Supplemental Information: The counterintuitive effect of summer-to-fall mixed layer
deepening on eukaryotic new production in the Sargasso Sea
Sarah E. Fawcett*, Michael W. Lomas, Bess B. Ward, and Daniel M. Sigman
*Corresponding author: sfawcett@princeton.edu
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1. Supplemental Text
S1. Effect of sample storage on measured ammonium concentrations
Ammonium concentrations were measured in the laboratory at Princeton according to the
method of Holmes et al. [1999]. The samples were stored frozen at -20°C in HDPE Nalgene
bottles that were acid-washed and copiously rinsed with DIW prior to the cruises, and then rinsed
at least three times with sample seawater just prior to sample collection. Owing to concern about
potential storage artifacts, we collected two different samples from each depth, and stored and
analyzed them separately. As evidenced by the relatively small magnitude of the error bars in
Fig. 2e (indicative of one standard deviation from the mean ammonium concentration of the two
samples), the measured ammonium concentration is representative of ammonium in the water
column at the time of sampling, and is not the result of analytical error or storage artifact.
Furthermore, on subsequent cruises (e.g., February 2012; Fig. S1), we have measured
ammonium concentrations at sea, and then re-measured them (from separately-collected samples,
stored as described above) in the laboratory at Princeton University, and have found little
difference in the results. At sea, samples were collected in acid-washed 60 mL HDPE bottles that
were rinsed copiously with sample seawater before filling, and then immediately amended with
OPA reagent, as per Holmes et al. [1999] (see manuscript text section 2.2.4. for methodological
details).
S2. Nitrate supply to the euphotic zone
S2.1. Nitrate concentration as a predictor of nitrate supply
The average concentration of nitrate in the upper 200 m (which incorporates the
subsurface nitrate supply) yields relationships with Δδ15Ne-P (Fig. S3a) and MLD (Fig. S3c) that,
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while relatively weak, are consistent with our interpretation that the deepening of the mixed layer
into the fall reduces the nitrate supply to the euphotic zone as a whole, and that this results in less
nitrate assimilation by eukaryotes. One might predict a positive correlation between Δδ15Ne-P and
the average nitrate concentration in the euphotic zone, yet this relationship is not significant (R2
= 0.14; Fig. S3b). While sampling resolution for nitrate concentration may be partly to blame
(i.e., in the case of December), a weak relationship is perhaps to be expected given that nitrate
supplied across the base of the euphotic zone will be rapidly assimilated, compromising euphotic
zone nitrate concentration as an indicator of nitrate supply. Similarly, the observation of
significant levels of nitrate in the euphotic zone (e.g., at the October BATS station, Fig. 2d) may
reflect a very recent supply event, such that nitrate assimilation may not yet be evident in the
15N of phytoplankton biomass, the turnover time of which is on the order of a week [Goldman,
1993].
S2.2. Effect of ammonium availability on eukaryote nitrate assimilation
Given that it is less energetically expensive to assimilate reduced N forms, ammonium
should be preferentially assimilated over nitrate by phytoplankton [Cochlan and Harrison, 1991;
Dortch, 1990; Harrison et al., 1996]. Indeed, one possible explanation for the different N
sources that are preferentially consumed by prokaryotes and eukaryotes is that prokaryotes are
the superior competitors for ammonium and other reduced N species, such that eukaryotic
phytoplankton are left to consume the energetically expensive N form, nitrate [Fawcett et al.,
2011]. Thus, it might be expected that the availability of ammonium may also affect the 15N of
eukaryotic phytoplankton.
One example where ammonium availability might be predicted to play a role is in
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October at BATS. Low levels of nitrate were detectable as shallow as 60 m (0.05 M), and
earlier nitrate consumption is evident in the high 15N of shallow nitrate (Fig. 2b), yet the 15N of
eukaryotic phytoplankton was low. It is possible that a nitrate supply event had just occurred,
such that nitrate assimilation was not yet evident in the 15N of eukaryotic biomass.
Alternatively, the low eukaryote 15N may derive from ammonium consumption, and indeed, at
100 m near the base of the euphotic zone, the ammonium concentration was high (Fig. 2e).
However, in the BATS October profile, at the depths from which eukaryotes were
sampled, the ammonium concentration was not elevated, and this is a strong argument against an
effect from ammonium availability. Unlike nitrate, the ammonium supply is dominantly by in
situ production rather than upward mixing. Furthermore, ammonium cycles rapidly in surface
waters. An f-ratio for the oligotrophic season at BATS of 0.2 [Fawcett, 2012] and a nitrate flux
of 0.25 mol N m-2 over the same time period [Jenkins, 1988; Spitzer and Jenkins, 1989;
McGillicuddy et al., 1998] imply a euphotic zone (upper 100 m) ammonium demand of 1.25 mol
N m-2 (~70 nM d-1), which suggests an average residence time for ammonium of <4 hours, and in
most cases <2 hours. Such a residence time is too short to allow for transport across the seasonal
thermocline; the relevant measure of ammonium availability at any given depth is thus the
ammonium concentration at that depth. A comparison of the ammonium concentration at each
depth from which we have 15N measurements with Δδ15Ne-P at that depth yields no significant
relationship (R2 = 0.07; Fig. S4a), suggesting that ammonium availability does little to regulate
N utilization by eukaryotic phytoplankton in this environment. For example, at 27N in October,
ammonium was virtually undetectable throughout the euphotic zone, yet the low 15N of
eukaryotes at every depth suggests complete reliance on ammonium (Fig. 3c). At SS #12 in
December, eukaryotes at 30 m had a 15N indicative of nitrate utilization (3.2‰; Fig. 3f) even
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though the ammonium concentration was relatively high (>25 nM). While ammonium is clearly
an important N source to eukaryotic phytoplankton, our data suggest that density-controlled
nitrate availability is the major determinant of eukaryotic nitrate utilization in this system.
It should be noted that a comparison of the nitrate concentration at each depth from
which we have 15N measurements with Δδ15Ne-P at that depth also yields no significant
relationship (R2 = 0.01; Fig. S4b). However, unlike ammonium, nitrate is predominantly
supplied to the euphotic zone by upward mixing from depth, such that a more appropriate
measure of nitrate availability is the average concentration of nitrate in the upper 200 m (which
incorporates the subsurface nitrate supply; Fig. S3a). Furthermore, nitrate supplied across the
base of the euphotic zone will be rapidly assimilated, compromising euphotic zone nitrate
concentration as an indicator of nitrate supply to the euphotic zone (Fig. S3b; see also Lewis et
al. [1986]).
S2.3. Mesoscale drivers of nitrate supply
Mesoscale eddies are ubiquitous features in the subtropical ocean and are important for
surface ocean ecology and biogeochemistry because they affect vertical nutrient transport and
light availability [Robinson, 1983; Falkowski et al., 1991; McGillicuddy et al., 1998; Siegel et
al., 2011]. “Submesoscale pumping” is one mechanism by which eddies influence primary
production, causing elevated chlorophyll concentrations in the high velocity regions surrounding
the eddy (rather than at its center; [Calil and Richards, 2010; Siegel et al., 2011]). It has been
hypothesized that such increases in chlorophyll result from submesoscale injections of nutrients
at the outer frontal regions of eddies due to physical instabilities induced by high velocity
currents [Calil and Richards, 2010].
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Within our dataset, there is tentative evidence of submesoscale pumping driving
seasonally anomalous eukaryote 15N through an increase in the nitrate supply to the euphotic
zone. The unusually shallow, low-density surface layer that we observed at SS #12 in December
(Fig. 2c) likely resulted from the passage of an anticyclonic eddy to the southwest of BATS (Fig.
S5a) that generated a strong, southeastward-flowing jet directly over SS #12 (Fig. S5b). The high
eukaryote 15N at SS #12, as well as an increase in 60-100 m fluorescence at this station (Fig.
S5c), are likely a response to nitrate supplied by submesoscale pumping at the front between the
eddies, which is apparent in the ADCP velocity on December 9-10 (Fig. S5b).
McGillicuddy et al. [1998] estimate that the vertical flux of nutrients induced by
mesoscale eddy dynamics can account for a third of annual new production in the Sargasso Sea.
The observation of these eddies year-round implies that a significant fraction of new production
occurs stochastically throughout the year, even during times of intense stratification.
Anticyclonic eddies are typically thought to decrease productivity because convergence at their
centers depresses the nutricline. However, eddy perimeter interactions with other mesoscale
features are hypothesized to result in the net upward transport of nutrients [McGillicuddy et al.,
1998; Calil and Richards, 2010], which, given the time required for biological removal (days;
Goldman [1993]), seldom manifest as an increase in surface nitrate concentrations [McGillicuddy
and Robinson, 1997].
The similarity between average eukaryote 15N (2.2‰) and the 15N of subsurface nitrate
(2.2‰) at SS #12 in December is consistent with an episodic nitrate supply event that has been
rapidly and completely drawn down by eukaryotic phytoplankton. The observed pattern of
increasing eukaryote 15N from the bottom of the euphotic zone up towards the base of the
mixed layer can be explained by isotope discrimination during nitrate assimilation as the
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consumption of newly supplied nitrate progresses upwards [Sigman et al., 1999]. It should be
noted that the eddy interaction driving the nitrate flux coincided with a shoaling of the mixed
layer, leading to consistency of the December inter-station 15N differences with the broader
mixed layer/nitrate assimilation correlation described above. This may not be coincidental: the
eddy-induced input of dense subsurface water that supplied nitrate to the euphotic zone can also
lead to shoaling of the base of the mixed layer [McGillicuddy et al., 1998]. Eukaryotes collected
from within the mixed layer had the highest 15N of all depths sampled at this station, suggesting
that the shoaling of the pycnocline resulted in the upward transport of nitrate, via physical or
biological means, into the mixed layer where it was assimilated by eukaryotic phytoplankton.
The difference in eukaryote 15N between BATS and SS #12, two stations located less
than 40 km apart that were sampled within days of one another, underlines the dynamic nature of
this system, the potential importance of mesoscale features for driving new production through
changes in euphotic zone density structure, and the rapidity with which phytoplankton can
respond to a nutrient supply event. It appears that our coupled flow cytometry-N isotope
approach has the potential to directly capture the biogeochemical and ecological effects of such
dynamic features.
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2. Supplemental table and figure captions
Table S1: Comparison of the 15N of low concentration nitrate standards (IAEA N3 and USGS
34) and samples before (15Nunconcentrated) and after (15NBÜCHI) concentration via the BÜCHI
parallel vortex evaporation technique. The 15N of the concentrated standards and samples was
corrected by referencing to 7.5 μM IAEA N3 and USGS 34 nitrate standards in seawater,
whereas the unconcentrated standards and samples, which were analyzed in a separate run, were
referenced to 1 μM and 2 μM IAEA N3 and USGS 34 nitrate standards in seawater. The true
15N of IAEA N3 is 4.7‰ and USGS 34 is -1.8‰ [Böhlke et al., 2003].
Sample
IAEA N3 (standard)
IAEA N3 (standard)
IAEA N3 (standard)
IAEA N3 (standard)
USGS 34 (standard)
USGS 34 (standard)
USGS 34 (standard)
December SS#12 150 m
October 23N 200 m
October 27N 200 m
October BATS 96 m
July PITS 100 m
15Nunconcentrated (‰)
4.64
4.78
4.81
4.75
-1.85
-1.65
-1.76
2.22
3.98
4.55
8.08
8.36
[NO3-] (μM)
0.5
0.5
0.5
0.5
0.5
0.5
0.5
1.42
1.40
0.60
0.38
0.35
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15NBÜCHI (‰)
4.57
4.61
4.93
4.77
-1.86
-1.41
-1.90
2.23
3.88
4.36
7.89
8.61
Figure S1: A comparison of ammonium concentrations measured at sea in February 2012 and
then re-measured in the laboratory at Princeton in March 2013. The solid blue line indicates a
relationship of 1:1.
Figure S2: Biomass N content ([N]) of flow cytometrically sorted components of the PN from
the Sargasso Sea in a) July at BATS, b) October at BATS, c) October at 27N, d) October at
23N, e) December at BATS, and f) December at SS #12. The mixed layer depth at the time of
sampling is indicated on the plots (MLD; dashed grey line). “Total cyanos” (purple triangles)
represents a combined population of Prochlorococcus plus Synechococcus, sorted and analyzed
independently of the individual genera. “Autotrophic sum” (grey pluses) denotes the sum of all
sorted autotrophic populations. Error bars indicate the full range of values measured for replicate
samples, commonly duplicates, collected, sorted, and analyzed independently.
Figure S3: Cross plot showing the difference between the concentration-weighted euphotic zone
average 15N of eukaryotic phytoplankton and Prochlorococcus (Δ15Ne-P = 15Neuk – 15NPro)
relative to a) average upper water column (0-200 m) nitrate concentration, b) euphotic zone (0100 m) nitrate concentration for all stations sampled in July (squares), October (circles), and
December (triangles). The relationship between average upper water column (0-200 m) nitrate
concentration and MLD is also shown (c). Data from PITS in July 2009 (“July PITS”) and Hydro
Station S in July 2008 (“July Hydro S”) are from Fawcett et al. [2011].
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Figure S4: Cross plots showing the difference between eukaryotic and Prochlorococcus 15N
(Δ15Ne-P = 15Neuk – 15NPro) at each depth relative to the corresponding a) ammonium
concentration (nM) and b) nitrate concentration (µM) at the same depth. Sorted PN 15N and
nitrate concentration data from PITS in July 2009 (“July PITS”) and Hydro Station S in July
2008 (“July Hydro S”) are from Fawcett et al. [2011].
Figure S5: a) Sea level anomaly showing the position of an anticyclonic eddy to the southwest of
BATS in December 2009, b) Acoustic Doppler Current Profiler (ADCP) measurements of
current speed (0-300 m) during the December cruise, and c) CTD-derived fluorescence (0-300
m) for the duration of the December cruise [http://bats.bios.edu] indicating an increase in
chlorophyll (and inferred increase in phytoplankton biomass) in response to the water column
instability induced by the passage of the eddy. The vertical line of open circles indicates the
timing of the BATS cast, the open rectangle shows the ship’s first transit across SS #12, and the
vertical line of open diamonds indicates the timing of the SS #12 cast that occurred a day later.
The high-velocity “jet” generated at the outer frontal region of the eddy is clearly visible in the
ADCP measurements at SS #12 (panel b, within the open rectangle).
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