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Figure 3.1. Specific growth rate of P. inermis and E. antarctica at different dissolved inorganic iron concentrations (Fe') (All values are means ±1SD, n ≤ 6).

The specific growth rate of both Southern Ocean diatoms in relation to Fe′ is presented in Figure 3.1 and a summery of the results can be found in Tables 3.1 and 3.2. A major consequence of Fe-limitation on diatom physiology is by definition a decrease in growth rate. The growth of E. antarctica and P. inermis was highly dependent on the concentration of Fe′ in the media, with growth rates decreasing up to four-fold at Fe′ concentrations of 0.2 pmol L-1 and 0.09 pmol L-1, respectively. Growth in Southern Ocean diatoms follows a Monod saturation function, with maximum specific growth

rates (μmax) under saturating light and nutrient conditions ranging from 0.02 – 0.62 d-1 (Timmermans et al., 2004). Previous studies show that Southern Ocean diatoms attain near maximum growth rates in EDTA-buffered media (≥ 0.9 of μmax) (Strzepek et al., 2011). As such, we assumed diatoms in EDTA-media were growing at or near their maximum rates, and their physiological behaviour in the media adequately reflected Fe- replete conditions. Tables 3.1 and 3.2 compare growth rates measured here to those rreported by Strzepek et al. (2011, 2012) under similar Fe′ concentrations. Although the value of μmax for E. antarctica compares well with previously reported values by Strzepek et al. (2011, 2012), (μ:μmax = 0.89 compared to 2011 study, 0.97 compared to 2012 study), it displayed significant variation ( μ:μmax = 0.74 compared to 2011 study, 0.69 compared to 2012 study) for P. inermis when compared to previously reported values by Strzepek et al. (2011, 2012). Explanations for this can be found in section 3.4.1 (Variations in specific growth rate and cell size in response to Fe-stress).

Table 3.1. Growth rates (μ) of P. inermis grown in Fe-EDTA and Fe-DFB media. Growth rates from studies by Strzepek et al. (2011, 2012) are listed below for comparison.

Fe Treatment Fe' (pmol L-1_{) μ day}-1 _{S.E n μ:μmax}

P.inermis (This study)

58.3 nmol L-1 _{Fe; 10 μmol L}-1 _{EDTA 3369 0.35 0.00 6 1}

4.4 nmol L-1 _{Fe; 40 nmol L}-1 _{DFB 0.2 0.16 0.02 3 0.45}

4.4 nmol L-1 _{Fe; 80 nmol L}-1 _{DFB 0.09 0.09 0.00 6 0.25}

P.inermis (Strzepek et al., 2011)

58 nmol L-1 _{Fe; 10 μmol L}-1 _{EDTA 3450 0.47 0.01 27 1.00}

4.4 nmol L-1 _{Fe; 10 μmol L}-1 _{EDTA 258 0.47 0.01 48 1.00}

2.4 nmol L-1 _{Fe; 10 μmol L}-1 _{EDTA 140 0.44 0.01 12 0.94}

1.8 nmol L-1 _{Fe; 10 μmol L}-1 _{EDTA 106 0.45 0.01 6 0.96}

1.8 nmol L-1 _{Fe; 100 μmol L}-1 _{EDTA 10.6 0.44 0.01 6 0.95}

0.8 nmol L-1 _{Fe; 10 μmol L}-1 _{EDTA 47.1 0.44 0.03 3 0.90}

4 nmol L-1 _{Fe; 4 nmol L}-1 _{DFB 30.1 0.42 0.04 12 0.90}

4 nmol L-1 _{Fe; 40 nmol L}-1 _{DFB 0.17 0.42 0.02 12 0.99}

4 nmol L-1 _{Fe; 80 nmol L}-1 _{DFB 0.08 0.46 0.01 6 0.61}

4 nmol L-1 _{Fe; 200 nmol L}-1 _{DFB 0.03 0.29 0.02 18 0.38}

4 nmol L-1 _{Fe; 400 nmol L}-1 _{DFB 0.02 0.18 0.00 3 0.67}

P.inermis (Strzepek et al., 2012)

4.4 nmol L-1 Fe; 10 μmol L-1 EDTA 277 0.51 0.02 - - 4 nmol L-1 Fe; 200 nmol L-1 DFB 0.03 0.31 0.02 - -

Table 3.2. Growth rates (µ) of E .antarctica grown in Fe-EDTA and Fe-DFB media. Growth rates from studies by Strzepek et al. (2011, 2012) are listed below for comparison.

Fe Treatment Fe' (pmol L-1_{) μ day}-1 _{S.E N μ:μmax}

E.antarctica (This study)

58.3 nmol L-1 _{10 μmol L}-1 _{EDTA 3369 0.32 0.01 4 1}

4.4 nmol L-1 _{Fe; 4 nmol L}-1 _{DFB; 10}

μmol L-1 _EDTA 83 0.16 0.02 3 0.51

4.4 nmol L-1 Fe; 40 nmol L-1 DFB 0.2 0.07 0.00 6 0.23

E.antarctica (Strzepek et al., 2011)

58 nmol L-1 _{Fe; 10 μmol L}-1 _{EDTA 3450 0.36 0.01 6 1}

4.4 nmol L-1 _{Fe; 10 μmol L}-1 _{EDTA 258 0.34 0.02 8 0.93}

2.4 nmol L-1 _{Fe; 10 μmol L}-1 _{EDTA 140 0.36 0.02 3 1}

1.8 nmol L-1 Fe; 10 μmol L-1 EDTA 106 0.33 0.02 3 0.92 4 nmol L-1 _{Fe; 4 nmol L}-1 _{DFB 30.1 0.23 0.01 9 0.64}

4 nmol L-1 _{Fe; 40 nmol L}-1 _{DFB 0.17 0.19 0.01 3 0.54}

E.antarctica (Strzepek et al., 2012)

4.4 nmol L-1 Fe; 10 μmol L-1 EDTA 277 0.33 0.01 - - 4 nmol L-1 _{Fe; 40 nmol L}-1 _{DFB 0.17 0.2 0.02 - -}

Cell size variations for E. antarctica and P. inermis in relation to Fe′ concentration are presented in Figures 3.2 - 3.4, and a summary of the results can be found in Table 3.3. Both E. Antarctica (Figure 3.3) and P. inermis (Figure 3.4) increased their cell volume and cell surface area in response to Fe-limiting conditions by increasing both their apical lengths (AL) and trans-apical widths (TW) (p < 0.01). Variations in AL and TW with growth rate are presented in Figure 3.4, and were not consistent between species. Both species exhibited different morphological adaptations to decreasing Fe′ even though their growth rates decreased in a similar fashion. For example, in Figure 3.3, E. antarctica increased its cell volume 2-fold at Fe′ = 83 pmol L-1 and almost 3-fold at Fe′ = 0.2 pmol L-1. This variation exhibited a linear relationship with the specific growth rate (Figure 3.5) (r2 = 0.99, n = 9) and was driven by a uniform linear change in both the AL (r2 = 0.99) and TW (r2 = 0.99) (Figure 3.5), thus similar relationships were evident between the specific growth rate and variations in the Surface Area and SA:V ratio (r2 ≥ 0.99). Consequently, there was no change in the cell aspect ratio (aspect ratio = AL/TW) in E. antarctica across all Fe-concentrations, due to the cell AL scaling with the TW between Fe-replete and Fe-limiting conditions. In contrast, P. inermis exhibited two significantly different morphological adaptations at both Fe-limiting concentrations (Figures 3.3 and 3.4). While cell volume and surface area increased under Fe-limiting conditions, a plateau was reached where Fe′ = 0.2 pmol L-1 as a result of P. inermis increasing its AL (p < 0.01), but not changing its TW significantly (p = 0.04) relative to its TW under Fe-replete conditions. At Fe′ = 0.09 pmol L-1, the AL drops and the TW increases. Despite a minor increase in cell volume, the surface area between both Fe- limiting conditions remain similar (p = 0.61). Subsequently the minor variation in the cell SA:V ratio is driven mainly by an increase in the AL at 0.2 pmol L-1 Fe′ and TW at 0.09 pmol L-1 Fe′. Similarly, variations in the valve aspect ratio are also directly proportional to changes in the AL and TW under both Fe-limiting conditions. Consequently, E. antarctica exhibits a linear increase in size with decreasing Fe′, while P. inermis exhibits a phased morphological change with decreasing Fe′, becoming more elongated at Fe′ = 0.2 pmol L-1, and then decreasing its length and increasing its width at Fe′ = 0.09 pmol L-1_.

Figure 3.2. Light microscope images of P. inermis ( a and c) and E. antarctica (b and d) under Fe-replete conditions (a and b) and Fe-limiting conditions (c and d).

Figure 3.3. Mean cell size parameters for different Feʹ concentrations for E. antarctica. Error bars represent the standard error of total cell measurements for each Fe′ value (n ≥

Figure 3.4. Cell size parameters at different Fe’ concentrations for P. inermis. Error bars represent the standard error of total cell measurements for each Fe′ value (n ≥ 300)

Figure 3.5. Apical width and trans-apical length variations in response to growth rate in E. Antarctica (a) and P. inermis (b).

Table 3.3. Summary of cell size variations in response to varying Fe-conditions. All values are means ±1SE, n ≥ 300.

Fe Treatment Fe' (pmol L-1) Valve apical

length (μm) Valve transapical width (μm) Cell volume (μm3) Cell Surface area (μm2) Surface area- to-volume ratio Valve aspect ratio P. inermis

58.3 nmol L-1 _{Fe; 10 μmol L}-1

EDTA 3369 118 ± 1.7 15.2 ± 0.09 21976 ± 431 6072 ± 96 0.28 ± 0.002 7.9 ± 0.1 4.4 nmol L-1 _{Fe; 40 nmol L}-1 _{DFB 0.2 181 ± 2.3 15.5 ± 0.1 34677 ± 624 9224 ± 127 0.27 ± 0.002 12 ± 0.2}

4.4 nmol L-1 _{Fe; 80 nmol L}-1 _{DFB 0.09 163 ± 2.2 17 ± 0.1 37038 ± 592 9137 ± 117 0.25 ± 0.001 9.8 ± 0.1}

E. Antarctica

58.3 nmol L-1 _{10 μmol L}-1 _{EDTA 3369 56 ± 0.9 15 ± 0.2 12744 ± 380 3766 ± 66 0.32 ± 0.003 4.1 ± 0.09}

4.4 nmol L-1 _{Fe; 4 nmol L}-1 _DFB;

10 μmol L-1 _EDTA 83 69 ± 1 18 ± 0.3 22335 ± 609 5491 ± 88 0.27 ± 0.003 4.1 ± 0.09