Deep-Sea Research II 47 (2000) 1023}1053
Nutrient regulation of phytoplankton
productivity in Monterey Bay, California
R.M. Kudela!,*, R.C. Dugdale"
!University of California Santa Cruz, Ocean Sciences Department, 1156 High Street, Santa Cruz,
CA 95064, USA
"Romberg Tiburon Center for Environmental Studies, San Francisco State University, P.O. Box 855, Tiburon,
CA 94920, USA
Received 16 June 1997; received in revised form 10 April 1998; accepted 15 December 1998
Abstract
A series of nutrient enrichment grow-out experiments were conducted in Monterey Bay,
California, to assess the relative importance of nutrient availability on growth rates and
biomass accumulation of the natural phytoplankton assemblage. During a series of four cruises,
enrichments with nitrogen (as nitrate and ammonium), silicate, and Guillard's `f a medium
consistently demonstrated that the phytoplankton were nitrogen limited, and that the addition
of nitrate provided the most potential for growth and biomass accumulation. Contrary to
previous reports for Monterey Bay, silicate was not found to limit the accumulation of biomass
in this diatom-dominated system, although there was evidence that silicate additions can
modify the uptake rates of the biomass-limiting substrate (nitrogen). We conclude that silicate is
a regulating, but not limiting, nutrient in this study site. Our results are consistent with both the
`shift-upa and `detritala explanations for changes in speci"c uptake rates. During upwelling
periods (May, September) when the biomass was dominated by phytoplankton, a shift-up type
physiological response was observed that was not dependent on the uptake normalization
procedure (e.g. chlorophyll versus PN). During the winter months (March, November), characterized by deep mixing, low light, and higher detrital N levels, the apparent shift-up response
could be attributed to a change in the PN : Chl ratios and alleviation of light limitation due to
the stable light regime provided by the enclosures. ( 2000 Elsevier Science Ltd. All rights
reserved.
* Corresponding author. Fax: 001-831-459-4882.
E-mail address: [email protected] (R.M. Kudela)
0967-0645/00/$ - see front matter ( 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 9 6 7 - 0 6 4 5 ( 9 9 ) 0 0 1 3 5 - 6
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R.M. Kudela, R.C. Dugdale / Deep-Sea Research II 47 (2000) 1023}1053
1. Introduction
In both marine and freshwater aquatic environments, it has been hypothesized that
one of the primary factors controlling new and primary production is the availability
of some limiting substrate, most often nitrogen or phosphorous. Typically, the
determination of this most limiting factor (sensu Liebig, 1840) has been tested through
the use of nutrient ratios (Red"eld et al., 1963) or through the use of bioassay
experiments, where one or more nutrients are added to a controlled volume of whole
water (e.g. Hecky and Kilham, 1988). This latter technique has been particularly
e!ective in determining the limiting e!ects of phosphorous in fresh water through the
use of whole-lake manipulations (Schindler, 1974). Until recently (Martin et al., 1994;
Coale et al., 1996), there have been no corresponding true mesocosm manipulation
experiments in the marine environment. Instead, researchers have utilized experimental enclosures, ranging from small bottles with volumes of a few liters or less, to
massive free-#oating enclosures of thousands of liters. Although the validity of these
enclosure experiments in relation to real-world ecosystem processes has been questioned (e.g. Smith, 1991), they remain the only way to reliably manipulate and evaluate
ecosystem responses to controlled perturbations in a systematic manner.
One of the primary limitations of experimental enclosures is that they eliminate
many of the gain and loss processes that occur in nature, such as advection and
di!usion, zooplankton grazing, and the e!ects of mixing. Several of these factors can
be mitigated through the appropriate choice of enclosure volume (Kuiper et al., 1983).
This same limitation also may be an asset, since it allows for the separation
of `top-downa (ecosystem level) and `bottom-upa (physiological level) controls.
Although these two types of control will still appear in enclosure experiments
(e.g. species succession, a form of top-down control), the di!erent time-scales they
operate on (hours to days for physiological responses, days to weeks for ecosystem
responses) allow one to isolate the various processes.
In the marine environment, and in eastern boundary current upwelling regimes in
particular, it is generally assumed that biomass accumulation (as phytoplankton) is
controlled by the availability of nitrogen (through mediation of N concentrations by
the physical e!ects of upwelling). The experiments presented here were designed to
determine whether nitrogen is the physiologically controlling factor of new production in Monterey Bay, CA. To test this hypothesis, a series of 20-l enclosure experiments were conducted with varying levels of nutrient additions (nitrate, ammonium,
silicate and other nutrients) during four cruises in 1992, encompassing upwelling,
non-upwelling, and winter conditions. By conducting these experiments both on
a short time-scale (4}5 days) and throughout an annual cycle (March}November), we
believe it is possible to discriminate between physiological responses as well as to
broader species and community changes associated with seasonal transitions.
Of particular interest in these experiments was the determination of whether the
phytoplankton assemblage demonstrates a speci"c physiological response to increased nitrate concentrations (in the presence of su$cient light levels) referred to as
`shift-upa (Dugdale and Wilkerson, 1989; Dugdale et al., 1990; MacIsaac et al., 1985;
Smith et al., 1992; Zimmerman et al., 1987). According to this hypothesis, an upwelling
R.M. Kudela, R.C. Dugdale / Deep-Sea Research II 47 (2000) 1023}1053
1025
zone exhibits long-term temporal changes in phytoplankton physiology and community structure as the phytoplankton assemblage is exposed to rapid changes in
ambient light and nutrient "elds. This phenomenon has been hypothesized to result in
measurable increases in the biomass-speci"c nutrient uptake (V, h~1) of the phytoplankton assemblage in response to changes in the physical environment associated
with the downstream transport of an upwelling plume of water. Dugdale et al. (1990)
used this response to formulate a predictive model of new production (sensu Dugdale
and Goering, 1967), which they compared to empirical data from various upwelling
regions to estimate the predicted versus realized new production values (see also
Probyn et al., 1995). According to this model, realized new production values will be
directly dependent on the initial nitrate concentration.
Dugdale and Wilkerson (1989) tested this hypothesis in a more controlled situation
through the use of whole-water barrel experiments (large-volume grow-outs) during
upwelling, and Smith et al. (1992) demonstrated in the laboratory that there is
a molecular basis for the shift-up response. Several authors (e.g. Garside, 1991;
Dickson and Wheeler, 1995), however, have provided arguments for why the shift-up
hypothesis remains invalid for explaining "eld observations. The principal argument
against the use of the shift-up model in "eld work is that the physical environment
associated with upwelling is simply too complex to attribute the observed responses to
a single phenomenon, and that the entire shift-up response may be readily explained
by the relative contribution of detrital matter to the N-speci"c uptake rates as
upwelling occurs. Nutrient-enrichment experiments can help to bridge the gap
between laboratory and "eld experiments, since they allow the use of natural assemblages, but in a more controlled manner than is typically possible with "eld results.
The purpose of these experiments was to move from observational (e.g., the barrel
experiments of Dugdale and Wilkerson, 1989) to manipulative experiments, to
determine whether the observed phytoplankton physiological responses result in
measurable changes in N-speci"c uptake rates as a function of nitrate concentration
(shift-up).
We also examined the e!ects of silicate concentration on the utilization of nitrogenous substrates, since it has been hypothesized that silicate may replace nitrate as
the controlling factor in many ecosystems or in some way modify the utilization of
N by siliceous organisms such as the diatom-dominated communities associated with
upwelling (e.g. Dugdale et al., 1995; Dugdale and Wilkerson, 1998; Minas et al., 1986).
It generally has been assumed that nitrogen limits both new and primary production
in coastal waters (e.g. Carpenter and Capone, 1983). Three lines of evidence support
the possibility of phytoplankton productivity limited by silicate in these areas. First,
diatoms have an obligate requirement for silicic acid to complete cell division (e.g.
Lewin, 1962; Brzezinski et al., 1990; Brzezinski, 1992), and it can be demonstrated
(e.g. Michaels and Silver, 1988) that enhanced levels of productivity and export require
progressively larger size classes of organisms (e.g. diatoms). Therefore, in highly
productive coastal waters such as Monterey Bay, a nutrient that limits diatom
production (e.g. silicate) also will limit total productivity. Second, the half-saturation
values for silicate uptake (K ) are typically many times greater than for nitrate uptake,
4
including for Monterey Bay (e.g. White and Dugdale, 1997; Brzezinski et al., 1997),
1026
R.M. Kudela, R.C. Dugdale / Deep-Sea Research II 47 (2000) 1023}1053
while typical nitrate K values for the same region under similar conditions range
4
from 0.5 to 1.5 (Kudela, 1995) and ammonium K values are even lower
4
(ca. 0.1}0.5 mg at m~3; Kudela, unpublished data). As a result, even though Si : N
ratios in upwelling waters are typically 1 : 1 (Codispoti et al., 1982; Dugdale and
Wilkerson, 1989) or greater (Brzezinski et al., 1997), silicate can become limiting at
much higher ambient concentrations than N. The third line of reasoning is that
silicate regeneration occurs much deeper in the water column than does N regeneration, causing selective exportation of Si relative to N from the euphotic zone, and
thereby lowering the Si : N ratio and the competitiveness of siliceous organisms (e.g.
Dugdale et al., 1995). The net result of these Si : N interactions is that silicate has the
potential to limit new and primary productivity in regions such as Monterey Bay, but
this hypothesis has not been extensively tested.
2. Materials and methods
2.1. Sample collection
Experiments were conducted in Monterey Bay, CA aboard the R/V Point Sur
during three one-week cruises in March, September and November 1992; a fourth
cruise took place aboard the R/V New Horizon during May 1992 (Table 1). Discrete
water samples were collected from 3 to 5 m depth (ca. 50% light penetration depth:
LPD) using acid-cleaned 10-l PVC Niskin bottles equipped with silicone tubing as
internal springs on an instrumented rosette equipped with a 4p light meter (Biospherical Instruments QSP-100). The water was dispensed (randomly from several Niskin
bottles for each container) into acid-cleaned 20-l polyethylene containers (Reliance
Products Ltd.) after rinsing several times. All water dispensing was carried out using
acid-cleaned silicone tubing and polyethylene gloves to minimize potential sources of
contamination. Water samples for initial nutrients and pigments were subsampled
directly from the control container, with the assumption that these samples were
representative of the initial nutrient and pigment concentrations for all of the containers. The enclosures were then spiked with varying levels of isotopically unlabeled
(cold) nutrients, and resampled for initial nutrient concentrations. Water was collected
from the enclosures for the initial nitrogen and carbon uptake experiments, and the
enclosures were transferred to deck incubators. The enclosures were maintained at ca.
50% surface irradiance with neutral density screening and at ambient surface seawater temperatures ($23C) with running seawater.
2.2. Nutrient additions
Three types of additions were conducted: nitrate (as KNO ), ammonium as
3
(NH Cl), and Guillard's medium (Guillard and Ryther, 1962) diluted to 1/20 ( f/20)
4
with no nitrogenous substrates added, as summarized in Table 2. Guillard's medium
was designed for the maintenance of phytoplankton cultures under laboratory conditions, and provides all of the essential macro- and micronutrients, including trace
27/3}2/4/92
23}31/5/92
24}30/9/92
17}23/11/92
March
May
Sept.
Nov.
Dates
37303.55@N
122316.37@W
36346.36@N
122301.07@W
36346.60@N
122300.92@W
36355.37@N
121353.45@W
Latitude,
Longitude
1565
1000
1638
1640
PAR
(lE m~2 s~1)
14.60
13.27
14.21
14.08
Sea surface
Temperature
(3C)
[40]
10
[3]
30
Mixed-layer
septh (m)
&1
4}5
1}2
1}2
Chl a
(mg m~3)
0.99
(2.96;
1.97
(1.88;
5.51
(3.72;
1.54
(2.27;
65)
85)
50)
76)
[Nitrate]
(SD; n)
4.64
(2.69;
8.11
(3.05;
6.49
(3.31;
3.31
(1.66;
64)
85)
50)
73)
[Silicate]
(SD; n)
0.23
(0.12;
0.37
(0.26;
0.25
(0.23;
0.32
(0.19;
62)
85)
50)
74)
[Ammonium]
(SD; n)
Table 1
General characteristics of the Monterey Bay, CA study site for each cruise. Nutrient and pigment data were collected from near surface ()5 m) samples collected
as part of routine mapping during the cruise period. PAR values are averaged for the duration of the cruise for the hours 09:00}15:00 Paci"c Local Time. Mixed
layer depth values in brackets indicate periods when the thermocline was very weak; those cruises were characterized by weakly strati"ed mixed layers
R.M. Kudela, R.C. Dugdale / Deep-Sea Research II 47 (2000) 1023}1053
1027
Table 2
Relevant initial parameters for bioassay nutrient enrichment experiments conducted in Monterey Bay, California. The grey bars indicate those experiments
conducted in collaboration with White and Dugdale (1997). PN values were taken from 15NO uptake experiments conducted during the "rst day of sampling,
3
and have not been corrected for PN accumulation occurring during the time period of the incubation. Values labeled NA were not available
1028
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R.M. Kudela, R.C. Dugdale / Deep-Sea Research II 47 (2000) 1023}1053
1029
metals and vitamins, necessary for phytoplankton growth. In addition to these
primary experiments, during the May and September cruises three additional enclosures (during each cruise) were maintained; two of these were treated with silicate
enrichments (as NaSi(OH) ) as part of a series of experiments conducted in collabora4
tion with other investigators (White and Dugdale, 1997), while the third was maintained as a control. These four types of additions were designed to test the e!ects of
various initial nitrate concentrations, the e!ect of ammonium on nitrate uptake rates,
and whether or not other nutrients such as silicate. All additions were prepared with
reagent-grade stocks and distilled, de-ionized water. Enclosure experiments were
maintained for 4}6 days. For the silicate enrichment experiments, no Chl a or
ammonium data were available. Because of the di$culty of maintaining large shipboard enclosures, no true replicates were conducted; the experimental design relies
instead on analysis of trends occurring during several cruises under similar nutrient
bioassay conditions.
2.3. Tracer uptake experiments
Water was dispensed into 280-ml polycarbonate incubation bottles daily and
maintained brie#y under low light prior to being inoculated with either carbon or
nitrogen isotopes. Uptake rates of NO~ and NH` were measured by adding K15NO
4
3
3
or 15NH Cl (Cambridge Isotope Laboratories; both 99 atom% 15N). Inoculations
4
were intended to be trace additions ()10%; Dugdale and Goering, 1967). Carbon
uptake rates were measured in September and November by adding trace amounts
(ca. 10% of ambient TCO concentrations) of NaH13CO (Cambridge Isotope
2
3
Laboratories; 99 atom% 13C) to the water samples which were inoculated with
K15NO . After inoculation, the bottles were placed back into the deck incubators.
3
Incubations were typically initiated at 09:00 Paci"c local time.
Carbon and nitrogen incubations were terminated after ca. 4}6 h by "ltration
(pressure di!erential(150 mmHg) onto pre-combusted Whatman GF/F "lters, or
onto 5.0 or 1.2 lm silver "lters (Poretics Inc.). The "lters were stored frozen (!203C)
and transported to the laboratory where they were placed in a drying oven ((603C,
'24 h). The samples were then prepared and analyzed for particulate nitrogen (PN)
or particulate organic carbon (POC) and isotopic enrichment on a Europa
Tracermass mass spectrometer. Nitrogen uptake rates were calculated using Eq. (3) in
Dugdale and Wilkerson (1986). Although Legendre and Gosselin (1997) have suggested a modi"cation to this equation (leaving the ambient nutrient concentration out of
the denominator), there is no practical di!erence in the calculated rates when using
tracer additions. Samples for 13C analysis were not treated to remove excess inorganic
carbon, and so may represent a slight overestimate of POC, with a corresponding
underestimate of carbon-speci"c uptake rates. However, a subset of POC samples
during the September cruise were treated with 0.5 ml sulfurous acid to volatilize
inorganic carbon. No signi"cant di!erence was found between treated and untreated
samples (data not shown). Carbon uptake rates were calculated using the same
equation as for nitrogen, modi"ed for carbon by substituting the natural abundance
of 13C (1.112%; PeeDee Belemnite standard) and the ambient TCO concentration in
2
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R.M. Kudela, R.C. Dugdale / Deep-Sea Research II 47 (2000) 1023}1053
seawater (assumed to be 2000 lM). No corrections were applied for isotopic dilution
from remineralization of 14N}NH` during the 15N}NH` incubations. Although this
4
4
potential experimental artifact is likely minimized by the relatively short (ca. 4}6 h)
incubation periods, our ammonium uptake rates should still be considered conservative estimates. Similarly, we were not equipped to determine the relative fraction of
15N incorporation that was subsequently lost as DON (e.g. Bronk and Glibert, 1994;
Bronk et al., 1994; Slawyk and Raimbault, 1995). Therefore, these rates must also be
considered net rather than gross estimates of nitrogen utilization. Samples for isotopic
enrichment from the March experiments were irretrievably contaminated during
processing, making it impossible to characterize the temporal trends associated with
the uptake rates for that cruise. Individual samples have been included in other data
analyses where appropriate. Replicate samples were not normally conducted except
for the November cruise because of water limitations; emphasis instead was placed on
the ability to provide multiple time points over a longer experimental period.
To determine the relative proportion of the size-fractionated uptake, nitrogenuptake parameters were determined by di!erence for the various size fractions
collected ('5 lm, '1.2 lm, 'GF/F). This was accomplished by treating the signal
from each larger size class as `excessa material. Values for the PN concentration and
atom% enrichment were determined for each size class, and the signal from the larger
size class was removed from the next smaller size class (which includes the PN and
atom% enrichment from the larger size class) before calculation of rates. For example,
values for PN and atom% enrichment were determined from the GF/F and 1.2-lm
"lters. Assuming that the GF/F "lter represents a PN and enrichment signal due to
organisms '0.4 lm, the `excessa fraction of the signal was determined from the PN
and enrichment values for the corresponding 1.2-lm "lter. This `excessa signal was
subsequently subtracted for the GF/F "lter, and the new values, equivalent to the PN
and atom% enrichment for the size class (1.2 lm and 'GF/F, were determined as
described above.
2.4. Analytical methods
Samples for automated nutrient analysis were frozen (!203C) for analysis ashore
using a Technicon AutoAnalyzer II for nitrate#nitrite (Wood et al., 1967), ammonium (SoloH rzano, 1969), and silicate (Armstrong et al., 1967) during the "rst two
cruises (March, May). For the remaining cruises (September, November) ammonium
samples were collected in disposable polyethylene centrifuge tubes and were analyzed
fresh with a Hewlett-Packard (HP 8452A) spectrophotometer equipped with a 10-cm
cuvette according to SoloH rzano (1969). Samples not immediately analyzed were
preserved with the phenolic reagent and refrigerated until analysis ((24 h). Samples
for chlorophyll a (Chl a) were "ltered onto uncombusted GF/F or polycarbonate
"lters (1.2 and 5.0 lm; Poretics Inc.) and analyzed for Chl a and phaeopigments by in
vitro #uorometry (Parsons et al., 1984). Samples for phytoplankton analysis were
visually inspected during the cruise to determine broad classes of organisms (e.g.
zooplankton, centric and pennate diatoms, #agellates, etc.).
R.M. Kudela, R.C. Dugdale / Deep-Sea Research II 47 (2000) 1023}1053
1031
2.5. Data analysis
Curve-"tting analyses were performed by plotting the data using a Michaelis}
Menten formulation. Data were initially linearized and plotted using a double
reciprocal method (Hanes-Woolfe; Segel, 1975). Values determined from that analysis
were then entered into an iterative, non-linear curve "tting package (Deltagraph;
Deltapoint Inc.). Statistical signi"cance was determined for the curve using a s2 goodness-of-"t test (Press et al., 1992). In this communication, <
will refer to the
.!9
maximum predicted value of < from the curve "t, while Max < will refer to the
maximal uptake rate (<) observed.
3. Results
3.1. General physical characteristics
Monterey Bay has been characterized as having upwelling, non-upwelling, and
winter periods (e.g. Breaker and Broenkow, 1994), and the four cruises were consistent
with this classi"cation. Nutrient concentrations were low during March and November, with characteristically deep-mixed layers associated with the winter period
(Table 1). General conditions during the beginning of 1992 indicated the in#uence of
an El Nin8 o event, resulting in elevated temperatures and decreased ambient nutrient
concentrations in Monterey Bay (Chavez, 1996). May was characterized by low but
variable nutrients at the time of water collection, with sporadic upwelling occurring
during the duration of the cruise. September exhibited a moderately strong upwelling
event, with high surface nutrient concentrations and elevated biomass relative to the
other sampling periods (Table 1). During all cruises initial biomass concentrations
('5.0 lm Chl a fraction) and phytoplankton samples indicated a prevalence of
diatoms, primarily Chaetocerous and Nitschia spp. Ammonium concentrations were
low but measurable (ca. 0.5 mg at m~3) at the beginning of all of the enclosure
experiments. With the exception of the September experiments, which took place with
recently upwelled waters, there were relatively high concentrations of silicate in the
initially collected waters relative to nitrate concentrations. Typically, silicate : nitrate
(Si : NO ) ratios at the initiation of the enrichment experiments were from (5 to 10) : 1,
3
while the source water ratio was 1 : 1 (ratio taken from below the mixed layer depth).
3.2. Phytoplankton biomass
For all of the enrichment experiments regardless of treatment, the biomass as
determined by Chl a and particulate nitrogen (PN) concentrations increased from
initial values (Fig. 1). Addition of a nitrogen source (as NO~ except for March, when
3
NH` also was added) maintained these increases relative to the controls; maximum
4
biomass attained was dependent on the concentration of nitrogen provided, with
increasing N sources resulting in increased maximal biomass attained. The addition of
Guillard's f/20 medium and no N source initially enhanced biomass accumulation
1032
R.M. Kudela, R.C. Dugdale / Deep-Sea Research II 47 (2000) 1023}1053
Fig. 1. Nutrient and Chl a concentrations are plotted versus time for the primary enclosure experiments
conducted in Monterey Bay, CA during 1992. The rows correspond to: March (top), May (upper middle),
September (lower middle) and November (bottom). Each graph represents a single enclosure experiment;
the type of enclosure is indicated in the upper right corner for each. Plotted are: nitrate (h), silicate (n)
ammonium (L) and Chl a (r) concentrations. Note the di!erent scales used for those enclosures with high
levels of added nutrients. Enclosures with additions of silicate are denoted by the gray background of the
panel.
relative to the other (N addition) treatments, but peaked at lower maximal values of
biomass. Guillard's f/20 added with high concentrations of NO~ initially reduced
3
biomass accumulation, but maximal values were second only to the same concentration of added NO~ with no Guillard's medium. In contrast, the silicate addition
3
experiments demonstrated a decrease in biomass (PN) relative to the control.
The ratios of PN to Chl a concentrations were typically between 0.5 and 3.0
(mg at N : mg Chl a m~3) at the initiation of the bioassay experiments (Table 2). In
those enclosures that had measurable concentrations of nitrogen at the termination of
the experiments, the PN : Chl ratio tended to decrease, while in those containers
which exhausted the ambient N this ratio increased. Healthy phytoplankton populations typically maintain a PN : Chl ratio of about 1 : 1 (e.g. Garside, 1991 and
references therein); this is consistent with our results, where the ratio approached 1 : 1
during periods of apparent logarithmic growth. At a similar study site Dickson and
Wheeler (1995) reported a similar ratio of between 1 and 2 for PN : Chl o! the coast of
R.M. Kudela, R.C. Dugdale / Deep-Sea Research II 47 (2000) 1023}1053
1033
Oregon, and estimated that ca. 80% of the PN was due to autotrophic phytoplankton.
In these experiments, we estimate that phytoplankton N was the dominant source of
PN during May and September for the initial time points (84}100% of PN in May,
70}100% in September,). During March and November, this decreased to 54}85%
and 40}44%, respectively.
3.3. Nutrient dynamics
Maximal biomass concentrations were attained temporally very close to the point
in the experiments where nitrogenous nutrients were reduced to near undetectable
limits (Fig. 1). For those enclosures that never exhausted the ambient N, biomass,
concentrations continued to rise until the end of the experiment, and presumably
would have continued until N depletion occurred. In the enclosure that had Guillard's
medium added with no nitrogenous sources, there was a slight increase in biomass
relative to the control (but note that a slight nitrogen contamination was introduced
as well; see Table 2). In contrast to these results, the addition of silicate to two
enclosures (10 and 50 mg at m~3) in both May and September resulted in a decrease
in biomass accumulation (May) or no signi"cant di!erence (September) between the
control, with no additions, and the silicate enrichments.
All of the enclosure experiments were conducted with water collected from a site
that is typically representative of upwelling waters in Monterey Bay, and is characterized by a Si : NO ratio in the source water of 1 : 1 (White and Dugdale, 1997). The
3
surface waters collected for these experiments started with Si : NO ratios much
3
greater than one (ca. [2}9]:1; see Table 2), except for September (during active
upwelling). Depletion rates of silicate and nitrate indicated that nitrate was depleted
from the enclosures 1.5}3 times faster than silicate (Table 3). This depletion ratio was
not a!ected by additions of nutrients (silicate, nitrate, ammonium or f/20). To test this,
an analysis of covariance (ANCOVA) was performed on the slopes of the individual
regression experiments for each enrichment within each cruise. Although one of the
experimental enclosures (#10 mg at m~3 Si) provided depletion ratios that indicated
enhanced silicate uptake (N : Si(1) with the addition of silicate, no signi"cant
di!erence (P'0.05) among the treatments was found, including those enclosures
from the same cruise to which more silicate was added (ca. 50 mg at m~3 Si).
3.4. Nitrogen uptake rates
Numerous investigators (e.g. Garside, 1991; Dickson and Wheeler, 1995; Legendre
and Gosselin, 1997) have suggested normalizing biomass-speci"c uptake rates to Chl
a concentrations rather than to PN to avoid problems associated with detrital
nitrogen, particularly in freshly upwelled waters where a large fraction of the total PN
may be detrital matter swept up from the sediments. Functionally, this involves
calculating the volume-speci"c uptake rate for the substrate, o (mg at m~3 h~1):
o "< zPN,
x
x
(1)
1034
R.M. Kudela, R.C. Dugdale / Deep-Sea Research II 47 (2000) 1023}1053
Table 3
Nitrogen (nitrate#ammonium):silicate depletion ratios for enclosure experiments conducted in Monterey
Bay, CA Ratios were determined from linear regression analysis of the available data which demonstrated
a linear trend; data were discarded when non-linearity was observed (i.e. at undetectable levels of nutrients
or if ambient concentrations of nutrients were not decreasing with time). The Standard Error column
represents the standard error of the slope of the regression (used to determine the depletion ratio). The grey
bars indicate those enclosures to which additional silicate was added, as either silicate or in the f/20
medium.
where o is the volume-speci"c uptake rate for some substrate, x, < is the associated
x
x
biomass-speci"c uptake rate (h~1), and PN is the particulate nitrogen concentration
(mass/volume) for that sample (see Dugdale and Wilkerson, 1986; Legendre and
Gosselin, 1997). Given this value, the chlorophyll-speci"c uptake rate, <C)x
(mg at N mg Chl~1 h~1), is calculated as
(2)
<C)-"ox/Chl,
x
where o is the same as described above and Chl is the associated Chl a concentration
x
with units of mass/volume. Following the conventions of Dickson and Wheeler
(1995), notation for uptake rates will di!erentiate between PN-speci"c (<PN; h~1),
Chl a-speci"c (<C)-; mg at N mg Chl~1 h~1) and absolute, or volume transport
(o; mg at N m~3 h~1) uptake rates. References to < imply either Chl a speci"c or PN
speci"c rates. Figs. 2 and 3 provide plots of these variables for each enclosure
R.M. Kudela, R.C. Dugdale / Deep-Sea Research II 47 (2000) 1023}1053
1035
Fig. 2. Nitrate uptake rates are plotted versus time for enclosure experiments conducted in Monterey Bay,
CA during 1992. Each row represents a single cruise, corresponding to May, September and November as
indicated. The "rst column represents the biomass-speci"c uptake rate, the second column the Chl
a-speci"c uptake rate, and the third column the absolute transport rate. Similar plots for silicate experiments conducted in collaboration with White and Dugdale (1997) may be found in their Fig. 8, and are not
presented here. Symbols represent di!erent treatments for each panel, as summarized in Table 2, progressing from control (G) and f/20 with no N addition (#), through the varying levels of nitrate or silicate
enrichment. The enrichments are denoted by low ( ) medium ( ) and high (m) nitrate or silicate
enrichments, both by the symbol style and gray density (highest is black).
experiment for nitrate and ammonium, respectively. Speci"c nitrate uptake
rates were similar for all treatments with added N. These nitrogen additions
generally elevated the speci"c nitrate uptake rates relative to the control
(but see below), although the maximal < 3 observed was frequently associated with
NO
1036
R.M. Kudela, R.C. Dugdale / Deep-Sea Research II 47 (2000) 1023}1053
Fig. 3. Ammonium uptake rates are plotted versus time for enclosure experiments conducted in Monterey
Bay, CA during 1992. Each row represents a single cruise, corresponding to May, September and
November as indicated. The "rst column depicts the biomass-speci"c uptake rate, the second column the
Chl a-speci"c uptake rate, and the third column the absolute transport rate. Within each panel, the symbols
represent control (h), f/20 with no N addition (#), and then varying levels of nitrogen addition, with the
shade of grey indicating lowest ( ) to highest (m) nutrient additions. No ammonium rates were available for
the silicate enrichment experiments.
a moderate nitrate addition rather than with maximal additions. The silicate
enrichments, which did not intentionally provide an additional source of nitrogen,
also demonstrated an enhancement in the maximal < 3 observed. These results
NO
were quite variable, with the maxima being associated with the largest silicate
addition in May, while the September maxima was associated with a moderate
silicate addition.
During the May enclosure experiments, < 3 peaked on the second day of the
NO
experiment in all but one of the enclosures. The September enclosures were characterized by steadily increasing < 3 values, regardless of the treatment, reaching maxima
NO
R.M. Kudela, R.C. Dugdale / Deep-Sea Research II 47 (2000) 1023}1053
1037
Fig. 4. Biomass speci"c nitrate uptake rates normalized to either PN (x-axis) or Chl a (y-axis) from
enclosure experiments conducted in Monterey Bay, CA during 1992 are plotted. The solid line indicates
a 1 : 1 correspondence. Values plotted are from March (r), May (L), September (j), and November (j).
Error bars for the September and November experiments indicate one standard deviation.
between days 2}4. The nitrate and ammonium uptake rates observed in September
were similar for all treatments. Those enclosures with additions of silicate (as either
f/20 or silicate) achieved the highest speci"c nitrate uptake rates, which declined with
the depletion of nitrogenous substrates. In contrast, the enclosure which had both f/20
and proportional amounts of nitrate added peaked early (day 2) and then steadily
declined. The November enclosures with the addition of nitrate again exhibited an
enhancement in <PN 3 relative to the control. Normalization of nitrate uptake to
NO
Chl a versus PN provided essentially the same results (Fig. 4) for the upwelling periods
May and September. During March and November, however, it is apparent that
<PN 3 values were consistently lower than <C)-3 . Volume-speci"c uptake rates (o 3 ),
NO
NO
NO
were generally similar to the < values except for November, where there was a major
discrepancy between these three rate measurements. From both the o 3 and the
NO
<PN 3 data (Fig. 2), it would appear that the enclosure enriched with nitrate exhibited
NO
much higher rates of nitrate utilization relative to the control. When the <C)-3 data are
NO
used, however, there is no apparent di!erence between the enriched and control
enclosures. This appears to be a classic example of a detritally dominated quasi-shiftup pattern.
Ammonium biomass-speci"c uptake rates (<PN 4 ; h~1) typically increased (Fig. 3).
NH
The exception was the September experiment, which demonstrated a decrease in
<PN 4 for some enclosures, and an increase for others. However, the Chl a-speci"c
NH
uptake rates consistently increased from days 1 to 2 for all enrichments. The rates for
1038
R.M. Kudela, R.C. Dugdale / Deep-Sea Research II 47 (2000) 1023}1053
the two enrichments to which large amounts of nitrate (18 and 36 mg at m~3,
respectively), was added proceed to decrease with time, while the remainder of the
enclosures continued to demonstrate an increase in ammonium uptake rates. This is
somewhat surprising, since it is generally assumed that ammonium is the preferred
substrate (relative to nitrate), while these data indicate a suppression of ammonium
uptake rates with the addition of high concentrations of nitrate (see also Dortch,
1990).
3.5. Carbon uptake rates
The September and November enclosure experiments, for which there are both
carbon and nitrogen uptake rates, were conducted during two very di!erent time
periods in Monterey Bay; September's experiments occurred during an active
upwelling event, while November's experiments were characteristic of the deepmixed layer, low-light, low-nutrient winter period. All of the enclosures demonstrated similar trends in N and C, with increasing uptake rates and increasing
biomass accumulation as a function of time (Fig. 5). It is apparent that none of the
phytoplankton assemblages were in steady-state growth (Shuter, 1979); uptake rates
were nitrogen-biased, with an assimilation ratio of ca. 4.5 : 1 rather than the
expected elemental composition of 6.6 : 1 C : N (Red"eld et al., 1963). Although not
entirely unexpected (a perturbation experiment is clearly not a steady-state
system), the positive nitrogen bias (which would increase if N uptake rates
were underestimated due to isotope dilution or DO15N loss) suggests preferential
uptake of N relative to C. The elemental composition of the phytoplankton
was also unexpected, with initial values greater than 60 : 1 (C : N by atoms). The
C : N ratio decreased with time in the enclosures, to a "nal value of (7}25) : 1 (C : N)
depending on the treatment (Fig. 5), consistent with the preferential uptake of
N. Those enclosures enriched with high concentrations of N achieved the
lowest "nal C : N ratio. In September, the C : N ratio initially increased (time 0,
relative to the control) with the addition of excess N; the opposite trend was seen in
November.
3.6. f-Ratios
Ammonium uptake rates generally increased slightly with time in all of the enclosures, while nitrate uptake rates were more dynamic, with both increases and decreases
as a function of time of sampling, time of year and treatment. Previous studies
(Kudela, 1995; Olivieri, 1996) have demonstrated that Monterey Bay typically exhibits
f-ratios that are characteristic of upwelling regimes (e.g. f-ratio'0.5; Eppley and
Peterson, 1979). The results from the enclosure experiments indicate that nitrate
provided the greatest proportion of nitrogen to the phytoplankton assemblage. f-ratio
values ranged between 0.35}0.80, with a mean value of 0.58$0.11, where the lowest
f-ratios were associated with no addition of N. Although the addition of nitrate
typically increased the f-ratio values, the relatively high values for the
R.M. Kudela, R.C. Dugdale / Deep-Sea Research II 47 (2000) 1023}1053
1039
Fig. 5. Carbon and nitrogen (NO~#NH`) uptake rates for enclosures conducted in September and
3
4
November of 1992 in Monterey Bay California are plotted. A. Absolute uptake rates of carbon versus
nitrogen; the solid line indicates the linear regression analysis (R2"0.77) for the data. B. C : N composition
of the samples plotted in panel A are plotted versus elapsed time since initiation of the enclosure. Symbols
for both panels indicate: September (open symbols) control (j), #f/20 (.), #8 mg at m~3 NO~(n),
3
#18 mg at m~3 NO~ (L), #36 mg at m~3 NO~ (e), and November (closed symbols) control (B),
3
3
#8 mg at m~3 NO~ (v). Values plotted at time zero were determined at the end of the initial short-term
3
uptake experiment, and so do not represent the true C : N ratio before any nutrient manipulations occurred.
1040
R.M. Kudela, R.C. Dugdale / Deep-Sea Research II 47 (2000) 1023}1053
control enclosures dampened this response; f-ratio values remained similarly high in
all enclosures.
3.7. Shift-up characterization
Several parameters must be known or estimated before the shift-up model may be
applied (Dugdale and Wilkerson, 1989; Dugdale et al., 1990; Zimmerman et al., 1987).
Using the model described by Zimmerman et al. (1987), the phytoplankton assemblage will approach a maximal speci"c nitrate uptake rate (Max <; h~1) as a function
of the initial nutrient conditions. This change in the measured < values as a function
of time is referred to as acceleration, A, with units of h~2:
d<
A" NO3 .
dt
(3)
This term was hypothesized to be dependent on the initial nutrient concentration, and
ranged (empirically determined) from 0.001 to 0.1 h~1 for initial and "nal values in an
upwelling plume. The value for A was empirically predicted to be
A"(4]10~5)zN#(4]10~5),
(4)
where N is equal to the initial nitrate concentration. The results from these enclosure
experiments agree well with the proposed values for <; our data provide a minimum
value of 0.001 and a maximum value of 0.057 h~1, associated with ambient nitrate
concentrations (at time zero) of 0.83 and 12.5 mg at m~3 NO~, respectively. The
3
results of these experiments indicate that both Max < and A increase as a function of
nitrate concentrations, as predicted. However, these data demonstrate that the change
in A with initial nutrient concentration follows a hyperbolic function, as does
Max < (Fig. 6) rather than the previously reported linear increase in A as a function
of N concentration. These data also demonstrate that the values of both Max <
and A reach a maximum at approximately the same nutrient concentration (ca.
10}15 mg at m~3 NO~). These data are summarized in Table 4.
3
An estimate of the uptake versus concentration values for nitrate also was determined by plotting <PN 3 values versus the ambient nitrate concentration (using those
NO
enclosures which were not enriched with nitrogen). The results of such a plot are
similar to a nutrient kinetics plot, except that a natural assemblage is used (including
multiple phytoplankton spp. as well as zooplankton, bacteria, detritus, etc.); this type
of plot has been utilized to determine nutrient kinetics parameters in previous studies
(e.g. Dickson and Wheeler, 1995; Probyn et al., 1995). As discussed by Garside (1991)
and Dickson and Wheeler (1995), if the shift-up response were to be observed in "eld
studies, it would be expected that low values of < would be measured at both low and
high-nitrate concentrations (Fig. 7). The low values associated with low-nutrient
concentrations are an e!ect of the kinetics of nutrient transport, while the low values
at high-nutrient concentrations are predicted to be a result of the phytoplankton
R.M. Kudela, R.C. Dugdale / Deep-Sea Research II 47 (2000) 1023}1053
1041
Fig. 6. Results from enclosure experiments conducted in Monterey Bay, CA, demonstrating the relationship between initial nitrate concentration and speci"c nitrate uptake rates. A. Maximum < observed
(Max <) as a function of initial nitrate concentration. B. Maximum acceleration (A) observed as a function
of initial nitrate concentration. Symbols represent: '5.0 mg at m~3 (A), 1.2}5.0 mg at m~3 (L) and
(1.2 mg at m~3 (v) fractions. The solid line represents the best "t for the the data (calculated using the
unfractionated GF/F uptake rates) as described in the main text.
assemblage not being `shifted-upa yet (as high-nutrient, freshly upwelled water and
the associated phytoplankton assemblage is upwelled into the stable, well lit surface
waters). Calculated K values for these data provide estimates of 1.22 or
4
4.14 mg at m~3 NO~ for these data, where the latter value is determined by excluding
3
from the calculation those points (from September) where low values of < were
associated with high ambient NO~ concentrations. Although these data (excluding
3
the low < high NO~ points) do not demonstrate strong statistical signi"cance
3
(P'0.05 using a s2 goodness-of-"t test), the resulting trends are as predicted for the
shift-up model and as discussed by Dickson and Wheeler (1995). Identical results are
obtained if <C)-3 values are substituted.
NO
1042
R.M. Kudela, R.C. Dugdale / Deep-Sea Research II 47 (2000) 1023}1053
Table 4
Kinetics parameters for nitrate uptake rates. All parameters were determined for mixed assemblages of
plankton and were determined using ambient concentrations of nitrate. The "rst 4 rows provide data from
this study for Monterey Bay, CA; two representative data sets from other areas are also provided. The
Parameter column provides the type of data used for each calculation; see the main text for more details
Location
Parameter
Ks
(mg at m~3)
< ;A *
.!9 .!9
(]10~3 h~1;
]10~3 h~2)
R2(n)
Authors
Monterey Bay
<PN (NO )
3
<PN (NO )
3
Max <PN (NO )
3
Max A (NO )
3
<C)- (NO )
3
1.22
4.14
2.51
2.59
48.84
98.85
55.00
1.09*
0.65
0.55
0.73
0.65
This
This
This
This
1.26
26.04
NA (10)
Dickson and
Wheeler (1995)
<PN (NO )
3
2.02
42.8
0.80 (27)
Probyn et al.
(1995)
Oregon Coast
Eastern Agulhas
Bank (Africa)
(24)
(22)
(10)
(8)
study
study
study
study
Fig. 7. Values for the PN-speci"c uptake rate of nitrate versus the nitrate concentration are plotted for
enclosure experiments to which no nitrogenous sources had been added for Monterey Bay, California. The
solid line represents a best "t using the Michaelis}Menten formulation, with the (m) symbols omitted; the
dashed line represents the same "t with both the (m) and (v) omitted. The open symbols provide the
Chl-speci"c uptake rates for comparison. Note that the symbols do not denote cruise date or treatment, as
in the other plots, but are representative of which data were used in the curve "t procedure. See the main
text for more details.
R.M. Kudela, R.C. Dugdale / Deep-Sea Research II 47 (2000) 1023}1053
1043
4. Discussion
Microcosm experiments are deceptively simple in design; ideally, the nutrient
concentrations are manipulated while all other relevant factors are maintained,
allowing for the discrimination of controlling factors (of growth and nutrient utilization) in natural assemblages of phytoplankton. Despite this simple design, numerous
pitfalls remain. Microcosm experiments are most often criticized because of the
potential `bottle e!ecta, which encompasses e!ects due to a large increase in population with no changes in the underlying physiological processes (e.g. Banse, 1990;
Dugdale and Wilkerson, 1990), the exclusion of the rare larger zooplankton species
(e.g. Banse, 1992; Frost, 1991; Welschmeyer et al., 1991), the e!ects of characterizing
mixed assemblages of organisms potentially dominated by detritus and bacterioplankton (e.g. Dickson and Wheeler, 1995; Fuhrman et al., 1989; Garside, 1991; Hobson
et al., 1973; Malone et al., 1993), the elimination of other important physical e!ects
such as advection, di!usion, and light limitation, the e!ects of species succession
during the incubation (e.g. Chavez et al., 1991; DiTullio et al., 1993; Martin et al.,
1989), and the elimination of other biogeochemically controlling processes such as
nitrogen "xation (e.g. Smith, 1991).
Despite these problems, nutrient-addition bioassay experiments are frequently
employed in oceanography because they allow a degree of control over physical and
biological parameters that are not readily manipulated in true "eld experiments, but
still provide a more realistic evaluation of natural plankton assemblages than laboratory culture work. Numerous investigators have found that microcosm enclosures are
reasonably accurate at tracking the events occurring in natural "eld assemblages (e.g.
Kuiper et al., 1983; Maranon et al., 1995; Oviatt et al., 1995; Pitcher et al., 1993;
Wilkerson and Dugdale, 1987). In the experiments presented here, it was not necessary
to mimic exactly the conditions occurring in Monterey Bay, since the goal was to
manipulate the underlying physiological processes associated with nutrient availability rather than to reproduce the ambient conditions under which the water was
collected. Nevertheless, the use of 20-l enclosures (ca. 70]larger than the bottles used
for the short-term uptake experiments) and relatively short incubations (days rather
than weeks) have hopefully minimized adverse e!ects due to containment. The
changes and di!erences observed unfortunately remain a mixture of the desired
physiological response as well as ecological in#uences and within-species adaptations
caused by changes in species composition (e.g. Fernandez et al., 1992; Geider and
La Roche, 1994; Rivkin, 1985). We have attempted to minimize this by evaluating
nutrient bioassay enrichments during several seasonal periods in Monterey Bay; this
provides a natural range of species composition and ecological conditions against
which the speci"c nutrient additions may be evaluated.
Nitrogen recycling due to rapid remineralization (regeneration) rates of 14N}NH`
4
and the resulting isotopic dilution, and/or the loss of 15N label to the DON pool
during long (ca. 24 h) incubations can result in a signi"cant underestimation of
measured uptake rates (e.g. Harrison and Harris, 1986; Bronk and Glibert, 1994). The
error associated with isotopic dilution during short (&8 h) incubation periods for
coastal planktonic assemblages is generally less severe (ca. a factor of 1.3}1.6;
1044
R.M. Kudela, R.C. Dugdale / Deep-Sea Research II 47 (2000) 1023}1053
Harrison and Harris, 1986; Kanda et al., 1987). In Monterey Bay, our use of short,
daytime incubations likely minimized the isotopic dilution e!ects. However, without
concurrent measurements of NH` remineralization, it is prudent to consider the
4
reported rates as conservative estimates. Similarly, we have insu$cient data to assess
adequately the role of DO15N release in these experiments. We can approximate
a mass balance for NO~ disappearance (using values from days 0 and 1) and
3
uptake by using a conversion factor for the initial o 3 estimates to estimate a daily
NO
uptake rate. Nitrate uptake rates could account for between ca. 50 and 100% of the
disappearance (March, 100%; May, 85%; September, 52%; November, 50%), consistent with what other investigators have reported for coastal waters (e.g. Bronk and
Glibert, 1994; Bronk et al., 1994; Slawyk and Raimbault, 1995). This is, of course, only
a crude estimate of the potential loss of DO15N, and is subject to numerous errors (e.g.
the use of a conversion factor, errors in the analyses, etc.). Therefore, following the
recommendations of Bronk et al. (1994), these reported values should be considered
net uptake rates, and are the lower boundary for the true (or gross) uptake rates.
Under steady-state conditions, uptake rates of the elements C, N and Si (for
diatoms) must remain more or less at constant proportions (Shuter, 1979), if the
elemental composition of the phytoplankton is constant (e.g. Red"eld, 1963). Conversely, under sub-optimal conditions, algae have been shown to vary their cellular
composition dramatically (e.g. Goldman et al., 1979; Sakshaug and Holm-Hansen,
1977). These changes in the cellular composition of C : N ratios in particular have
been used as bio-indicators of the physiological status of the phytoplankton assemblage (e.g. Goldman, 1980,1986; Goldman et al., 1979; Laws et al., 1989), although the
interpretation of these ratios may be hindered by many of the methodological
problems associated with the `bottle e!ecta problem, as well as other physiological
processes (see Geider and La Roche, 1994) when applied to "eld data.
The results for Monterey Bay clearly demonstrate that the assemblages assayed
during the September and November cruises were not in balanced growth, as the high
proportion of N}C utilization and the high initial C : N compositional ratio both
point to N and/or light limitation of this assemblage (note that any underestimation of
N uptake rates would further exaggerate these trends). All enclosures demonstrated
a decrease in biomass C : N ratios with time, with all of the September enclosures and
the N-enriched November enclosure near Red"eld proportions at the termination of
the experiments. In contrast, the November control was still substantially above
Red"eld proportions after 3 days. The initial elevation of biomass C : N ratios in
September with the addition of N (Fig. 5, day 0), in conjunction with N concentrations
substantially in excess of typical N K values (Table 1), suggests that stabilization in
4
the enclosures caused an immediate increase in carbon "xation, which we attribute to
release from light limitation. It is unclear what role N availability played;
co-limitation of light and N on the carbon assimilatory pathways or the temporary
suppression of N uptake (in favor of C) during the "rst day would both explain these
results. No increase in assimilation ratios was seen during day 0, however, which leads
us to believe that the phytoplankton assemblages either increased biomass-speci"c
C assimilation or decreased C loss in response to the stable light regime and the
availability of excess N. Similar changes in C : N composition and assimilation were
R.M. Kudela, R.C. Dugdale / Deep-Sea Research II 47 (2000) 1023}1053
1045
observed along a drifter track during an upwelling event in Monterey Bay (Kudela
et al., 1997).
Other explanations for the observed trends in assimilation and composition ratios,
such as the presence of a large amount of organic detrital (or bacterial) carbon at the
initiation of the experiments, or the competition for inorganic N by a bacterial
assemblage, can not be entirely ruled out, but do not adequately explain the data. The
presence of a large pool of organic, carbon-rich detritus can not explain the low
(4.5 : 1) C : N assimilation ratios observed during the time course of the experiments,
and would be diluted out with the accumulation of phytoplankton biomass. Although
the observed decrease in C : N composition is supportive of a dilution in carbon
detritus, we would expect to see a corresponding increase in C : N assimilation ratio
with time, which was not the case. Similarly, the presence of a competing population
of heterotrophic bacteria is not supported by the results, since this putative population should also be diluted out as phytoplankton biomass increased. Additionally,
heterotrophic bacteria in coastal waters typically obtain 90}100% of their N from
dissolved free amino acids (Billen and Fontigny, 1987; Kirchman, 1994), and are
generally out competed by autotrophs at elevated substrate concentrations (Suttle
et al., 1990). Finally, note that the C : N composition of the biomass decreases within
the 6 h of the initial incubation as a function of N concentration during November
(Fig. 5). This rapid decrease suggests luxury or surge uptake during the initial
experimental period. The slower but steady decrease in the C : N composition of the
control enclosures suggests that light limitation was also a factor, since the enclosures
e!ectively minimized changes in the ambient light "eld (e.g. due to mixing). It is clear
that the change in C : N composition was not solely driven by availability of light,
however, as demonstrated by the di!erential response associated with the increased
N concentrations in both the short (6 h) and long (days) term. We conclude that
although there may have been some detrital organic C contamination of the samples,
the phytoplankton assemblages exhibited both light and nitrogen limited during these
experiments.
Examination of the N : Si depletion ratios (Table 3) again demonstrates that
nitrogen was in high demand. Even in the enclosures enriched with elevated concentrations of silicate, the ratio remained disproportionately weighted towards nitrogen
assimilation. This could be explained in part by di!erences in species composition,
since only diatoms (and silico#agellates) have an absolute requirement for silicic acid
(Lewin, 1962). However, the observed increase in biomass was consistently due to the
'5 lm fraction of the phytoplankton assemblage, which was composed predominantly of diatoms. Assuming that diatoms composed roughly 50% of the biomass by
weight at the beginning of the enclosure (based on size-fractionated chlorophyll), then
it would be expected that N : Si depletion ratios would start at around 2 : 1, assuming
that nitrate is the only source of nitrogen and that all cells are growing optimally, and
at approximately the same growth rate. As the enclosures age and the diatom fraction
becomes increasingly dominant, the N : Si depletion ratio should approach 1 : 1, if
balanced growth is observed. In contrast, the ratios observed were consistently
between (1.5 and 3) : 1, regardless of sampling time. Diatoms may attain higher
speci"c growth and uptake rates and greater biomass than other species of algae (see
1046
R.M. Kudela, R.C. Dugdale / Deep-Sea Research II 47 (2000) 1023}1053
review by Chisholm, 1992), which also would suggest that the N : Si depletion ratio
should approach 1 : 1, especially in enclosures to which large amounts of silicate were
added, as the diatom community should quickly out-compete the smaller phytoplankton species if N were not limiting. The consistently higher depletion ratio again
demonstrates that N depletion was ultimately controlling the accumulation of
biomass (rather than the availability of silicic acid).
Although this type of analysis can identify the limiting nutrient with respect to the
system (or in this case to each enclosure), it does not necessarily represent the limiting
nutrient at the cellular level (see Dugdale et al., 1995 for a further discussion). The
enhanced speci"c nitrate uptake rates observed in the May enclosures to which
silicate was added, and in the September enclosure to which 10 mg at m~3 Si(OH)
4
was added, suggest that for a subset of the phytoplankton assemblage (i.e. diatoms)
the limiting nutrient in fact may be silicate. However, calculation of the acceleration
values (A) for those experiments with additional nutrients indicates no statistically
signi"cant di!erence between treatments (P'0.05; ANCOVA). Although the maximal value of < observed in the silicate addition enclosures (May) increased, the
associated acceleration rates were not signi"cantly di!erent. Similarly, the enclosures
to which silicate was added demonstrated a rapid decrease in their < 3 rates as
NO
nitrate approached zero (Fig. 8), although enclosures with N added showed no similar
decrease as silicate concentrations approached zero. We must conclude from these
results that nitrogen remains the limiting nutrient (in the sense of Liebig) to the
phytoplankton assemblage, and that silicate availability acts as a controlling factor
(using the terminology of Thingstad and Sakshaug, 1990); in this role, silicate serves to
modify the limiting factor (nitrogen) without actually demonstrating true limitation of
the phytoplankton regime. As such, the concentration of silicate may adjust the rate at
which N limitation occurs, but does not directly control the accumulation of biomass.
Since the introduction of the shift-up model (MacIsaac et al., 1985) several authors
have provided persuasive arguments for why this phenomenon does not exist or
cannot be measured in "eld populations. Garside (1991) has provided an alternative
model, which invokes changes in the ratio of `detritala (including true detritus as well
as heterotrophic organisms) and autotrophic PN as an upwelling plume (or enclosure
experiment) ages. According to this hypothesis, the acceleration in speci"c uptake
rates is a result of the increasing dominance of autotrophic biomass with time. It is
assumed that the autotrophic biomass increases logarithmically (proportional to
phytoplankton growth and accumulation) while the detrital PN does not increase at
the same rate. As a result, it appears that the speci"c uptake rates (which are
normalized to PN) change with time. More recently, Dickson and Wheeler (1995)
have suggested that speci"c uptake rates should be normalized to Chl a rather than
PN; this was reiterated by Legendre and Gosselin (1997) from the standpoint of the
mathematical assumptions inherent in the uptake calculations. Although Legendre
and Gosselin recommend not using < values, it is unavoidable if we are to examine the
physiological component of uptake. We concur with these authors that normalization
to a phytoplankton-speci"c product such as chlorophyll, as is commonly done for
carbon, is preferable to using uncorrected <PN values, and we have analyzed our
results using both methods.
R.M. Kudela, R.C. Dugdale / Deep-Sea Research II 47 (2000) 1023}1053
1047
Fig. 8. PN-speci"c NO~ uptake rates for enclosures with silicate additions, conducted in Monterey
3
Bay, CA. A. May control (h) and f/20 enclosure (v); B. May control (h) #10 mg at m~3 Si (J) and
#50 mg at m~3 Si (H) enclosures; C. September control (G) #10 mg at m~3 Si (v) and #50 mg at m~3
Si (m) enclosures. The solid line in each panel represents the approximate time when nitrate concentrations
approached undetectable levels. To the right of each panel the "nal silicate concentration for each enclosure
is given.
It is clear that artifacts due to changes in the relative proportion of detritus may
play a role during the non-upwelling periods (March, November) when the Chl : PN
ratio suggests that 40}50% of the initial PN was detrital. During May and September,
the trends reported for these data are equally apparent when values of <PN 3 , <C)- or
NO
NO
o 3 are used. Although <PN 3 values may be diluted by detritus, the values Êfor
NO
NO
o 3 are not (Eq. (1); also see discussion by Legendre and Gosselin, 1997). Similarly,
NO
1048
R.M. Kudela, R.C. Dugdale / Deep-Sea Research II 47 (2000) 1023}1053
values normalized to Chl a concentrations are independent of detrital nitrogen
content. Results from the November enclosures provide an example of the di$culties
in isolating the cause of the apparent shift-up response. When rates are normalized to
chlorophyll, it is clear that there is no di!erence in speci"c uptake between the control
and the enriched enclosures, despite the massive increase in biomass, and increase in
non-normalized rates. Due to the likelihood of N and light limitation during September and November, it is probable that available nitrogen was channelled to pigment
products. Nutrient (nitrogen) stress has been shown to dramatically reduce cell
chlorophyll concentrations over a period of days, while relief from this stress causes
a similar (days) recovery (e.g. La Roche et al., 1993). This, together with the elevated
detrital N concentrations, would result in a noticeable di!erence between <PN and
NOÊ
<C)-3 in enclosure experiments conducted during periods of low ambient nitrate
NO
concentrations and deep mixing (November and March, Fig. 4), and would cause
a quasi-shift-up response in the assemblage if not normalized to chlorophyll. Trends
in the <C)-3 values also may be related to changes in cell chlorophyll concentrations,
NO
which would cause an arti"cial dampening of any changes in < when normalized to
chlorophyll. Clearly, without additional information it is not possible to attribute the
observed trends to any speci"c physiological response.
A "nal possibility that should be explored with respect to alternative explanations
for these data is that the apparent shift-up response observed in May and September is
due to a change in species composition, with corresponding changes in algal physiology. For these experiments, diatoms consistently accounted for '50% of the initial
biomass. In those enclosures that demonstrated increased biomass, the '5 lm
fraction (con"rmed by microscopy to be diatoms) consistently increased at the
greatest rate. However, the patterns of observed changes in < and acceleration were
independent of the size of the "lter used (Fig. 6), which demonstrates that all size
classes were exhibiting an up-regulation of nitrate assimilation. Thingstad and Sakshaug (1990) have presented a model based on analysis of steady-state systems using
simple Lotka}Volterra predator}prey interactions. These authors demonstrated that
it is possible for a subset of the community (e.g. picoplankton) to be growing at near
maximal growth rates, while larger organisms (e.g. diatoms) do not appear in the
system until su$cient excess nutrients have been introduced to stimulate growth; at
that point, the larger organisms will rapidly accumulate due to their higher potential
growth rates. Although upwelling zones are not what is typically considered to be
steady-state conditions, a two-layer system such as this may explain why the shift-up
hypothesis accurately describes cellular processes in monocultures during simulated
upwelling (Smith et al., 1992), but is di$cult to measure in the "eld. Assuming the
shift-up response occurs in all size-classes of phytoplankton (as indicated from our
results), this phenomenon may be blurred due to the rapid increase of diatoms, which
were presumably at a much lower physiological state initially, and hence were capable
of a more obvious shift-up phenomenon. At the same time, the rapid accumulation of
diatom biomass would tend to confuse the results by providing an apparent species
succession as well as increases in both Chl a and PN. It is important to distinguish,
however, the artifactual nature of the shift-up response when detrital N is dominant,
as occurred in November and March.
R.M. Kudela, R.C. Dugdale / Deep-Sea Research II 47 (2000) 1023}1053
1049
Previous investigators have suggested that the relationship between maximum
values of < and ambient nitrate concentrations is a linear function (Dugdale and
Wilkerson, 1991). The intercept, at ca. 6 mg at m~3, was hypothesized to represent
a threshold below which a phytoplankton bloom will not develop. Other authors
(Dickson and Wheeler, 1995) have found a hyperbolic relationship, as was demonstrated for these data. These authors suggested that the `critical nitrate pointa may
in fact be the concentration at which maximal uptake rates are observed (equal to the
<
value for the curve), from 10 to 15 mg at m~3. Based on the results presented
.!9
here, we would suggest that the critical value probably lies somewhere between the
K and <
values (for nitrate), or between ca. 2}4 and 10}15 mg at m~3 NO~. By
3
4
.!9
de"nition, the K value implies that the utilization of the substrate in question is only
4
occurring at 50% e$ciency. The maximal uptake rates, acceleration, and observed
uptake rates plotted as a function of nitrate concentration all demonstrate
a Michaelis}Menten-type relationship and provide a half-maximum value of ca.
2}4 mg at m~3 NO~. This would seem to indicate that below this threshold, nitrate
3
utilization will quickly decline; accompanying this we would expect to see a transition
from large, bloom-producing diatoms to smaller, regeneration-based phytoplankton
systems. Under certain conditions, such as the vigorous upwelling encountered in
May and September, the shift-up hypothesis would appear to be the most consistent
explanation of both the gross ecological and underlying physiological changes observed in these experiments, and is preferable to the instantaneous adaptation to
ambient upwelling conditions previously described for the coast of Oregon (Dickson
and Wheeler, 1995).
We conclude that this eastern boundary current regime is regulating (in the Liebig
sense) on nitrogen availability during most of the year, although deep mixing and low
light are likely as important during the low-productivity winter months. Following
the conventions of Thingstad and Sakshaug (1990), silicate must be relegated to
a controlling, but not limiting factor, despite the prevalence of diatoms in the
phytoplankton assemblage. Nitrate provides the majority of the nitrogenous nutrition
during most of the year, supporting a predominantly large, diatom dominated
community. Both the C : N composition and utilization ratios point towards nitrogen
limitation. Not surprisingly, both the shift-up model and the detrital hypothesis do
well at explaining the observed results at varying times of the year. We conclude that
there is no a priori reason to discount the shift-up model, but that it can adequately
explain the observed results only during phytoplankton dominated periods such as
occur during vigorous upwelling.
Acknowledgements
We thank Klane White for his participation in the design and analysis of the silicate
experiments as well as the other members of the Shift-Up program, and both the
crew and our co-workers aboard the R/V Pt Sur and New Horizon. Helpful
discussions and critical reviews were provided by members of RMK's dissertation
committee, Jed Fuhrman, Doug Hammond and Burt Jones, and two anonymous
1050
R.M. Kudela, R.C. Dugdale / Deep-Sea Research II 47 (2000) 1023}1053
reviewers. Funding for the data collection and initial analysis was provided by NASA
Global Change Research Fellowship 1694-GC92-0052 (RMK) and National Science
Foundation grant OCE-91-15929 (RCD). Subsequent analysis and publication was
supported by the David and Lucile Packard Foundation and NASA grant NAG56563 (RMK).
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