Light Intensity Modulates the Response of Two Antarctic Diatom Species to Ocean Acidification

It is largely unknown how rising atmospheric CO2 concentrations and changes in the upper mixed layer depth, with its subsequent effects on light availability will affect phytoplankton physiology in the Southern Ocean. Linking seasonal variations in the availability of CO2 and light to abundances and physiological traits of key phytoplankton species could aid to understand their abilities to acclimate to predicted future climatic conditions. To investigate the combined effects of CO2 and light on two ecologically relevant Antarctic diatoms (Fragilariopsis curta and Odontella weisflogii) a matrix of three light intensities (LL = 20, ML = 200, HL = 500 μmol photons m−2 s−1) and three pCO2 levels (low = 180, ambient = 380, high = 1000 μatm) was applied assessing their effects on growth, particulate organic carbon (POC) fixation and photophysiology. Under ambient pCO2, POC production rates were highest already at low light in Fragilariopsis, indicating saturation of photosynthesis, while in Odontella highest rates were only reached at medium irradiances. In both species ocean acidification did not stimulate, but rather inhibited, growth and POC production under low and medium light. This effect was, however, amended under high growth irradiances. Low pCO2 levels inhibited growth and POC production in both species at low and medium light, and further decreased absolute electron transport rates under high light. Our results suggest that Southern Ocean diatoms were sensitive to changes in pCO2, showing species-specific responses, which were further modulated by light intensity. The two diatom species represent distinct ecotypes and revealed discrete physiological traits that matched their seasonal occurrence with the related physical conditions in Antarctic coastal waters.


INTRODUCTION
Concentrations of atmospheric carbon dioxide (CO 2 ) are predicted to rise from 400 µatm today to over 750 µatm by the end of this century affecting carbonate chemistry in ocean surface waters by increasing dissolved inorganic carbon availability and decreasing pH (IPCC, 2014).This will potentially affect the physiology and ecology of primary producers (Tortell et al., 2008;Trimborn et al., 2013Trimborn et al., , 2014)).Elevated concentrations of CO 2 in surface waters can be beneficial to phytoplankton, because at present-day the amount of aqueous CO 2 accounts for ∼1% within the total inorganic carbon pool (Zeebe and Wolf-Gladrow, 2001).As the enzyme Ribulose-1,5-bisphosphate carboxylase-oxygenase (RubisCO) requires CO 2 as substrate for the production of particulate organic carbon (POC) during photosynthesis, most phytoplankton, including several Southern Ocean (SO) species, operate so-called carbon concentrating mechanisms (CCMs) (Reinfelder, 2011;Trimborn et al., 2013).CCMs are energetically costly as they include active transport of CO 2 and bicarbonate (HCO − 3 ) across cell membranes, operation of the enzyme carbonic anhydrase (CA, interconverting CO 2 and HCO − 3 ) and prevention of diffusive CO 2 efflux (Raven and Johnston, 1991;Hopkinson et al., 2011).Increasing pCO 2 in surface waters will lead to an increase in the fraction of aqueous CO 2 available to phytoplankton, allowing cells to potentially down-regulate their CCMs, which can save up to ∼20% of CCM-related energy expenditures (Hopkinson et al., 2011;Reinfelder, 2011).Downregulation of CCMs is expected to promote growth through energy savings especially under limiting conditions such as light-or iron-limitation (Hopkinson et al., 2011).Increased CO 2 concentrations have been shown to influence Southern Ocean phytoplankton growth and community composition (Tortell et al., 2008;Feng et al., 2010;Hoppe et al., 2013).Laboratory studies on Antarctic phytoplankton species, however, did not find stimulating effects by elevated pCO 2 on growth (Boelen et al., 2011;Hoppe et al., 2015).
Alongside elevated atmospheric CO 2 concentrations, sea surface temperatures are on the rise, thus influencing water column stratification.The expected outcome is a shallower upper-mixed layer (UML), in which phytoplankton cells become exposed to higher mean light intensities (Boyd et al., 2015b).As major parts of the Southern Ocean are generally assumed to be light limited, these changes could be beneficial for phytoplankton species (Mitchell and Brody, 1991;Mitchell and Holm-Hansen, 1991;Nelson and Smith, 1991).Yet, increased westerly winds (predicted trends for the Southern Annular Mode) could lead to opposite effects, causing a deepening of the UML (Hauck et al., 2015) thereby decreasing light availability to phytoplankton and thus creating an unfavorable environment.
The amount of available light is substantial for phytoplankton growth and photosynthesis.Light limiting conditions can be stressful for phytoplankton species as light-harvesting has to become more efficient in order to sustain electron transport between photosystems (MacIntyre et al., 2002;Dubinsky and Stambler, 2009) and thereby generation of energy equivalents necessary for carbon fixation in the Calvin-Benson-Cycle (Falkowski and Raven, 2007).If the generation of energy equivalents and carbon fixation through photosynthesis gets restricted by a deficit in excitation energy capture, the energy demands within the phytoplankton cell cannot be met, leading to reduced metabolic rates (Shi et al., 2015).
Oversaturating light intensities on the other hand can lead to photoinactivation of photosystem II (PSII) reaction centers and, when not fully counteracted by repair mechanisms, can cause photoinhibition and cell damage (Murata et al., 2007;Raven, 2011).Therefore, most phytoplankton species possess various dissipation pathways such as non-photochemical quenching (NPQ).The NPQ involves pigment de-epoxidation that can be induced within seconds upon changes in light intensity (Krause and Weis, 1991;Müller et al., 2001).Antarctic diatoms rely on the xanthophyll cycle for photoprotection (Kropuenske et al., 2009;Arrigo et al., 2010;Mills et al., 2010) involving the pigment diadinoxanthin (DD), which can be de-epoxidised to diatoxanthin (DT), thereby dissipating excess energy captured by light-harvesting pigments (reviewed in Goss and Lepetit, 2014).The adjustment of the functional absorption cross section of PSII (σ PSII ) rather than the number of photosynthetic units per cell is considered to act as an important photoacclimation strategy particularly in polar diatoms (Kropuenske et al., 2009(Kropuenske et al., , 2010;;Strzepek et al., 2012).Photoacclimation strategies of polar phytoplankton could be related to species-specific occurrences during the season, as changes in vertical mixing with its effects on light availability influence the seasonal succession of phytoplankton species (Mitchell and Holm-Hansen, 1991;Garibotti et al., 2005).
Next to light availability, also the partial pressure of CO 2 (pCO 2 ) of Antarctic coastal waters substantially varies over the season with reported values from 190 to 560 µatm (Kapsenberg et al., 2015).During winter time, sea ice prevents gas exchange between ocean surface waters and the atmosphere, causing the pCO 2 to exceed atmospheric levels after sea-ice retreat in spring (Sweeney, 2003;Arrigo et al., 2008).While in spring the UML is deep and phytoplankton needs to cope with light-limiting conditions, during summer the increasing surface warming creates a stable and shallow upper water body with high irradiances (Mitchell and Holm-Hansen, 1991;Nelson and Smith, 1991).Solar irradiances increase during the season promoting phytoplankton blooms.Through high rates of photosynthetic carbon fixation, especially towards the end of the bloom, pCO 2 can be drawn down to or even below preindustrial values in Antarctic surface waters (Sweeney, 2003;Arrigo et al., 2008;Kapsenberg et al., 2015).Thus light and pCO 2 are important factors influencing phytoplankton in Antarctic coastal waters, which unlike the open waters of the Southern Ocean display high iron concentrations and thus exhibit regular and extensive bloom events over the season (Holm-Hansen et al., 1989;Martin et al., 1990;Prézelin et al., 2000;Garibotti et al., 2003;de Jong et al., 2012).The frequent occurrence of pronounced blooms in coastal waters and their immense productivity make them very important for the global carbon cycle and thus important to study (Comiso et al., 1993;Arrigo et al., 2008).
At present it is largely unknown what the combined effects of pCO 2 and light on Southern Ocean phytoplankton physiology are.For various temperate phytoplankton species, elevated CO 2 concentrations in combination with low light were found to stimulate growth and carbon fixation, while the combination with high light showed reduced primary production and increased light stress in a natural phytoplankton community from the South China Sea (Gao et al., 2012).Whether similar responses can be expected for Southern Ocean phytoplankton species remains yet unclear.Studies on the combined effects of light and pCO 2 on Antarctic phytoplankton are sparse and results are diverging.The diatom Proboscia alata showed elevated particulate organic carbon content with increasing pCO 2 when combined with a high constant irradiance, whereas the opposite was true for low light conditions (Hoogstraten et al., 2012).For the Antarctic diatom Chaetoceros brevis, growth remained unaffected by pCO 2 irrespective of the dynamic light regime applied (Boelen et al., 2011).Yet, in the diatom C. debilis, under dynamic light, elevated pCO 2 decreased primary production (Hoppe et al., 2015).Experiments on multiple stressors including not only pCO 2 and light, but also temperature, nutrients and iron, found that light increased growth, when nutrients were not limiting, while pCO 2 did not have any effect (Xu et al., 2014;Boyd et al., 2015a).There are great differences in the definition of high and potentially stressful irradiances that are used in studies investigating effects of constant irradiances in temperate and Antarctic phytoplankton species (125 to 380 µmol photons m −2 s −1 ; Kropuenske et al., 2010;Mills et al., 2010;Norici et al., 2011;Hoogstraten et al., 2012).However, applied irradiances often are below the extremes that phytoplankton cells can encounter in shallow mixed coastal waters with reported values of integrated daily mean irradiances ranging between 77 and 740 µmol photons m −2 s −1 under constant light in a 16:8 light:dark cycle (Lancelot et al., 1993;Moline and Prézelin, 1996;Venables et al., 2013).Studies on fluctuating light regimes applied higher irradiances which reach levels between 250 and 1200 µmol photons m −2 s −1 .Yet, exposure to these high irradiances occured for short and repeated periods (Kropuenske et al., 2009;Mills et al., 2010;Boelen et al., 2011;Xu et al., 2014;Boyd et al., 2015a;Hoppe et al., 2015).In a future ocean, the mixed layer depth of Antarctic coastal waters may decrease potentially implying higher integrated daily irradiances including longer periods of light exposure that phytoplankton encounter (Boyd et al., 2015b).Therefore, it is important to study also the effects of exposure to very high and persisting light conditions on phytoplankton physiology.
This study aims to investigate the interactive effects of pCO 2 and light availability on two Antarctic bloom-forming species typically occurring within spring (Fragilariopsis curta) and summer (Odontella weisflogii) in iron replete Antarctic coastal waters (Martin et al., 1990;Garibotti et al., 2005;Annett et al., 2010;de Jong et al., 2012).To better understand their physiological responses under different light and CO 2 scenarios a matrix of three pCO 2 conditions (low = 180, ambient = 380 and high = 1000 µatm) was applied in order to span the range of late-bloom to future ocean acidification (OA) conditions.These were combined with light conditions chosen to represent low, medium and high irradiances (LL = 20, ML = 200 and HL = 500 µmol photons m −2 s −1 , respectively).The physiological responses of both species were studied on growth, carbon fixation, pigment content and photophysiology.We aimed to investigate whether increased pCO 2 levels, at limiting light conditions, may promote growth and productivity due to lowered energy expenditures for CCM operation.Also we tested whether high pCO 2 in conjunction with high light availability may synergistically trigger light stress.This study further tried to link the seasonal occurrence of the two phytoplankton species with respect to their physiological characteristics.

Culture Conditions
Semi-continuous cultures of the two diatom species Fragilariopsis curta (isolated from the Weddell Sea by Thomas Mock during ANT XVI/3 in 1999) and Odontella weisflogii (isolated in the Atlantic sector of the Southern Ocean by Bank Beszteri during ANT-XXIX/5, 2013) were grown at 4 • C in sterile-filtered (0.2 µm) unbuffered natural Antarctic seawater (30.2) enriched with silicate, trace metals and vitamins according to F/2 medium (Guillard and Ryther, 1962).Nitrate and phosphate were added in concentrations reflecting the Redfield N:P ratio of 16:1, 100 and 6.25 µmol L −1 respectively (Redfield, 1958).
Based on photosynthesis-irradiance-curves (PE-curves) with both species acclimated to ambient pCO 2 and 150 µmol photons m −2 s −1 light treatments were chosen.Both species were grown in triplicates under low, medium and high light (LL = 20, ML = 200, HL = 500 µmol photons m −2 s −1 ) conditions at a 16:8 h light:dark light cycle using light-emitting diodes (LED) lamps (SolarStinger LED SunStrip Marine Daylight, Econlux).The three light treatments were further continuously bubbled through a frit with humidified air of the three CO 2 partial pressures (pCO 2 ) of 180, 380, and 1000 µatm (low, ambient and high pCO 2 ).A gas flow controller (CGM 2000, MCZ Umwelttechnik) was used to generate the CO 2 gas mixtures from CO 2 -free air (< 1 ppmv CO 2 , Nitrox CO2 RP280, Domnick Hunter ltd.) and pure CO 2 (Air Liquide Deutschland ltd., Germany).Triplicates of both species were exposed to all light and pCO 2 treatment combinations.All replicates of one experimental treatments were run in parallel.
For each light treatment chlorophyll a (Chl a) fluorescence was calibrated against cell number and pH, allowing us to monitor the status of the culture using fluorescence alone.Cultures were grown in 1 L glass bottles (custom made), kept in exponential phase and harvested at cell densities of 224,000 ± 97,000 and 732 ± 170 cells per mL for the small Fragilariopsis (∼5 µm) and the much larger Odontella (> 50 µm) respectively to prevent drift of carbonate chemistry.
Only during the pre-acclimation phase to all experimental conditions of at least 10 days, cultures were diluted two to three times using pre-equilibrated medium.During the main experiment, cells grew for 3.8 ± 1.7 days until all parameters were sampled.

Carbonate Chemistry
The pH was measured every other day and at the final sampling day using a pH-ion meter (pH-Meter 827, Metrohm), calibrated (3 point calibration) with National Institute of Standards and Technology-certified buffer systems.The pH remained constant at 8.39 ± 0.03, 8.02 ± 0.03, and 7.76 ± 0.02 for the low, ambient and high pCO 2 treatments respectively (Table 1).Total Alkalinity (TA) was measured by duplicate potentiometric titrations (TW alpha plus, SI Analytics, Brewer et al., 1986) of filtrated samples (Whatman GF/F glass fiber filters, ∼ 0.6 mm), having been stored at 4 • C in 150 mL borosilicate bottles until analysis.Certified reference material (CRMs provided by Prof. A. Dickson, Scripps, USA; batch no.111; reproducibility ±13 µmol kg −1 ) was used to correct TA for systematic errors.Dissolved inorganic carbon (DIC) samples were sterile-filtered (0.2 µm) and stored at 4 • C in 5 mL gas-tight borosilicate bottles without headspace until analysis.DIC was measured colourimetrically in duplicates with a QuAAtro autoanalyzer (Seal Analytical, Stoll et al., 2001).The analyzer was calibrated with NaHCO 3 solutions (salinity of 35, achieved by NaCl addition) with concentrations ranging between 1800 and 2300 mmol DIC kg −1 .Certified reference materials (reproducibility ± 8 µmol kg −1 ) were used to correct for errors in instrument performance such as baseline drifts.The carbonate system was calculated based on TA, pH, silicate, phosphate, temperature and salinity using the CO2Sys program (results shown in Table 1, Pierrot et al., 2006) choosing the equilibrium constant of Mehrbach et al. (1973) refitted by Dickson and Millero (1987).

Growth
Samples for cell counts of every treatment were fixed with acid Lugol's solution (10% final concentration) and stored at 3 • C in the dark until analysis.Cells were counted on an inverted light microscope (Axio Observer.D1; Zeiss) after sedimentation for 24 h in 10 mL Utermöhl chambers (Hydro-Bios).Growth rates were determined from samples taken right after at the beginning of the experiment (N 0 ) and at the day of sampling (N fin ) and for each sample >400 cells were counted.Cell specific growth rate (µ, day −1 ) was calculated as where t is the incubation duration in days.

Particulate Organic Carbon and Particulate Organic Nitrogen
Particulate organic carbon (POC) and nitrogen (PON) were determined from gently filtered (< 20 mmHg) subsamples of each treatment using pre-combusted GF/F filters (15h, 200 • C; Whatman).Samples were stored at −20 • C and defrosted prior to analysis (> 12 h, 60 • C), acidified with 0.1 mol HCl L −1 and dried over night at 60 • C. Samples were analyzed using an elemental analyzer (EURO EA Elemental Analyzer, Euro Vector).Contents of POC and PON were corrected using blank measurements and normalized to filtered volume and cell densities to yield cell quotas.Production rates of POC and PON, were calculated by multiplication of the cellular quota with the specific growth rate of the respective treatment.

Pigments
Pigment concentrations of chlorophyll a, chlorophyll c2, fucoxanthin, diadino-and diatoxanthin were determined using High-Performance-Liquid-Chromatography (HPLC).Samples were gently filtered onto GF/F (Whatman) filters, immediately frozen and stored at −80 • C for later analysis.Pigments were extracted in 90:10 acetone:water for 24 h at 4 • C in the dark and filtered (4 mm nylon syringe filters, 0.45 µm pore size, NalgeneC; Labware) prior to analysis.Analyses were performed on a LaChromElite R system consisting of a chilled autosampler L-2200, a DAD detector L-2450 (VWR-Hitachi International GmbH) and a Spherisorb ODS-2 column (25 cm × 4.6 mm, 5 µm particle size; Waters).The system used a LiChrospher R 100 RP-18 guard cartridge for pigment separation applying a gradient following Wright et al. (1991) detecting peaks at 440 nm which were identified and quantified via co-chromatography of pigment standards obtained from DHI Lab Products (ORT, Denmark) using the software EZChrom Elite ver.3.1.3.From concentrations of diadinoxanthin (DD) and its deepoxidised equivalent diatoxanthin (DT) the de-epoxidation state (DES) was calculated according to (2)

Chlorophyll a Fluorescence
Photophysiological parameters were measured using a Fast Repetition Rate fluorometer (FRRf, FastOcean PTX; Chelsea Technologies) in combination with a FastAct Laboratory system (Chelsea Technologies).Measurements were conducted at growth temperature (4 • C).Samples were dark-acclimated for 10 min prior to measurement.The duration of the dark acclimation phase was chosen after pre-testing different time intervals (5, 10, 20, 30 min) to ensure that all photosystem II (PS II) reaction centers were fully oxidized and nonphotochemical quenching was relaxed.Excitation wavelength of the fluorometer's LED was 450 nm with an automated adjustment of the light intensity (between 0.66 and 1.2 × 10 22 photons m −2 s −1 ).The single turnover mode was used with 100 flashlets saturation phase on a 2 µs pitch and 40 flashlets relaxation phase on a 40 µs pitch in order to cumulatively saturate PS II.Estimation of minimum (F 0 ) and maximum chlorophyll a (Chl a) fluorescence (F m ) was based on iterative algorithms for induction (Kolber et al., 1998) and relaxation phase (Oxborough et al., 2012).Minimum and maximum Chl a fluorescence were used to calculate the apparent maximum quantum yield of photochemistry in PS II (F v /F m ) according to the following equation: Additional Chl a fluorescence measurements of every treatment were performed in response to increasing incident irradiances (E) generating photosynthesis-irradiance-curves (PE-curves; irradiances ranged between 0 and 1800 µmol photons m −2 s −1 ) using 15 steps with an acclimation duration of 5 min per light step and six subsequent Chl a fluorescence measurements.From these fluorescence measurements, the light-adapted minimum (F ′ ) and maximum (F ′ m ) fluorescence of the single turnover acquisition was estimated.The effective PSII quantum yield under ambient light (F ′ q /F ′ m ) was derived according to the equation (F ′ m /F ′ )/F ′ m (Genty et al., 1989).Absolute electron transport rates (absETR, e − PSII −1 s −1 ) at the different light steps of the PE-curve were calculated as (Suggett et al., 2004(Suggett et al., , 2009;;Huot and Babin, 2010): where σ PSII is the functional absorption cross section of PSII photochemistry (in nm 2 quanta −1 ) and E denotes the instantaneous irradiance (µmol photons m −2 s −1 ).Light-use characteristics were analyzed by fitting irradiance-dependent ETRs according to Ralph and Gademann (2005), including maximum absolute ETR (ETR m ), minimum saturating irradiance (I K ) and maximum light utilization efficiency (α).The PE-Curve was followed by another 10 min of dark acclimation with single turnover flashlets in order to assess PSII recovery (yield recovery).Yield recovery was calculated from the F v /F m measured before and after the PE-curve and given as % of the initial F v /F m (before the PE-curve).Non-photochemical quenching (NPQ, Equation 5) was calculated following the Stern-Volmer equation.
From the single turnover measurement of dark-adapted cells, also the time constant for electron transport at the acceptor side of PSII (τ Qa , µs), the connectivity factor of adjacent PSII light-harvesting pigment matrices (p, dimensionless) and the concentration of functional PSII reaction centers ([RCII], nmol m −3 ), were derived according to Oxborough et al. (2012), using FastPro8 software (Version 1.0.50,Kevin Oxborough, CTG Ltd.).

Statistics
All data are given as replicate means (n = 3) ± SE.To test for significant differences between treatments, two-way analyses of variance (ANOVA) with additional normality (Shapiro-Wilk) and post hoc (Holm-Sidak method) tests were performed (α = 0.05).In addition to this, to test for direct effects between two particular treatments standard t-tests were used.All statistical analyses were carried out with SigmaPlot 12.3 (SysStat Software Inc.).Different letters in figures and tables indicate statistical differences between treatments based on post-hoc tests.

Growth Rates
Despite the large difference in cell size both investigated species showed comparable growth rates (Figures 1A,B).Several studies, investigating polar diatom species, of very different cell sizes, reported growth rates that are comparable to those found in this study (Gilstad and Sakshaug, 1990;Kropuenske et al., 2009;Arrigo et al., 2010;Boelen et al., 2011;Petrou et al., 2014).Hence, within polar diatoms growth rates generally seem not to be greatly affected by cell size, but much more by abiotic factors such as temperature and light availability.
Growth rates of Fragilariopsis curta (Fragilariopsis) generally increased from low (LL) to medium light (ML) conditions and slightly decreased toward high light (HL, Figure 1A).At ambient pCO 2 (380 µatm), Fragilariopsis increased growth by 20% from LL to ML, whereas it decreased growth by 58% between ML and HL conditions.Under high pCO 2 (1000 µatm), no significant light effects were detectable in growth.At low pCO 2 (180 µatm), however, a strong increase in growth (86%) occurred between LL and ML, with no detectable changes from ML to HL.There were no overall trends with increasing pCO 2 discernible within all light treatments.At LL, growth of Fragilariopsis was suppressed in low pCO 2 treatments compared to the other two pCO 2 treatments, whereas at ML both low and high pCO 2 treatments grew less than the ambient treatment, yet only the former was statistically significant.The reverse pattern occurred at HL, exhibiting decreased growth at ambient pCO 2 , 22% compared to low and 39% toward high pCO 2 treatments.
In Odontella weisflogii, (Odontella), in the ambient and high pCO 2 treatments growth increased from LL to ML by 58% and 79%, respectively, yet both pCO 2 treatments showed no changes between ML and HL (Figure 1B).Within the low pCO 2 treatments, no light effects on growth were found from ML to HL, yet at LL despite several attempts Odontella could not grow.At LL and ML, growth of Odontella decreased from ambient to high pCO 2 .No differences between pCO 2 treatments were detectable at HL.At ML and HL, low pCO 2 acclimated cells grew slower than the ones acclimated to ambient pCO 2 while they reached similar growth rates as cells grown at high pCO 2 .

Elemental Composition
The production rate of particulate organic carbon (POC) revealed species-specific trends (Figures 1C,D).In Fragilariopsis, at ambient pCO 2 POC production significantly decreased (84%) from LL to HL.Under high pCO 2 , POC production decreased by 51% from LL to ML and showed no further changes from ML to HL.Under low pCO 2, POC production increased about 86% from LL to ML and decreased about 67% from ML to HL.At LL, POC production rates increased about 91% from low to ambient pCO 2 and decreased by 39% in response to high pCO 2 .At ML, POC production decreased about 53% from low to high pCO 2 , whereas at HL no differences between pCO 2 treatments were found.
In Odontella, POC production rates increased from LL to ML by 86 and 93% at ambient and high pCO 2, respectively.At ambient pCO 2, POC production rates decreased from ML to HL.Within high and low pCO 2 treatments, no changes were detected between ML and HL.At LL and ML, the ambient pCO 2 treatments had higher POC production rates than high pCO 2 treatments.Low pCO 2 treatments revealed lower POC production rates at ML, when compared to the two other pCO 2 treatments.No differences between pCO 2 treatments were observed at HL.
In Fragilariopsis, the ratio of cellular carbon to nitrogen (C:N, mol mol −1 ) showed effects of both light and pCO 2 .Under all three pCO 2 treatments C:N ratios decreased from LL to ML, but increased from ML to HL under high pCO 2 (Figure 1E).Under LL, effects of pCO 2 became evident as C:N ratios were reduced under high compared to low and ambient pCO 2 , whereas the opposite was true under HL with increased C:N ratios under high pCO 2 .Odontella, however, showed no effects of light on the C:N ratio (Figure 1F).Only under ML, the ratio of C:N increased from low to ambient pCO 2 with no further increase toward high pCO 2 .

Chl a Fluorescence-Based Physiology
Photosynthesis-irradiance curves (PE-curves; Figure 2) displayed a strong light effect as maximum electron transport rates (ETRm), light saturation point (I K ) and light use efficiency (α, Table 2) increased with increasing irradiance in both Fragilariopsis and Odontella, but there were no differences in I k and α between LL and ML in both species.The pCO 2 did not affect absolute ETRs (absETR) within LL treatments in both species (Figures 2A,B).Under ML, ETRm and α increased from low to ambient pCO 2 in both species, but remained unaltered between ambient and high pCO 2 .Only at HL, an effect of pCO 2 on absETR (Figures 2E,F) and I K (Table 2) was evident in Fragilariopsis and Odontella, with decreased ETRm and I K values in the low compared to ambient and high pCO 2 treatments, respectively.
The maximum quantum yield (F v /F m ) is an indicator for overall fitness and revealed species-specific patterns under the different experimental treatments (Figures 3A,B).In Fragilariopsis, F v /F m decreased from LL to ML and remained unchanged between ML and HL in the ambient pCO 2 treatments.At LL, the low pCO 2 treatments exhibited the lowest yield.Besides this, no pCO 2 effects were found.In Odontella, F v /F m did not change between LL and ML in the ambient pCO 2 treatments, whereas it decreased from LL to ML in the high pCO 2 treatments.Between ML and HL, F v /F m decreased in all pCO 2 treatments.
To test whether both species experienced short-term light stress or any damage in PSII, potentially induced during the PE-curve, a second dark acclimation phase right after the PE-curve was carried out followed by another F v /F m measurement, named yield recovery (given as % of initial F v /F m , Figures 3C,D).The two species showed diverging patterns of yield recovery.In Fragilariopsis, the recovery potential increased with increasing light intensity.In Odontella, a reversed bell shape was observed with a decrease in recovery from LL to ML and an increase from ML to HL. Yield recovery was not affected by pCO 2 under all light conditions in Odontella, whereas in Fragilariopsis the recovery increased from low to high pCO 2 conditions at LL and ML, but remained unaffected by pCO 2 at HL.

Light Harvesting Pigment Content
Cellular pigment content generally decreased with increasing light intensity (Table 3).In both species and for all lightharvesting pigments (Chl a, Chl c2, fucoxanthin, β-carotene, Table 3), a significant decrease between LL and ML was observed in all pCO 2 treatments and only minor or no decreases were observed between ML and HL conditions.Besides the light effect, Fragilariopsis showed a pCO 2 effect on all light-harvesting pigments (Chl a, Chl c2, fucoxanthin and β-carotene, Table 3).To prevent repetition only the CO 2 -dependent change in Chl a will be described exemplarily for all light-harvesting pigments.At LL, cellular Chl a concentrations increased from low to ambient pCO 2 and decreased toward high pCO 2 .At ML and HL, no pCO 2 effects were detected.In contrast to Fragilariopsis, in Odontella cellular Chl a concentrations remained unaltered in response to changes in pCO 2 (Table 3).

Adjustments of Photosystem II
The functional absorption cross section of PSII (σ PSII , Table 4), is a measure of the size of the functional "target area" of light-harvesting antenna.In Fragilariopsis, σ PSII decreased with increasing light intensity, whereas there was no change detectable in Odontella.However, effects of pCO 2 on σ PSII were found in both species.Under low compared to ambient and high pCO 2, σ PSII was significantly lower at ML and HL in both diatoms.
Light had an influence on re-oxidation times of the primary electron acceptor Q a (τ Qa , Table 4), but with different effects for the two investigated species.In Fragilariopsis, at LL, low pCO 2 treatments showed the highest re-oxidation times with no differences between ambient and high pCO 2 , whereas at ML the re-oxidation time increased from low to high pCO 2 , and at HL no differences between pCO 2 treatments were detectable.In Odontella, at ambient pCO 2, τ Qa decreased from ML to HL.In the high pCO 2 treatments of Odontella τ Qa increased with light.There was no light effect present in low pCO 2 treatments.When light was limiting (LL), τ Qa was lower at high than at ambient pCO 2 .At ML and HL, in Odontella τ Qa was less at low compared to ambient and high pCO 2 .At HL, the high pCO 2 treatments showed a higher τ Qa than ambient treatments (results are insignificant due to a lack of statistical power).
The connectivity between PSIIs (p, Table 4) was influenced by pCO 2 in Fragilariopsis.Low pCO 2 treatments showed decreased connectivity compared to ambient and high pCO 2 treatments at LL and ML.In Odontella, in all pCO 2 treatments p decreased from ML to HL.At ML, p increased from low to ambient pCO 2 and remained constant between ambient and high pCO 2 .At HL the high pCO 2 treatments showed the lowest p. Yet, statistical results for Odontella were not significant.
In Fragilariopsis, the concentration of PSII reaction centers ([RCII]) varied with light, decreasing significantly between LL and ML in the ambient and high pCO 2 treatments, respectively and increasing between ML and HL at ambient, but not at high pCO 2 (Table 4).Only in the low pCO 2 treatments, [RCII] was not altered in response to increasing light intensities.At LL, [RCII] increased with increasing pCO 2 in Fragilariopsis, whereas at ML the ambient pCO 2 treatments showed higher concentrations than the low pCO 2 treatments and no difference from the high pCO 2 treatments.Compared to Fragilariopsis, in Odontella [RCII] exhibited a different pattern with light.Concentrations of RCII increased from LL to ML in ambient and high pCO 2 treatments, but decreased from ML to HL in all pCO 2 treatments.At LL, [RCII] did not change in response to pCO 2 in Odontella.At ML, [RCII] increased from low to ambient pCO 2 , but not from ambient to high pCO 2 .Only at HL, the low and high pCO 2 treatments displayed lower concentrations of RCII than the ambient pCO 2 treatments.

Cellular Protective Pigment Content
The two diatom species showed different light-dependent trends in the de-epoxidation state (DES), an indicator for the dissipation of excess light energy (Table 3).In Odontella DES increased with increasing light intensity in all pCO 2 treatments, whereas Fragilariopsis showed a bell shaped pattern.DES increased from LL to ML in Fragilariopsis, and decreased toward HL in all pCO 2 treatments.Besides the low pCO 2 treatments, no de-epoxidation was observed in Fragilariopsis at LL. Furthermore, in Fragilariopsis at ML and HL DES was lower in the low compared to ambient pCO 2 treatments.Odontella exhibited elevated DES only in the low pCO 2 treatments at ML and HL.

Non-photochemical Quenching (NPQ)
The NPQ in Fragilariopsis showed an increase with increasing light intensity, while Odontella displayed a bell shaped pattern with highest quenching rates at ML (Figure 4).For the low pCO 2 treatments, NPQ reached higher values at LL in Fragilariopsis and at HL in Odontella.

DISCUSSION
This study investigated the physiological responses of two Antarctic key diatom species over a range of low to high light intensities in combination with low, ambient and elevated pCO 2 levels.As future changes in water column stratification are considered to go in hand with ocean acidification, the potential for acclimation of Southern Ocean phytoplankton to these two factors can indicate potential future shifts in species composition.
Next to climate change, light availability during summer and early spring greatly differs, with lower mean irradiances in spring (Dubinsky and Stambler, 2009) and higher incident light during summer (Mitchell and Brody, 1991;Mitchell and Holm-Hansen, 1991;Nelson and Smith, 1991).Moreover, large seasonal variation in pCO 2 is frequently observed.The pCO 2 in ocean surface waters is slightly elevated in early spring and can decrease tremendously during phytoplankton blooms (Sweeney, 2003;Arrigo et al., 2008).Our study was designed to link abiotic conditions to the physiological traits of phytoplankton species, in order to understand spatial and temporal distribution patterns under future light and pCO 2 scenarios.The two diatom species studied here were found to occur in high abundances during different times of the year.Odontella weisflogii (Odontella) forms blooms in Antarctic coastal waters in summer (Garibotti et al., 2005;Annett et al., 2010), whereas elevated abundances of Fragilariopsis curta (Fragilariopsis) were observed rather early in season when the sea ice retreats (Garibotti et al., 2005;Annett et al., 2010).In our study, the observed physiological responses of Fragilariopsis and Odontella were clearly species-specific and varied largely in response to the applied CO 2 and light scenarios and could further be related to their seasonal occurrence.
Light Optima for Photosynthesis Are Species-Specific Under ambient pCO 2 , an increase in light intensity from low light (LL) to medium light (ML) stimulated growth by 42% in Odontella, with no further stimulation between ML and high light (HL, Figure 1B), indicating that growth was saturated at ML in this species.Other studies also reported that growth was stimulated by light when increased from lower to higher irradiances in various diatoms and prymnesiophytes (Bartual and Gálvez, 2002;Arrigo et al., 2010;Boelen et al., 2011).Congruently, growth of Fragilariopsis was also stimulated by 20% from LL to ML, but declined by 58% from ML to HL (Figure 1A).Such a decline at high light intensities was already found in the diatom Phaeodactylum tricornutum and the prymnesiophyte Phaeocystis antarctica (Arrigo et al., 2010;Li et al., 2014).In Fragilariopsis, particulate organic carbon (POC) production rates and quantum yield of photosynthesis (F v /F m ), a measure of a cell's photosynthetic performance, were highest under LL and decreased from LL to ML, indicating saturation of photosynthesis already at LL conditions and an onset of light stress under ML (Figures 1C, 3A).In contrast, POC production increased Values represent mean ± SE (n = 3).Odontella did not grow at low light in combination with low pCO 2 as indicated by /.Statistical differences between two treatments (p = 0.05) derived from post-hoc tests are indicated by different letters.
from LL to ML in Odontella.This finding suggests that even under LL conditions Fragilariopsis was able to fix POC at a maximum rate while for Odontella this was only the case at ML, thus indicating species-specific light optima for photosynthesis.Despite the decrease in POC production from LL to ML in Fragilariopsis, growth increased suggesting that cell volumes might have changed.However, cell size measurements did not show any changes in both species and in all treatments (data not shown).Considering further that cell volume of Odontella was on average 2000 times larger than of Fragilariopsis (cell volumes estimated based on Hillebrand et al. (1999), data not shown) this potentially implied a much higher carbon demand in the former.
For both species from ML to HL, we observed a significant decline in POC production (Figures 1C,D) and in Odontella a decrease in F v /F m (Figure 3B).In contrast to this observation, ETRm increased in both species from LL to HL, indicating no light saturation (Table 2).Due to this, the observed reduced POC production rates, between LL and HL in Fragilariopsis and between ML and HL in Odontella, might have resulted from a saturation of the Calvin-Benson-Cycle.The Calvin-Benson-Cycle is considered to be the rate-limiting step of photosynthesis under excessive light conditions, thus creating the demand of alternative electron pathways such as Mehler reaction, midstream oxidase pathways and cyclic electron transport around PSI to dissipate excess electrons (Behrenfeld and Milligan, 2012).Cyclic electron transport would rise the transthylakoid pH gradient and consequently lead to a higher production of ATP at the expense of NADPH and thereby would result in lower POC fixation rates (Falk and Palmqvist, 1992).Congruently with the changes in POC production the photosynthetic performance (F v /F m ) declined from LL to ML in Fragilariopsis and from ML to HL in Odontella (Figures 1C,D, 3A,B).With increasing irradiance, F v /F m was commonly found to decrease in various temperate and Antarctic phytoplankton species (Boelen et al., 2011;Hoogstraten et al., 2012;Li et al., 2014) implying a lower photosynthetic capacity through photoinhibition and eventually photodamage of PSII.The observed species-specific changes in F v /F m may have resulted from a decreased ratio of photons absorbed per electrons generated in PSII.The amount of electrons generated depends on the cell's ability to capture light energy via lightharvesting pigments and the amount of functional PSII reaction centers.Less active PSII can result from closure of reaction centers (Schreiber, 2004) through photodamage (e.g., damage of D1 protein of PSII; Alderkamp et al., 2010), which can be an initial stage of photoacclimation process yielding reduced concentrations of PSII (Sakshaug et al., 1997).
For both diatoms, cellular concentrations of light-harvesting pigments (Ch a, Chl c2, fucoxanthin) significantly decreased similarly between LL and ML conditions, suggesting an acclimation to increased light by reducing light-harvesting pigment concentration for both species (Table 3).Yet, between ML and HL concentrations of light-harvesting pigments remained similar in both species, indicating a lower limit of pigment content for light absorption.We, however, found that concentrations of functional reaction centers ([RCII]) and F v /F m decreased significantly between ML and HL in Odontella while the absorption cross section of PSII (σ PSII ) remained unchanged (Figure 3B, Table 4).This shows that Odontella experienced stress at HL compared to ML.In comparison, F v /F m , [RCII] and σ PSII already declined from LL to ML in Fragilariopsis (Figure 3A, Table 4).This finding suggests a higher susceptibility of this species to light stress already at moderate light intensities, yet these did not affect growth rates until HL conditions were applied.
To counteract photodamage, phytoplankton cells possess various strategies such as non-photochemical quenching (NPQ) including the operation of the xanthophyll cycle (XC), therefore dissipation of excess light energy in Odontella and Fragilariopsis was investigated via the concentrations of cellular diadinoxanthin (DD), its de-epoxidised form diatoxanthin (DT) and the deepoxidation state (DES).An increase in DES (Table 3) with incident light is a response commonly found in phytoplankton (Casper-Lindley and Björkman, 1998;Arrigo et al., 2010;Petrou et al., 2011).NPQ increased from LL to HL in Fragilariopsis while in Odontella it increased mainly from LL to ML, but decreased from ML to HL (Figure 4).In Odontella, DES rose from LL to HL whereas there was no XC activity detectable in Fragilariopsis at LL, but there was a decrease from ML to HL (Table 3).The discrepancy in the trends of NPQ and DES from ML to HL in both species could result from smaller and much larger cellular DD+DT pools in Odontella and Fragilariopsis (−60 and +300%; Table 3), respectively under these conditions changing the general capacity for energy dissipation.
Overall, the two diatom species showed diverging responses to increasing light at ambient pCO 2 during acclimation.Fragilariopsis was characterized by highest POC production rates and photosynthetic performance (F v /F m ) under LL, but experienced light stress with increasing light intensity, as POC production rates decreased and NPQ increased from LL to HL.Hence, it showed good ability for energy dissipation even under HL conditions and thus seemed better able to cope with light stress.In comparison, Odontella showed an increase in POC production rates from LL to ML, indicating no saturation at LL.With increasing light intensities, Odontella was susceptible to light stress as POC production, F v /F m and NPQ declined indicating a limited potential to tolerate HL conditions.

Low pCO 2 Inhibits Growth and Carbon Fixation Regardless of the Light Conditions
Phytoplankton blooms in Antarctic coastal waters occur annually and have been found to sometimes coincide with a severe drawdown of inorganic carbon through high photosynthetic carbon fixation rates in surface waters (Sweeney, 2003;Arrigo et al., 2008).In order to sustain photosynthesis under these low CO 2 conditions, phytoplankton cells need to up-regulate their carbon concentrating mechanisms (CCMs) leading to increased energy demands and potentially impeding carbon fixation (Raven et al., 2014).
Low pCO 2 in combination with LL was found to have the strongest impact on growth and carbon fixation in comparison to all other treatments.Under these conditions, both factors light and CO 2 were limiting, causing lowest growth and POC production rates in Fragilariopsis while Odontella did not grow (Figures 1A-D).Commonly, the CCM is up-regulated under low pCO 2 in order to supply CO 2 to RubisCO, which is highly energy demanding (Hopkinson et al., 2011).Yet, in conjunction with LL conditions, the production of energy equivalents during photosynthesis might not be sufficient to maintain carbon fixation at a maximum, potentially causing the here observed minimal growth and POC production in Fragilariopsis.Additionally, light-harvesting pigment concentrations (Table 3), [RCII] and p (Table 4) were lowest under these conditions, indicating a small efficiency of individual PSII reactions centers with a low probability of excitation distribution among them.Furthermore, an excess of excitation energy was clearly visible as NPQ during PE-curves (Figure 4) was highest and yield recovery thereafter was lowest (Figure 3C).Overall, Fragilariopsis revealed great difficulties to grow under LL in conjunction with low pCO 2 .Nonetheless, it grew whereas Odontella could not cope with these highly stressful conditions.With increasing light availability the energetic constraints under LL were alleviated in both species, as potentially a larger share of photosynthetically generated energy (ATP and NADPH) was available to fuel their CCMs.Hence, at ML in conjunction with low pCO 2 Odontella was able to grow and fix POC.This stimulative light effect was also found in Fragilariopsis, strongly increasing growth and POC production from LL to ML (Figures 1A,C).
Within the ML treatments, pCO 2 was also found to stimulate growth rates of Odontella and Fragilariopsis by 8 and 42% from low to ambient pCO 2 , respectively (Figures 1A,B), suggesting optimal growth conditions for both species at ambient pCO 2 .A similar CO 2 -dependent increase in growth was previously reported for the two polar diatoms Rhizosolenia cf.alata and C. debilis (Riebesell et al., 1993;Trimborn et al., 2013).POC production was enhanced from low to ambient pCO 2 levels in Odontella (Figure 1D) potentially resulting from a higher diffusive CO 2 supply and therewith a lower energy demand.However, for Fragilariopsis we observed a CO 2 -dependent decline in POC production (Figure 1C).This decline was accompanied by a reduced capability for light absorption as cellular light-harvesting pigment concentrations were decreased at ambient compared to low pCO 2 (Chl a, Chl c2, fucoxanthin, Table 3).Please note that Fragilariopsis experienced light stress already at ML when grown at ambient pCO 2 , hinting toward a higher susceptibility to light stress in this species.Through the down-regulation of the CCM from low to ambient pCO 2 , it may serve less as an energy sink and thereby increasing light stress exerted by excessive light (Gao et al., 2012).This was, however, not reflected in any change in F v /F m .In Odontella, under ML absETRs, ETRm, I K and α decreased from low to ambient pCO 2 while POC production increased indicating that carbon uptake might not have been sufficient to saturate RubisCO and the Calvin-Benson-Cycle.The reason for this might be its higher cell volume compared to Fragilariopsis and thereby higher amount of carbon necessary to saturate the Calvin-Benson-Cycle and to sustain growth.Increased absETR under these conditions could further indicate cycling of electrons around PSI and thus generation of energy equivalents that could be funneled into carbon uptake.At HL, we observed an increase of the photosynthetic efficiency from low to ambient pCO 2 treatments of both species, displaying higher absETR (Figures 2E,F), ETRm, I K , α (Table 2), σ PSII , τ Qa (Odontella only), p and [RCII] (Fragilariopsis only, Table 4) while NPQ and DD+DT pools decreased in both species, indicating lowered energy dissipation with increasing pCO 2 .Hence, in the low pCO 2 treatments the HL was not utilized, but cells rather dissipated the excess light energy absorbed.As the CCM of both species was potentially downregulated from low to ambient pCO 2 , the higher photosynthetic efficiencies as well as lowered energy dissipation at HL suggest that the CCM does not represent an important sink for excess energy under high light conditions (Gao et al., 2012).
Overall, this study revealed that low pCO 2 concentrations were stressful for both Antarctic diatoms, but especially in conjunction with light limiting conditions.According to our results, the hypothesis that the regulation of the CCM aids in the dissipation of excess energy at high light intensities cannot be conclusively answered for the two species tested here.

OA Effects Are Modulated by Light Availability
Under OA, Antarctic diatoms might benefit from diffusive CO 2 uptake as has been shown for temperate species (Burkhardt et al., 2001;Rost et al., 2003;Trimborn et al., 2009), decreasing the demand for CCM operation.Especially under limiting light conditions, the lowered energy demand through downregulation of the CCM was found to stimulate growth and carbon fixation in temperate phytoplankton species (Wu et al., 2010;Li et al., 2014).Similarly, the temperate coccolithophorid Emiliania huxleyi showed increased POC production rates under OA when irradiances are low, yet under higher light intensities this effect was amended (Rokitta and Rost, 2012).In our investigated species, however, growth and POC production remained either unaffected or declined under OA and LL (Figures 1A-D) contradicting the aforementioned assumption of a stimulative effect of OA, but going in line with other findings for Antarctic phytoplankton species (Hoogstraten et al., 2012).We even observed for Fragilariopsis negative effects of OA under LL on light-harvesting pigment concentrations (Chl a, Chl c2, and fucoxanthin; Table 3) potentially compensated by an increased number of active RCII (Table 4).Interestingly, in Odontella light harvesting pigments (Chl c2, fucoxanthin, Table 3) were increased under elevated pCO 2 , indicating a maximization of light absorption under LL.In agreement with this, an OA-dependent induction of chlorophyllfucoxanthin protein genes were reported for the temperate diatom Phaeodactylum tricornutum (Li et al., 2015).In addition to this, enhanced rates of mitochondrial respiration were previously reported under OA in combination with relatively LL levels in the temperate diatom Thalassiosira pseudonana (Yang and Gao, 2012).Together with the species-specific photoacclimation responses under OA, one could hypothesize that the two investigated species may have experienced higher metabolic costs, potentially causing the observed decline in POC production.
For both species, the negative OA effects were amplified under ML conditions.Next to negative responses in growth and POC production (Figures 1A-D), the two species displayed OA-dependent photoacclimation.While Odontella revealed lowered photochemical efficiencies (Figure 3B), Fragilariopsis had less active RCIIs with reduced connectivity between PSIIs (Table 4) potentially counteracted by increased light harvesting pigmentation (Chl a and fucoxanthin, Table 3).For both species, acclimation to high light conditions was indicated by a larger cellular DD+DT pool size (Table 3).This is in line with previous suggestions that down-regulated CCMs serve less as a sink for excess light energy under OA in conjunction with saturating light intensities (Gao et al., 2012).However, this positive effect of CCM operation under excessive irradiances was not found when comparing low and ambient pCO 2 treatments under HL (see discussion above).Unexpectedly, this negative OA effect was alleviated under HL conditions in our two tested species.In this case, growth rates and POC production remained unaffected (Figures 1A-C).Only for Fragilariopsis, POC production was stimulated by OA and HL.For the latter, C:N ratios were also found to raise (Figure 1E).For the two coccolithophores Gephyrocapsa oceanica and Coccolithus pelagicus ssp.braarudii, OA also increased POC production rates, but C:N ratios were either stimulated (200 µmol photons m −2 s −1 , Rickaby et al., 2010) or reduced (100 µmol photons m −2 s −1 , Jin et al., 2013) in G. oceanica.In our study, the CO 2 -dependent increase in C:N ratios under HL was mainly due to lowered cellular PON content (data not shown), the underlying reason for this, however, remains unclear.
From our results, we can conclude that OA in conjunction with all tested irradiances did not lead to stimulation in growth or POC production in any of the tested species.It was, however, evident that increasing light intensities during acclimation caused OA-dependent photoacclimation responses and negatively impacted POC production.Only under HL, this OA effect was alleviated in Odontella, while in Fragilariopsis POC production was even increased, potentially resulting from a shift in carbon allocation.Congruently we cannot support the finding that a down-regulation of the CCM and consequent decrease of energy dissipation therein increases light stress under OA in conjunction with HL in these Antarctic diatoms.

Ecological Implications and Conclusion
Antarctic coastal waters were found to have high iron concentrations and form highly productive and extensive blooms over the season (Holm-Hansen et al., 1989;Martin et al., 1990;Prézelin et al., 2000;Garibotti et al., 2003;de Jong et al., 2012).Annual patterns of phytoplankton species succession and their presence in either spring or summer blooms can be related to species-specific abilities to acclimate, utilize and tolerate different light intensities.The physiological characteristics of both species we observed match well their seasonal occurrence.In line with its presence in spring (Garibotti et al., 2005;Annett et al., 2010), Fragilariopsis showed greater ability than Odontella to grow under LL conditions.Accordingly, it was characterized by highest POC production rates under limiting light as well as a good ability to endure high light stress when mixed to the water surface.Odontella was still light limited under LL, requiring higher irradiances coinciding with its occurrence in high abundances in summer when the upper mixed layer is shallow and stable (Mitchell and Holm-Hansen, 1991;Nelson and Smith, 1991).Congruently, it was able to endure longer periods of high irradiances.Yet, at the peak of a bloom when cell density is high, light is reduced and CO 2 gets drawn down by high photosynthetic activity, this creates very unfavorable growth conditions for Odontella.
Strong species-specific physiological responses were apparent in response to different future climate scenarios, mimicking either OA in conjunction with increased irradiances due to shallower UML depth or OA associated with decreased daily irradiances due to deepening of the UML through increased winds.Under ML, both species experienced light stress, which was further amplified by OA.Yet, in the HL OA-scenario Fragilariopsis was able to tolerate the HL conditions.Also under the LL OA-scenario, neither of the investigated species showed an OA-dependent stimulation in growth or POC production.Hence, OA mainly showed negative effects on growth and carbon fixation in both diatom species, implying that OA could potentially reduce the strength of the biological carbon pump under the tested light scenarios.
The results of this study indicate that physiological traits can help to explain the spatial distribution of diatom species in the current Southern Ocean.We further demonstrate that the here tested future climatic scenarios could negatively affect growth and carbon fixation of both diatom species with potential implications for a future SO.Furthermore, the results of this study demonstrated that the effect of OA was strongly modulated by the irradiance regime, pointing out the need to conduct multiple stressor experiments to better understand the impact of climate change on Southern Ocean key phytoplankton species.
bValues represent mean ± SE (n = 3).Odontella did not grow at low light in combination with low pCO 2 as indicated by /.Statistical differences between two treatments (p = 0.05) derived from post-hoc tests are indicated by different letters.