Ocean acidification and high irradiance stimulate the photo-physiological fitness, growth and carbon production of the Antarctic cryptophyte Geminigera cryophila

Ecophysiological studies on Antarctic cryptophytes to assess whether climatic changes such as ocean acidification and enhanced stratification affect their growth in Antarctic coastal waters in the future are lacking so far. This is the first study that investigates the combined effects of the increasing availability of pCO₂ (400 and 1000 µatm) and irradiance (20, 200 and 500 µmol photons m⁻² s⁻¹) on growth, elemental composition and photophysiology of the Antarctic cryptophyte Geminigera cryophila. Under ambient pCO₂, this species was characterized by a pronounced sensitivity to increasing irradiance with complete growth inhibition at the highest light intensity. Interestingly, when grown under high pCO₂ this negative light effect vanished, and it reached the highest rates of growth and particulate organic carbon production at the highest irradiance compared to the other tested experimental conditions. Our results for G. cryophila reveal beneficial effects of ocean acidification in conjunction with enhanced irradiance on growth and photosynthesis. Hence, cryptophytes such as G. cryophila may be potential winners of climate change, potentially thriving better in more stratified and acidic coastal waters and contributing in higher abundance to future phytoplankton assemblages of coastal Antarctic waters.

1 Introduction

Even though Antarctic coastal waters comprise a relatively small area relative to the open ocean, these waters are highly productive due to the constant supply of macronutrients and iron (Arrigo et al., 2008). Shelf waters adjacent to the western Antarctic Peninsula (WAP) are currently undergoing rapid physical changes, exhibiting more rapid warming rates than anywhere in Antarctica over the last decades (Ducklow et al., 2007, 2013). Rising air temperature resulted in shorter sea ice seasons (Smith and Stammerjohn, 2001) with contrasting effects on phytoplankton biomass, composition and productivity between the northern and southern WAP. For the latter, the earlier retreat of sea ice together with the observed increase in surface water temperature led to shallow water column stratification, which favored phytoplankton growth and productivity. In the northern part of the WAP on the other hand, the earlier disappearance of sea ice was associated with greater wind activities and more cloud formation. As a consequence, a deepening of the upper mixed layer was found, providing less favorable light conditions. Next to reduced chlorophyll a accumulation (Montes-Hugo et al., 2009) and primary production (Moreau et al., 2015), a decline in large phytoplankton such as diatoms relative to the whole community was observed (Montes-Hugo et al., 2009; Rozema et al., 2017). Accordingly, a recurrent shift from diatoms to cryptophytes and small flagellates was reported for waters north of the WAP, with important implications for food web dynamics (Moline et al., 2004; Ducklow et al., 2007; Montes-Hugo et al., 2009; Mendes et al., 2017). The frequent occurrence of cryptophytes was previously reported after diatom blooms (Moline and Prézelin, 1996) and was related to surface meltwater stratification (Moline et al., 2004). As a result of rising global air temperatures, surface water freshening is expected to shallow the upper water layer, exposing phytoplankton to higher light intensity (Moreau et al., 2015). Relative to diatoms and the prymnesiophyte Phaeocystis antarctica, cryptophytes were found to be the main contributors to biomass in stratified and warm WAP coastal waters potentially resulting from their high tolerance to withstand high irradiances (Mendes et al., 2017). Considering the lack in ecophysiological studies carried out with Antarctic ecologically relevant cryptophyte species it remains unclear as yet whether the projected climatic changes could promote cryptophyte growth in Antarctic coastal waters in the future. Hence, higher abundances of cryptophytes could have important implications for the biogeochemistry of these waters, as they are considered to be inefficient vectors of carbon and thus could reduce the efficiency of the biological carbon pump.

Light availability strongly influences the rate of growth and carbon fixation of phytoplankton (Falkowski and Raven, 2007). With increasing irradiance, Antarctic phytoplankton species exhibited increased growth and carbon fixation, but only until photosynthesis was saturated (Fiala and Oriol, 1990; Heiden et al., 2016). When exposed to excessive radiation, phytoplankton cells can become photo-inhibited and even damaged (Falkowski and Raven, 2007). Cryptophytes are exceptional among eukaryotic microalgae as, similar to diatoms, they contain chlorophyll a∕c proteins, the carotenoid alloxanthin and phycobiliproteins homologous to red algal phycobiliproteins (Gould et al., 2008). This pigment composition allows cryptophytes to cope well under limited irradiance. Unlike diatoms and prymnesiophytes, cryptophytes have no photo-protective de-epoxidation–epoxidation cycling of xanthophyll pigments (e.g., diadinoxanthin, diatoxanthin), instead they rely on chlorophyll a∕c proteins, which dissipate excess light energy, as another type of non-photochemical quenching (NPQ; Funk et al., 2011; Kana et al., 2012). While laboratory studies so far have mainly concentrated on disentangling the physiological response of Southern Ocean key species of diatom and prymnesiophytes to different environmental factors, almost nothing is known on Antarctic cryptophytes. Apart from field studies (Moline et al., 2004; Ducklow et al., 2007; Montes-Hugo et al., 2009), almost nothing is known on how climate change could influence the ecophysiology of Antarctic cryptophytes. Due to the increased solubility of CO₂ in cold water, ocean acidification (OA) is anticipated to strongly affect polar waters (Orr et al., 2005; Pachauri et al., 2014). For various Antarctic diatoms and the prymnesiophyte P. antarctica, growth and/or carbon fixation remained unaltered by OA alone (Riebesell et al., 1993; Boelen et al., 2011; Hoogstraten et al., 2012; Trimborn et al., 2013; Hoppe et al., 2015; Heiden et al., 2016). Recent studies suggest that Southern Ocean diatoms are more prone to OA especially in conjunction with high light than the prymnesiophyte Phaeocystis antarctica (Feng et al., 2010; Trimborn et al., 2017a, b; Beszteri et al., 2018; Heiden et al., 2018, 2019; Koch et al., 2018). The response of cryptophytes to OA is hitherto almost unexplored. The few studies on temperate phytoplankton communities suggest that cryptophytes were not affected by OA (Schulz et al., 2017), even when exposed in combination with increased ultraviolet radiation (Domingues et al., 2014) or warming (Sommer et al., 2015). Similarly, no discernible OA effect on cryptophyte abundance was found in subantarctic (Donahue et al., 2019) and Antarctic (Young et al., 2015) natural phytoplankton communities composed of diatoms, cryptophytes and Phaeocystis spp.

Based on previous studies on the single effects of light or CO₂ alone, we hypothesize that cryptophytes are able to cope well with OA and high light conditions. Due to the limited information available on Antarctic cryptophyte physiology, this study assessed OA effects and their interaction with increasing irradiance on the physiology of the Antarctic cryptophyte Geminigera cryophila. To this end, G. cryophila was grown under two pCO₂ levels (400 and 1000 µatm) in combination with three irradiance levels (20, 200 and 500 µmol photons m⁻² s⁻¹) and their interactive effects on growth, elemental composition and photo-physiology were assessed.

2 Material and methods

2.1 Culture conditions

Triplicate semicontinuous cultures of the Antarctic cryptophyte Geminigera cryophila (CCMP 2564) were grown in exponential phase at 2 ^∘C in sterile-filtered (0.2 µm) Antarctic seawater (salinity 30.03). This seawater was enriched with phosphate (final concentration of 100 µmol L⁻¹), nitrate (final concentration of 6.25 µmol L⁻¹) (N:P ratio of 16:1; Redfield, 1958), and trace metals and vitamins according to F/2 medium (Guillard and Ryther, 1962). G. cryophila cells were grown under a 16 h light and 8 h dark cycle at three constant light intensities (LL = 20, ML = 200 and HL = 500 µmol photons m⁻² s⁻¹: LL – low light; ML – medium light; HL – high light) using light-emitting diode (LED) lamps (SolarStinger LED SunStrip Marine Daylight, Econlux). Light intensities were adjusted using a LI-1400 data logger (Li-Cor, Lincoln, NE, USA) with a 4π sensor (Walz, Effeltrich, Germany). The three light treatments were further combined with two CO₂ partial pressures (pCO₂) of 400 (ambient pCO₂ treatment) or 1000 µatm (OA treatment, Table 1). All pCO₂ treatments and the respective dilution media were continuously and gently bubbled through a frit with humidified air of the two pCO₂ levels, which were generated from CO₂-free air (<1 ppmv CO₂; Dominick Hunter, Kaarst, Germany) and pure CO₂ (Air Liquide Deutschland ltd., Düsseldorf, Germany) with a gas flow controller (CGM 2000, MCZ Umwelttechnik, Bad Nauheim, Germany). The CO₂ gas mixtures were regularly monitored with a nondispersive infrared analyzer system (LI6252; Li-Cor Biosciences) calibrated with CO₂-free air and purchased gas mixtures of 150±10 and 1000±20 ppmv CO₂ (Air Liquide Deutschland). Dilutions with the corresponding acclimation media ensured that the pH level remained constant (±0.07 units, Table 1) and that the cells stayed in the exponential growth phase. G. cryophila cells were acclimated to the matrix of three light intensities (LL = 20, ML = 200 and HL = 500 µmol photons m⁻² s⁻¹) and two pCO₂ levels (ambient = 400 and OA = 1000 µatm) for at least 2 weeks prior to the start of the main experiment. Despite several attempts, G. cryophila did not grow at ambient pCO₂ in conjunction with HL. For the main experiments, cells grew in exponential phase and final sampling took place between 7280 and 17 161 cells mL⁻¹.

Table 1 Partial pressures of CO₂ (pCO₂) for the ambient and ocean acidification (OA) treatments were calculated from measured pH, concentrations of dissolved inorganic carbon (DIC), silicate and phosphate, temperature, and salinity using the CO2Sys program (Pierrot et al., 2006). For all parameters, values are given for the abiotic control bottles (abiotic, bubbled seawater without cells) and the culture bottles at the end of the experiment (biotic). Different letters indicate significant differences between treatments (p<0.05). Values represent the means ± SD (n=3).

2.2 Seawater carbonate chemistry

The pH of the different cultures and the culture medium was measured every other day and on the final sampling day using a pH ion meter (pH meter 827, Metrohm), calibrated (three-point calibration) with National Institute of Standards and Technology-certified buffer systems. The pH remained constant at 8.13±0.07 and 7.82±0.06 for the ambient pCO₂ and OA treatments, respectively (Table 1). Dissolved inorganic carbon (DIC) samples were sterile-filtered (0.2 µm) and stored at 4 ^∘C in 5 mL gastight borosilicate bottles without headspace until analysis. DIC was measured colorimetrically in duplicates with a QuAAtro autoanalyzer (Seal Analytical, Stoll et al., 2001). The carbonate system was calculated based on DIC, pH, silicate (97 µmol kg⁻¹), phosphate (6.1 µmol kg⁻¹), temperature (2.0 ^∘C) and salinity (30.03) 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).

2.3 Growth, elemental stoichiometry and composition

Cell count samples of every G. cryophila treatment were taken on a daily basis at the same time of day and were determined immediately after sampling using a Coulter Multisizer III (Beckmann-Coulter, Fullerton, USA). Cell-specific growth rate (μ, unit d⁻¹) was calculated as

$$\begin{array}{}\text{(1)}& {\displaystyle }\mathit{\mu }=(\ln N_{\mathrm{fin}}-\ln N_{\mathrm{0}})/\mathrm{\Delta }t,\end{array}$$

where N₀ and N_fin denote the cell concentrations at the beginning and the end of the experiments, respectively, and Δt is the corresponding duration of incubation in days.

Particulate organic carbon (POC) and particulate organic nitrogen (PON) were measured after filtration onto precombusted (12 h, 500 ^∘C) glass fiber filters (GF/F, pore size ∼0.6 µm, Whatman). Filters were stored at −20 ^∘C and dried for >12 h at 64 ^∘C prior to sample preparation. Analysis was performed using a Euro Vector CHNS-O elemental analyzer (Euro Elemental Analyzer 3000, HEKAtech GmbH, Wegberg, Germany). Contents of POC and PON were corrected for blank measurements and normalized to filtered volume and cell densities to yield cellular quotas. Production rates of POC and PON were calculated by multiplication of the cellular quota with the specific growth rate of the respective treatment.

2.4 Chlorophyll a fluorescence

The efficiency of photochemistry in photosystem II (PSII) was assessed in all treatments using a fast repetition rate fluorometer (FRRf; FastOcean PTX; Chelsea Technologies Group Ltd., West Molesey, United Kingdom) in combination with a FastAct Laboratory system (Chelsea Technologies Group Ltd., West Molesey, United Kingdom). Cells of the respective treatment were dark-acclimated for 10 min, before minimum chlorophyll a (Chl a) fluorescence (F_o) was recorded. Subsequently, a single turnover flashlet (1.2×10²² photons m⁻² s⁻¹, wavelength 450 nm) was applied to cumulatively saturate photosystem II (PSII), i.e., a single photochemical turnover (Kolber et al., 1998). The single turnover saturation phase comprised 100 flashlets on a 2 µs pitch and was followed by a relaxation phase comprising 40 flashlets on a 50 µs pitch. This sequence was repeated 24 times within each acquisition. The saturation phase of the single turnover acquisition was fitted according to Kolber et al. (1998). From this measurement, the minimum (F_o) and maximum (F_m) Chl a fluorescence was determined. Using these two parameters, the dark-adapted maximum PSII quantum yield (F_v∕F_m) was calculated according to the equation ($F_{\mathrm{m}}-F_{\mathrm{o}})/F_{\mathrm{m}}$. During the fluorescence light curve (FLC), cells were exposed for 5 min to eight actinic light levels ranging from 35 to 1324 µmol photons m⁻² s⁻¹. From these measurements, the light-adapted minimum (F^′) and maximum ($F_{\mathrm{m}}^{\prime }$) fluorescence of the single turnover acquisition was estimated. The effective PSII quantum yield under ambient light ($F_{\mathrm{q}}^{\prime }/F_{\mathrm{m}}^{\prime }$) was derived according to the equation ($F_{\mathrm{m}}^{\prime }-F^{\prime })/F_{\mathrm{m}}^{\prime }$ (Genty et al., 1989). From this curve, absolute electron transport rates (ETRs, e⁻ PSII⁻¹ s⁻¹) were calculated following Suggett et al. (2004, 2009):

$$\begin{array}{}\text{(2)}& {\displaystyle }\mathrm{ETR}={\mathit{\sigma }}_{\mathrm{PSII}}\times (F_{\mathrm{q}}^{\prime }/F_{\mathrm{m}}^{\prime }/F_{\mathrm{v}}/F_{\mathrm{m}})\times E,\end{array}$$

where σ_PSII is the functional absorption cross section of PSII photochemistry and E denotes the applied instantaneous irradiance (photons m⁻² s⁻¹). Light-use characteristics were analyzed by fitting irradiance-dependent ETRs according to Ralph and Gademann (2005), including maximum ETR (ETR_max), minimum saturating irradiance (I_k) and maximum light utilization efficiency (α). Using the Stern–Volmer equation, NPQ of chlorophyll a fluorescence was calculated as $F_{\mathrm{m}}/F_{\mathrm{m}}^{\prime }-\mathrm{1}$. From the single turnover measurement of dark-adapted cells, σ_PSII, the energy transfer between PSII units (i.e., connectivity, P), the re-oxidation of the electron acceptor Q_a (${\mathit{\tau }}_{Q_{\mathrm{a}}}$) and the concentration of functional photosystem II reaction centers ([RII]) were assessed from iterative algorithms for induction (Kolber et al., 1998) and relaxation phase (Oxborough et al., 2012). [RII] represents an estimator for the content of PSII in the sample and was calculated according to the following equation:

$$\begin{array}{}\text{(3)}& {\displaystyle }\left[\mathrm{RII}\right]=(F_{\mathrm{o}}/{\mathit{\sigma }}_{\mathrm{PSII}})\times (K_R/E_{\mathrm{LED}}),\end{array}$$

where K_R is an instrument-specific constant and E_LED is the photon flux from the FRRf measuring LEDs. After the completion of the FLC, an additional dark-adaptation period of 10 min was applied, followed by a single turnover flashlet to check for recovery of PSII. Using the F_v∕F_m measured before and after the FLC, the yield recovery was calculated and given as the percentage of the initial F_v∕F_m (before the FLC). All measurements (n=3) were conducted at the growth temperature of 2 ^∘C.

2.5 Pigments

Samples for the determination of pigment concentration were filtered onto GF/F filters and immediately frozen at −80 ^∘C until further analysis. Pigments samples were homogenized and extracted in 90 % acetone for 24 h at 4 ^∘C in the dark. After centrifugation (5 min, 4 ^∘C, 13 000 rpm) and filtration through a 0.45 µm pore size nylon syringe filter (Nalgene^®, Nalge Nunc International, Rochester, NY, USA), concentrations of chlorophyll a and c₂ (Chl a and c₂), and alloxanthin (Allo) were determined by reversed phase high-performance liquid chromatography (HPLC). The analysis was performed on a LaChromElite^® system equipped with a chilled autosampler L-2200 and a DAD detector L-2450 (VWR-Hitachi International GmbH, Darmstadt, Germany). A Spherisorb^® ODS-2 column (25 cm × 4.6 mm, 5 µm particle size; Waters, Milford, MA, USA) with a LiChropher^® 100-RP-18 guard cartridge was used for the separation of pigments, applying a gradient according to Wright et al. (1991). Peaks were detected at 440 nm and identified as well as quantified by co-chromatography with standards for Allo, Chl a and c₂ (DHI Lab Products, Hørsholm, Denmark) using the software EZChrom Elite ver. 3.1.3. (Agilent Technologies, Santa Clara, CA, USA). Pigment contents were normalized to filtered volume and cell densities to yield cellular quotas.

2.6 Statistics

Combined effects of the two pCO₂ (ambient and OA) and light (LL, ML and HL) treatments on all experimental parameters were statistically analyzed using two-way analyses of variance (ANOVAs) with Bonferroni's multiple comparison post tests. To test for significant differences between light treatments of the OA-grown cells of G. cryophila cells one-way ANOVAs with additional Bonferroni's multiple comparison post tests were applied. All statistical analyses were performed using the program GraphPad Prism (Version 5.00 for Windows, Graph Pad Software, San Diego California, USA), and the significance testing was done at the p<0.05 level.

3 Results

3.1 Seawater carbonate chemistry

The two target pCO₂ levels of 400 and 1000 µatm were successfully achieved for abiotic control (abiotic, bubbled seawater without cells) and culture bottles (biotic, one-way ANOVA: p<0.0001, Table 1). As the pCO₂ of the abiotic control and culture bottles of the same pCO₂ treatment were similar, this indicates that final cell numbers of G. cryophila did not alter the pCO₂ of the culture bottles relative to the culture medium. Similar trends as for the pCO₂ were also apparent for the measured pH values, which yielded 8.13±0.07 and 7.82±0.06 in the culture bottles of the ambient and OA treatments, respectively (Table 1). While DIC concentrations were significantly enhanced for OA relative to the ambient pCO₂ treatments (one-way ANOVA: p<0.0001), they also significantly differed between abiotic control (abiotic, bubbled seawater without cells) and culture bottles (biotic, Table 1).

3.2 Growth, elemental stoichiometry and composition

Growth rates were significantly affected by light (two-way ANOVA: p<0.01) but not by OA or the interaction of both factors (Fig. 1a). In response to increasing irradiance, growth rates of cells grown under ambient pCO₂ remained unchanged between LL and ML but were negatively influenced by HL as they were unable to grow. Under OA, however, growth rates significantly increased between LL and ML by 89 % (post hoc: p<0.05) and between ML and HL by 32 % (post hoc: p<0.05). Irrespective of changes in irradiance, pCO₂ or their combination, cellular POC contents did not change (Fig. 1b). Daily POC production rates, were, however, significantly altered by increasing irradiance (two-way ANOVA: p<0.01) but not by OA or the interaction of both factors (Fig. 1c). While increasing light intensity did not affect POC production rates of the ambient pCO₂ treatments, there was a significant OA-dependent enhancement of 69 % between LL and ML (post hoc: p<0.01) and of 39 % between ML and HL (post hoc: p<0.05). Molar C:N ratios were only significantly influenced by the interaction of both factors together (two-way ANOVA: p<0.01; Fig. 1d). From LL to ML C:N ratios did not change for both pCO₂ treatments, whereas from ML to HL the ratio declined by 12 % for the OA treatment (one-way ANOVA: p<0.05). In response to increasing pCO₂, C:N decreased by 10 % when grown under LL (post hoc: p<0.05) but remained unaltered at ML.

Figure 1 Growth rate (a), cellular content (b), and production rate (c) of particulate organic carbon (POC) and the molar ratio of carbon to nitrogen (C:N, d) for Geminigera cryophila acclimated to ambient (black bars) or high CO₂ (gray bars) conditions combined with low (LL), medium (ML) or high light (HL). G. cryophila did not grow under ambient pCO₂ and HL as indicated by ng. Values represent the means ± SD (n=3). Different letters indicate significant differences between treatments (p<0.05).

3.3 Pigments

For all pCO₂ treatments, cellular concentrations of the measured pigments (Allo, Chl a and c₂) showed a strong and significant decline between LL and ML (two-way ANOVA: p<0.0001, Table 2). Between ML and HL, however, different effects were seen, with a significant enhancement for Chl a (post hoc: p<0.01) and Allo (post hoc: p<0.01) and no effect for Chl c₂. Increasing pCO₂ had generally no effect on cellular pigment quotas (Allo, Chl a and c₂) except for the Allo quotas of the LL treatments, which displayed a significant OA-dependent decline of 26 % (post hoc: p<0.01). A significant interaction between light and CO₂ was only found for Allo quotas (two-way ANOVA: p<0.05).

Table 2 Cellular concentrations of chlorophyll a and c₂ (Chl a and c₂) as well as alloxanthin (Allo) were determined for Geminigera cryophila acclimated to ambient or high CO₂ conditions combined with low (LL), medium (ML) or high light (HL). G. cryophila did not grow under ambient pCO₂ and HL as indicated by ng. Photosynthetic parameters were derived from at least three independent measurements. Different letters indicate significant differences between treatments (p<0.05). Values represent the means ± SD. Here, “fg” denotes femtogram.

3.4 Chlorophyll a fluorescence

The dark-adapted maximum quantum yield of PSII (F_v∕F_m) was strongly influenced by irradiance (two-way ANOVA: p<0.0001) and CO₂ (two-way ANOVA: p=0.0012) and their interaction (two-way ANOVA: p<0.05; Fig. 2a). With increasing irradiance F_v∕F_m generally declined, whereas OA increased it at LL (17 %, post hoc: p<0.01) or did not change it at ML. Noticeably, the interaction of HL and OA resulted in the lowest F_v∕F_m value. Comparing the F_v∕F_m measured before and after the FLC, the F_v∕F_m recovery was calculated and given as the percentage of the initial F_v∕F_m. Neither high pCO₂ nor the interaction of light and CO₂ affected F_v∕F_m recovery, whereas the increase in irradiance had a significant effect (two-way ANOVA: p<0.01, Fig. 2b), being increased by 11 % between LL and ML under ambient pCO₂ (post hoc: p<0.05).

Figure 2 The dark-adapted maximum PSII quantum yield F_v∕F_m (a) and the yield recovery after short-term light stress (% of initial F_v∕F_m) (b) for Geminigera cryophila acclimated to ambient (black bars) or high CO₂ (gray bars) conditions combined with low (LL), medium (ML) or high light (HL). G. cryophila did not grow under ambient pCO₂ and HL as indicated by ng. Values represent the means ± SD (n=3). Different letters indicate significant differences between treatments (p<0.05).

The increase in CO₂ or light alone had no effect on cellular concentrations of functional photosystem II reaction centers ([RCII]), while the interaction of both factors strongly altered [RCII] (two-way ANOVA: p<0.0001; Fig. 3a). From LL to ML [RCII] decreased under ambient pCO₂ (39 %, post hoc: p<0.001) while the combination of ML with OA synergistically increased it (44 %, post hoc: p<0.01, Fig. 3). [RCII] was reduced by OA at LL (37 % post hoc: p<0.01), whereas the combined effect of OA and ML led to an increase (49 %, post hoc: p<0.01). While CO₂ and the interaction of CO₂ and light together did not change the energy transfer between PSII units (i.e., connectivity, P), only the increase in irradiance had a significant effect (two-way ANOVA: p<0.05), reducing P by 22 % between LL and ML under OA (post hoc: p<0.05, Fig. 3b). While the increase in CO₂ or light did not alter the functional absorption cross sections of PSII (σ_PSII), the interaction of both factors, however, had an effect (two-way ANOVA: p<0.05; Fig. 3c). σ_PSII values were similar under LL and ML at ambient pCO₂. The interaction of OA and ML, however, lowered them (post hoc: p<0.05, Table 3). On the other hand, when grown under OA σ_PSII was larger under HL than under ML (one-way ANOVA: p<0.01). Re-oxidation times of the primary electron acceptor Q_a (${\mathit{\tau }}_{Q_{\mathrm{a}}}$) significantly changed with increasing irradiance (two-way ANOVA: p<0.05) but not with high CO₂ or the interaction of both factors together (Fig. 3d). ${\mathit{\tau }}_{Q_{\mathrm{a}}}$ of OA-acclimated cells was much shorter at HL than at ML (one-way ANOVA: p<0.05).

Table 3 Maximum absolute electron transport rates (ETR_max), minimum saturating irradiances (I_k) and maximum light utilization efficiencies (α) were determined for Geminigera cryophila acclimated to ambient or high CO₂ conditions combined with low (LL), medium (ML) or high light (HL). G. cryophila did not grow under ambient pCO₂ and HL as indicated by ng. Photosynthetic parameters were derived from at least three independent measurements. Different letters indicate significant differences between treatments (p<0.05). Values represent the means ± SD.

Figure 3 Cellular concentrations of functional photosystem II reaction centers [RCII] (a), energy transfers between PSII units (i.e., connectivity, P) (b), functional absorption cross sections of PSII photochemistry (σ_PSII) (c) and re-oxidation times of the primary electron acceptor Q_a (${\mathit{\tau }}_{Q_{\mathrm{a}}}$) (d) were determined for Geminigera cryophila acclimated to ambient or high CO₂ conditions combined with low (LL), medium (ML) or high light (HL). G. cryophila did not grow under ambient pCO₂ and HL as indicated by ng. Different letters indicate significant differences between treatments (p<0.05).

Absolute ETRs differed in amplitude and shape in response to the applied changes in irradiance and pCO₂ (Fig. 4a–c). Both maximum absolute electron transport rates (ETR_max) and minimum saturating irradiances (I_k) followed the same trend and were significantly changed by CO₂ (two-way ANOVA: p<0.05) but not by light or the interaction of both factors (Table 3). OA significantly enhanced both parameters under LL (ETR_max: post hoc: p<0.05; I_k: post hoc: p<0.05) but not under ML. The maximum light utilization efficiency (α) was significantly affected by light (two-way ANOVA: p<0.01) and the interaction of CO₂ and light (two-way ANOVA: p<0.01) but not by CO₂ alone (Table 3). α significantly increased from LL to ML at ambient pCO₂ (53 %, post hoc: p<0.01) while such an effect was absent under ML and OA. Between ML and HL, α did not differ when grown under OA.

Figure 4 Absolute electron transport rates (ETR, a–c) and non-photochemical quenching (NPQ, d–f) were measured in response to increasing irradiance in Geminigera cryophila acclimated to ambient (black circles) or high CO₂ (white circles) conditions combined with (a) low (LL), (b) medium (ML) or (c) high light (HL). G. cryophila did not grow under ambient pCO₂ and HL. ETRs were obtained in three individual measurements. Values represent the means ± SD (n=3).

NPQ generally went up with increasing actinic irradiance during the FLC (Fig. 4d–f). Compared with the LL treatments, NPQ values of the ML and HL treatments were as twice as high. There were no differences in the NPQ pattern between the ML and HL treatments observed. Much higher NPQ values were determined in the ambient pCO₂ relative to the OA treatment under LL while such a pCO₂ effect was absent under ML.

4 Discussion

Ecophysiological studies on Antarctic cryptophytes to assess whether climatic changes such as ocean acidification and enhanced stratification affect their growth in Antarctic coastal waters in the future are lacking so far. This study can show that the Antarctic cryptophyte G. cryophila may be a potential winner of such climatic conditions as it reached the highest rates of growth and POC production when grown under HL and OA.

4.1 Geminigera cryophila is sensitive to increasing irradiance under ambient pCO₂

The cryptophyte G. cryophila was well adapted to grow under LL and ML at ambient pCO₂, yielding similar growth, POC quotas and production rates as well as C:N ratios (Fig. 1). In line with this, the exposure of the cryptophyte Rhodomonas salina to 30 up to 150 µmol photons m⁻² s⁻¹ did not lead to any changes in growth rate at 5 ^∘C (Hammer et al., 2002). Even though growth and biomass remained unchanged in G. cryophila between LL and ML, acclimation to the even higher light intensity of 500 µmol photons m⁻² s⁻¹ was indicated by the reduction in F_v∕F_m and [RCII] (Figs. 2a, 3a). Such a decline in the number of photosystems is a typical photo-acclimation response of most microalgae to increasing light and is usually accompanied by a decrease in cellular concentrations of light-harvesting pigments (MacIntyre et al., 2002), as seen here for cellular Chl a and c₂ quotas (Table 2). Even though most studies on temperate cryptophytes report a photo-protective function of Allo, with higher amounts of this carotenoid toward high irradiance (Funk et al., 2011; Laviale and Neveux, 2011), the reduction in cellular Allo quotas from LL to ML in our tested species (Table 2) rather suggests its role in light absorption. Similarly, cellular Allo contents also declined between 40 and 100 µmol photons m⁻² s⁻¹ in the temperate cryptophyte Rhodomonas marina (Henriksen et al., 2002). In this study, various photo-physiological parameters (P, σ_PSII, ${\mathit{\tau }}_{Q_{\mathrm{a}}}$, Fig. 3) did not change between LL and ML in G. cryophila while other photo-acclimation processes such as higher ETR_max, I_k and α took place (Table 3). Such a light-dependent, apparent higher electron flow was accompanied by similar high POC quotas and production rates between LL and ML (Fig. 1b, c) and suggests saturation of the Calvin cycle and therewith the requirement for alternative electron cycling to dissipate the excessive light energy. In the temperate R. salina, the onset of NPQ was induced after saturation of the Calvin cycle and was found to be located in the chlorophyll a∕c proteins and not in the phycobiliproteins (Kana et al., 2012). In fact, NPQ was strongly enhanced in G. cryophila between LL and ML at ambient pCO₂ (Fig. 4). As ML-acclimated cells relative to LL-acclimated ones also exhibited a higher potential of F_v∕F_m recovery after the FLC (Fig. 2b), it appears that all these adjustments allowed a reduction in the excitation pressure on the photosynthetic apparatus and protected G. cryophila well against short-term high light exposure. Unexpectedly, G. cryophila was, however, unable to grow at the highest light intensity of 500 µmol photons m⁻² s⁻¹ under ambient pCO₂, pointing towards its vulnerability of coping with HL under present-day pCO₂. On the other hand, long-term field observations have shown that cryptophytes mainly occur under stratified conditions along the WAP (e.g., Moline and Prézelin, 1996; Moline et al., 2004; Mendes et al., 2013). A connection of this group with highly illuminated conditions was first suggested by Mendes et al. (2017), but this data set lacks information on which cryptophyte species were present and their photosynthetic responses. The reason for this difference could be related to species- or strain-specific differences. On the basis of our results, the G. cryophila strain tested here was indeed able to cope well with medium, but not high irradiances. More tests with other cryptophytes are certainly required to be able to better evaluate cryptophytes' abilities to cope with high irradiances.

4.2 OA alters the physiological response of G. cryophila to high irradiance

In line with previous studies on Antarctic diatoms and P. antarctica (Hoogstraten et al., 2012; Heiden et al., 2016; Trimborn et al., 2017b), OA in conjunction with low irradiance did not alter growth, cellular contents or production rates of POC in G. cryophila (Fig. 1a–c). There was, however, an OA-dependent decline in C:N under LL (Fig. 1d), resulting from a significant enhancement of PON quotas between ambient and high pCO₂ (23.1±1.4 and 29.0±1.6 pg N cell⁻¹, respectively). Hence, G. cryophila cells most probably benefitted from lower energy investments to acquire inorganic carbon under high pCO₂. Whereas the two temperate cryptophytes Rhodomonas sp. and Chroomonas sp. (Burns and Beardall, 1987; Camiro-Vargas et al., 2005), similar to other Antarctic phytoplankton taxa (Trimborn et al., 2013), were able to actively take up CO₂ and ${\mathrm{HCO}}_{\mathrm{3}}^{-}$, the operation of a carbon concentrating mechanism in G. cryophila has not been tested so far. As typically observed for temperate phytoplankton (Hopkinson et al., 2011; McCarthy et al., 2012; Yang and Gao, 2012), the higher pCO₂ was not used to fix more POC per cell by G. cryophila at LL (Fig. 1b, c) but instead fueled protein buildup through conversion of carbohydrate skeletons to proteins. N assimilation is energetically costly due to the reduction steps involved (Sanz-Luque et al., 2015); therefore, the finding of elevated PON buildup under OA and LL is surprising. In line with this, ETR_max per PSII increased by 121 % from ambient to high pCO₂ (Table 3), likely used to reduce nitrite to ammonium. Calculating overall maximum ETRs per cell (cETR_max = ETR_max × [RCII], given in amol cell⁻¹ s⁻¹), there was also an OA-dependent increase in cETR_max of 41 % (mean value of 76 and 107 amol cell⁻¹ s⁻¹ under ambient and high pCO₂, respectively), but this increase was comparably lower relative to ETR_max per PSII. The reason for this comes from the strong reduction in [RCII] between ambient and high pCO₂ at LL (Fig. 3). Next to the positive OA effect on N metabolism, the photochemical efficiency (F_v∕F_m) of G. cryophila was also significantly increased by 17 % by OA under LL (Fig. 2a). Unexpectedly, this effect was not the result of reduced cellular quotas of the light-harvesting pigments (Chl a and c₂), as they remained the same under these conditions. Instead a significant OA-dependent decrease of 26 % in the carotenoid Allo was found (Table 2), which could explain the positive OA effect on F_v∕F_m. This is in line with the OA-dependent reduction in NPQ observed in LL-acclimated G. cryophila cells (Fig. 4), pointing towards a reduced need to dissipate excess light energy following short-term high light exposure. Overall, OA in conjunction with LL was beneficial for G. cryophila, with positive effects in particular on N metabolism.

The beneficial OA effect on N assimilation under LL, however, vanished at ML (Fig. 1d; 23.2±3.2 and 23.2±2.3 pg N cell⁻¹), probably as a result of the higher N metabolism cost to maintain photosynthesis under these conditions (Li et al., 2015). Based on our results, the physiology of the cryptophyte G. cryophila remained more or less unchanged between ambient and high pCO₂ at ML (Figs. 1, 2, 4; Tables 2 and 3). At the highest irradiance (HL), G. cryophila could not grow under ambient pCO₂, but surprisingly grew well under the same light intensity in conjunction with OA, displaying the highest growth rates compared to all other treatments (Fig. 1a). Similarly, this species also showed the highest production rates of POC and PON (Fig. 1). Compared with the Antarctic diatoms and P. antarctica tested so far, which exhibited either negative or neutral effects in response to OA and high irradiance on growth and/or photosynthesis (Feng et al., 2010; Heiden et al., 2016, 2019; Trimborn et al., 2017a, b; Koch et al., 2019), growth and photosynthesis of the cryptophyte benefitted synergistically from OA and HL. Looking at the significantly higher amounts of Chl a and Allo per cell and its larger σ_PSII between ML and HL under OA (Table 2, Fig. 3c), this species even reinforced its capacity to absorb light. Faster electron drainage into downstream processes was evident by the shorter Q_a re-oxidation time between ML and HL under OA (Fig. 3d), supporting the idea that this species indeed managed to cope well with these conditions.

4.3 Implications for the ecology of G. cryophila in future coastal Antarctic waters

Along the coast of the WAP, diatoms, prymnesiophytes and cryptophytes represent the main phytoplankton groups, which form prominent blooms and therefore strongly contribute to carbon biomass buildup (Garibotti et al., 2005; Trimborn et al., 2015). The occurrence of cryptophytes in this region was associated with low salinity and warm stratified surface waters (Moline and Prézelin, 1996; Moline et al., 2004; Mendes et al., 2013, 2017). Only recently, it was suggested by Mendes et al. (2017) that a high tolerance of cryptophytes to withstand high irradiances could potentially explain their occurrence in well-illuminated surface waters. Our results from a short-term CO₂ light experiment point towards a high ability of G. cryophila to acclimate to such conditions and to cope well with medium, but not high irradiances; whether this applies to other Antarctic cryptophyte species as well needs further testing. Also, it remains unclear whether similar responses would be found when exposed in the longer term. In general, with respect to the projected climatic changes little is known about the potential CO₂ sensitivity of cryptophytes. Previous studies mainly assessed the response to OA of cryptophytes at the community level, and this showed no discernible effects on their abundance (Domingues et al., 2014; Sommer et al., 2015; Young et al., 2015; Schulz et al., 2017; Donahue et al., 2019). This study is the first at the species level to show that the combination of OA and high irradiance promoted growth and biomass production in the Antarctic cryptophyte G. cryophila. In fact, while HL conditions inhibited growth of this species under ambient pCO₂, the combination of OA and HL, on the other hand, enabled it to grow and to cope even better with the applied environmental conditions, reaching highest growth and POC production rates (Fig. 1). This was also accompanied with a high photo-physiological capacity of this species when exposed in the short term to increasing irradiances. The beneficial effect of OA and HL for G. cryophila is contrary to previous observations, where growth and/or photosynthesis were inhibited in several diatoms, but without any effect on the prymnesiophytes P. antarctica (Feng et al., 2010; Trimborn et al., 2017a, b; Beszteri et al., 2018; Heiden et al., 2018, 2019; Koch et al., 2018). Hence, G. cryophila could be a potential winner of climate change, with higher abundances and an increased contribution to the productivity of future stratified, acidified and well-illuminated coastal Antarctic waters. This study further confirms previous results (Moline et al., 2004; Ducklow et al., 2007; Montes-Hugo et al., 2009; Mendes et al., 2017), which point to a stronger importance of flagellates in the future. A functional shift away from efficient carbon sinkers such as diatoms to less efficient carbon vectors such as flagellates including cryptophytes and prymnesiophytes could, however, diminish the strength of the biological carbon pump of future Antarctic coastal waters.