Microbial and biogeochemical responses to projected future nitrateenrichment in the California upwelling system

  Katherine Rose Marie Mackey, Chia-Te Chien and Adina Paytan

Journal Name: Frontiers in Microbiology

ISSN: 1664-302X

Article type: Original Research Article

First received on: 19 Jun 2014

Revised on: 24 Oct 2014

Frontiers website link: www.frontiersin.org

Aquatic Microbiology

Microbial and biogeochemical responses to projected future nitrate enrichment 1

in the California upwelling system 2

3

Katherine RM Mackey1*

, Chia-Te Chein2, and Adina Paytan

2,3 4

5

6

7

1 Earth System Science, University of California Irvine, Irvine CA 92697 8

2 Earth and Planetary Sciences, University of California, Santa Cruz, California 95064, United 9

States 10

3 Institute for Marine Science, University of California, Santa Cruz, California 95064 , United 11

States 12

13

*corresponding author, kmackey@uci.edu, 301.356.4041 14

Acknowledgements 15

16

We thank Alanna Lechter, Kimberley Null, Zhongwu Ma, and Nicole McInerney for 17

assistance with sampling, Joe Jennings and Rob Franks for assistance analyzing samples, and 18

John Ryan for useful input on monitoring methodologies. We also thank the three reviewers for 19

their insightful comments to help improve the manuscript. K.R.M.M. was supported by a 20

National Science Foundation Postdoctoral Research Fellowship in Biology (Grant No. NSF 21

1103575) during the writing of the manuscript. This work was supported by a NSF-OCE grant 22

(#0850467) and a California Sea Grant to A.P. 23

Abstract 24

Coastal California is a dynamic upwelling region where nitrogen (N) and iron (Fe) can both limit 25

productivity and influence biogeochemistry over different spatial and temporal scales. With 26

global change, the flux of nitrate from upwelling is expected to increase over the next century, 27

potentially driving additional oceanic regions toward Fe limitation. In this study we explored the 28

effect of changes in Fe/N ratio on native phytoplankton from five currently Fe-replete sites near 29

the major California upwelling centers at Bodega Bay and Monterey Bay using nutrient addition 30

incubation experiments. Despite the high nitrate levels (13-30 M) in the upwelled water, 31

phytoplankton at three of the five sites showed increased growth when 10 M nitrate was added. 32

None of the sites showed enhanced growth following addition of 10 nM Fe. Nitrate additions 33

favored slow sinking single-celled diatoms over faster sinking chain-forming diatoms, 34

suggesting that future increases in nitrate flux could affect carbon and silicate export and alter 35

grazer populations. In particular, solitary cells of Cylindrotheca were more abundant than the 36

toxin-producing genus Pseudonitzschia following nitrate addition. These responses suggest the 37

biogeochemistry of coastal California could change in response to future increases in nitrate, and 38

multiple stressors like ocean acidification and hypoxia may further result in ecosystem shifts. 39

40

Introduction 41

Coastal upwelling regions along eastern boundary currents are the most productive 42

marine ecosystems, supporting complex ecological networks and economically important 43

fisheries. These systems experience a high degree of natural spatial and temporal variability with 44

respect to biological, chemical, and physical characteristics. Upwelling is typically a seasonal 45

phenomenon, where alongshore winds drive sub-surface, nutrient rich waters toward the sunlit 46

surface layers, enriching them with the macronutrients nitrogen (N) and phosphorus (P) (Chavez 47

and Messie 2009). Other factors, such as the width and depth of the continental shelf at the 48

upwelling site and internal cycling of elements play a role in determining the flux of trace metals 49

to surface waters (Bruland et al. 2001; Biller and Bruland 2013; Biller et al. 2013). 50

Coastal upwelling regions face threats from anthropogenic global change, including 51

changes in stratification patterns due to sea surface temperature warming, altered nutrient 52

chemistry, increased hypoxia, and ocean acidification (as reviewed in Capone and Hutchins 53

2013). For example, decreased ventilation of the Pacific Ocean due to increased stratification in 54

the gyres is expected to alter seawater chemistry, increasing the nitrate inventory and decreasing 55

the oxygen content in waters that are upwelled (Rykaczewsk and Dunne 2010). As a result, the 56

flux of nitrate in the coastal California upwelling system is expected to be 64% greater in the 57

year 2100 compared to preindustrial times (or 28% over modern day values; Rykaczewsk and 58

Dunne 2010). 59

Iron (Fe) availability governs nitrate drawdown in many coastal upwelling systems and is 60

strongly influenced by the physical and bathymetric characteristics of each site. Along the 61

California coast, biomass at locations with narrow continental shelves can become Fe-limited 62

due to the low suspended sediment levels and high nitrate concentrations from upwelling 63

(Hutchins and Bruland 1998; Firme et al. 2003). Recent work by Biller and Bruland (2014) 64

expanded these regions to include the coastal California transition zone (TZ, Fig. 1C), which is 65

an offshore region with high nitrate from upwelled waters advected offshore. As the water moves 66

offshore, labile Fe is consumed leading to Fe limitation and excess nitrate. These Fe-limited 67

regions typically have iron-to-nitrogen (Fe/N) ratios below 0.2 nmol Fe/ 1mol N, and have been 68

designated as high nutrient low chlorophyll (HNLC) regions due to the relatively high residual 69

nitrate and lower than expected chlorophyll levels. Other studies in the region have shown that 70

despite high nitrate concentrations in the water phytoplankton biomass remains nitrogen-limited 71

(Kudela and Dugdale 2000), with other nutrients like phosphorus influencing physiology and 72

competition between taxonomic groups (Nicholson et al. 2006; Mackey et al. 2012). 73

Iron availability in coastal California and other HNLC regions is also known to influence 74

phytoplankton community structure and biogeochemistry. Large scale iron addition experiments 75

conducted in the Southern Ocean and equatorial Pacific HNLC regions favored blooms of 76

diatoms over other types of phytoplankton (Martin et al. 1994; Coale et al. 1996; Boyd et al. 77

2000; Smetacek et al. 2012), affected diatom speciation (Assmy et al., 2007; Tsuda et al., 2005; 78

Tsuda et al., 2003), and altered grazing rates (Tsuda et al 2006). Iron additions have also been 79

shown to stimulate the toxin producing diatom Pseudo-nitzschia spp. in the equatorial Pacific 80

Ocean (Silver et al. 2010, Trick et al. 2010). In Fe-limited areas of coastal California, selective 81

enrichment of chain-forming diatoms occurred following iron addition, and diatoms formed 82

more heavily silicified cells (Hutchins and Bruland 1998). Iron availability therefore has the 83

potential to influence the type of phytoplankton that dominate blooms following upwelling, 84

affect the uptake and cycling of other nutrients, and alter the relative proportions of silica and 85

carbon sequestration by the biological pump. In addition blooms with different types of 86

phytoplankton (size, density, TEP production etc.) have different sinking velocities also 87

impacting carbon sequestration by the biological pump (De La Rocha and Passow 2007). Larger 88

cells or chain-forming cells have faster sinking rates and therefore sequester carbon more 89

efficiently than smaller cells. 90

A number of factors sensitive to global change influence the supply and bioavailability of 91

Fe in coastal upwelling regions. Biological ligand production, ocean acidification, hypoxia, 92

rainfall, groundwater discharge, and deposition of anthropogenic aerosols influence Fe supply 93

and biogeochemistry in coastal waters. The predicted increased supply of nitrate in the future 94

(Rykaczewsk and Dunne 2010) could likewise shift Fe limitation regimes for biomass by 95

changing the Fe/N ratio in the water. For example, a 50% increase in nitrate supply could drive 96

certain Fe-replete California waters below the Fe/N limitation threshold (Capone and Hutchins 97

2013), potentially expanding Fe limitation in the California upwelling region. 98

To understand how Fe-replete waters in coastal California could respond to changing 99

nitrate fluxes (and hence changing Fe/N ratios) in the future, we conducted nutrient addition 100

incubation experiments with water collected at five Fe replete sites from Monterey Bay to Point 101

Reyes several days after upwelling when Fe and nitrate levels remained elevated. The goal of the 102

study was to expand our understanding of how N and Fe availability and ratios could influence 103

phytoplankton growth and physiology in modern-day and future coastal California waters and to 104

assess the spatial variability in this response. These coastal sites were selected to encompass 105

various distances from shore and represent major upwelling centers such that a range of nutrient 106

conditions could be tested. We used phytoplankton cell counts to determine if there is a 107

phytoplankton community shift toward faster sinking chain-forming diatoms as observed by 108

Hutchins and Bruland (1998) and Silver et al. (2010) in Fe-limited regions following Fe 109

fertilization. Finally, based on nutrient measurements, we show that only in some cases do Fe 110

and N cause shifts in the drawdown ratios of N:P and Si:N. We discuss the implications of these 111

findings to the export of C and Si. 112

113

Materials and Methods 114

115

Remote sensing and mooring data. In situ wind speed, wind direction, sea surface 116

temperature (SST) and nitrate concentrations at station M1 were obtained from 117

http://www.mbari.org/oasis/. Satellite images of sea surface temperature were obtained from 118

NOAA POES AVHRR, LAC, 0.0125 degrees, day and night, courtesy of NOAA NWS 119

Monterey and NOAA CoastWatch. Oceanographic and atmospheric conditions were monitored 120

prior to the experiment to identify a period of upwelling followed by relaxation. The incubation 121

experiments were conducted several days after upwelling occurred when upwelling-favorable 122

winds had relaxed and SST values indicated surface waters were warming (Fig. 1A,B). 123

Incubation setup and sampling. Incubation experiments were conducted to determine the 124

effect of N and Fe on phytoplankton using seawater collected from five sites as shown in Fig. 1C 125

a few days after the relaxation of upwelling. Water temperature and salinity were used to identify 126

water masses by assuming that upwelled water warms ~0.5-1oC per day (K. Bruland, personal 127

communication) and is more saline than water from the North Pacific. Water from Drakes Bay 128

near Point Reyes (PR; 37o59’23.2”N, 122

o58’52.2”W) and north of the mouth of San Francisco 129

Bay (SF; 37o55’30.7”, 122

o49’41.7”W) was collected on June 20, 2011 (Fig. 1C) aboard the R/V 130

Shana Rae. These two sites are located to the south and downstream of the major upwelling 131

center located at Bodega Bay. Water was collected from the Monterey Bay moorings M0 132

(36o49.442N 121

o56.967W) and M1 (36

o45.190N 122

o01.525W) and offshore of Monterey Bay 133

near mooring M2 on June 22, 2011 using a small motor boat. In this study we refer to the 134

offshore site as “M2”, although its actual location (36o42.382N 122

o13.798W) differs slightly 135

from the official location of the offshore mooring. Station M0 is located within Monterey Bay. 136

Station M1 is situated directly downstream of the Monterey Bay upwelling center. 137

Surface (5m) seawater was collected at each site into trace metal clean, seawater rinsed 138

carboys. Water was transported in the dark back to Long Marine Laboratory in Santa Cruz, CA 139

where the experiments were conducted. The following protocol was followed while setting up 140

the experiments for each site. Three baseline samples to characterize the collected seawater were 141

immediately collected and processed for each of the measurements described below. Water was 142

then dispensed into acid cleaned, sample rinsed, transparent polycarbonate bottles (500 mL per 143

bottle, 9 bottles per treatment). All materials used in the experiment were rendered trace metal 144

clean prior to use. Plastic ware (including incubation bottles, carboys, and sample bottles) was 145

soaked overnight in heated, ultrapure 10% hydrochloroic acid (HCl; Optima), rinsed several 146

times with MilliQ water, and stored individually inside clean plastic bags prior to use. During 147

sampling, bottles were opened within a laminar flow hood to minimize contamination from 148

airborne particles. 149

The Fe/N ratio of the seawater was manipulated by making additions of Fe or nitrate. 150

Different treatments used in the experiments included control (no addition), 10 M sodium 151

nitrate (hereafter "nitrate"), or 10 nM iron prepared from atomic absorption standard stock in 152

HCl (Sigma). The nitrate addition was intended to mimic the increase in nitrate projected for the 153

California upwelling system in the year 2100 (Rykaczewsk and Dunne 2010), and the Fe was 154

intended to at least double ambient concentrations of Fe in this area (Bruland et al. 2001). 155

Following the addition of nutrient spikes (time zero), three bottles were immediately sampled for 156

each of the measurements described below. The remaining bottles (6 per treatment) were placed 157

in a large pool through which ocean water was continually circulated. A neutral density shade 158

cloth was placed over the pool to decrease the irradiance by half, and we note the shading does 159

not alter the spectral quality of the light. Three bottles from each treatment were collected 48 and 160

96 hours after time zero and were processed as described below. 161

Bottle incubation experiments are useful for examining short term changes in 162

phytoplankton community composition and physiology, particularly with respect to changing 163

water chemistry including nutrients. The impact of grazers is more difficult to assess from bottle 164

experiments, particularly when the incubation volume is low. This is particularly true for large 165

grazers, like copepods, that are far less abundant than phytoplankton and may not survive in the 166

bottles over time. Bottle experiments also have difficulty capturing variability in light due to 167

mixing of surface water. These factors must be taken into consideration when interpreting bottle 168

experiment results, where grazing and other factors do not perfectly mimic the environment. 169

Water chemistry. Nutrient samples were 0.45 m filtered and frozen until analysis. 170

Nutrient analyses for nitrate (with nitrite), soluble reactive phosphorus (SRP), and silicate were 171

carried out on a Lachat Autoanalyzer (FIA, Lachat Instruments Model QuickChem 8000). Initial 172

seawater total dissolved Fe samples were 0.2 m filtered in a laminar flow hood and acidified to 173

pH<2.0 with concentrated trace metal grade nitric acid (final concentration 0.02 M) at least 48 174

hours before analysis. The pH was adjusted to 6 with ammonium acetate and ammonium 175

hydroxide, and samples were concentrated using Nobias Chelate-PA1 resin (HITACHI High 176

Technologies, Japan) to remove the seawater matrix (Sohrin et al., 2008; Biller and Bruland 177

2012). The Fe was eluted with 1 M trace metal grade nitric acid and analyzed for by HR-ICPMS 178

(Thermo Element XR). The detection limit for Fe was 0.91 nM. 179

Phytoplankton growth. Phytoplankton growth was assessed based on measurements of 180

chlorophyll a (chl a) and direct enumeration of cells using flow cytometry and microscope cell 181

counts. For chl a measurement, 50mL of incubation water was filtered through GFF filters 182

(Whatman) and the filters were flash frozen and stored at -80oC until analysis. Filters were 183

extracted in 5 mL 90% acetone (Optima) in the dark at -20oC for 24 hr. Fluorescence was 184

measured on a Turner Designs AU10 and converted to concentration via a standard curve 185

calibration. 186

Flow cytometry samples were collected and fixed with a final concentration of 0.1% 187

glutaraldehyde and flash frozen and stored at -80oC until analysis. Samples were thawed on ice 188

and run on an Influx flow cytometer (Becton Dickinson). Using FlowJo software, phytoplankton 189

cells were identified from their red (692 nm) chlorophyll autofluorescence signal. Synechococcus 190

cells were identified by their phycoerythrin signal (572 nm). 191

Samples for microscope analysis were fixed with a final concentration of 2% formalin 192

and stored in the dark at 4oC in glass bottles. A settling chamber was used to concentrate 2-10 193

mL of sample, and cells were viewed on an inverted microscope. Typically ~1000 cells were 194

counted per sample, but never less than 300, and replicate samples were randomly included to 195

check for accuracy. Cells >4 m in diameter were identified and enumerated as pennate diatoms, 196

centric diatoms, or "others" (Supplemental Fig. 1-8). Pennate and centric diatoms were further 197

grouped according to whether they occurred as single cells or in chains of two or more cells. In 198

the text their relative abundances are categorized as rare (<1%), present (1-9%), common (10-199

49%) or abundant (>50%). Statistical significance was determined using ANOVA followed by 200

pairwise comparisons using a Bonferroni correction. Cell counts in the Fe and N treatments at 201

each site were compared to the control (two comparisons per site). 202

203

Results 204

Remote monitoring. Wind velocity recorded at the M1 mooring indicated that upwelling 205

favorable winds reached speeds up to ~8 m s-1

during the period of June 10-16. The winds 206

consistently blew from the northwest to the southeast during this period (Fig. 2A). Cold water 207

masses were observed from the AVHRR satellite during these dates along the California coast, 208

indicating upwelling of deep water was occurring. By June 15, a large, cold (9-10oC) upwelled 209

water mass was located surrounding Point Reyes and was centered offshore of Bodega Bay, and 210

smaller upwelled water masses were located north of Monterey Bay and at Point Sur (Fig. 1A). 211

Upwelling favorable winds relaxed from June 17-23, and the wind direction became more 212

variable (Fig 2A). By June 20 the amount of cold water being upwelled had declined and was 213

more restricted to the coastline north of Point Reyes (Fig. 1B). The water masses south of Point 214

Reyes and near Monterey Bay continued to warm once upwelling ceased (Fig. 1B). 215

Temperature, salinity, and nitrate data from the mooring at M1confirmed the pattern of 216

upwelling and relaxation observed from the satellite images (Fig. 2, Supplemental Figure 9). At 217

M1, surface waters became progressively colder and more saline from June 10-16 while 218

upwelling favorable winds prevailed (Fig 2B,C, supplemental Fig. 9). By June 15 a cold 219

(~11.5oC), saline (~33.6) water mass was present at the mooring. Upon relaxation of upwelling 220

favorable winds (June 17-23), the water mass began to warm. Relatively stable salinity indicated 221

that the warmer temperatures were associated with the same upwelled water mass rather than 222

from intrusion of waters from the less saline California current. 223

The nitrate concentration at mooring M1 reached its highest level (~15 mol L-1

; Fig 2D) 224

in surface waters by the end of the upwelling period (June 15-16). Nitrate declined gradually by 225

~5 mol L-1

from June 17 to June 23 once upwelling favorable winds relaxed; however, nitrate 226

levels were still relatively high (~10 mol L-1

) and had not been consumed entirely by June 23. 227

Water was collected for the incubation experiments once upwelling favorable winds had 228

relaxed for several days and surface water had warmed 1-2oC based on remote monitoring data 229

as described above (e.g., June 20 for stations PR and SF, and June 22 for stations M0, M1, and 230

M2). Nutrient and Fe levels were lower at stations M0, M1, and M2 than at stations PR or SF 231

(Fig. 3A-D). The ratio of N/P was similar among all five sites (Fig. 4A,B), whereas the ratio of 232

Si/N was higher at stations PR and SF than at M0, M1, or M2 (Fig. 4E,F). The Fe/N ratio was 233

not statistically different among the five sites at p<0.05 (Fig. 4 C,D). Station SF had a lower Fe/ 234

N ratio than the nearby station PR. The incubation results for each of the stations are discussed 235

below. 236

237

Incubation Experiments 238

Drakes Bay at Point Reyes (Station PR). Initial nutrient concentrations at station PR 239

were 29.5 ± 3.3 mol L-1

nitrate, 2.2 ± 0.0 mol L-1

SRP, 40.8 ± 0.1 mol L-1

silicate, and 9.2 ± 240

0.65 nmol L-1

Fe, with a N/P ratio of 13.4 ± 1.5, a Si/N ratio of 1.4 ± 0.1, and a Fe/N ratio of 241

0.33 ± 0.0 nM/M (Fig. 3, 4). The range of N/P drawdown ratios for all treatments was 13 to 19, 242

and the range for Si/N was 0.8 to 1.2 (Fig. 5). 243

The initial chl a concentration was 1.9±0.1 mg m-3

, and the range of final concentrations 244

for all treatments was 32.2-43.0 mg m-3

. The N treatment resulted in a higher chl a increase than 245

other treatments (Fig. 6). The phytoplankton population at Point Reyes was dominated by several 246

species of Thalassiosira, and the majority of these were chain-forming in all treatments by the 247

end of the incubation. Pennate diatoms were present at this site across all treatments and between 248

40-57% of them were chain-forming (Fig. 7, 8; cell concentration data is given in Supplemental 249

Table 1). Among the picophytoplankton, picoeukaryotes (range across treatments = 78-104 x103 250

cells mL-1

) outnumbered Synechococcus (range across treatments = 5-7 x103 cells mL

-1) (Fig. 7). 251

Coastline north of San Francisco Bay (Station SF). Initial nutrient concentrations at 252

station SF were 27.9 ± 3.8 mol L-1

nitrate, 2.1 ± 0.0 mol L-1

SRP, 49.5 ± 0.2 mol L-1

silicate, 253

and 6.2 ± 0.61 nmol L-1

Fe with a N/P ratio of 13.3 ± 1.8, a Si/N ratio of 1.8 ± 0.2, and a Fe/N 254

ratio of 0.24 ± 0.04 nM/M (Fig. 3, 4). The range of N/P drawdown ratios for all treatments was 255

13 to 18, and the range for Si/N was 0.7 to 1.0 (Fig. 5). 256

The highest initial chl a concentration (3.3±0.0 mg m-3

) was observed at this site. By the 257

end of the experiment chl a levels in the Fe treatments clustered with the control (16.0-18.4 mg 258

m-3

), and N caused an increase in chl a (29.4±1.1 mg m-3

) above the control (Fig. 6). The highest 259

picophytoplankton abundances were observed at this site, where picoeukaryote abundances 260

ranged from 198-276 x103 cells mL

-1, and Synechococcus abundances ranged from 6-9 x10

3 cells 261

mL-1

(Fig. 7). Among phytoplankton larger than 4 m, small centric diatoms were abundant and 262

the majority existed as single cells (Fig. 7,8). Some of these small centric cells fell below the 263

4m size category and are instead included in the picoeukaryote category (enumerated by flow 264

cytometry). Treatment with Fe caused an increase in chain-forming centric diatoms relative to 265

the control (Fig. 7,8). Coscinodiscus was also observed at this site, though rare. Pennate diatoms 266

were observed, and chain-forming pennate cells were very rare (<<1%). The majority were 267

Cylindrotheca spp. 268

Inner Monterey Bay (Station M0). Initial nutrient concentrations at station M0 were 13.5 269

± 3.7 mol L-1

nitrate, 1.1 ± 0.0 mol L-1

SRP, 12.7 ± 0.0 mol L-1

silicate, and 3.6 ± 0.93 nmol 270

L-1

Fe, with a N/P ratio of 12.5 ± 3.4, a Si/N ratio of 0.94 ± 0.3, and a Fe/N ratio of 0.28 ± 0.11 271

nM/M (Fig. 4). The range of N/P drawdown ratios for all treatments was 17 to 19, and the 272

range for Si/N was 0.1 to 0.2 (Fig. 5). 273

The initial chl a concentration was 1.6±0.1 mg m-3

, and all treatments had similar final 274

concentrations as the control (8.2-9.3 mg m-3

; Fig. 6). The phytoplankton population at M0 was 275

dominated by Pseudonitzschia spp., and the majority of cells were chain-forming in all 276

treatments. Bottles receiving N additions had a lower percentage (60%) of chain-forming 277

pennate diatoms compared to the control and Fe addition treatments (78-82%, Fig. 7). Centric 278

diatoms were common at this site across all treatments, and between 34-57% were chain-forming 279

(Fig. 7,8). Picoeukaryotes (range across treatments = 49-57 x103 cells mL

-1) outnumbered 280

Synechococcus (range across treatments = 6-7 x103 cells mL

-1) (Fig. 7). 281

Outer Monterey Bay (Station M1). Initial nutrient concentrations at station M1 were 15.8 282

± 3.2 mol L-1

nitrate, 1.3 ± 0.0 mol L-1

SRP, 16.8 ± 0.0 mol L-1

silicate, and 4.5 ± 0.17 nmol 283

L-1

Fe, with a N/P ratio of 12.6 ± 2.5, a Si/N ratio of 1.1 ± 0.2, and a Fe/N ratio of 0.30 ± 0.06 284

nM/M (Fig. 3, 4). The range of N/P drawdown ratios for all treatments was 16 to 21, and the 285

range for Si/N was 0.1 to 0.2 (Fig. 5). 286

The initial chl a concentration was 1.9±0.0 mg m-3

, and the range of final concentrations 287

for all treatments was 8.2-12.4 mg m-3

. Nitrogen additions caused the greatest increase in chl a 288

(12.4 mg m-3

, Fig. 6). The phytoplankton population at M1 was dominated by Pseudonitzschia 289

spp., and the majority of cells were chain-forming. Bottles receiving N additions had a lower 290

percentage (56%) of chain-forming pennate diatoms compared to the control and Fe addition 291

treatments (64-70%, Fig. 7). Centric diatoms were common at this site across all treatments, and 292

between 64-72% were chain-forming (Fig. 7,8). Picoeukaryotes (range across treatments = 61-65 293

x103 cells mL

-1) outnumbered Synechococcus (range across treatments = 6-9 x10

3 cells mL

-1) 294

(Fig. 7,). Treatment with Fe additions caused a decline in Synechococcus abundance relative to 295

the control, whereas picoeukaryote abundances were not sensitive to any of the treatments (Fig. 296

7) 297

Offshore of Monterey Bay (Station M2). Initial nutrient concentrations at station M2 298

were 13.0 ± 4.0 mol L-1

nitrate, 1.1 ± 0.0 mol L-1

SRP, 9.2 ± 0.2 mol L-1

silicate, and 3.7 ± 299

1.0 nmol L-1

Fe, with a N/P ratio of 12.3 ± 3.8, a Si/N ratio of 0.7 ± 0.2, and a Fe/N ratio of 0.30 300

± 0.12 nM/M (Fig. 3, 4). The range of N/P drawdown ratios for all treatments was 16 to 21, and 301

the range for Si/N was 0.2 to 0.4 (Fig. 5). 302

The initial chl a concentration was 0.92±0.0 mg m-3

. All treatments had final 303

concentrations similar to the control (6.1-7.2 mg m-3, Fig. 6). The phytoplankton population at 304

M2 was dominated by Pseudonitzschia spp. Bottles receiving N additions had a lower percentage 305

(39%) of chain-forming pennate diatoms compared to the control and Fe addition treatments (69-306

71%, Fig. 7,8). Centric diatoms were common at this site across all treatments, and the majority 307

(77-87%) were chain-forming (Fig. 7,8). Picoeukaryotes (range across treatments = 49-57 x103 308

cells mL-1

) outnumbered Synechococcus (range across treatments = 6-7 x103 cells mL

-1) (Fig. 7). 309

310

Discussion 311

Coastal California nutrient availability creates a mosaic of nutrient limitation patterns that 312

vary with shelf width, distance from shore, and timing and extent of upwelling. In this study we 313

examined five Fe replete sites close to the central California coastline as upwelling relaxed (Fig. 314

2), when N and Fe levels were both elevated (Fig. 2,3). Even within this relatively small 315

geographical area, the waters in this study showed a high degree of spatial heterogeneity with 316

respect to nutrient levels, phytoplankton populations, and biomass nutrient limitation 317

characteristics. Concentrations of nitrate, SRP, silicate, and Fe were approximately twice as high 318

at stations PR and SF than at stations M0, M1, and M2 (Fig. 2,3), likely because they are 319

influenced by the very strong upwelling center at Bodega Bay (Fig 1). Stations PR and SF also 320

had higher ratios of Si:N, although the ratios of N:P were similar among all five sites (Fig. 4). 321

The plankton community compositions differed considerably among sites. The 322

phytoplankton community near Monterey Bay at stations M0, M1, and M2 was dominated by 323

Pseudonitzschia spp. (Fig. 9). Picophytoplankton comprised the vast majority of cells in station 324

SF, whereas station PR was populated by Thalassiosira spp., and virtually no pennate diatoms 325

were observed (see supplemental figures). Grazer populations also varied by location; stations 326

M0, M1, and M2 supported diverse populations of tintinnids (see Supplemental Fig. 7), whereas 327

station SF samples contained copepods and copepod fragments, as well as Protoperidinium sp. 328

No grazers were observed in samples from station PR. The patchy distribution of populations is 329

consistent with prior observations in Monterey Bay, where phytoplankton species trade off 330

dominance over small spatial scales and time periods (Mackey et al. 2012, Ryan et al 2011). 331

Nutrient limitation patterns for biomass also varied by location. All sites had N:P ratios 332

of approximately 12; this would typically suggest N limitation (because it is lower than the 333

Redfield Ratio of 16:1), however, only three stations (PR, SF, and M1) showed evidence of N 334

limitation based on chl a responses in the incubation experiments (Fig. 6). At those sites, more N 335

tended to be preferentially drawn down relative to P in bottles receiving additional N (higher N:P 336

drawdown ratio, Fig. 5). This suggests that N uptake rates were not saturated even though 337

ambient nitrate levels were high, because added nitrate increased the N uptake rate. This could 338

indicate luxury uptake of N by the diatoms, many of which store nitrate in large vacuoles (Lomas 339

and Gilbert 2000). In contrast, at stations M0 and M2 all treatments led to similar chl a increases, 340

suggesting that nutrient levels were high enough to saturate phytoplankton at these sites and 341

biomass was not nutrient limited. 342

As expected for the broad shelf regions tested in this study, phytoplankton were not Fe-343

limited in our experiments. This differs from the nearby California transition zone, where Fe 344

levels are routinely below 1 nM (Biller and Bruland 2014). The Fe levels at sites in this study 345

ranged from ~4-10 nM (Fig. 3) comparable to the range of total dissolved Fe reported previously 346

for this part of the California coast (0.3-10 nM; Bruland et al. 2001). Additionally, Fe/N ratios in 347

the transition zone were generally below 0.05 nM/M (Biller and Bruland 2014), which is well 348

below the threshold for Fe limitation in oceanic (0.07) and coastal (0.20) diatoms (Sunda and 349

Huntsman, 1995). Only in the stations closest to shore did Biller and Bruland report Fe/N ratios 350

exceeding the Fe-replete threshold, where values reached up to 0.20, similar to ratios we 351

observed in this study (0.24-0.33; Fig. 4C,D). Moreover, natural populations of diatoms can take 352

up excess iron to go through a number of cell divisions when Fe becomes scarce (Sugie et al., 353

2011; Yoshida et al., 2006), which could further explain the lack of biomass Fe limitation in our 354

bottles. 355

Changes in the chain forming behavior of diatoms has been linked to Fe and N 356

availability (Hutchins and Bruland 1998) and temperature (Takabayashi et al., 2006). In this 357

study Fe addition did not cause deviations from the control with respect to chain forming 358

behavior in pennate diatoms (Fig. 8). However, Fe additions did appear to enhance chain 359

forming morphology in centric diatoms slightly, though the effect was not significant at p<0.05 360

(Fig. 8). This behavior is consistent with the findings of Hutchins and Bruland (1998), where Fe 361

additions caused more fast sinking, chain forming centric diatoms to bloom. The incubation 362

results suggest that the Fe/N ratios at sites in this study were already high enough (0.24-0.33 363

nM/M) to support chain forming behavior in pennate diatoms in the control and Fe addition 364

samples, thus further Fe additions did not cause any change (Fig 7,8). 365

Major differences in the drawdown of Si relative to N were observed among the sites 366

(Fig. 5C,D). Stations M0, M1, and M2 all had Si/N drawdown ratios well below the 1:1 Si/N 367

drawdown ratio expected for diatoms (even though these sites were dominated by the diatom 368

Pseudo-nitzschia spp.), while stations SF and PR had drawdown ratios close to 1:1. Therefore, 369

less Si than expected was taken up in Monterey Bay compared to the other sites. Interestingly, 370

Si/N drawdown did not show a consistent response to either Fe or N additions (and therefore 371

Fe/N ratios). This response differs from Fe-limited sites along the CA coast where Fe additions 372

caused diatoms to incorporate up to 50% less Si into their frustules relative to N during 373

incubation experiments (Hutchins and Bruland 1998). The discrepancy in Si/N drawdown among 374

sites suggests factors other than Fe availability influence the drawdown of Si relative to N. One 375

possibility could be differences in Si drawdown between different diatom species, as stations 376

M0, M1 and M2 were dominated by pennate diatoms, whereas SF and PR had more centric 377

diatoms. Alternately, as Si was depleted at M0, M1 and M2, cells may have begun taking up Si 378

and N at different rates, where luxury N uptake could drive lower Si:N drawdown. 379

Global change presents the possibility for a number of alterations to the biogeochemistry 380

of coastal California (Capone and Hutchins 2013 and references therein). The flux of nitrate is 381

expected to increase over the next century, and areas that are currently Fe replete could transition 382

toward Fe limitation as the Fe/N ratio decreases (Rykaczewski and Dunne 2010). In this study, 383

the transition toward higher nitrate flux (lower Fe/N ratios) was simulated by nitrate additions in 384

the incubation experiment. These additions of 10 mol/L nitrate are within the range of predicted 385

future N increases, and brought Fe/N ratios into Fe-limited range (range of Fe/N = 0.16-0.18 386

nmol/ mol) at each site except for station PR (Fe/N = 0.25 nmol/ mol). 387

We compared our data to published values of Fe and N for the region spanning from 388

Monterey Bay to Point Reyes (Biller et al. 2013), and calculated the potential shift in Fe/N ratio 389

assuming a 50% increase in nitrate at each site as projected by Rykaczewsk and Dunne (2010) 390

(Fig. 10). While biomass at many of the locations in this region are currently Fe replete, others 391

are closer to becoming Fe-limited. Coastal diatoms become Fe-limited below a Fe/N ratio of 0.20 392

nmol/mol, and oceanic species have a slightly lower threshold ratio (0.07 nmol/mol; Sunda 393

and Huntsman, 1995). Thirteen of the 23 sites we compared would be considered Fe replete 394

under present day conditions (>0.2 nmol Fe/ mol N); however, biomass at five of these 395

locations (e.g., within Monterey Bay, south of Half Moon Bay, and along the coast near San 396

Francisco Bay) would cross into the Fe-limited range in the future if nitrate flux increases. The 397

ratios of the other ten sites that are presently Fe-limited would of course also decline further. 398

These shifts could lead to succession of phytoplankton species better adapted to dealing with Fe 399

limitation, as well as changes in chain forming behavior as observed in this study. 400

Increased nitrate flux is also anticipated to cause a concomitant increase in productivity 401

in coastal California (Rykaczewsk and Dunne 2010). Addition of nitrate increased phytoplankton 402

biomass in three of the five sites assayed in this study, leading to a 28-60% increase in chl a 403

(Fig. 6) and increasing the drawdown of N relative to P (Fig. 5A,B). Nitrogen addition also 404

affected the chain-forming behavior of phytoplankton by encouraging the growth of slower 405

sinking, single-celled pennate diatoms in all of the sites (Fig. 8A). This was due to both a 406

physiological shift in Pseudonitzschia spp. toward a single-celled growth habit, as well as a 407

community shift toward species like Cylindrotheca spp. that naturally tend to exist as solitary 408

cells. 409

These changes in phytoplankton physiology and community structure suggest that 410

enhanced N flux could lead to ecological and biogeochemical shifts in the California upwelling 411

system in the future. First, the sinking rate of cells, and hence the export of C, Si, and other 412

elements could decline with the shift to smaller chains of cells with slower sinking rates. Second, 413

these shifts have the potential to propagate up the food web because different, potentially smaller 414

grazers could be favored by the increase in single cells. And finally, if increased N flux favors 415

pennate species other than Pseudonitzschia spp. as our experiments suggest, then blooms of this 416

potentially toxin-producing genus could become less prevalent in the future. Indeed, the toxin 417

producing species Pseudonitszchia pungens is more competitive at low N:P ratios of ~10:1 418

compared to more N rich waters (Hu et al. 2008). The N:P ratios in our unamended water ranged 419

from 12.3 to 13.4, whereas the nitrate-treated water ranged from 18.0 to 21.8. It is therefore not 420

surprising that the slightly N limited conditions and low N/P ratios in the present-day water 421

would favor Pseudonitszchia, which leaves open the possibility that Pseudonitszchia could 422

become less competitive due to excess N in the future. 423

Changes in ecosystem services, which are the benefits people derive from marine 424

ecosystems, could also be affected by the changes described here. In California, a major 425

ecosystem service provided by coastal waters is fishery yield. Many studies have shown a link 426

between the amount of upwelling that occurs in a given year and the production of fisheries 427

(Gunsolus 1978; Nickelson 1986). The relationship is also apparent in comparing fishery 428

production along the west coast of North and South America during El Nino (low upwelling) and 429

La Nina (high upwelling) years. If future primary productivity increases affect fisheries in a 430

similar manner to natural increases in upwelling, it is possible that CA fisheries could become 431

more productive in the future. The phytoplankton population shift toward smaller cells, which 432

decreases export production due to a decrease in sinking rates, could likewise increase fisheries 433

yields by providing more carbon biomass to grazers. 434

The projected increase in N flux by the year 2100 is expected to coincide with an 18% 435

decrease in oxygen concentration (Rykaczewsk and Dunne 2010) and a decrease in pH by 0.5 436

units due to anthropogenic ocean acidification (Doney et al. 2009). These multiple stressors 437

could exacerbate or mitigate the effects observed in this study. In particular, the effect of 438

changing seawater chemistry on Fe solubility is difficult to predict. Ocean acidification has the 439

potential to reduce Fe bioavailability by protonating Fe ligands, causing them to retain Fe ions 440

(Shi et al. 2010). In contrast, expansion and shoaling of hypoxic zones would serve to increase 441

Fe solubility, because the reduced Fe(II) in hypoxic waters is more soluble than the Fe(III) in 442

oxygenated waters. In this study Fe additions caused little change in the phytoplankton 443

community because cells were already Fe replete; however, future shifts toward Fe limitation 444

could enhance the importance of soluble Fe supply in these regions (Fig. 10). Moreover, large 445

blooms of phytoplankton increase the pH of seawater, which could partially or wholly offset the 446

local effects of ocean acidification on Fe availability. It is therefore not clear whether Fe 447

availability will increase or decrease in the future, or what effect, if any, this will have on marine 448

biota in upwelling regions. The Canary and Benguela upwelling systems, which are more Fe 449

replete than the California and Peru systems, are likely to be less sensitive to these changes in Fe 450

biogeochemistry, and changes in N flux may be more important. 451

Upwelling systems are naturally variable regions inhabited by organisms that thrive 452

under changing conditions. In regions like coastal California where phytoplankton biomass is 453

already on the cusp between N and Fe limitation, modest shifts in nutrient supply ratios could 454

elicit important changes in cell physiology, community composition, and nutrient uptake. This 455

study shows that increased N availability in recently upwelled water leads to faster cell growth, 456

greater nitrate drawdown, and favors solitary cells over chain-formers. Upwelling centers 457

throughout the world's oceans are morphologically and biogeochemically diverse, and future 458

studies should help determine if similar responses could occur in other upwelling regions. 459

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Figure 1: Sea surface temperature indicated that colder upwelled water was present on 567

June 15 (A) and that upwelling had relaxed and surface water began to warm by June 20 568

(B). Temperature scale is in oC. Areas of intense upwelling in this study are circled on 569

each map. (C) Map showing the locations where incubation water was collected (see 570

methods for latitude and longitude). Shaded region in (C) indicates the approximate 571

location of the "transition zone" (TZ) described by Biller and Bruland (2014). 572

573

574

575

PR

SF

M0M1

M2

_ 37o 55’

_ 37o 05’

_ 36o 15’

|-121o 40’

|-122o 30’

|-123o 20’

BA C

TZPoint Sur

Point Reyes

San Francisco

Bay

Monterey Bay

15 June 2011 20 June 2011

Figure 2: In situ mooring data for station M1 showing (A) hourly gridded wind velocity, (B) sea 576

surface temperature, (C) salinity, and (D) nitrate concentration. Upwelling favorable winds 577

occurred in the days prior to sampling and were accompanied by cold, saline water with high 578

nitrate levels. Winds relaxed beginning June 17, causing upwelling to cease and allowing surface 579

waters to warm as nitrate was drawn down. Incubation water was collected on June 20 and 22. 580

581

582

10.5

11.5

12.5

13.5

14.5

33.2

33.4

33.6

33.8

0.0

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Win

d s

pe

ed

(m s

-1) 8.0

-8.0

0.0

1 5 10 15 20 25 30

Day in June 201122

1 5 10 15 20 25 3022

Upwelled water warmed as it aged

Consistent salinity indicated upwelled water mass source

NO3- was consumed gradually

as upwelled water aged

Upwelling favorable winds

Sampling datesW

ate

r te

mp

era

ture

(o C)

Salin

ity

(-)

NO

3- c

on

cen

trat

ion

(m

olL

-1)

A

B

C

D

Figure 3: Initial concentrations of (A) soluble reactive phosphorus, SRP, (B) Fe, (C) silicate, and 583

(D) nitrate at the five sampling locations. Error bars show standard deviation. 584

585

0.0

10.0

20.0

30.0

40.0

M0 M1 M2 PR SF

0.0

1.0

2.0

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60.0

M0 M1 M2 PR SF

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8

12

M0 M1 M2 PR SF

A) SRP

Nit

rate

(

M)

SRP

(M

)Si

licat

e (

M)

Fe (n

M)

B) Fe

C) Si

D) Nitrate

Figure 4: Nutrient ratios of (A,B) SRP: nitrate, (C,D) Fe: nitrate, and (E,F) silicate: nitrate at the 586

five sampling locations when the incubation water was collected. Legend for (B,D,F shown in 587

panel F). Error bars show standard deviation. Broken lines in (A,C,E) and solid lines in (B,D,E) 588

show typical ratios of diatom cellular nutrient quotas. 589

590

591

592

593

0

2

4

6

8

10

12

5 10 15 20 25 30 35

0.0

5.0

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0

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20

30

40

50

5 10 15 20 25 30 35

Nitrate (M)

0.0

1.0

2.0

3.0

0 5 10 15 20 25 30 35Fe

(nM

)Si

licat

e (

M)

SRP

(M

)

B)

D)

F)

1:1

1:16

PR

SF

M0

M1

M2

A) N/P

0

0.1

0.2

0.3

0.4

0.5

M0 M1 M2 PR SF

0.0

0.5

1.0

1.5

2.0

M0 M1 M2 PR SF

Nit

rate

/SR

P (

M/

M)

Silic

ate

/Nit

rate

(

M/

M)

Fe/N

itra

te (

nM

/

M)

C) Fe/N

E) Si/N

0.2 nM/ M

P limited

N limited

N limited

Fe limited

N limited

Si limited

Figure 5: Nutrient drawdown ratios of (A,B) N/P and (C,D) Si/N for the five stations in this 594

study. For (B,D) values are shown for all three treatments for each station.Values are calculated 595

as the ratios of final minus initial nutrient concentrations. Error bars show standard error. Broken 596

lines in (A,C) and solid lines in (B,D) show typical ratios of diatom cellular nutrient quotas. 597

598

599

600

0.5

1.0

1.5

2.0

2.5

0 10 20 30 40 50

PR

SF

0

10

20

30

40

0 10 20 30 40 50

0

10

20

30

C Fe N

0

10

20

30

C Fe N

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30

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20

30

C Fe N

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20

30

C Fe N

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1.5

2.0

C Fe N0.0

0.5

1.0

1.5

2.0

C Fe N

0.0

0.5

1.0

1.5

2.0

C Fe N

0.0

0.5

1.0

1.5

2.0

C Fe N

PR SF M0 M1 M2

Si:N

dra

wd

ow

n (

M/

M)

N:P

dra

wd

ow

n (

M/

M)

0.5

1.0

1.5

2.0

2.5

0 10 20 30 40 50

PRSFM0M1M2x

Nitrate drawdown (M)

1:161:1

A

C

B D

Treatments

0

10

20

30

40

0 10 20 30 40 50

Silic

ate

dra

wd

ow

n (

M)

SRP

dra

wd

ow

n (

M)

Nitrate drawdown (M)

Figure 6: Growth curves showing chlorophyll a over time. Nitrogen additions increased growth 601

at stations PR, SF, and M1. Error Bars show standard error. 602

603

604

0

15

30

45

0 2 4

CFeN

0

15

30

45

0 2 4

0

5

10

15

0 2 4

0

5

10

15

0 2 4

0

5

10

15

0 2 4

Ch

loro

ph

yll a

(mg

m-3

)

Time (day) Time (day)

PR SF

M0 M1 M2

Ch

loro

ph

yll a

(mg

m-3

)

Time (day)

Figure 7: Cell count data from the final time point of each incubation, showing concentrations of 605

(A) pennate diatoms, (B) centric diatoms, (C) dinoflagellates and other cells >4 m, (D) 606

picoeukaryotes, and (E) Synechococcus. Error bars show standard error. Light-colored stacked 607

bars in (A,B) show single celled diatoms and dark-colored stacked bars show chain-forming 608

cells. Symbol † denotes p<0.05; symbol * denotes p<0.10; symbol ǂ denotes p<0.15. 609

610

0

5

10

15

20

C Fe N C Fe N C Fe N C Fe N C Fe N

0

200

400

600

C Fe N C Fe N C Fe N C Fe N C Fe N

0

20

40

60

80

C Fe N C Fe N C Fe N C Fe N C Fe N

C Fe N C Fe N C Fe N C Fe N C Fe N

C Fe N C Fe N C Fe N C Fe N C Fe N

A

B

C

D

E

1

2

3

4

8

12

Penn

ate

diat

oms

(cel

ls m

L-1)

Cen

tric

dia

tom

s

(cel

ls m

L-1)

Pico

euka

ryot

es(1

05

cell

s m

L-1)

Synechococcus

(10

3ce

lls

mL-1

)O

ther

cel

ls(c

ells

mL-1

)

PR SF M0 M1 M2

* *

*

ǂ

**

*

†

Figure 8: Observations of chain-forming behavior for pennate diatoms (above) and centric 611

diatoms (below). At right data from all sites has been (grey bars, n=15 for each treatment). Error 612

bars show standard error. Symbol † denotes p<0.05; symbol * denotes p<0.10; symbol ǂ denotes 613

p<0.15. 614

615

616

617

0

25

50

75

100

C Fe N C Fe N C Fe N C Fe N C Fe N

0

25

50

75

100

C Fe N C Fe N C Fe N C Fe N C Fe N

A) Pennate diatoms

B) Centric diatoms

PR SF M0 M1 M2

25

50

75

25

50

75

0

25

50

75

100

C Fe N C Fe N C Fe N C Fe N C Fe N

Average all sites

Pe

rcen

t chain

-form

ing (%

)Pe

rce

nt c

hai

n-f

orm

ing

(%)

†

†

ǂ

Figure 9: Micrographs of (A) Pseudonitzschia spp.; (B) Thalassiosira sp. with dividing cells; (C) 618

Thalassiosira sp.; (D) Thalassiosira rotula; (E) Pseudonitzschia sp.; (F) Asterionellopsis 619

glacialis; (G) Cylindrotheca closterium dividing cells; (H) unknown chain forming centric 620

diatom; (I) Cylindrotheca closterium; (J) Thalassionema sp. with Thalassiosira sp. 621

622

623

GF

C

E

D

B

A

IH

J

Figure 10: Range of Fe/N ratios for the region between Monterey Bay and Point Reyes. Boxes 624

show the present day (right hand side of box) and projected future Fe/N ratio (left hand side of 625

box). Shaded region denoted the range of Fe/N ratios (0.07-0.2) over which oceanic and coastal 626

diatoms become Fe-limited. Data are from this study for stations PR, SF, M0, M1, and M2, and 627

from table 1 of Biller et al (2013) for all other latitudes. Projected future ratios were calculated 628

assuming a 50% increase in nitrate as predicted by Rykaczewsk and Dunne (2010). 629

630

631

0.01 0.1 1 10 100

M0M1M2

36.929737.038337.157337.183937.187037.249837.280237.299337.304137.416337.418037.603437.758337.781037.842937.870537.8882

SF37.9308

PR

Fe/N ratio (nmol Fe/ mol N)

Lati

tud

e

Fe limited

Fe replete

tran

siti

on

San Francisco Bay

Monterey Bay

Point Reyes

Half Moon Bay

Point Año Nuevo

SUPPLEMENTAL INFORMATION 632

Supplemental table 1: 633

Detection limit and CRM analyses. Units are nmol/kg if not mentioned.

Metal Detection limit*

SAFe_S (n=6)**

measured consensus

av ± SD

Al 2.28

BD 1.67 ± 0.10

Mn 0.023

0.84 ± 0.05 0.79 ± 0.06

Fe 0.74

BD 0.093 ± 0.008

Co 0.005

0.003 ± 0.002 0.005 ± 0.001

Ni 0.11

2.45 ± 0.17 2.28 ± 0.09

Cu 0.013

0.37 ± 0.02 0.52 ± 0.05

Zn 0.24

BD 0.069 ± 0.010

Cd 0.0004

0.0017 ± 0.0008 0.0011 ± 0.0003

Pb 0.0009 0.044 ± 0.001 0.0480 ± 0.0022

* Detection limit calculated by 3×standard deviation of the blank.

**no UV treatments

634

635

Supplemental Figure 1: Chain forming Thalassiosira spp. 636

637

A

B

C

D

E

F

G H I

Supplemental Figure 2: Single and chain-forming pennate diatoms. 638

639

640

641

A B C

D

E F

G

H

I

J

K

L

M N

Supplemental Figure 3: Thalassionema spp. 642

643

644

645

646

647

A B C

D E

F G

H

Supplemental Figure 4: Chain-forming centric diatoms. 648

649

650

651

A B

C D E

F G H

Supplemental Figure 5: Chain-forming centric diatoms. 652

653

654

655

A B

C D

E F

G H I J

Supplemental Figure 6: Single cell centric diatoms. 656

657

658

A B

C D E

F G H

Supplemental Figure 7: Dinoflagellates, tintinnids, and other cell types. 659

660

661

A B C

D

E

F

G H I

J K L M N

Supplemental Figure 8: Picoplankton from station SF. 662

663

664

665

Figure 9: Relationships among temperature, salinity, and nitrate at the M1 mooring for June 666

2011. 667

668

Nitrate (

M)

Temperature (oC)

Salin

ity

(psu

)

Supplemental Table 1: Mean ± standard error cell concentration data; n=3 replicates for each treatment at each site. Concentration

units are in (cells mL-1

).

Station Treatment

Single-

cell

pennate

diatoms

Chain-

forming

pennate

diatoms

% Chain-

forming

pennate

diatoms

Single-

cell

centric

diatoms

Chain-

forming

centric

diatoms

% Chain-

forming

centric

diatoms Other Picoeukaryotes Synechococcus

PR Control 30 ± 4 21 ± 7 40 ± 8 112 ± 11 499 ± 128 81 ± 2 8 ± 7 78294 ± 10706 4781 ± 499

PR Fe 30 ± 7 32 ± 8 51 ± 7 98 ± 12 439 ± 68 82 ± 1 4 ± 2 89404 ± 6615 5022 ± 212

PR N 32 ± 6 30 ± 8 47 ± 7 103 ± 23 298 ± 100 71 ± 8 4 ± 3 96652 ± 5787 7053 ± 1619

SF Control 4 ± 1 0 ± 0 6 ± 1 34 ± 1 16 ± 6 30 ± 7 3 ± 1 197639 ± 37446 9367 ± 1312

SF Fe 5 ± 1 0 ± 0 6 ± 4 39 ± 7 29 ± 7 41 ± 3 2 ± 0 264084 ± 20738 8916 ± 1855

SF N 5 ± 1 0 ± 0 0 ± 0 46 ± 9 13 ± 1 23 ± 4 2 ± 0 276031 ± 8551 7547 ± 224

M0 Control 5 ± 0 25 ± 3 82 ± 2 11 ± 4 8 ± 1 46 ± 9 7 ± 3 53841 ± 2624 6087 ± 347

M0 Fe 8 ± 1 28 ± 4 78 ± 2 13 ± 2 18 ± 4 57 ± 2 6 ± 2 56271 ± 6157 7375 ± 504

M0 N 14 ± 4 20 ± 2 60 ± 6 19 ± 3 10 ± 1 34 ± 2 6 ± 2 56654 ± 6790 6100 ± 198

M1 Control 13 ± 1 29 ± 3 70 ± 3 6 ± 1 15 ± 5 67 ± 10 13 ± 2 63766 ± 4873 9135 ± 288

M1 Fe 28 ± 5 49 ± 4 64 ± 6 8 ± 2 15 ± 4 64 ± 5 16 ± 1 65479 ± 4092 7106 ± 640

M1 N 25 ± 4 34 ± 9 56 ± 8 6 ± 1 21 ± 7 72 ± 8 14 ± 2 64716 ± 3577 8562 ± 545

M2 Control 16 ± 5 40 ± 7 71 ± 7 6 ± 0 22 ± 6 77 ± 6 2 ± 1 25341 ± 1224 3220 ± 91

M2 Fe 22 ± 7 40 ± 10 65 ± 5 6 ± 0 33 ± 7 84 ± 3 1 ± 1 27302 ± 4312 3391 ± 48

M2 N 44 ± 3 28 ± 2 39 ± 3 6 ± 1 46 ± 13 87 ± 3 1 ± 0 24485 ± 3145 2962 ± 688