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Submitted August 29th 2009 to Marine Ecology Progress Series
Responses of Different Antarctic Genotypes of Phaeocystis antarctica to
Abstract:
The polar prymnesiophyte, Phaeocystis antarctica, often dominates phytoplankton blooms occurring in either deeply relatively weakly stratified waters or shallow mixed water layers in the Southern Ocean. Although the Southern Ocean seems to be a unique large water body circling around the Antarctic continent, it consists of several geographical regions and different oceanographic regimes. Preliminary genetic AFLP analysis revealed a high genetic diversity within P. antarctica populations obtained from the Weddell Sea, Prydz Bay and the Antarctic Circumpolar Current. Some isolates from the Weddell Sea Gyre and Prydz Bay isolates were closely related to each other and the two ecosystems were not isolated from each other genetically. Thus, we asked whether genetically closely related strains originating from different geographic regions in the Southern Ocean would react similarly to changes in environmental conditions or whether the environment would be more important to set physiological constraints (phenotype) than the genotype. Here we tested the salinity tolerance of five different isolates from three different locations. Genetically close isolates did not respond similarly to conditions tested. Rather, a geographical based response could be shown, and we conclude that certain ecotypic responses predominate over genotypic differences in each geographic region sampled. Each ecosystem had developed a highly diverse population and we conclude that this diversity provided the populations in each ecosystem the ability to respond to the environmental parameters of that ecosystem.
Keywords: ecosystem resilience, physiological responses, Phaeocystis antarctica, salinity regimes, genetic diversity
157 Introduction
The Southern Ocean (SO) is one of the harshest environments on earth. Temperatures can be very low; the light regime is rather extreme with no or little light in winter and extensive solar radiation during the summer months. Thus, photoperiods are highly variable, showing wide ranges in photon flux densities (Tang et al. 2009). The SO can be covered by sea ice up to approximately 60° S during the austral winter and ice cover decreases southward in summer.
Antarctic sea ice coverage has a much greater seasonal variation than Arctic sea ice and is an important parameter in the global climate system (King and Turner, 1997; DeLiberty et al., 2004). Because of its circulation patterns, the SO exhibits a unique oceanographic regime. It is the only ocean that connects the Atlantic, Pacific and Indian Oceans. The Antarctic Circumpolar Current (ACC), encircling the Antarctic driven partly by the vigorous mid-latitude westerly winds and affected by adjacent landmasses and submarine topography, is the main current, transporting a water volume of 130 Sv (1 Sverdrup = 1 Sv = 1x106 m3 s-1) along a 24 000 km path, varying in depth and width (Rintoul et al. 2001, Boning et al. 2008, Thompson 2008). The ACC is also unique because no continental barriers exist in the latitudes spanning the Drake Passage (the gap between South America and the Antarctic Peninsula), which allows the current to close upon itself in a circumpolar loop (Thompson 2008). Closer to the Antarctic continent, smaller ocean currents appear, which are more complex and form two well defined gyres: the Weddell Gyre and the Ross Gyre. Ocean boundaries in the SO caused by the current patterns delimit specific Antarctic marine ecosystems, such as the ACC system. These specific ecosystems offer a high diversity of life but also the capability of establishing genetically distinct populations within a species. The role of the oceanic currents in the dispersal/distribution of Antarctic marine organisms has been investigated for a long time, but species gene flow around the Antarctic and between gyres is documented only in a few cases (Marr 1962; Amos 1984; Deacon 1984, Patarnello et al. 1996, Zane et al. 1998, Rogers et al. 1998, Bargelloni et al. 2000, Copley and Young 2006, Copley et al. 2007).
Most studies show that oceanographic barriers could be sufficiently strong and temporally stable to restrict gene flow between distinct areas (Zane et al. 1998). But because the effects of the water mass movements are greater on small organisms than larger ones, small organisms, such as phytoplankton, can have a distribution largely shaped by water circulation and thus a global gene flow (Patarnello et al. 1996).
Diatoms are generally regarded as the main contributors for biomass build-up in the SO, but nanoflagellates and other phytoplankters, such as the prymnesiophyte, Phaeocystis antarctica, sometimes dominate in certain regions and contribute to primary produced biomass in the Antarctic pelagic system.
The genus Phaeocystis plays an important role in ecology and biogeochemical cycles in almost all marine ecosystems. It contains two species that are dominant in polar regions: P.
antarctica Karsten and P. pouchetii Scherffel. P. antarctica is the most abundant primary producer in the Southern Ocean, where it regularly forms large, virtually, monospecific blooms. It can contribute >90% of total phytoplankton abundance and up to 65% of the annual primary production. Its ability to form monospecific blooms could indicate a life-history strategy that may be more successful than the competition of single cells with other phytoplankton (Verity et al. 2007). Blooms of P. antarctica have been shown in the SO to dominate both deeply relatively weakly stratified waters (Arrigo et al. 1999) and shallow mixed layers (von Bodungen et al. 1986). This illustrates the ability of this species to adapt easily to varying environmental conditions. P. antarctica shows also a much higher drawdown of carbon dioxide and nitrate per mole of phosphate and rate of new production than do diatoms. It is one of the major organisms within the biological community capable of drawing down atmosphericCO2 and transporting it to the deep ocean (Arrigo et al. 1999). P.
antarctica is also known to be a major producer of dimethylsulphoniopropionate (DMSP), which is known not only to act as cryoprotectant in algae (Kirst et al. 1991, Karsten et al.
1996, Stefels 2000), to serve as an antioxidant system (Sunda et al. 2002) or possibly to maintain intracellular osmotic pressure (Dickson et al. 1982, Vairavamurthy et al. 1985, Dickson and Kirst 1986, 1987a, 1987b), but also to deter herbivores (Wolfe et al. 1997).
DMSP is the precursor of dimethylsulfide (DMS) known as a cooling gas for the atmosphere serving as cloud condensation nuclei for cloud formation (Charlson et al. 1987).
The distribution of P. antarctica varies throughout different regions of the SO. Populations of P. antarctica within Antarctic continental boundary water masses appear to be well-mixed because currents move around the Antarctic continent rather quickly, which may prevent a defined population structure (Medlin and Zingone 2007). Because these various regions are ecologically diverse, P. antarctica may also be genetically diverse to accommodate to these broad ecological conditions.
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Genetic diversity is most commonly measured by fingerprinting techniques, of which Amplified Fragment Length Polymorphisms (AFLPs) and microsatellites (MS) are most those often used (Chavarriaga-Aguirre et al. 1999, Mariette et al. 2001, De Bruin et al. 2003, Lamote et al. 2005). AFLPs can reveal higher diversity than MS in recently diverged populations, because they show polymorphic genetic loci over the entire genome (Alpermann et al. 2009). A subset of isolates of four Antarctic Regions: Prydz Bay, Scotia Sea, Weddell Sea and Antarctic Circumpolar Current were used for AFLP analysis. Preliminary genetic analysis demonstrated an extremely high diversity within P. antarctica (Fig. 7.1; Gäbler et al.
2007, Gäbler-Schwarz unpubl. data) with isolates from different regions being genetically closely related to isolates from other regions. Thus, the four regions sampled were not genetically isolated from one another. This finding led us to assess (1) whether genetically closely related strains originating from different geographic regions in the SO react similarly to changes in environmental conditions or (2) whether the environment is more important to set physiological constraints (phenotype) than the genotype. In order to test both hypotheses, we conducted experiments on growth, photosynthetic efficiency (PAM) and DMSP content from five P. antarctica strains from three different Antarctic regions: Prydz Bay, Ross Sea (both ice covered most of the year) and Scotia Sea (open water, little influence from sea ice).
Material and Methods
Cultures and Culture Conditions for the physiological experiments
Five unialgal cultures of the prymnesiophyte P. antarctica (two of which were isolated from Prydz Bay in 1989, one from Ross Sea in 2003 and two from Scotia Sea in 2005, Antarctica) were maintained at 0°C in sterile filtered GP5 media (33 psu, Loeblich and Smith, 1968, Loeblich 1975) (Table 1, Fig. 7.2a). In each case, isolates from the same region originated from the same bloom and even the same bucket of water. Cultures contained both flagellate and colonial stages of P. antarctica and salinity responses were tested in GP5 media under three different salinity conditions: 18psu, 33psu and 70psu over a 60 days period. Isolates were either directly shocked on day 0 by inoculation directly into 18psuD and 70psuD GP5 media from 33psu (D=direct) or gradually accommodated to 70psu over a week (5.3psu*d-1).
The latter isolates will be hereafter referred to as 70psuG (G=gradual). A gradual acclimation to 18psu was not performed to the tested strains, because pre-experimental tests showed that isolate PrydzBay_1 was not inhibited by hyposaline conditions. Five replicates per sample were used for each salinity treatment. Samples were taken for the analysis described below
three times on day 0 (at 0h, 2h and 6h after inoculation), once on day 1 and day 2 and then once every 2nd day until day 20. Next measurements started on day 24, day 28, day 36 and day 60.
Vitality assessment via microscopy
The investigated strains were checked each sampling day microscopically by bright field microscopy with a ZEISS Axioplan microscope, magnification 63x to 400x and photographed using a digital camera (ColorView softimaging system) to document their vitality and fitness.
Chlorophyll a measurement
In order to follow the biomass development algal subsamples were filtered onto GF/C Filters (Whatman). The concentration of chlorophyll a was determined fluorometrically using a Turner Fluorometer after extraction in 90% acetone according to Evans and O'Reilly (1983).
Pulse Amplitude Modulated (PAM) fluorometry
Variable chlorophyll a fluorescence, measured with Pulse Amplitude Modulated (PAM) fluorometry, was applied as a proxy to monitor physiological integrity of the photosynthetic apparatus. Maximum quantum yield (Fv/Fm) was determined using a Xenon-PAM Fluorometer (WALZ GmbH Germany) equipped with a temperature control unit and a magnetic stirrer and controlled by a personal computer with the Fluorwin 3.5 Software and a FL-100 control unit (Photon System Instruments s.r.o. Czech republic) . The maximum quantum yield was calculated from fluorescence readings of dark acclimated samples as:
Fv/Fm = Fm – F0/Fm where Fm and F0 denote the maximum and minimum fluorescence in a dark acclimated sample (Krause and Weis 1991, Maxwell and Johnson, 2000). Dark adaptation was 12 min at 4°C, sufficient to attain stabilization of the fluorescence signal for all light regimes.
Gas chromatography – DMSP measurements
Algal subsamples were filtered onto GF/C Filters (Whatman), frozen instantly in liquid nitrogen, and stored at – 80°C until further processing. To detect DMSP, an indirect method after White (1982) was used. 5 ml 6.25M NaOH solution was added to each frozen GF/C filter, and the vial was immediately sealed with a Teflon-lined crimp cap seal (Latek, Eppelheim). After 20 to 24 h of alkalinisation, DMS derived from cellular DMSP, which was trapped in the headspace of the vial. This gas was then injected with a gas-tight syringe
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(Hamilton Bonaduz AG, Bonaduz, Schweiz) into a gas chromatograph (GC-9A, Shimadzu, Kyoto) (GC). GC analysis conditions were as follows: injector temperature: 150°C; detector temperature: 175°C; oven and the column temperature: 60°C. A Teflon column (3 m ´ 3 mm i.d., Hefu and Kirst 1997), filled with 60/80 mesh Chromosil 330, was used with N2 (quality:
4.6) as carrier gas at a flow rate of 60 ml/min. Hydrogen (quality: 5.0) pressure was 0.75 kg/cm2 and air pressure was 0.75 kg/cm2. The GC was equipped with a Flame Photometric Detector, which had a minimum sensitivity of 10 pmol DMS. Quantification was made by comparison with a DMSP standard (produced by Research Plus, Bayonne, N.J.).
Statistics
Statistical analyses were performed using R (R Development Core Team, 2008). Data of chlorophyll a fluorescence parameters, PAM parameters and their possible connection to Sea ice coverage are presented as means±SD of five replicates for each sample. Data were analyzed by repeated measurement ANOVA.
Results
Responses of the five P. antarctica isolates to three different salinity regimes: 18psu, 33psu, and 70psu, were tested over a period of 60 days. Isolates were either shocked on day 0 by direct inoculation into 18psuD and 70psuD GP5 media (D=direct) from 33psu or gradually accommodated to 70psu over a week (5.3psu*d-1). The latter 70psuG (G=gradual) isolates showed the same growth patterns and responses as the 70psuD isolates. Because of this, only data of the direct salinity experiments were analyzed further. All five replicates in this study reacted similarly.
Vitality assessment via microscopy
Figure 7.3 shows an overview of the investigated strains within the exponential growth phase for the different salinities (18psuD, 33psu and 70psuD). Except for PrydzBay_1 (18psu, day 8), RossSea_1 (18psu, day 36), ScotiaSea_1 (18psu, day 16), and PrydzBay_1 (70psu, day 36), all photos were taken on day 12 of the experiment to document cell integrity, because these strains had not yet reached or already left their exponential growth phase on day 12. The two strains from Scotia Sea (ScotiaSea_1 and ScotiaSea_2) were an exception, because they did not survive treatment in low and high salinities (marked by a cross in the photo). Cells at higher salinity appeared more granulated and more deeply pigmented than controls at 33 psu (see Fig. 7.3, PrydzBay_1 70psu). Also a phenotypic difference could be seen between Scotia
Sea isolates and those from the other regions. Scotia Sea isolates consisted of many, very small and condensed colonies, whereas the isolates from Prydz Bay and Ross Sea had larger colonies in their cultures.
Chlorophyll a measurements
Figure 7.4 shows box plots of chlorophyll a (Chl a). All control isolates (33psu) of all three sampling regions started their exponential phase on day eight and went into stationary phase around day 18. The chlorophyll a concentration varied among the isolates. The isolate PrydzBay_1 directly shocked with 18psu had a shorter but prolonged lower growth, starting its exponential growth phase shortly before day 8. The isolate, PrydzBay_2, directly shocked with 18psu, also showed a shorter but prolonged lower growth. The Ross Sea isolate, directly shocked with 18psu, appeared to have a much longer lag phase and prolonged lower growth, starting its exponential growth phase around day 28. The isolates of these two regions directly shocked with 70psu showed a nearly similar pattern, but at this salinity isolate PrydzBay_1 showed a much longer lag phase and started its exponential growth phase around day 28 and the two other isolates PrydzBay_2 and RossSea_1 showed a prolonged lower growth, starting their exponential growth before day 12.
Both isolates from Scotia Sea did not survive the low and high salinity treatments.
Pulse Amplitude Modulated (PAM) fluorometry
Figure 7.5 shows box plots of PAM measurements. The photosynthetic efficiency (Fv/Fm) varied among the isolates and showed the same trend as the chlorophyll data described above.
All control isolates (33psu) showed a very high photosynthetic efficiency. All of them reached at least a maximum quantum yield between 0,5 and 0,6 (mean) on minimum one day within their growth period. The Prydz Bay isolates showed hereby the highest amount of time (five days) keeping the maximum quantum yield from 0,5-0,6. Isolate PrydzBay_2 even reached a maximum quantum yield between 0,6 and 0,7 (four days). The Prydz Bay and Ross Sea samples directly shocked with 18psu and 70psu showed a lower photosynthetic efficiency and a prolonged lag phase. Isolates from Scotia Sea did not survive the salinity treatments.
To determine the statistical significance of the difference in the photosynthetic efficiency responses of the strains isolated from temporally ice covered (Prydz Bay, Ross Sea) vs. not covered (Scotia Sea) areas, we performed a repeated measurement ANOVA. The p value of the comparison (<2e-16) showed that the differences described above were also statistically highly significant.
163 Gas chromatography – DMSP measurements
Figure 7.6 shows boxplots of DMSP measurements. Samples for DMSP measurements were taken regularly, but we only analyzed data from the following days: day 0; 0+2h; 2; 12; 20;
28; 36 and day 60. The DMSP content in all isolates from Prydz Bay and Ross Sea showed similar results within all salinity treatments, increasing while the culture aged and reaching the highest point on day 60. The DMSP content of the control isolate (33psu) from Ross Sea was the highest, but it also had the lowest chlorophyll a content in the control. Prydz Bay and Ross Sea isolates within the lower or higher salinities showed a much lower DMSP content without increasing over time, except isolate PrydzBay_1 in 70psu, which showed a slight DMSP content increase on day 60. All Scotia Sea isolates had a much lower DMSP content in the control isolates and none to very little in the lower and higher salinities, because they died.
Discussion
We tested the phenotypic response of five genetically distinct P. antarctica isolates originating from three different Antarctic regions: Prydz Bay, Ross Sea and Scotia Sea (PrydzBay_1, PrydzBay_2, RossSea_1, ScotiaSea_1 and ScotiaSea_2). We tested their growth, fitness (based on photosynthetic efficiency, PAM) and DMSP content within three different salinity regimes (18psu, 33psu, 70psu). In each case, isolates from the same region originated from the same bloom or bucket of water and it could be assumed that these isolates were closely related genetically. However an AFLP analysis with 34 P. antarctica isolates showed that the Weddell Sea Gyre (including Scotia Sea isolates) and Prydz Bay isolates tested, were not genetically isolated from each another, because some isolates from both sites were closely related to each other (Fig. 7.1). One Scotia Sea isolate (ScotiaSea_1) was genetically close the Prydz Bay isolates (PrydzBay_1), and both of these were distinct from the other isolates from the same gyre. Thus, our main aim was to test if genetically closely related isolates would react similarly to environmental changes in salinity although coming from different environments (sea ice covered – open water) being geographically apart (Table 7.1, Fig. 7.2a). The results indicate that these genetically close isolates did not react similarly to certain changes of environmental parameters. Rather a geographical based response occurred, because isolates from the same region, although genetically distant, reacted similarly. The two isolates from the Scotia Sea died nearly instantly after being shocked with either a lower or higher salinity, whereas the Prydz Bay and Ross Sea samples only showed a delayed growth in comparison to their control at 33psu. This was supported from chlorophyll
a and PAM measurements (Fig. 7.4 and 7.5). The three regions differ in their environmental characteristics, especially in their sea ice coverage (Fig. 7.7). Stroeve and Meier (1999, 2008) documented the mean sea ice coverage in the entire Antarctic Region from 1979 to 2007, and the Scotia Sea has not been covered with sea ice for at least the last ~30 years, whereas the two other regions, Prydz Bay and Ross Sea, are only ice free during the Antarctic summer months (Fig. 7.7b-c). We assume that the ability of a strain to adapt to salinity variations is dependent on the dynamics of sea ice coverage. This is likely the main reason why the Scotia Sea isolates died in lower and higher salinities. These isolates do not regularly experience any melting or freezing events, resulting in a low adaptation potential to survive at lower or higher salinities. Of course, the salinity regime in the SO open water regions would not change as rapidly as the salinity regimes used in this study, but nevertheless, only the three isolates originating from sea-ice-covered regions survived all treatments. This is because during sea ice formation and sea ice melting processes strong gradients in the salinity regimes can be observed. Fairly high salinities are found in the brine channels during freezing and throughout the winter. Salinity increase of dense shelf water is recorded because of brine rejection during the further formation of Antarctic sea ice (Toggweiler and Samuels, 1995). Low salinities occur when sea ice melts, with surface salinities near freshwater.
Along the western Antarctic Peninsula, changes in the phytoplankton community have been associated with rapid regional climate changes, with sea ice dependent species being replaced by ice avoiding ones (Montes-Hugo et al. 2009). We could infer the same scenario for P.
antarctica populations under climate change scenarios. As climate change proceeds and the ice coverage diminishes, ice tolerant strains in the Weddell Sea could be replaced by the more northerly ice intolerant strains from the Scotia Sea.
But what happens to P. antarctica cell when it experiences sudden salinity increases? DMSP supposedly has many different functions within cells, such as a cryoprotectant (Kirst et al.
1991, Karsten et al. 1996, Stefels 2000), an antioxidant (Sunda et al. 2002) or a herbivore determent (Wolfe et al. 1997), but also a possible osmolyte (Vairavamurthy et al. 1985, Dickson and Kirst 1986, 1987a, 1987b). DMSP measurements were carried out in our study because P. antarctica is known to be a major producer. Our measurements showed an increase at a salinity of 33psu as the cultures aged. We only measured significant DMSP concentrations in all five isolates when nutrient depletion occurred (Fig. 7.6). Chl a concentrations already decreased (Fig. 7.4). DMSP increase was most pronounced in the isolates from the seasonally ice covered regions Prydz Bay and Ross Sea. Our observations agree with those made by Groene and Kirst (1992) and Keller and Korjeff-Bellows (1996)
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who showed that nitrogen deficiency leads to a higher cellular DMSP accumulation. The different salinity treatments had no effect on the DMSP production in Phaeocystis because there was no real increase to be seen in lower and higher salinities. This could be because we only processed the filtered cells and not the culture media, so we assume that the DMSP could have been excreted from the cells to maintain intracellular osmotic pressure at 18 psu as described for other algae (Vaivaramurthy et al. 1985, Dickson and Kirst 1986, Dickson and Kirst 1987a, b) and we did not measure it. No increase of DMSP occurred in Phaeocystis with higher salinity tested; therefore DMSP content is not correlated to higher salinity in P.
antarctica. This is in contrast to Stefels et al. (1996) who found DMSP accumulation in Phaeocystis sp., which were grown up to a salinity of 50 psu. However our data agree with several other studies in marine algae and higher plants that showed no change in DMSP concentrations under hypersaline conditions (Van Diggelen et al., 1986; Edwards et al., 1987;
Colmer et al., 1996, Stefels et al. 2000). We therefore assume, that from a certain salinity concentration, other osmolytes, such as proline, glycine or betaine, which were not measured in our study, were more likely produced as the main osmolytes in P. antarctica as already described for other microalgae (Kirst 1995, Kirst 1996, Nothnagel 1995).
The three sampling regions are ecologically different, and we find different geographical based responses from our tested isolates. From our previous genetic studies, we conclude that each ecosystem is genetically diverse. However there is an overall phenotypic response by the isolates from each ecosystem. This would suggest that the ecosystem resilience is an important factor maintaining population structure in Antarctic continental waters. Ecosystem resilience was firstly defined by Holling (1973) and is the amount or magnitude of disturbance that an ecosystem can experience before it shifts into a different state (stability domain) with different controls on structure and function (Gunderson 2000, Folke et al. 2004). We assume that each ecosystem has evolved genetically diverse populations in parallel in some extent as shown by our AFLP analyses. Similiar AFLP patterns just shows that the same genetic loci are present but not how often specific loci, i.e. those responsible for salinity acclimation, is expressed and therefore making the cell able to react to changing conditions in time. P.
antarctica is very successful in the Antarctic ecosystem and a key algae in the phytoplankton community. Each bloom consists of many different genetic clones but we assume that all clones from a single ecosystem/gyre, although genetically distinct and possibly closely related to isolates from a different ecosystem/gyre, are capable of reacting in a similar manner to changing environmental conditions.
In order to understand, how these geographical responses could have originated, we need to examine the geological history of the Antarctic. The Antarctic, as we know it today, originated 30 million years ago (MA) in the late Oligocene with the opening of the Drake Passage between the Antarctic Peninsula and Southern America. This opening allowed the development of the ACC around the Antarctic between 40° and 60° degrees south. This stopped the transport of surface water from south to north and led to a temperature decrease in the southern hemisphere followed by a glaciation of Antarctica. Prior to this, the temperature distribution was symmetrical from the equator to the poles. Both the Weddell Sea Gyre and the Ross Sea Gyre existed and water was exchanged between this part of the globe and the northern hemisphere (Lawver and Gahagan 1998, Bjornsson and Toggweiler 2001). After the Drake Passage opened, these gyres became isolated and the phytoplankton populations had to adapt to the new environmental conditions caused by the glaciations of Antarctica. So from the scenario described above by Montes-Hugo et al. (2009) that changes in the phytoplankton community are associated with rapid regional climate changes such that sea ice dependent species are replaced by ice avoiding ones, we could speculate that should climate change be reversed, the ice intolerant strains that could move from the Scotia Sea to the Weddell Sea could be driven to extinction in one season following ice cover and the entire ecosystem would collapse. This implies further consequences to climatic changes because the carbon cycle can become even more imbalanced for a certain period of time. Thus, Phaeocystis antarctica is one of the major organisms within the biological community capable of drawing down atmosphericCO2 and transporting it to the deep ocean in ice covered regions (Arrigo et al. 1999). The same holds true for the DMS production, which will be reduced leading towards a hotter world.
Conclusions
Our analysis has shown that the isolates from the Scotia Sea and Prydz Bay were genetically similar indicating gene flow between the two gyres. However, because genetically similar isolates from Scotia Sea and Prydz Bay were phenotypically different, this would suggest that each gyre has diversified genetically in parallel while maintaining the phenotypic responses needed to survive the ecological conditions characteristic of each gyre. We believe this to be one of the few examples published documenting the genetic potential of an ecosystem to maintain resilience.
167 Acknowledgements
This research was founded by the German Science Foundation (DFG) through a postgraduate research fellowship (ME 1480/2).
We want to thank N. Hoch and E. Allhusen for help with PAM setup, S. Murawski for help with Chl a filter processing and. Prof. Dr. K. Bischof for offering help and laboratory capacities at University of Bremen (Germany) for the DMSP measurements. We acknowledge the source `The GEBCO_08 Grid, version 20081212, http://www.gebco.net`.
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