prevented by the addition of Si leading to greater Si use when Si and Fe were added together than when Fe was added alone. The depletion of both N+N and orthophosphate was greater in the +Fe+Si treatment than in the +Fe treatment over the first 72 h with the difference between treatments increasing over time. However, by 96 h both N+N and orthophosphate were exhausted in both the +Fe+Si and +Fe treatments, masking the difference in depletion rates between these treatments. These patterns suggest that much larger differences in N+N and orthophosphate use would have been manifested between the +Fe and +Fe+Si treatments had these nutrients not been exhausted. Changes in chlorophyll biomass were congruent with the patterns of nutrient use. Controls and +Si treatments each showed similar increases in the concentrations of both chlorophyll size fractions (Fig. 3G, H) with the concentrations increasing from ca mg L 1 to mg L 1 over the 120 h experiment (Fig. 3G, H). Chlorophyll levels increased substantially more in the +Fe and +Fe+Si treatments with concentrations increasing in both treatments until 96 h (Fig. 3) after which time they declined. At peak chlorophyll levels the addition of Fe resulted in a ten-fold increase in the concentration of the 43.0-mm chlorophyll fraction and a doubling of the chlorophyll biomass in the mm size fraction relative to controls. Addition of both Fe and Si increased the 43.0-mm chlorophyll concentration to a greater extent than did the addition of Fe alone while the increase in the mm size fraction with added Fe and Si was similar to that when Fe was added by itself. The addition of Ge, with or without Fe, eliminated the increase in chlorophyll in both size classes (noting that the smaller size class likely included non-diatom representatives) such that these treatments contained significantly less chlorophyll than the controls by the end of the experiment. Biogenic silica concentrations increased from 0.13 to 1.2 mmol Si L 1 in controls with modestly greater increases in both the +Fe and +Si treatments (Fig. 3F). In this experiment the addition of Si produced slightly more biogenic silica than did the addition of Fe by 120 h. However, this difference is biased by the fact that the accumulation of biogenic silica in the +Fe treatment was truncated by silicic acid exhaustion at 96 h while silicic acid exhaustion was prevented in the +Si treatment due to the addition of Si. Siliceous biomass reached its highest level in the +Fe+Si treatments and continued to increase after N+N and orthophosphate were exhausted. No increase in biogenic silica concentration was observed in the +Ge treatment (Fig. 3F). With the exception of a 48-h lag in the +Si and +Ge treatments, PIC concentrations increased regardless of treatment from 0.05 to 0.25 mmol L 1, over 120 h (Fig. 3I). By the end of the experiment, PIC had increased most in the controls followed by the +Ge and +Si treatments, which had nearly identical final PIC concentrations (Fig. 3I). Interestingly, both treatments receiving Fe (+Fe and +Fe+Si) had lower final PIC concentrations compared to both the controls and the +Ge and +Si treatments (Fig. 3I) Response of nutrient use and biomass accumulation across all nine experiments The mean changes in nutrient and biomass concentrations in the controls and in treatments for all nine microcosm experiments are presented in Table 3 with the results of a q-fdr pairwise t-test analysis for each response variable and treatment combination given in Table 4. Average nutrient use and biomass accumulation across all experiments is illustrated graphically in Fig. 4. Each value presented in Fig. 4 is the mean change for all nine experiments except for the +Si treatment where only the results of the six experiments using clean Si stock were averaged. Changes in the controls represent the mean change from time zero whereas changes in treatments are the mean change in each treatment after subtraction the mean change in the corresponding controls. Changes in PIC are those occurring over 120 h because [PIC] was measured only at 0, 48 and 120 h. For all other parameters, the time interval used to calculate changes in treatments and in controls was that between time zero and the time of nutrient exhaustion. In each case, nutrient exhaustion occurred simultaneously in the +Fe and +Fe+Si treatments. For the experiments where no macronutrient went to exhaustion, changes in concentration were calculated as the difference between those at time zero and those at the end of the experiment. As was the case for the single experiment illustrated in Fig. 3, changes in ammonium concentration across all experiments were generally more than an order of magnitude less that those of N+N averaging o0.1 mm in both the controls and in all treatments, and there was no statistical difference in net ammonium use among treatments or between treatments and

9 M.A. Brzezinski et al. / Deep-Sea Research II 58 (2011) Table 3 Changes in nutrient and biomass concentrations in the microcosm experiments. Values are the mean changes from time zero until the time of nutrient exhaustion or 120 h (see text for details). Errors are standard deviations. PIC measurements were not replicated. Cruise date Exp. Treatment [Fe] (nm) ½NO 3 Šþ NO 2 Š [H 3 PO 4 ] [Si(OH) 4 ] [Chl-a] 43 mm (mg L 1 ) [Chl-a] mm (mg L 1 ) [bsio 2 ] (mmol Si L 1 ) PIC (mmol C L 1 ) December 1 Control Fe Si * Ge Fe & Si * Fe & Ge Control Fe Si * Ge Fe & Si * Fe & Ge Control Fe Si * Ge Fe & Si * Fe & Ge Control Fe Si Ge Fe & Si Fe & Ge Control Fe Si Ge Fe & Si Fe & Ge Control Fe Si Ge Fe & Si Fe & Ge September 1 Control Fe Si Ge Fe & Si Fe & Ge Control Fe Si Ge Fe & Si Fe & Ge Control Fe Si Ge Fe & Si Fe & Ge n Stock solution of Si(OH) 4 found to be contaminated with Fe in these experiments. Table 4 Results of False Discovery Rate analysis of pairwise t-tests among treatments. Means with the same letter are not statistically different based on q-values with p¼0.05. N¼9 sites for all treatments except for the +Si treatment where N¼6. Treatment Total Fe (nm) NO 3 +NO 2 Si(OH) 4 H 3 PO 4 43 mm Chl a (mg L 1 ) mm Chl a (mgl 1 ) bsio 2 (mmol Si L 1 ) PIC (mmol C L 1 ) Control 0.14 A 2.8 C 1.3 B 0.18 B 0.6 B 0.3 A 0.7 C 0.24 A +Si 0.04 A 3.0 C 3.6 D 0.23 B 0.6 B 0.4 A 1.2 B 0.23 A +Fe 1.51 B 5.3 D 2.3 C 0.35 C 3.3 A 0.5 A,C 1.6 B 0.20 A +Ge 0.03 A 1.4 A 0.6 A 0.07 A 0.1 D 0.0 D 0.1 D 0.26 A +Fe+Si 1.52 B 5.4 D 6.3 E 0.37 C 3.7 A 0.5 A,B 3.8 A 0.25 A +Fe+Ge 1.36 B 1.6 B 0.6 A 0.09 A 0.2 C 0.1 B,C 0.1 D 0.19 A

10 502 M.A. Brzezinski et al. / Deep-Sea Research II 58 (2011) Fig. 4. Average change in nutrient concentrations and phytoplankton biomass across all nine microcosm experiments. Changes in the controls are the average change in paired controls from time zero up until the time when one or more nutrients were exhausted in those treatments receiving Fe or until the end of the experiment at 120 h for those experiments where no nutrients were exhausted in any treatment (see text for details). Changes in the experimental treatments are expressed as the difference between the mean change in a given experimental treatment and that in the corresponding controls. The response in the +Si treatment is the average response for the six experiments using the clean silicic acid stock solution. In all other cases the bars represent the average change for all nine experiments. Errors are standard errors. Note that the changes in orthophosphate concentration have been multiplied by 15 (the Redfield N:P) to facilitate comparison with patterns of (nitrate+nitrite) and silicic acid depletion. PIC concentrations are arbitrarily multiplied by ten to allow the use of a common concentration axis. controls (q-fdr, P40.16 for all pairwise comparisons). Thus, changes in ammonium concentration are not discussed further. Average nutrient use and biomass accumulation across all experiments was significant in the controls (Fig. 4, Table 4). On average, 4474% of the N+N, 47710% of the orthophosphate and 5679% of the silicic acid was consumed in the controls (uncertainty terms are standard errors). Biogenic silica concentration increased by an average of mmol Si L 1, with the concentration of PIC increasing by an average of mmol CL 1. The 43-mm and mm chlorophyll size fractions increased by mg L 1 and mgl 1, respectively. On average, [Fe] increased by nm in the controls. Both Si addition and Fe addition had consistent and significant effects on average nutrient consumption and biomass accumulation across all experiments, but those effects were very different. On average, Si addition increased silicic acid use and biogenic silica accumulation, but it had no significant effect on N+N or orthophosphate use, PIC production or the accumulation of chlorophyll (Fig. 4, Table 4). Similarly, Fe addition resulted in significant increases in mean silicic acid use and biogenic silica production, but in contrast to Si additions, Fe also significantly enhanced mean N+N and orthophosphate drawdown and increased average chlorophyll accumulation compared to the average response in both controls and in the +Si treatment. The average increase in chlorophyll across experiments with the addition of Fe was overwhelmingly dominated by the accumulation of 43-mm chlorophyll with Fe having far less of an affect on the average accumulation of chlorophyll in the mm size fraction. Fe did not stimulate PIC production over that occurring in the controls. Average silicic acid depletion was less in the +Fe treatments than in the +Si treatments, mainly as a result of Si exhaustion in the +Fe treatments; Si exhaustion was prevented in the +Si treatments by the +20 mm additions of silicic acid. Average total dissolved Fe concentrations showed no significant change with added Si, but declined significantly in the +Fe treatments. The addition of Si and Fe together had synergistic effects on macronutrient use and biomass accumulation. The addition of both Si and Fe clearly resulted in the greatest average yield of biogenic silica, and it enhanced mean silicic acid depletion compared to the addition of either nutrient alone (Fig. 4, Table 4). This was especially significant when comparing the +Fe+Si with