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Appendix S1 Relationship per-capita nitrogen uptake and medium nitrogen
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Supporting Information
2
4
Nitrate
Ammonium
0
Per-Cell N uptake (10 -7 µM-N cell-1 L-1)
1
0
200
400
600
800
-1
3
Medium Nitrogen (µM-N L )
4
Relationship between per-capita nitrogen uptake and medium nitrogen, divided between
5
nitrate and ammonium. Rates are calculated by dividing the amount of depleted nitrogen
6
medium for the mean population density between each two successive observations. Only
7
data from single-nitrogen experiments were included in the plot. Model selection indicates
8
that per-cell nitrate and ammonium uptakes are better represented by a linear functional
9
response, as a more complex Michaelis-Menten saturating functional response does not
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significantly improve the model goodness-of-fit (Likelihood ratio test: 2=0.295, df=2,
11
p=0.86). Excluding the three outliers in the top-right corner did not change the conclusions
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(solid and dashed lines; 2=1.774, df=2, p=0.41).
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Appendix S2 Details on laboratory procedures
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Culture maintenance
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To avoid self-shading and light limitation, cultures were continuously suspended with
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magnetic stirrers at 300 rpm (IKA RCT Basic, IKA Labortechnik, Germany) and aerated with
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0.45 µm filtered air (Durapore, Millipore). Furthermore, risks of carbon limitation and
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dissociation of ammonium ion (NH4+ ) into volatile un-ionised ammonia (NH3 ) were
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minimized by ensuring pH levels below 7 by buffering the modified nitrate-BBM and
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ammonium-BBM media with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)
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at 8 mmol L-1 and adding NaHCO3 at 2.38 mmol L-1 as a carbon source (Vaddella, Ndegwa
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& Jiang 2011). We further ensured that N was the limiting factor for population growth rate
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by checking that cells returned to divide at exponential rates after adding nitrogen at the end
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of the experiments.
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Medium nitrogen analysis
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Medium nitrate (NO3-) was quantified using the ultraviolet spectrometric screening
28
method (Collos et al. 1999; Lanoul, Coleman & Asher 2002; Malerba, Connolly & Heimann
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2016), with optical density measured at 230 nm (OD230; EnSpire® Multimode Plate Reader;
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Perkin-Elmer, Waltham MA, US) on ultraviolet transparent 96-wellplate (Ultraviolet-Star®,
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Greiner Bio-One GmbH). As certain types of dissolved organic matter can also absorb at 230
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nm and NO3- does not absorb at 275 nm, a second measurement at 275 nm (OD275) was used
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to correct each OD230 reading (Clescerl et al. 1999). Medium ammonium (NH4+) was
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measured using the Salicylate–Hypochlorite method (Bower & Holm-Hansen 1980) with
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optical density measured at 670nm (EnSpire® Multimode Plate Reader) on standard 96-
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wellplates (CellStar®, Greiner Bio-One GmbH). The standard curves for both nitrate (OD230
Article: Nutrient utilization traits vary systematically with intraspecific cell size plasticity
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- OD275) and ammonium were linear across the range of nitrogen concentrations used in the
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experiments (R2 ≥ 0.995) and the minimum detection limits were 1 µM-N.
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40
References (not present in main text)
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Clescerl, L. S., Greenberg, A. E. & Eaton, A. D. (1999) 4500 NO3 Nitrogen (nitrate).
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Standard Methods for Examination of Water and Wastewater. pp. 120-122. Washington, DC:
43
American Public Health Association (APHA).
44
Vaddella, V.K., Ndegwa, P.M. & Jiang, A. (2011) An empirical model of ammonium ion
45
dissociation in liquid dairy manure. Transactions of the American Society of Agricultural and
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Biological Engineers, 54, 1119–1126.
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Appendix S3 Experimental design
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Diagram for the 3 × 2 factorial experimental design.
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Appendix S4 Relationship cell size and forward scatter
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10
15
Mean Cell Area (µm2)
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30
55
3000
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57
3500
4000
4500
5000
5500
6000
Mean Forward Scatter (unitless)
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Relationship between mean cell size in the culture and mean forward scatter quantified with a
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flowcytometer. Each point represents a sample from a culture. Mean cell area was calculated
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from measuring at least 10 randomly selected cells in the sample, while mean forward scatter
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was calculated from 3 replicate flowcytometric readings of the sample. Error bars represent
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1 standard error. Regression analysis: F1, 17 = 64.9, p<0.001, R2 = 0.79. Linear equation:
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𝑀𝑒𝑎𝑛 𝐶𝑒𝑙𝑙 𝐴𝑟𝑒𝑎 = 0.28 + 7.7 × 10−5 × 𝑀𝑒𝑎𝑛 𝐹𝑜𝑟𝑤𝑎𝑟𝑑 𝑆𝑐𝑎𝑡𝑡𝑒𝑟
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Appendix S5 Phenomenological models of observed rates
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Material and Methods
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Specific growth rates were computed for each day of each experiment as:
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𝜇𝑡 = ln(𝐵𝑡 ) − ln(𝐵𝑡−1 )
(eq. S1)
69
where B represents the total population density either at time t or t-1 (units of day-1). Per-cell
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nitrogen uptake for nitrate and ammonium were computed for each day of each experiment
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as:
𝑁𝑂 (𝑡)−𝑁𝑂 (𝑡+1)
1
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3
3
𝑣𝑁𝑂3,𝑡 = 𝑚𝑒𝑎𝑛(𝐵(𝑡),𝐵(𝑡+1))
× 𝑁𝑂
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4
4
𝑣𝑁𝐻4,𝑡 = 𝑚𝑒𝑎𝑛(𝐵(𝑡),𝐵(𝑡+1))
× 𝑁𝐻
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3 (𝑡)
𝑁𝐻 (𝑡)−𝑁𝐻 (𝑡+1)
1
4 (𝑡)
(eq. S2)
(eq. S3)
where NO3 and NH4 represent the mean nitrate and ammonium concentrations at time t or
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t+1. Hence, nitrogen uptake is computed as the total nitrogen uptake divided by the mean
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population density (left fraction in eq. S2-3), and further divided for the mean nitrogen
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concentration (right fraction in eq. S2-3). In this way, 𝑣𝑁𝑂3 and 𝑣𝑁𝐻4 quantified per-cell
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nitrogen uptake rates while accounting for total medium nitrogen availability, with units of
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cell-1 day-1.
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B
-22.5
-21.5
N-Replete
N-Deplete
1
0.0
0.5
C
-0.5
Specific culture growth rate
(µt ; loge+1 day-1)
-23.5
Ammonium uptake
(vNH4,t ; loge day-1 cell-1)
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Nitrate uptake
(vNO3,t ; loge day-1 cell-1)
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2.6
2.8
3.0
3.2
3.4
Cell Area (µm )
2
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Figure Appendix S5: Effects of cell size and nutrient history on the observed per-cell rates for
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(A) nitrate uptake, vNO3,t , (B) ammonium uptake, vNH4,t , and (C) specific culture growth rate,
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µt . Each point is the mean between three replicate measurements for each replicate culture,
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for each day, for each experimental treatment. All plotted lines represent significant linear
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models at p<0.05.
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Results
Statistical linear models revealed a significant effect of cell size and nutrient history on the
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observed rates of nitrogen uptake and specific growth rates, calculated between each two
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successive days with eq. 6 and 7 a-b. Specifically, nitrate uptake showed a weak positive
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effect of cell size (F1,48 = 6.59, MS = 2.30, p=0.033), but no significant effect of nutrient
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history (F1,48 = 0.97, MS = 0.34, p>0.5; Fig. Appendix S5 A). Conversely, rates for
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ammonium uptake were significantly higher in N-deplete cells compared to N-replete cells
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(F1,40 = 41.90, MS = 5.69, p<0.001) and displayed a significant positive effect of cell size
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(F1,40 = 43.47, MS = 5.9, p<0.001; Fig. Appendix S5 B). The effect of cell size was constant
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across nutrient histories for both nitrate (F1,48 = 0.27, MS = 0.1, p>0.05) and ammonium
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uptake (F1,40 = 0.01, MS = 0.01, p>0.05; Fig. Appendix S5 A-B). Finally, growth rate
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increased with cell area at N-deplete conditions, not at N-replete conditions (F1,149 = 8.07, MS
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= 0.51, p<0.01; Fig. Appendix S5 C). Overall, these results are mostly consistent with
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findings from fitting the process-based models. The only detectable difference is the effect of
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cell size on per-cell nitrate uptake, which was not detected by the process-based models.
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However, the very weak coefficient of determination (R2<0.13) and the 95% confidence
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intervals for the slope coefficient almost including zero (0.12 - 2.6) suggest some care should
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be taken in the interpretation of this result (Fig. Appendix S5 A).
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Appendix S6 Demographic parameters with process noise-only likelihood functions
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Comparison for the best-estimates for demographic parameters (median ±95% credible
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intervals) calculated with process noise-only likelihood functions for the “baseline” model
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(dashed lines) and with the best-fitting “Allometric N-history” model (solid lines).
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Parameters represent rates of per-cell uptake for nitrate (A) and ammonium (B), growth rate
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at infinite stored internal nitrogen quota (C), and minimum internal nitrogen quota (D). Two
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solid lines in the same pannel represent the effect of cell size on the demographic parameters
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between N-replete (red) and N-deplete (blue) previous N-history. Dashed line is the
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corresponsive parameter estimated with the “baseline” model, which assumes independence
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with cell size and nutrient history. See Fig. 1 in main text for more details.
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Appendix S7 Correlation between internal quota and cell size
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Correlation between internal quota dynamics extracted from the best-fitting “Allometric N-
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history” model and observed mean population cell size. Different symbols represent nitrogen-
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replete and nitrogen-replete experimental conditions. Also reported is the Spearman’s rho
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correlation coefficient (r s).
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Appendix S8 Ammonium inhibition on nitrate uptake
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Calibrated functional response for the inhibitory effects of available ammonium in the
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medium on the nitrate uptake of a cell (eq. 3). finhib = 1 indicates that the nitrate uptake is
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independent of ammonium, while finhib = 0 means that nitrate uptake is fully inhibited by the
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presence of ammonium. The estimates for parameters a and b in eq. 3 were taken from Table
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1. Shaded area represents the 95% credible intervals.
Article: Nutrient utilization traits vary systematically with intraspecific cell size plasticity