A comparison of species specific sensitivities to changing light and carbonate chemistry in calcifying marine phytoplankton

Coccolithophores are unicellular marine phytoplankton and important contributors to global carbon cycling. Most work on coccolithophore sensitivity to climate change has been on the small, abundant bloom-forming species Emiliania huxleyi and Gephyrocapsa oceanica. However, large coccolithophore species can be major contributors to coccolithophore community production even in low abundances. Here we fit an analytical equation, accounting for simultaneous changes in CO2 and light intensity, to rates of photosynthesis, calcification and growth in Scyphosphaera apsteinii. Comparison of responses to G. oceanica and E. huxleyi revealed S. apsteinii is a low-light adapted species and, in contrast, becomes more sensitive to changing environmental conditions when exposed to unfavourable CO2 or light. Additionally, all three species decreased their light requirement for optimal growth as CO2 levels increased. Our analysis suggests that this is driven by a drop in maximum rates and, in G. oceanica, increased substrate uptake efficiency. Increasing light intensity resulted in a higher proportion of muroliths (plate-shaped) to lopadoliths (vase shaped) and liths became richer in calcium carbonate as calcification rates increased. Light and CO2 driven changes in response sensitivity and maximum rates are likely to considerably alter coccolithophore community structure and productivity under future climate conditions.

. Scanning electron microscope images of a E. huxleyi, S. apsteinii and a Coccolithus species showcasing size and cellular calcium carbonate quota differences between species. Species shown were either isolated or cultured by the authors.  Table 1. Fit coefficients (k 1 , k 2 , k 3 , k 4 , k 5 , k 6 ), R 2 , p-values, F-values and degrees of freedom obtained from fit equation (2) for calcification (pg C cell −1 d −1 ), photosynthetic carbon fixation (pg C cell −1 d −1 ) and growth rate (d −1 ) fits to all data. Note that for calcification and photosynthetic carbon fixation rates the unit of k 1 is pg C cell −1 day −1 and for growth rates day −1 .
Scientific RepoRts | (2019) 9:2486 | https://doi.org/10.1038/s41598-019-38661-0 upon CO 2 level and light intensity, rates varied between 28-234 pg C cell −1 d −1 for calcification, 60-243 pg C cell −1 d −1 for photosynthesis and 0.14-0.57 d −1 for growth (Table S1). At all light intensities, particulate inorganic to organic carbon ratios PIC:POC ratios increased with CO 2 to an optimal point before declining with further increases in CO 2 (Table S1). PIC:POC ratios varied from 0.42 to 1.44 depending upon CO 2 level and light intensity (Table S1). Calcification, photosynthetic carbon fixation and growth rates had similar CO 2 requirements to achieve half saturation ( ) K sat 1 2 CO 2 and optimal rates ( Responses to changing light intensity. Maximum rates for growth, calcification and photosynthetic carbon fixation were observed to increase up to an optimum light intensity before declining with further increases in light (Fig. 3). Calcification rates increased 39% between 50 and 100 μmol photons m −2 s −1 before declining by 45% at 515 μmol photons m −2 s −1 . Photosynthetic carbon fixation rates increased 62% between 50 and 200 μmol photons m −2 s −1 and declined by 30% at 515 μmol photons m −2 s −1 . Growth rates increased 24% between 50 and 200 μmol photons m −2 s −1 and declined by 23% at 515 μmol photons m −2 s −1 ( Table 2). The effect of carbonate chemistry (i.e. the combined change in concentrations of CO 2 , − HCO 3 , − CO 3 2 and H + as a result of rising f CO 2 ) on metabolic rates was greatest between 100-400 μmol photons m −2 s −1 and decreased towards more extreme light intensities (Fig. 3).
Increasing f CO 2 resulted in an increase in light half-saturation intensities ( ) K sat 1 2 PAR , a decrease in optimal light requirements, and an increase in sensitivity to high light ( ) K inhib 1 2 PAR for all rates (Table 3). Calcification reached optimal rates at lower light intensities (120-157 μmol photons m −2 s −1 ) than growth (122-196 μmol photons m −2 s −1 ) or photosynthesis (128-263 μmol photons m −2 s −1 ), though differences between the rates decreased with increasing f CO 2 (Table 3). Inhibiting light levels were lowest for calcification (385-1050 μmol  Table S2). The number of muroliths per cell varied between five and 23 (average 12.4), while the number of lopadoliths per cell varied between zero and six (average 2.3). The ratio of muroliths to lopadoliths on a cell varied from zero to 22 (average 6.4). The length of muroliths was 6.1-10.9 μm (average 8.3) while the width was 4.0-9.1 μm (average 6.1). The length of lopadoliths varied between 2.8-13.9 μm (average 8.1) and the width 6.1-16.3 μm (average 10.5). The ratio of muroliths to lopadoliths was observed to increase with increasing light at 100 (n = 479 df = 2, Chi-sq = 14.34, p = 7.69E-4) and 400 μatm CO 2 (n = 357 df = 2, Chi-sq = 11.44, p = 3.28E-3). There was also a significant effect of calcification rate on PIC per lith, with liths containing more PIC in treatments with higher calcification rates (n = 8 R 2 = 0.80, F = 23.45 p = 2.87E-3, Fig. 4). Other than this, no environmentally significant patterns were observed in any morphological features with changing CO 2 or light (see supplementary information for discussion on these results).

Discussion
Increasing CO 2 concentrations resulted in an optimum curve response in all physiological rates for S. apsteinii (Fig. 2). This response pattern has now been observed for multiple coccolithophore species 16,18 , and is most likely driven by the combined effects of physiological rate stimulation by increasing substrate (CO 2 and − HCO 3 ) availability and physiological rate inhibition by increasing [H + ] 14,17,18,36 .
Larger species generally require more substrate than smaller species to sustain growth. However, larger species have less surface area, relative to their volume, over which to take up essential materials and nutrients 37 . As such, it is expected that larger species, with a lower surface area to volume ratio, would require either a higher substrate concentration or a faster uptake rate in order to support their higher cellular requirements 37 . If higher substrate concentrations were needed then it could be expected that S. apsteinii might have higher CO 2 half-saturation and optimum requirements than E. huxleyi and G. oceanica for all rates. However, S. apsteinii had a similar K sat 1 2 CO 2 and CO 2 optimum as the two smaller species for growth and photosynthetic carbon fixation rates and a slightly higher CO 2 optimum requirement for calcification rates ( Table 2, 20,30 ). This suggests that S. apsteinii would need to support its greater substrate demand by fixing more carbon per unit surface area than smaller species. To see if this was the case, daily carbon fixation per unit surface area was calculated using carbon fixation rates (POC, PIC and TPC from 16,19,20 and this paper at 20 °C) and average cell diameters of 5.59, 9.33, and 17.59 μm for E. huxleyi, G. oceanica and S. apsteinii, respectively. Based on these calculations, S. apsteinii fixes more carbon per unit surface area per day than either E. huxleyi or G. oceanica under most CO 2 conditions at low to moderate light intensities ( Fig. 5a and b). However, at higher light intensities it fixes approximately the same amount of carbon per unit surface area per day (Fig. 5c). The relative decrease in carbon fixation at these higher light intensities in comparison to E. huxleyi and G. oceanica is likely due to a higher light sensitivity (see below). Meanwhile below this threshold, the generally higher fixation of carbon per unit surface area per day by S. apsteinii can be achieved by higher inorganic carbon transporter density (higher substrate uptake), or by less diffusive CO 2 leakage from the cell. It may be that by having a proportionally lower amount of the cells internal volume interacting with the surrounding media (due to a much lower surface area to volume ratio) S. apsteinii loses proportionally less CO 2 through leakage. While decreased leakage in larger coccolithophore species has been suggested previously 37 , this difference in leakage was thought to be driven by the uptake of different proportions of CO 2 and − HCO 3 between the species rather than size alone. While evidence suggests that carbon uptake efficiency is higher in some species and phytoplankton groups (i.e. 38,39 ), it is not yet clear if cell size has a consistent effect on carbon uptake efficiencies.
Sensitivity to high CO 2 /low pH varied between the three rates with calcification being more sensitive than photosynthesis and growth ( Table 2). This agrees with previous work on G. oceanica, E. huxleyi and C. pelagicus, and provides additional evidence for the general notion that calcification by coccolithophores will be negatively impacted by future ocean changes 16,[18][19][20] . Sensitivity of calcification rates to high CO 2 ( ) K inhib 1 2 CO 2 are similar between S. apsteinii and E. huxleyi, with G. oceanica being much more sensitive 20,30 . The steeper decline of calcification rates, beyond optimum CO 2 , of G. oceanica compared to E. huxleyi has previously been speculated to be connected to a higher degree of inorganic versus organic carbon production (PIC:POC) in G. oceanica 18,20 . The relatively greater production of intracellular H + , via calcification, makes it more difficult for species species with higher PIC content to maintain intracellular pH homeostasis 17 . In this regard, S. apsteinii with a similar PIC:POC as E. huxleyi would fit into this concept.
Significant inhibition of all rates by light was observed in S. apsteinii when CO 2 levels exceeded 100 μatm (Fig. 3, Table 3). That is in sharp contrast to rates in E. huxleyi and G. oceanica for which little light inhibition is observed   20,30 , unpublished E. huxleyi data). This suggests that S. apsteinii may be a lower light adapted species which is also supported by the fact that it has been observed to grow at light intensities as low as 5 μmol photons m −2 s −1 46 . Oceanic abundance data for S. apsteinii is sparse, as a result it is currently not possible to confirm if similar low light preferences are observed in the natural environment.
As f CO 2 levels increased S. apsteinii needed lower light intensities to sustain optimal rates. These results agree with those for G. oceanica, which also required less light to support optimum calcification and photosynthetic carbon fixation rates as f CO 2 increased 30 . The results also agree with those for E. huxleyi growth rates, which required less light with rising f CO 2 , but not calcification or photosynthetic carbon fixation rates which were insensitive to changing light intensity 20 (Table S3). In all three-above species, the increase in f CO 2 levels also resulted in an overall decrease in maximum rates 20,30 (Table 2). Most phytoplankton acquire inorganic carbon through a combination of passive CO 2 diffusion into the cell, and CO 2 concentrating mechanisms (CCMs) which actively transport inorganic carbon into the cell 47,48 . As CO 2 concentrating mechanisms are an active process, they require energy to function 39,48 . The efficiency of passive uptake depends upon the cell to seawater CO 2 concentration gradient (uptake versus leakage rates), membrane permeability and area 39 . So, assuming that cell size and membrane permeability stay constant, increasing seawater CO 2 concentrations increase the diffusive influx and reduce loss through leakage. This would reduce the need for an active CCM thus lowering the cell's energy requirements. As a result, higher substrate uptake efficiency could be indicated by a decrease in light levels needed to reach half-saturated and optimal rates (see Fig. 6 dotted line). Another explanation, for reduced light requirements could be the inhibiting effects of high CO 2 /H + . As CO 2 /H + concentrations increase to above optimum levels it suppresses maximum rates to lower and lower absolute rates. Lower rates result in decreased demand for resources such as carbon. This lower substrate demand could result in decreased CCM activity thereby decreasing optimum light requirements under elevated CO 2 /H + (Fig. 6 dashed line). All three species show decreased maximum rates under elevated CO 2 /H + . As a result, decreased energy requirements because of inhibited maximum rates could explain decreased optimum light requirements in E. huxleyi, G. oceanica and S. apsteinii. However, G. oceanica also shows indications of increased substrate uptake efficiency, through decreasing K sat 1 2 PAR and optimal light requirements 30 . So, for G. oceanica it appears that increasing CO 2 /H + may decrease light optimal requirements through a combination of increased substrate uptake efficiency and through inhibition of maximum rates (Fig. 6 dot-dashed line).
As well as decreasing light intensities for optimal rates, increasing f CO 2 also resulted in a narrowing of the light tolerances (light niche) of S. apsteinii. This was driven by a combined increase in light intensities required for half saturation of rates, and a decrease in light intensities required to half inhibit rates with rising f CO 2 ( Table 3). The CO 2 niche of this species was also observed to narrow with increasing light intensity, with an increased sensitivity to high CO 2 and a decrease in CO 2 requirements for optimal physiological rates ( Table 2). So S. apsteinii, when already under stress from one environmental condition, becomes less tolerant to other extreme environmental conditions, similar to E. huxleyi and G. oceanica for high CO 2 sensitivity under rising temperature 20,30 . However, when exposed to the same stress of rising light intensities, E. huxleyi becomes less sensitive to high CO 2 conditions, while the sensitivity of G. oceanica to high CO 2 depends upon the rate considered 20,30 . Similarly, when exposed to the stress of rising CO 2 conditions, neither E. huxleyi or G. oceanica show any change in sensitivity to high light conditions 20,30 (Table S3). So, it would appear that the response to multiple environmental stressors is species-specific. This adds weight to the suggestion that coccolithophore assemblages may undergo shifts in species composition, under future ocean change.
Depending on the emission scenario, p CO 2 levels are projected to reach between 420 to 985 μatm by 2100 resulting in ocean temperature increases of 2.6 to 4.8 °C (RCP 2.6-8.5) 49,50 . Ocean warming is expected to strengthen stratification of the water column, resulting in a shallower mixed layer and thus a higher average light availability within the mixed layer 2,51 . The dependence of light responses on f CO 2 levels, and vice-versa, could have important implications for S. apsteinii under future ocean conditions where both light and f CO 2 availabilities are expected to change. S. apsteinii already appears to be a low light adapted species with rates saturating at relatively low light intensities under current day f CO 2 conditions (<170 μmol photons m −2 s −1 ). Considering that light levels to saturate and inhibit rates for this species increase and decrease, respectively, with rising f CO 2 (Table 3), this species could become restricted to a narrower light range under predicted future ocean conditions.

Conclusion
S. apsteinii appears to be a low light adapted species, with a similar optimum curve response to rising CO 2 as E. huxleyi and G. oceanica, C. pelagicus and C. leptoporus. Calculations suggest that for S. apsteinii, a single unfavourable growth condition (CO 2 , or light) will result in increased sensitivity to changes in other environmental variables. This contrasts with E. huxleyi, which either becomes less sensitive or shows no change in sensitivity to changes in other environmental variables when at extreme CO 2 or light conditions, and G. oceanica whose change in sensitivity varies between different physiological rates. With light and CO 2 levels both set to increase over the coming century 2,51 S. apsteinii could become restricted to narrower light and CO 2 ranges while under Figure 6. Conceptual diagram depicting how the light intensities required to support half-saturated ( ) K sat 1 2 and optimum rates change with changing CO 2 . While the solid line represents the default response to rising light levels, the dotted line represents an increase in substrate uptake efficiency with rising CO 2 , the dashed line represents an increase in H + inhibition with rising CO 2 and the dot-dashed line represents an increase in both substrate uptake efficiency and H + inhibition with rising CO 2 .
Scientific RepoRts | (2019) 9:2486 | https://doi.org/10.1038/s41598-019-38661-0 rising temperature, E. huxleyi and G. oceanica could become more sensitive to high CO 2 . These species-specific changes in temperature/light/CO 2 sensitivity are likely to affect coccolithophore community composition under future ocean conditions. Data from E. huxleyi, G. oceanica and S. apsteinii indicate that rising CO 2 levels will also result in decreased optimum light requirements of all species as a result of reduced maximum rates and, in the case of G. oceanica, increased substrate uptake efficiency. This adds further evidence to the idea that rising CO 2 will not only result in changes in species composition, but also in community-wide shifts in total organic and inorganic carbon production by coccolithophores, with consequent effects on local carbon cycling and sequestration. 3 ] using the program CO2SYS 54 , the stoichiometric equilibrium constants (K 1 and K 2 ) for carbonic acid determined by 55 and refitted by 56 , K S for sulphuric acid determined by 57 and K B for boric acid following 58 . Cell densities and particulate and dissolved carbon. Cell densities for each treatment were checked every 2-3 days using a flow cytometer (Becton Dickinson FACSCalibur). Living cells were detected by scatter plots of red autofluorescence in relation to orange fluorescence of the cells (FL3 vs. FL2). Specific growth rate (μ) was determined using cell density counts from the beginning and at the end of the experiment. Growth rate was calculated as:

Methods
where C t and C 0 are the cell densities at the end and the beginning of the experiment respectively and d is incubation length in days. Calcification and photosynthetic rates were obtained by multiplying cellular quotas of particulate inorganic (calcification) or particulate organic (photosynthesis) carbon with growth rates. End of experiment sampling started approximately two hours after the onset of the light period and lasted no longer than 3 hours. Duplicate samples for total and organic particulate carbon (TPC and POC) were filtered (−200 mbar) onto pre-combusted (500 °C for 4 hours) Whatmann GF/F filters and stored in pre-combusted (500 °C for 4 hours) glass petri-dishes at −20 °C. Prior to analysis, POC filters were treated with 37% fuming HCl in a desiccator for 2 hours to remove all particulate inorganic carbon (PIC). TPC and POC filters were dried and analysed for carbon content and carbon isotopic signatures on an elemental analyser (Flash EA, Thermo Fisher) coupled to an isotope ratio mass spectrometer (IRMS, Delta V plus, Thermo Fisher) according to 59  where the metabolic rate (MR) of photosynthesis, calcification or growth is dependent on substrate (S = CO 2 and − HCO 3 ), [H + ] (H) and light (I) conditions, and fit coefficients k 1 , k 2 , k 3 , k 4 , k 5 and k 6 . For the experiment, a higher number of treatment levels was used at the expense of replication within treatments. This approach provides more information on the functional relationship, and tipping points, between carbonate chemistry and cellular rates without a significant loss of statistical power (see 60  reached), are presented in Tables 1, 2 and 3, respectively. Please note changes in CO 2 optima and half-saturation of less than 10 μmol kg −1 were considered to be within the uncertainties of the model fit.
Coccolith morphology. S. apsteinii is unusual in that it bears two types of coccoliths; the plate-like muroliths and the vase-shaped lopadoliths 61 . To assess potential changes in coccolith morphology under different treatments, samples for scanning electron microscopy (SEM) were filtered onto Nucleopore TM polycarbonate membrane filters (25 mm diameter, 0.8 μm pore size) and air-dried at room temperature over 12 hours. Samples were then stored in a desiccator until analysis. Samples were mounted onto metallic stubs using sticky tabs and sputter-coated with gold before being visualized using a ZEISS EVO/LS15 scanning electron microscope. For each treatment 80-100 cells were examined and the number of muroliths and lopadoliths per cell, average of murolith and lopadolith length and width per cell, and the ratio of muroliths to lopadoliths were recorded. Due to time constraints, only a sub-set of treatments could be examined. These were; 100, 400 and 2000 μatm f CO 2 at 50 and 515 μmol photons m −2 s −1 and 100, 200, 400 and 2000 μatm at 100 μmol photons m −2 s −1 of PAR. Comparisons between treatments were made using a Kruskal-Wallis ANOVA with an alpha level of significance of 0.05. The Kruskall-Wallis ANOVA was chosen firstly to avoid an inflated Type I error rate which can occur if making multiple comparisons between groups, and secondly to account for the slight non-normal distribution of the SEM data. The mass of PIC per coccolith was calculated by dividing PIC quota per cell by the total number of liths (muroliths + lopadoliths) for each treatment. Patterns in PIC per coccolith were tested using a linear regression with an alpha of significance of 0.05. Linear regressions were chosen for the reasons explained in Section 'Cell densities and particulate and dissolved carbon' above.

Data Availability
Datasets on physiological rate responses generated and compared during the current study are available in this published article (and its Supplementary Information files) and in 20,30 . Datasets containing the raw scanning electron microscopy values are available from the corresponding author on reasonable request.