Upload
others
View
2
Download
0
Embed Size (px)
Citation preview
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, [email protected], 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
References 460
Assmy, P., Henjes, J., Klaas, C. and Smetacek, V., 2007. Mechanisms determining species 461
dominance in a phytoplankton bloom induced by the iron fertilization experiment 462
EisenEx in the Southern Ocean. Deep-Sea Research Part I-Oceanographic Research 463
Papers, 54(3): 340-362. 464
Biller, DV, and KW Bruland. 2012. Analysis of Mn, Fe, Co, Ni, Cu, Zn, Cd, and Pb in seawater 465
using the Nobias-chelate PA1 resin and magnetic sector inductively coupled plasma mass 466
spectrometry (ICP-MS). Marine Chemistry. 120: 12–20 467
Biller DV, TH. Coale, RC. Till, GJ Smith, KW Bruland. 2013. Coastal iron and nitrate 468
distributions during the spring and summer upwelling season in the central California 469
Current upwelling regime. Continental Shelf Research 66: 58-72 470
Biller, DV, and KW Bruland. 2013. Sources and distributions of Mn, Fe, Co, Ni, Cu, Zn, and Cd 471
relative to macronutrients along the central California coast during the spring and 472
summer upwelling season. Mar. Chem. 155:50-70 473
Biller, DV and KW Bruland. 2014. The central California Current transition zone: A broad 474
region exhibiting evidence for iron limitation. Progress in Oceanography 120: 370–382 475
Boyd, PW, et al. 2000. A mesoscale phytoplankton bloom in the polar Southern Ocean 476
stimulated by iron fertilization. Nature 407, 695-702 477
Bruland KW, EL. Rue and G J. Smith (2001) Iron and Macronutrients in California Coastal 478
Upwelling Regimes: Implications for Diatom Blooms. Limnology and Oceanography, 479
46:1661-1674 480
Capone, DG and DA Hutchins. 2013. Microbial biogeochemistry of coastal upwelling regimes in 481
a changing ocean. Nature Geoscience 6: 711-717 482
Chavez, F. P., and M. Messie (2009), A comparison of eastern boundary upwelling ecosystems, 483
Progress in Oceanography, 83: 80–96 484
Coale, KH et al. 1996. A massive phytoplankton bloom induced by an ecosystem-scale iron 485
fertilization experiment in the equatorial Pacific Ocean. Nature 383: 495-501 486
Doney, SC, V J Fabry, R A. Feely, and J A. Kleypas (2009) Ocean Acidification: The Other 487
CO2 Problem. Annual Review of Marine Science. DOI: 488
10.1146/annurev.marine.010908.163834 489
De La Rocha, CL, and U Passow. 2007. Factors influencing the sinking of POC and the 490
efficiency of the biological carbon pump. Deep Sea Research II: Topical Studies in 491
Oceanography. 54: 639–658 492
Duce, R.A., LaRoche, J., Altieri, K., Arrigo, K.R., Baker, A.R., Capone, D.G., et al. (2008). 493
Impacts of atmospheric anthropogenic nitrogen on the open ocean. Science 320, 893–897 494
Firme GF, Rue EL, Weeks DA, Bruland KW, Hutchins DA (2003) Spatial and temporal 495
variability in phytoplankton iron limitation along the California coast and consequences 496
for Si, N, and C biogeochemistry. Global Biogeochemical Cycles 17:1016, 497
doi:1010.1029/2001GB001824 498
Gunsolus, R. T. 1978. The status of Oregon coho and recommendations for managing the 499
production, harvest, and escapement of wild and hatchery–reared stocks. Oregon 500
Department of Fish and Wildlife, Clackamas, OR. 501
Hu, Z-X, XU Ning, A-F Li, S-S Duan. 2008. Effects of different N:P ratios on the growth of 502
Pseudo-Nitzschia pingens, Prorocentrum donghaiense and Phaeocystis globosa. Acta 503
Hydrobiologica Sinica 32:482-487 DOI: 10.3724/SP.J.1035.2008.00482 504
Hutchins, D. A. & Bruland, K. W. 1998. Iron-limited diatom growth and Si:N uptake ratios in a 505
coastal upwelling. Nature 393, 561–564 506
Kudela, R. M., and R. C. Dugdale (2000), Nutrient regulation of phytoplankton productivity in 507
Monterey Bay, California, Deep Sea Res., Part II, 47, 1023–1053 508
Lomas, MW and PM Gilbert (2000) COMPARISONS OF NITRATE UPTAKE, STORAGE, 509
AND REDUCTION IN MARINE DIATOMS AND FLAGELLATES. J. Phycol. DOI: 510
10.1046/j.1529-8817.2000.99029.x 511
Mackey, KRM, CE Mioni, JP Ryan, A Paytan. 2012. Phosphorus cycling in the red tide 512
incubator region of Monterey Bay in response to upwelling. Frontiers in Microbiology 513
vol 3, article 33, doi: 10.3389/fmicb.2012.00033 514
Mackey, KRM, G van Djiken, S Mazloom, AM Erhardt, J Ryan, K Arrigo, A Paytan. 2010. 515
Influence of atmospheric nutrients on primary productivity in a coastal upwelling region. 516
Global Biogeochem. Cycles. 24: GB4027, doi:10.1029/2009GB003737 517
Marchetti, A, et al. Ferritin is used for iron storage in bloom-forming marine pennate diatoms. 518
Nature 457, 467-470 519
Martin, JH et al. 1994. Testing the iron hypothesis in ecosystems of the equatorial Pacific Ocean. 520
Nature 371: 123-129 521
Nicholson, DP, ST Dyhrman, F Chavez, and A Paytan. 2006. Alkaline phosphatase activity in 522
the phytoplankton communities of Monterey Bay and San Francisco Bay. Limnol. 523
Oceanogr. 51: 874-883 524
Nickelson, T. E. 1986. Influences of upwelling, ocean temperature, and smolt abundance on 525
marine survival of coho salmon (Oncorhynchus kisutch) in the Oregon Production Area. 526
Canadian Journal of Fisheries and Aquatic Sciences 43:527–535. 527
Pennington, J. T., and F. Chavez (2000), Seasonal fluctuations of temperature, salinity, nitrate, 528
chlorophyll, and primary production at station H3/ M1 over 1989–1996 in Monterey Bay, 529
CA, Deep Sea Res., Part II, 47, 947–973. 530
Ryan, J.P., Greenfield, D., Marin, R., Preston, C., Roman, B., Jensen, S., Pargett, D., Birch, J., 531
Mikulski, C., Doucette, G., and Scholin, C. (2011). Harmful phytoplankton ecology 532
studies using an autonomous molecular analytical and ocean observing network. Limnol. 533
Oceanogr. 56, 1255–1272. 534
Rykaczewski, R. R. & Dunne, J. P. Enhanced nutrient supply to the California current ecosystem 535
with global warming and increased stratification in an earth system model. Geophys. Res. 536
Lett. 37, L21606 (2010). 537
Shi, D., Xu, Y., Hopkinson, B. M. & Morel, F. M. M. Effect of ocean acidification on iron 538
availability to marine phytoplankton. Science 327, 676–679 (2010). 539
Silver, MW et al. 2010. Toxic diatoms and domoic acid in natural and iron enriched waters of the 540
oceanic Pacific. PNAS 107: 20762–20767, doi: 10.1073/pnas.1006968107 541
Sugie, K. et al., 2011. Importance of intracellular Fe pools on growth of marine diatoms by using 542
unialgal cultures and on the Oyashio region phytoplankton community during spring. 543
Journal of Oceanography: 1-14. 544
Takabayashi, M. et al., 2006. The effect of nutrient availability and temperature on chain length 545
of the diatom, Skeletonema costatum. J. Plankton Res., 28(9): 831-840. 546
Trick, CG et al. 2010. Iron enrichment stimulates toxic diatom production in high-nitrate, low-547
chlorophyll areas. PNAS 5887–5892, doi: 10.1073/pnas.0910579107 548
Tsuda, A. et al., 2005. Responses of diatoms to iron-enrichment (SEEDS) in the western 549
subarctic Pacific, temporal and spatial comparisons. Progress In Oceanography, 64(2-4): 550
189-205. 551
Tsuda, A. et al., 2006. Mesozooplankton response to iron enrichment during the diatom bloom 552
and bloom decline in SERIES (NE Pacific). Deep-Sea Research Part Ii-Topical Studies In 553
Oceanography, 53(20-22): 2281-2296. 554
Tsuda, A. et al., 2003. A Mesoscale Iron Enrichment in the Western Subarctic Pacific Induces a 555
Large Centric Diatom Bloom. Science, 300(5621): 958-961. 556
Smetacek, V, et al. 2012. Deep carbon export from a Southern Ocean iron-fertilized diatom 557
bloom. Nature 487, 313–319 558
Sohrin,Y.S., Urushihara,S., Nakatsuka, T., Kono, E., Higo, T., Minami, K.,et al. (2008). Multi-559
elemental determination of GEOTRACES key trace metals in seawater by ICPMS after 560
preconcentration using an ethylenediaminetriacetic acid chelating resin. Anal.Chem. 80, 561
6267–6273. 562
Sunda, W.G., Huntsman, S.A., 1995. Iron uptake and growth limitation in oceanic and coastal 563
phytoplankton. Marine Chemistry 50, 189–206. 564
Yoshida, M. et al., 2006. Effect of aging time on the availability of freshly precipitated ferric 565
hydroxide to coastal marine diatoms. Marine Biology, 149(2): 379-392.566
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
5.0
10.0
15.0
20.0
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
3.0
M0 M1 M2 PR SF
0.0
20.0
40.0
60.0
M0 M1 M2 PR SF
0
4
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
10.0
15.0
20.0
M0 M1 M2 PR SF
0
10
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
0
10
20
30
C Fe N
0
10
20
30
C Fe N
0
10
20
30
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 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