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Supplemental Information: Isotopic evidence for nitrification in the Antarctic
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winter mixed layer
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Sandi M. Smart*, Sarah E. Fawcett, Sandy J. Thomalla, Mira A. Weigand, Chris
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J. C. Reason, and Daniel M. Sigman
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*Corresponding author: sandimsmart@gmail.com
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1. Supplemental Text
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S1. Description of nitrate isotope depth profiles from the full wintertime transect
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between South Africa and the Antarctic sea-ice
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The dominant pattern in the profile data is one of decreasing [NO3-] towards the surface
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(Figure S2a), accompanied by rises in δ15N and δ18O (Figure S2b&c). In terms of the
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magnitude of these vertical changes, a clear meridional progression is evident; below, we
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describe each latitudinal zone (and relevant subsurface water masses) in turn.
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In the PAZ, the characteristic sub-surface [NO3-] maximum of UCDW is evident at 100-
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200 m with concentrations close to 34 μM, while in the OAZ, maximum concentrations
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of ~35 μM occur at 200-300 m. These maxima roughly correspond with, or fall slightly
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shallower than, the core potential density (1027.6 kg m-3) of UCDW described elsewhere
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[Orsi et al., 1995; Sigman et al., 1999]. The sub-surface [NO3-] minimum of LCDW is
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evident at 500-700 m (32-33 μM) in the PAZ deepening northwards to 1000-1500 m (30-
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32 μM) in the OAZ, following or occurring just shallower than the 1027.8 kg m-3
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isopycnal. LCDW is further distinguished from overlying UCDW by its elevated salinity
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and higher oxygen content [Orsi et al., 1995]. Although their nitrate concentrations
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differ, UCDW and LCDW are similar in isotopic composition throughout the AZ interior,
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with a δ15N of 4.83 ± 0.07‰ and 4.74 ± 0.07‰, and a δ18O of 1.89 ± 0.16‰ and 1.81 ±
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0.09‰ for UCDW and LCDW, respectively (concentration weighted mean ± standard
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deviation; n = 23 for UCDW and n = 30 for LCDW). As the δ15N elevation of UCDW
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derives from its exchange with the relatively high δ15N of Pacific Deep Water [Rafter et
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al., 2013], the weakness of this δ15N elevation at our section is likely a consequence of it
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being in the Atlantic sector. The lowest δ15N (< 4.75‰) values are observed in the
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deepest, most polar waters sampled (south of the SACCF). Overall, the mean δ15N and
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δ18O of nitrate in the AZ interior are 4.78 ± 0.08‰ and 1.84 ± 0.13‰, respectively (n =
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53).
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The PAZ profiles exhibit the smallest vertical changes from the sub-surface [NO3-]
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maximum into the mixed layer; [NO3-] decreases by 4.8-5.9 μM (Figure S2a), and δ15N
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and δ18O increase by 0.3-0.4‰ and 0.9-1.2‰ (Figure S2b&c), respectively. In the OAZ,
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a [NO3-] decrease of 8.5-8.8 μM from the [NO3-] maximum into the mixed layer is
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accompanied by rises in δ15N and δ18O of 0.7-1.0‰ and 1.4-1.7‰, respectively.
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In the PFZ, the [NO3-] minimum of LCDW decreases further to ~29 μM at 1700 m,
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deeper than in the AZ. Here, its δ15N is slightly higher than in the AZ (4.9‰), while its
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δ18O remains relatively constant (1.8‰). Further north, LCDW is typically located
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deeper than our maximum depth of sampling [Orsi et al., 1995]. Across the PFZ, the
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[NO3-] maximum of UCDW deepens from 500 m to 800 m and maintains nitrate
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concentrations in the 34-35 μM range. Its δ15N, however, increases from 4.8‰ in the AZ
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to ~5.2‰ and its δ18O increases from 1.9‰ to ~2.2‰. The PFZ profiles exhibit a 12-14
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μM decrease from the subsurface [NO3-] maximum into the mixed layer (Figure S2a),
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accompanied by δ15N and δ18O increases of 1.2-1.6‰ (Figure S2b) and 2.3-2.5‰
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(Figure S2c), respectively.
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To the north across the SAZ, the [NO3-] maximum (33-34 μM) deepens from 1000 m to
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1200 m, in parallel with a 200 m deepening of the 1027.6 kg m-3 core isopycnal. The ~1
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μM decrease in maximum concentration (relative to the adjacent PFZ) is accompanied by
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a slight increase in δ15N to 5.3‰, while δ18O remains unchanged at ~2.2‰ (Figure S2).
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Overall, [NO3-] decreases by 16-22 μM from the deep [NO3-] maximum of the SAZ
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upwards into the mixed layer (Figure S2a), over which interval δ15N and δ18O increase
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by 2.1-3.2‰ (Figure S2b) and 2.9-4.2‰ (Figure S2c), respectively. While the rises in
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δ15N and δ18O are largely focussed at the base of the mixed layer in the SAZ (122-189
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m), the concentration decrease occurs gradually from great depth (≥ 1000 m); this is in
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contrast to the AZ where the sharpest vertical gradients in both concentration and
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isotopes occur near the base of the mixed layer. PFZ profiles appear largely transitional
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between the SAZ and the AZ, although more similar to the SAZ.
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Profiles north of the STF deviate from the expected relationship of [NO3-] with 15N and
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18O observed across the rest of the transect (Figure S2). While [NO3-] either decreases
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or remains constant through the upper 200 m towards the surface, its corresponding 15N
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decreases and its 18O is variable. In general, such behavior is to be expected in this
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region, as nitrate consumption in the mixed layer is nearly complete and so new parcels
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of nitrate-bearing water from the SAZ or from below will dominate the 15N and 18O of
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the nitrate in the mixture [Sigman et al., 2000]. In specific, with more data from this
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region, the nitrate isotopes should be helpful in understanding the circulation of this
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region, which includes Agulhas rings.
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S2. Estimating the δ15N of nitrite effluxed from phytoplankton
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As a potential source of nitrite to the AZ winter mixed layer, we investigate the expected
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δ15N for nitrite effluxed from a phytoplankton cell during intracellular nitrate reduction
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(Figure S4) in this environment to compare with the nitrite δ15N inferred from our data.
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Assuming steady-state conditions, the δ15N of nitrate inside the cell (δ15NNO3inside) is
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calculated as follows:
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δ15NNO3inside = δ15NNO3outside – 15εNO3uptake + (1 – R) ∙ 15εNO3reduction + R ∙ 15εNO3efflux
(S1)
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δ15NNO3outside is the δ15N of nitrate supplied to the cell, for which we assume the δ15N of
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UCDW (4.8‰), with no prior assimilation-driven δ15N elevation to yield the most
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conservative (i.e., lowest) δ15N estimate for nitrite efflux.
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εNO3reduction (26.6‰) and
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εNO3uptake (2.0‰),
εNO3efflux (1.2‰) are the N isotope effects of nitrate uptake,
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reduction and efflux, respectively (as determined by Karsh [2013]). R is the ratio of
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cellular nitrate efflux to gross nitrate uptake, and is calculated by the following
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approximation (François et al. [1993]; as used by Granger et al. [2010]):
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R = (15εexpressed – 15εNO3uptake) / (15εNO3reduction – 15εNO3efflux)
(S2)
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where 15εexpressed is the net N isotope effect of nitrate assimilation that is expressed in the
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environment. Using an
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al., 2003; Granger et al., 2010] and field-based studies [e.g., DiFiore et al., 2010]
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suggest to be appropriate for the AZ, produces an R value of 0.16, and thus an estimate
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for δ15NNO3inside of 25.4‰. At steady-state, the δ15N of the intracellular nitrite pool will
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be ~26.6‰ lower than that of the intracellular nitrate from which it is produced (as
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determined by the value of
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efflux is unknown. If it is similar to that of nitrate efflux (i.e., assuming
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εexpressed of 6‰, which both diatom culture- [e.g., Needoba et
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εNO3reduction); i.e., -1.2‰. The N isotope effect of nitrite
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εNO2efflux =
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εNO3efflux = 1.2‰; from Karsh [2013]), then the δ15N of nitrite released to the
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environment will be around -2.4‰. If intracellular nitrite δ15N is elevated by nitrite
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reduction in the chloroplast (an unlikely scenario as this would require nitrite efflux
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across at least three chloroplast membranes) then the δ15N of the nitrite effluxed to the
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environment will be higher than -2.4‰. With a δ15N of roughly -2‰ or higher, nitrite
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efflux from phytoplankton cells cannot explain the extremely low δ15N (-40‰ to -20‰)
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of AZ winter mixed layer nitrite.
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2. Supplemental figure captions
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Figure S1: Comparison between filtered and unfiltered seawater nitrate a) δ15N (in ‰ vs.
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N2 in air) and b) δ18O (in ‰ vs. VSMOW) measurements. Both filtered and unfiltered
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samples were collected at 19 different underway sampling locations on a separate leg of
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the winter voyage (from the Antarctic sea-ice edge (56.7°S) to Marion Island (46.9°S)
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and back to South Africa). Blue squares denote seawater samples filtered through 0.4 μm
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polycarbonate (PC) filters, pink triangles indicate samples filtered through combusted 0.7
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μm glass fiber filters (GF/Fs) and grey crosses denote unfiltered samples. Error bars
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indicate one standard deviation from the mean of replicate measurements (n ≥ 3 for each
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sample).
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Figure S2: Vertical profiles of a) [NO3-] (in μM), b) nitrate δ15N (in ‰ vs. N2 in air) and
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c) nitrate δ18O (in ‰ vs. VSMOW) for the upper 2000 m from the wintertime transect
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between Cape Town (33.9°S) and the Antarctic sea-ice edge (56.7°S). Error bars indicate
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measurement standard deviation (n ≥ 3).
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Figure S3: Comparison between untreated samples (i.e., nitrate+nitrite; shown in grey)
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and the same samples with nitrite removed (i.e., nitrate-only; OAZ in pink, PAZ in green)
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in Rayleigh space (δ15N vs. ln([NO3-]) or ln([NO3- + NO2-])) for a) full AZ vertical depth
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profiles (and underway surface data); b) samples from the surface to the depth of the
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[NO3-] maximum of CDW. The N isotope effect estimates (15εassim) indicated on the plot
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for each station derive from the slopes of the linear trendlines; c) all AZ mixed layer data
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(note the change of x-axis scale for panel c)), yielding 15εassim estimates of 6.5‰ (dashed
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grey trendline) and 7.1‰ (solid grey trendline) for nitrate+nitrite and nitrate-only,
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respectively. Excluding mixed layer samples from the profile at 52.0°S and nearby
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underway samples (which appear to derive from a different subsurface source) decreases
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the slopes (15εassim estimates) to 5.2‰ (dashed black trendline) and 5.8‰ (solid black
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trendline), respectively. Despite the low nitrite concentrations in our AZ winter mixed
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layer samples, nitrite removal raises the measured δ15N substantially relative to untreated
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samples (as seen in a) to c) above), implying a very low δ15N for ambient nitrite.
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Figure S4: Schematic representation of the nitrogen pathways involved in the efflux of
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nitrite (NO2-) from a phytoplankton cell during nitrate (NO3-) reduction. The N isotope
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effects relevant to the δ15N of nitrite efflux are indicated; namely, the N isotope effects
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associated with nitrate uptake into the cell (15εNO3uptake), nitrate reduction to nitrite
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(15εNO3reduction), nitrate efflux (15εNO3efflux), and nitrite efflux from the cell (15εNO2efflux). If
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nitrite taken up into the chloroplast (with
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εNO2uptake) is not completely reduced (with
εNO2reduction) to ammonium (and finally organic nitrogen; Norg), there may be some
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amount of nitrite effluxed from the chloroplast (with 15εNO2efflux) which would act to raise
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the δ15N of the nitrite ultimately released to the environment. [source: based on Granger
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et al., 2004, 2010]
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3. Supplemental references cited
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DiFiore, P., D. Sigman, K. Karsh, T. Trull, R. Dunbar, and R. Robinson (2010), Poleward
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decrease in the isotope effect of nitrate assimilation across the Southern Ocean,
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Geophysical Research Letters, 37(L17601), doi: 10.1029/2010GL044090.
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François, R., M. Bacon, M. Altabet, and L. Labeyrie (1993), Glacial/interglacial changes
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in sediment rain rate in the SW Indian sector of sub-Antarctic waters as recorded by Th-
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230, Pa-231, U, and δ15N, Paleoceanography, 8(5), 611–629, doi: 10.1029/93PA00784.
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Granger, J., D. Sigman, J. Needoba, and P. Harrison (2004), Coupled nitrogen and
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oxygen isotope fractionation of nitrate during assimilation by cultures of marine
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phytoplankton,
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10.4319/lo.2004.49.5.1763.
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Granger, J., D. Sigman, M. Rohde, M. Maldonado, and P. Tortell (2010), N and O
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isotope effects during nitrate assimilation by unicellular prokaryotic and eukaryotic
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plankton cultures,
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10.1016/j.gca.2009.10.044.
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Karsh, K. (2013), Physiological and environmental controls on the nitrogen and oxygen
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isotope fractionation of nitrate during its assimilation by marine phytoplankton, Ph.D.
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thesis, Princeton University, USA, and CSIRO, Australia.
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Needoba, J., N. Waser, P. Harrison, and S. Calvert (2003), Nitrogen isotope fractionation
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in 12 species of marine phytoplankton during growth on nitrate, Marine Ecology
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