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0.0 02 0.4 0.6 0 8 1 0 1.2 1.4 1.8 18 ZO 2 2 Dissolved Iran (nM)

0.0 02 0.4 0.6 0 8 1 0 1.2 1.4 1.8 18 ZO 2 2 Dissolved Iran (nM)

Fig. 2 (a) Chl c3:Chl a and (b) Hex:Chl c3 ratios of Ross Sea colonial P. antarctica culture as a function of initial dissolved iron concentration, after incubation for 25 days (duplicate bottles sampled) and 31 days (triplicate bottles sampled) at an irradiance of 20 iE m-2 s-1. The data points show mean values ± one standard deviation are typically low (<0.2 nM) and high (>0.2 nM), respectively (Fitzwater et al. 2000; Sedwick et al. 2000; Coale et al. 2005). Thus, in the southern Ross Sea, there may be a general seasonal increase in the Chl c3:Chl a ratio of colonial P. antarctica that is associated with a corresponding seasonal decrease in dissolved iron availability, although these trends are not entirely consistent with our experimental results.

Hex:Chl c3 ratios

Since diatoms and P. antarctica dominate the algal community in the southern Ross Sea polynya (DiTullio and Smith 1996; Arrigo et al. 1999), a potentially unique pigment signature for P. antarctica populations in this region is the ratio of Hex to chlorophyll c3 (Hex:Chl c3 ratio), because diatoms produce neither of these accessory pigments. In our laboratory iron-addition experiment, during the early exponential growth phase (i.e., day 25), the Hex:Chl c3 ratio was relatively constant (-3.5) with respect to dissolved Fe concentration (Fig. 2b). However, by day 31, as Chl a biomass accumulated during the later exponential growth phase, lower Hex:Chl c3 ratios were observed at higher dissolved Fe concentrations (Fig. 2b). Van Leeuwe and Stefels (1998) have reported a similar relationship between the Hex:Chl c3 ratio and dissolved Fe concentration for a Weddell Sea P. antarctica culture grown at both high (110 iE m-2 s-1) and low (25 iE m-2 s-1) irradiance. Although the iron concentrations used in that study were significantly higher than those employed in our experiment, the results of van Leeuwe and Stefels (1998) are consistent with our experimental data, and there are no clear differences that might be attributed to the different P. antarctica strains (Weddell Sea versus Ross Sea) used in the two experimental studies.

The results of both studies indicate that Hex:Chl c3 ratios are greater than -3 for ambient dissolved Fe concentrations below -1 nM, an iron concentration at which the growth rate of colonial P. antarctica appears to be significantly reduced at an irradiance of 20 iE m-2 s-1 (Sedwick et al. this issue). The fact that Hex:Chl c3 ratios in the lower euphotic zone of the southern Ross Sea were -3-5 during the ROAVERRS III spring-early summer cruise (Fig. 4d) is suggestive of the importance of iron availability in limiting the growth and biomass of colonial P. antarctica, as discussed by Sedwick et al. (this issue). However, we note that the experiments of van Leeuwe and Stefels (1998) also demonstrate a significant effect of irradiance on the Hex:Chl c3 ratio of colonial P. antarctica, with growth under iron-replete conditions, producing Hex:Chl c3 ratios of 0.6 at high irradiance (110 iE m-2 s-1) and 0.06 at low irradiance (25 iE m-2 s-1). These values, however, were approximately an order of magnitude lower compared to the high Hex:Chl c3 ratios (3-5) observed under iron-limited conditions. Thus, it is clear that iron limitation results in significantly higher Hex:Chl c3 ratios in P. antarctica compared to iron-sufficient conditions. Because these two pigments are diagnostic pigments for P. antarctica in the Ross Sea, the Hex:Chl c3 ratio may serve as a physiological indicator of iron stress for P. antarctica in this region. Further work comparing this ratio with dissolved iron concentrations in the region should provide a good test for this hypothesis.

Fuco:Hex ratios

The results of our laboratory experiment show that sub-nanomolar iron additions to our low-iron (0.22 nM dissolved Fe) growth medium mediated an increase in the Fuco:Chl a ratio and a decrease in the Hex:Chl a ratio of colonial P. antarctica at an irradiance of 20 iE m-2 s-1. Not surprisingly, our data reveal a strong correlation (r2 = 0.82) between the ratio of Fuco to Hex (Fuco:Hex ratio) after 25 and 31 days incubation and the initial dissolved Fe concentration of our experimental treatments (Fig. 3a). This observed increase in the Fuco:Hex ratio with dissolved iron availability appears to extend well above the range of dissolved iron concentrations used in our laboratory experiment. For instance, when grown under iron-replete conditions (-500 nM dissolved Fe, in a 20-fold seawater dilution of L1 medium) at an irradiance of 20 iE m-2 s-1, the same strain of colonial P. antarctica exhibited a Fuco:Hex ratio of -0.45 (Fig. 3b), which was around 40 times higher than the Fuco:Hex ratio (ca. 0.01) in our experimental control treatments (0.22 nM dissolved Fe). In addition, two older P. antarctica isolates that had been maintained under iron-replete conditions for more than 15 years displayed Fuco:Hex ratios > 1 (Fig. 3b), which is consistent with other reported data for P. antarctica laboratory cultures (Buma et al. 1991).

Our experimental results also corroborate the findings of van Leeuwe and Stefels (1998), who have reported an increase in the Fuco:Hex ratio of a Weddell Sea P. antarctica isolate with increasing iron availability, based on experiments using growth media with EDTA-buffered iron concentrations on the order of 1 nM ('iron-deplete' conditions) and 1 iM ('iron-replete' conditions). However, in contrast to this earlier work, our experimental results may be directly

Fig. 3 (a) Fuco:Hex ratios of Ross Sea colonial P. antarctica culture as a function of initial dissolved iron concentration, after incubation for 25 days (duplicate bottles sampled) and 31 days (triplicate bottles sampled) at an irradiance of 20 iE m-2 s-1. The data points show mean values ± one standard deviation. A linear regression using all data points yields the following relationship: y = 0.073x + 0.002 (r2 = 0.82), where y is the Fuco:Hex ratio and x is the initial dissolved Fe concentration in nM. (b) Same data as figure (a), as well as Fuco:Hex ratios measured for two strains (1347 and 1871) of P. antarctica grown under iron-replete conditions (obtained from the Bigelow culture collection) and our Ross Sea P. antarctica culture (new strain) grown at a dissolved Fe concentration of 0.5 iM and an irradiance of 20 iE m-2 s-1. Note the scale break on the x-axis compared with field measurements of pigments in Antarctic surface waters. Specifically, we have used relatively low dissolved iron concentrations (-0.2-2 nM) that are typical of Antarctic surface waters during spring and summer, without using EDTA to control dissolved iron concentrations, thereby allowing iron speciation to be controlled by the natural iron-binding ligands present in Antarctic surface seawater (see discussion by

Gerringa et al. 2000). In addition, our experiment was conducted using a recently isolated strain of P. antarctica, in an effort to avoid any genetic and/or physiological biases that might arise when algal clones are maintained over a period of years under unnatural conditions (e.g., relatively high dissolved iron concentrations).

Our experimental estimates of growth rate as a function of iron concentration yielded a halfsaturation constant for growth of 0.45 nM dissolved Fe for colonial P. antarctica at an irradiance of -20 iE m-2 s1 (Sedwick et al., this issue). These results imply that at this relatively low irradiance, colonial P. antarctica was iron-limited (sensu Morel et al. 1991) at ambient dissolved Fe concentrations < 0.45 nM (Fuco:-Hex < 0.035), and was still significantly iron-stressed (sensu Morel et al. 1991) at dissolved iron concentrations of 1-2 nM (Fuco:Hex -0.0750.15). Thus, the mean Fuco:Hex ratio of 0.076 measured in subsurface, P. antarctica-dominated waters of the Ross Sea during mid spring and early summer 1998 (DiTullio et al. 2003b; Figs. 4a, b) would suggest that ambient dissolved Fe concentrations were limiting growth. Although no iron measurements were made during the ROAVERRS III cruise, the results of other studies in the southern Ross Sea suggest that dissolved Fe concentrations in the upper water column are typically subnanomolar during mid spring-early summer (Fitzwater et al. 2000; Sedwick et al. 2000; Coale et al. 2005).

When comparing our experiental data with field observations, an important caveat that must be considered is the potential effect of irradiance on the pigment composition of P. antarctica in relation to iron availability. The experiments of van Leeuwe and Stefels (1998) revealed relatively low Fuco:Hex ratios (ca. 0.3 and 0.7) for P. antarctica grown under low iron conditions at high irradiance (110 iE m-2 s-1) and low irradiance (25 iE m-2 s-1), respectively. Under

Fig. 4 Pigment ratios and P. antarctica abundance for 30-50 m depth in the Ross Sea during the ROAVERRS III expedition in November-December 1998: (a) Fuco:Hex ratios; (b) % P. antarctica as estimated by CHEMTAX analyses and corroborated by microscopy (see DiTullio et al. 2003b for details);

(d) Hex:Chl c3 ratios

Fig. 4 Pigment ratios and P. antarctica abundance for 30-50 m depth in the Ross Sea during the ROAVERRS III expedition in November-December 1998: (a) Fuco:Hex ratios; (b) % P. antarctica as estimated by CHEMTAX analyses and corroborated by microscopy (see DiTullio et al. 2003b for details);

(d) Hex:Chl c3 ratios

iron-replete conditions, the Fuco:Hex ratio in that study averaged 5 and 41 under high- and low-light conditions, respectively. Hence, similar to the trend in the Hex:Chl c3 ratios, the effects of irradiance on the Fuco:Hex ratios were relatively small compared to the observed changes due to iron availability. Similarly, the results of a shipboard bioassay experiment in the Ross Sea using a P. antarctica-dominated assemblage suggest that the Fuco:Hex ratio of colonial Phaeocystis is more sensitive to changes in iron availability than to changes in irradiance (Sedwick et al., this issue). However, the relationship between iron availability and the pigment composition of P. antarctica may be complicated by the interrelated influence of iron and light on phytoplankton growth, whereby algal iron requirements are expected to decrease under increased irradiance (Raven 1990; Sunda and Huntsman 1997). Indeed, the results of recent culture experiments in our laboratory indicate that the half-saturation constant for the growth of colonial P. antarctica with respect to iron is significantly less than 0.45 nM at irradiances greater than 20 iE m-2 s-1 (Garcia et al. manuscript in preparation). This finding has potentially important implications for the pigment composition of P. antarctica in the southern Ross Sea, where there are seasonal decreases in dissolved iron concentration and increases in mean irradiance between the early spring and mid summer (see discussion by Sedwick et al., this issue).

Thus there is a need for further experimental work to assess the relationship between the pigment composition of P. antarctica and dissolved iron concentrations as a function of irra-diance. To this end, we are currently undertaking the analysis of pigment samples from iron-addition culture experiments conducted at irradiances > 20 iE m-2 s-1. In addition, there are other factors that may limit the utility of pigment ratios such as Fuco:Hex in assessing the physiological status and/or ambient growth conditions of P. antarctica in Antarctic waters. These factors include growth limitation/co-limitation by other resources, such as zinc (Coale et al. 2003) or vitamin Bi2 (Bertrand et al. in press); luxury uptake of iron; integrated effects of prior changes in availability of iron and/or light; physiological differences between genetically distinct P. antarctica ecotypes; and life-stage of P. antarctica (i.e., colonial versus solitary cells, each of which may have quite different growth requirements).

Physiological and ecological implications

The use of algal pigment measurements in field samples as a chemotaxonomic tool begs a mechanistic understanding of the environmental factors that impact the pigment composition of relevant phytoplankton species. With regard to understanding the distribution and ecology of P. antarctica in the Ross Sea and the wider Southern Ocean, it is of interest to consider the physiological basis behind the significant changes in the Hex:Chl a, Fuco:Chl a, Hex:Chl c3 and Fuco:Hex ratios in response to sub-nanomolar iron additions that were observed in our culture experiment. These experimental trends are qualitatively consistent with the results of previous work, which has demonstrated decreases in both the cellular chlorophyll content and the Fuco:Hex ratio of colonial P. antarctica in response to decreased iron availability (van Leeuwe and Stefels 1998; Schoemann et al. 2005). This decrease in cellular chorophyll is readily explained by the involvement of iron in the synthesis of chlorophyll (Greene et al. 1992; Schoemann et al. 2005). With regard to the decrease in the Fuco:Hex ratio, van Leeuwe and Stefels (1998) have proposed that this reflects the conversion of Fuco to Hex, which acts as a photo-protective mechanism under conditions of low iron availability, when iron-deficient cells are more susceptible to photo-damage. Field measurements from the ROAVERRS cruises provide some support for this hypothesis, in that the Hex:Chl a ratio in P. antarctica-dominated waters was highest during the summer, when dissolved Fe levels are typically low and mean mixed-layer irradiance is relatively high (see discussion by Sedwick et al. this issue). Conversely, under conditions of high iron availability, Hex may be converted to the more-efficient light-harvesting pigment Fuco, in order to maximize the capture of light energy by the iron-replete cells (van Leeuwe and Stefels 1998). There is evidence for this in the observed increase in the Fuco:Hex ratio of our P. antarctica cultures with increasing dissolved iron concentration, with the trend extending to dissolved Fe concentrations as high as 0.5 iM (Fig. 3b).

At present, it is not clear whether the Ross Sea P. antarctica strain used in our experiments is genetically distinct from other P. antarctica strains, particularly those isolated from open-ocean surface waters of the Antarctic circumpolar current (ACC), where dissolved iron levels are likely to be low (<0.5 nM) year-round (Martin et al. 1990; Coale et al. 2005), and mean summer mixed-layer depths are typically greater (hence mean irradiance lower) than those over the Antarctic continental shelf (Trull et al. 2001). It may be that our experimental data are readily applicable to P. antarctica in these ACC waters, given that our experiment was conducted using sub-nanomolar iron additions and relatively low irradiance. On the other hand, long-term adaptation of open-ocean P. antarctica strains to the iron and light regimes of the ACC, which differ significantly from those in the southern Ross Sea, may limit the application of our experimental results to P. antarctica ecotypes from the Ross Sea/Antarctic shelf region. Ongoing molecular studies of relevant P. antarctica isolates, as well as further experimental work using open-ocean P. antarctica strains, are expected to provide answers to these questions.

Acknowledgements We are grateful to Mark Geesey, who performed the CHEMTAX and HPLC measurements for the ROAVERRS III samples, and Christopher Marsay, who performed the analyses of dissolved iron in the Ross Sea seawater and our experimental media. We also acknowledge the outstanding support of the officers, crew and support personnel aboard RV/IB Nathaniel B. Palmer. The primary funding for this research was provided by the United States National Science Foundation grants 0PP-0230513 (to GRD), OPP-0230559 (to PNS) and to DGE-0139313 (to NSG through M. VanSickle and G. Tempel).

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