2.1 Experimental design

The three phytoplankton species used in the experiments were the diatom P. tricornutum (SAG, 1090-1b; isolated from Plymouth, UK; De Martino et al., 2007), the cryptophyte Rhodomonas sp. (isolated from the Kiel Bight, Baltic Sea, and identified by the working group at GEOMAR; maintained and still available from GEOMAR for laboratory culture) and the haptophyte E. huxleyi (internal culture collection reference code: A8; isolated from waters off Terceira Island, the Azores). P. tricornutum and Rhodomonas sp. are model species widely used in studies of diatom genomes, cryptophyte photosynthesis and planktonic trophic dynamics (Bi et al., 2017), and E. huxleyi is one of the major calcifying organisms in the pelagic ocean (Winter et al., 2014). The algal cultures were non-axenic. Because the biomass of bacteria was very low, the bacterial influence on the chemical composition of phytoplankton was negligible. Over the course of the experiments, the cultures of all species were exposed to a salinity of 37 psu and a light intensity of 100 µmol photons m⁻² s⁻¹ following a light / dark cycle of 16 / 8 h in temperature-controlled rooms of 12, 18 and 24 ^∘C. The culture medium was prepared according to the modified Provasoli's medium (Provasoli, 1963; Ismar et al., 2008), with enrichment nutrient solutions added to sterile, filtered (0.2 µm pore size, Sartobran^® P 300; Sartorius, Göttingen, Germany) North Sea water. Sodium nitrate and potassium dihydrogen phosphate were added to achieve the molar ratios of 10 : 1 (35.2 µmol L⁻¹ N and 3.6 µmol L⁻¹ P), 24 : 1 (88 µmol L⁻¹ N and 3.6 µmol L⁻¹ P) and 63 : 1 (88 µmol L⁻¹ N and 1.4 µmol L⁻¹ P). Sodium silicate pentahydrate was also added to diatom cultures at a concentration of 88 µmol L⁻¹. Initial pCO₂ was manipulated by bubbling with CO₂-enriched air (560 and 2400 µatm). Subsequently, the culture medium was transferred into sealed cell culture flasks with a 920 mL culture volume. Each treatment was replicated three times. All culture flasks were carefully agitated twice per day at a set time to minimize sedimentation.

At the onset of the experiments, each species was grown in batch cultures across a fully factorial combination of three temperatures (12, 18 and 24 ^∘C), three N : P supply ratios (molar ratios 10 : 1, 24 : 1 and 63 : 1) and two pCO₂ levels (560 and 2400 µatm) (Fig. S1 in the Supplement). The chosen levels of temperature, N : P supply ratio and pCO₂ cover the ranges of typical changes of the three factors in natural environments, and they are in general agreements with projections. The temperature regimes broadly conform to sea surface temperatures in the source regions for the three taxa studied: Plymouth, UK, for P. tricornutum (∼9–17 ^∘C) (Highfield et al., 2010); the Kiel Bight for Rhodomonas sp. (∼3–18 ^∘C) (Hiebenthal et al., 2013); and the Azores for E. huxleyi (16–22 ^∘C; http://dive.visitazores.com/en/when-dive, last access: 17 September 2019). The 6 ^∘C elevation also mimics the largest projected warming under climate change scenarios (Sommer and Lengfellner, 2008). N : P molar ratio of 24 : 1 was selected as the balanced ratio under which phytoplankton cultures are typically maintained (Guillard, 1975). Surface ocean inorganic N and P concentrations are highly depleted throughout much of the low-latitude oceans (Moore et al., 2013). Nutrient availability in the future will be altered by increasing external nutrient inputs especially in coastal oceans, changes in surface ocean chemistry driven by anthropogenic increases in atmospheric CO₂ and changes in ocean circulation (Moore et al., 2013). Therefore, inorganic N : P ratios show a strong spatial variation in the oceans, e.g., a low ratio of 6 : 1 mol mol⁻¹ in the center of the South Pacific Gyre versus a global scenario of an increase driven by the high N : P atmospheric deposition of ∼ 370 : 1 mol mol⁻¹ (Bonnet et al., 2008; Peñuelas et al., 2012). N : P ratios of 10 : 1 and 63 : 1 selected in this study cover large-scale spatial patterns of nutrient status, and thus our prediction of lipid biomarker production changes is based on both future open-ocean and coastal conditions. Partial CO₂ pressure of 560 µatm is double the preindustrial value and is a standard level for determining climate model sensitivity to pCO₂ forcing (e.g., IPCC, 2014). The value of 2400 µatm is at the mid-range of the projected values (1371–2900 µatm) by 2150 (RCP8.5 scenario; IPCC, 2014). Also, a high pCO₂ has been observed in the areas where one of the studied algae was isolated, e.g., 375–2309 µatm in the Kiel Bight (Thomsen et al., 2010).

The observed maximal growth rate (μ_max, d⁻¹) was calculated from the changes of cell numbers within the exponential growth phase in batch cultures (Bi et al., 2012). Once the early stationary phase was reached, semicontinuous cultures were started with the algae from batch cultures, and the gross growth rate (μ, d⁻¹) was set as 20 % of μ_max. We calculated the volume of the daily renewal incubation water by multiplying daily renewal rate (D, d⁻¹; $D=\mathrm{1}-e^{-\mathit{\mu }\cdot t}$, where t is renewal interval; here t=1 d) with the incubation volume of 920 mL. According to the gross growth rate of 20 % of μ_max, the renewal volumes were about 10 % of the total culture volume (100–200 mL out of 920 mL), and renewal interval was 1 d. Steady state was reached at about 10 d with slight differences between the individual cultures. Renewal of the cultures was carried out at the same hour every day using fresh, filtered seawater pre-acclimated to target pCO₂- and CO₂-enriched water. The difference between the gross growth rate and the loss rate, i.e., the net growth rate (r (d⁻¹); $r=\mathit{\mu }-D$), was used to assess the steady state, at which r was zero and μ was equivalent to D.

2.2 Sampling and measurements

At steady state in semicontinuous cultures, samples were collected for the measurements of cell density, dissolved inorganic carbon (DIC), total alkalinity, pH, POC, FAs, sterols and alkenones. Cell density was measured daily in batch and semicontinuous cultures using an improved Neubauer hemacytometer (Glaswarenfabrik Karl Hecht GmbH, Rhön Mountains, Germany) under a microscope (Hund, Wetzlar, Germany). Also, pH measurements were carried out daily in semicontinuous cultures, and the electrode was calibrated using standard pH buffers (pH 4 and pH 7; WTW, Weilheim, Germany).

DIC samples were taken on sampling days with 10 mL glass vials (Resteck, Germany) filled using a peristaltic pump and an intake tube containing a single-use syringe filter (0.2 µm, Minisart RC25; Sartorius, Göttingen, Germany). Vials were immediately sealed and stored in the dark at 4 ^∘C. DIC was measured according to Hansen et al. (2013) using a gas chromatographic system (8610C; SRI-Instruments, California, USA). For total alkalinity analysis, samples were filtered (GF/F filters; Whatman GmbH, Dassel, Germany) and analyzed with the Tirino plus 848 (Metrohm, Filderstadt, Germany). The remaining carbonate parameter pCO₂ was calculated from DIC and total alkalinity using CO2SYS (Pierrot et al., 2006) and the constants of Hansson (1973) and Mehrbach et al. (1973) that were refitted by Dickson and Millero (1987) (Table S1 in the Supplement; Bi et al., 2017, 2018). DIC and pH did not differ substantially between different temperature and N : P ratio treatments in our study (Table S1).

POC and FA samples (15–50 mL) were taken on precombusted and prewashed (5 %–10 % HCl) GF/F filters (Whatman GmbH, Dassel, Germany). After filtration, filters for POC analysis were immediately dried at 60 ^∘C and stored in a desiccator, and those for FA measurements were stored at −80 ^∘C. For POC analysis in E. huxleyi, particulate inorganic carbon was removed by exposing filters to fuming hydrochloric acid for 12 h. POC was determined using an elemental analyzer (Thermo Flash 2000; Thermo Fisher Scientific, Schwerte, Germany) after Sharp (1974). FAs were measured as FA methyl esters (FAMEs) using a gas chromatograph (Trace GC Ultra; Thermo Fisher Scientific, Schwerte, Germany). Analytical procedure of FAs was modified after Christie (1989). Briefly, FAs were extracted with a solvent mixture of chloroform ∕ dichloromethane ∕ methanol (1 : 1 : 1, volume ratios). The FAME 19 : 0 was added as an internal standard and 21 : 0 as a esterification control. The extracted FAMEs were dissolved in 100 µL n-hexane for analysis. Sample aliquots (1 µL) were injected onto the gas chromatograph (GC) with hydrogen as the carrier gas. Individual FAs were identified with reference to the standards (Supelco 37 component FAME mixture and Supelco Menhaden fish oil), and the peaks were integrated using Chromcard software (Thermo Fisher Scientific, Schwerte, Germany), which included saturated, monounsaturated and polyunsaturated FAs (Bi et al., 2017, 2018).

Samples for sterol and alkenone analysis were filtered (GF/F filters; Whatman GmbH, Dassel, Germany) and measured according to the procedure of Zhao et al. (2006). The filtration volume of sterol and alkenone samples was 100–200 mL, while only 10 % of the extractions were used for GC quantification and the rest was stored for later use. Lipids were extracted ultrasonically eight times from the freeze-dried filter samples utilizing dichloromethane and methanol (3 : 1, volume ratios) as extraction solvent, with C₁₉ n-alkanol added for quantification. After hydrolysis with 6 % potassium hydroxide in dichloromethane, the lipids were separated into a polar fraction and a nonpolar fraction using silica gel chromatography. The polar lipid fractions containing sterols from the extracts of all the three species, as well as C₃₇–C₃₉ alkenones from the E. huxleyi extracts, were eluted with 22 mL dichloromethane and methanol (95 : 5, volume ratios). Subsequently, the polar lipid fractions were silylated with 80 µL N,O-bis(trimethylsilyl)-trifluoroacetamide at 70 ^∘C for 1 h. The sterol and alkenone fractions were analyzed and quantified using an Agilent 7890A GC with flame ionization detection (50 m HP-1 capillary column, 0.32 mm i.d., 0.17 µm film thickness) by comparing analyte peak area to known amount of the internal standard C₁₉ n-alkanol. The oven temperature held initially at 80 ^∘C for 1 min; it was then increased to 200 ^∘C at 25 ^∘C min⁻¹, to 250 ^∘C at 4 ^∘C min⁻¹, to 300 ^∘C at 1.7 ^∘C min⁻¹ (holding for 12 min), and finally to 315 ^∘C at 5 ^∘C min⁻¹ with an 8 min isothermal period.

The identification of brassicasterol ∕ epi-brassicasterol, the major sterol in all three species, was conducted with reference to laboratory standards. In marine systems, the most likely epimer for 24-methylcholesta-5,22E-dien-3β-ol is epi-brassicasterol (24α) synthesized by diatoms (Gladu et al., 1990, 1991), cryptophytes (Goad et al., 1983) and haptophytes (Maxwell et al., 1980). In contrast, 24β-methylcholesta-5,22E-dien-3β-ol (brassicasterol) has been also found, e.g., in a strain of the haptophyte Isochrysis (Gladu et al., 1990). As the stereochemistry at C-24 could not be determined in our study, we use the trivial name brassicasterol ∕ epi-brassicasterol for 24-methylcholesta-5,22E-dien-3β-ol.

Alkenones were identified using an Agilent GC 7890B (30 m HP-5MS column, 0.25 mm i.d., 0.25 µm film thickness) connected to an Agilent MSD 5977B mass selective detector (70 eV constant ionization potential, ion source temperature 230 ^∘C). The temperature program started with a 1 min hold time at 80 ^∘C and then increased to 200 ^∘C at 25 ^∘C min⁻¹, followed by a 4^∘ min⁻¹ ramp to 250 ^∘C and a 1.8 ^∘C min⁻¹ ramp to 300 ^∘C (holding for 10 min); finally the temperature increased to 315 ^∘C at 5 ^∘C min⁻¹ (holding for 5 min). Alkenone identifications were performed by comparing sample mass spectra generated by GC–MS to previously published EI (electron ionization) mass spectra, based on the molecular ion and prominent ions of each alkenone component (de Leeuw et al., 1980; Volkman et al., 1980c; Lopez and Grimalt, 2006). Note that the molecular ions for C_39 : 3 ethyl ketone (C_39 : 3 Et) (at m∕z 556) and C_39 : 2 ethyl ketone (C_39 : 2 Et) (at m∕z 558) were below detection; only prominent ion fragments were detected. In addition, the comparisons of the GC retention times of alkenone molecules between our study and previous work (Volkman et al., 1980c) indicated the presence of C_39 : 3 Et and C_39 : 2 Et.

2.3 Statistics

Generalized linear mixed models (GLMMs; Bolker et al., 2009) were used to test the effects of temperature, N : P supply ratios and pCO₂ on carbon-normalized and per-cell contents of brassicasterol ∕ epi-brassicasterol and C₃₇–C₃₉ total alkenones (as µg mg C⁻¹ and pg cell⁻¹); per-cell contents of C₃₇ alkenones, C₃₈ alkenones, C₃₈ ethyl ketones (C₃₈ Et), and C₃₈ methyl ketones (C₃₈ Me); C₃₇ ∕ C₃₈ alkenone ratios; and C₃₈ Et ∕ C₃₈ Me ratios (C₃₈ Et ∕ Me), with temperature, N : P supply ratios and pCO₂ as fixed effects. Target distributions were tested before GLMMs were taken. Subsequently, appropriate link functions were chosen, e.g., identity link function for any distribution except for multinomial and logit link function for the binomial or multinomial distribution. To find the model that best predicted targets, we tested models containing first-order effects and second- and third-order interactions of temperature, N : P supply ratios and pCO₂. The Akaike Information Criterion corrected (AICc) was used to select the best model for each response variable, with a lower AICc value representing a better fit of the model. When the changes of AICc values were 10 units or more, it was considered a reasonable improvement in the fitting of GLMMs (Bolker et al., 2009). In case AICc values were comparable (<10 units difference), the simpler model was chosen. Based on the differences in AICc values, models containing only first-order effects of temperature, N : P supply ratios and pCO₂ were chosen as the best models for all response variables (bold letters in Table S2), while those containing second- or third-order interactions were not selected.

Principal component analysis (PCA) was conducted to visualize the responses of carbon-normalized contents of brassicasterol ∕ epi-brassicasterol and total fatty acids (TFAs) in the three species and total alkenones in E. huxleyi to the changes of temperature, N : P supply ratios and pCO₂. Data for TFAs were from previous studies (Bi et al., 2017, 2018).

GLMMs were performed using SPSS 19.0 (IBM Corporation, New York, USA). PCA was conducted using R packages factoextra (Kassambara and Mundt, 2017) and FactoMineR (Le et al., 2008) in R version 3.5.1 (R Development Core Team, R Foundation for Statistical Computing, Vienna, Austria). All statistical tests were conducted at a significance threshold of p=0.05.

3.1 Sterol and alkenone composition

Brassicasterol ∕ epi-brassicasterol was the major sterol observed in all the three species, while alkenones were only detected in E. huxleyi. The alkenones in E. huxleyi consisted of four pairs of homologues, i.e., C₃₇ methyl ketones (C₃₇ Me including C_37 : 4 Me, C_37 : 3 Me and C_37 : 2 Me), C₃₈ Me (C_38 : 3 Me and C_38 : 2 Me), C₃₈ ethyl (C_38 : 3 Et and C_38 : 2 Et) and C₃₉ Et ketones (C₃₉ Et including C_39 : 3 Et and C_39 : 2 Et). C₃₇ Me compounds were the most abundant, followed by C₃₈ Et, C₃₈ Me and C₃₉ Et.

3.2 Responses of brassicasterol ∕ epi-brassicasterol to environmental changes

GLMM results showed that per-cell contents of brassicasterol ∕ epi-brassicasterol responded significantly to changes in N : P supply ratios in the three species (bold letters in Table 1). Moreover, per-cell contents of brassicasterol ∕ epi-brassicasterol in P. tricornutum also showed significant responses to temperature changes, while nonsignificant effects of pCO₂ were observed in all species. Specifically, higher per-cell contents of brassicasterol ∕ epi-brassicasterol were observed at higher temperatures and higher N : P supply ratios in P. tricornutum (Fig. 1a; Table S3), under the lowest and highest N : P supply ratios in Rhodomonas sp. (Fig. 1c) and under higher N : P supply ratios in E. huxleyi (Fig. 1e).

Table 1Results of the selected GLMMs testing for the effects of temperature, N : P supply ratios and pCO₂ on carbon-normalized and per-cell contents of brassicasterol ∕ epi-brassicasterol (brassi. ∕ epi-brassi. ∕ POC and brassi. ∕ epi-brassi. ∕ cell; µg mg C⁻¹ and pg cell⁻¹) in Phaeodactylum tricornutum, Rhodomonas sp. and Emiliania huxleyi; carbon-normalized and per-cell contents of C₃₇–C₃₉ total alkenones (Alkenones ∕ POC and Alkenones ∕ cell); per-cell contents of C₃₇ alkenones (C₃₇ ∕ cell), C₃₈ alkenones (C₃₈ ∕ cell), C₃₈ ethyl ketones (C₃₈ Et ∕ cell) and C₃₈ methyl ketones (C₃₈ Me ∕ cell); and the ratios of C₃₇ ∕ C₃₈ alkenones and C₃₈ Et ∕ C₃₈ Me (C₃₈ Et ∕ Me) in E. huxleyi. Significant p values are shown in bold; T represents temperature, and N : P represents N to P supply ratios.

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Carbon-normalized contents of brassicasterol ∕ epi-brassicasterol responded significantly to pCO₂ in Rhodomonas sp. and to temperature and N : P supply ratios in E. huxleyi (bold letters in Table 1) but with nonsignificant responses in P. tricornutum. Carbon-normalized contents of brassicasterol ∕ epi-brassicasterol in Rhodomonas sp. decreased as pCO₂ increased (Fig. 1d), while those in E. huxleyi were generally higher at higher temperatures and under the balanced N : P supply ratio (N : P = 24 : 1 mol mol⁻¹; Fig. 1e).

Figure 1Carbon-normalized (open circles) and per-cell (open triangles) contents of brassicasterol ∕ epi-brassicasterol (mean ± SE; µg mg C⁻¹ and pg cell⁻¹, respectively) in response to the changes in temperature, N : P supply ratios and pCO₂ in Phaeodactylum tricornutum (a, b), Rhodomonas sp. (c, d) and Emiliania huxleyi (e, f). Triplicates were set for each treatment.

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3.3 Responses of alkenones to environmental changes

Total alkenone content per cell in E. huxleyi increased with increasing N : P supply ratios (Fig. 2a; bold letters in Tables 1; S4). However, carbon-normalized contents of total alkenones showed nonsignificant responses to changes in temperature, N : P supply ratios and pCO₂.

Figure 2Carbon-normalized (open circles) and per-cell (open triangles) contents of C₃₇–C₃₉ total alkenones (mean ± SE; µg mg C⁻¹ and pg cell⁻¹, respectively) (a, b), C₃₇ ∕ C₃₈ alkenone ratios (c, d) and C₃₈ ethyl ∕ C₃₈ methyl ratios (C₃₈ Et ∕ Me alkenone ratios) (e, f) in Emiliania huxleyi in response to the changes in temperature, N : P supply ratios and pCO₂. Triplicates were set for each treatment.

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C₃₇ ∕ C₃₈ alkenone ratios responded significantly to all three environmental factors (bold letters in Table 1), showing a clear increase with increasing temperature, a higher value under the lowest and highest N : P supply ratios, and a slight decrease at high pCO₂ (Fig. 2c and d). C₃₈ Et ∕ Me ratios had significant responses only to temperature changes, which were clearly higher at the highest temperature (Fig. 2e and f).

3.4 Comparisons of sterol, alkenone and fatty acid responses

PCA extracted four axes with eigenvalues >1, and the first two axes in PCA explained 44.1 % of total variance (Table S5). PC axis 1 explains 26.4 % of the total variance and largely differentiates between the highest (N : P = 63 : 1 mol mol⁻¹) and lower N : P treatments (N : P = 10 : 1 and 24 : 1 mol mol⁻¹). Along PC axis 1, N : P supply ratios correlated positively with carbon-normalized contents of TFAs in P. tricornutum and Rhodomonas sp. but negatively with brassicasterol ∕ epi-brassicasterol in E. huxleyi (Fig. 3). PC axis 2 (17.7 % of the total variance) differentiates between the highest and both colder temperature treatments, along which temperature showed positive correlations with brassicasterol ∕ epi-brassicasterol in Rhodomonas sp. and alkenones in E. huxleyi but a negative correlation with TFAs in E. huxleyi. Both Dim1 and Dim2 loadings for pCO₂ were very low (0.185 and 0.151, respectively), showing a weaker effect of pCO₂ on lipid biomarkers compared to temperature and N : P supply ratios. Along PC axis 2, pCO₂ plays a negative role particularly on TFAs in E. huxleyi, while the contribution of pCO₂ to other lipid biomarkers was weak.

Figure 3PCA biplot based on the carbon-normalized contents of the major sterol (brassicasterol ∕ epi-brassicasterol) and total fatty acids (µg mg C⁻¹) in Phaeodactylum tricornutum, Rhodomonas sp. and Emiliania huxleyi, as well as C₃₇–C₃₉ total alkenones in E. huxleyi under different temperatures, N : P supply ratios and pCO₂. Blue, black and red symbols represent 12, 18 and 24 ^∘C, respectively. Open triangles, open circles and closed circles represent N : P molar ratios of 10 : 1, 24 : 1 and 63 : 1, respectively. The first two dimensions (Dim1 and Dim2) account for 44.1 % of the total variance. The length of each vector reflects the combined loading of each variable in the first two dimensions (Table S5).

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4.1 Sterol and alkenone composition

Brassicasterol ∕ epi-brassicasterol was the only major sterol in the three algal species under wide ranges of temperature, N : P supply ratios and pCO₂. It is well established that the diversity of sterols is low in most phytoplankton species (Martin-Creuzburg and Merkel, 2016; Volkman, 2016). The predominance of brassicasterol ∕ epi-brassicasterol has been reported for P. tricornutum (Orcutt and Patterson, 1975; Ballantine et al., 1979; Rampen et al., 2010), Rhodomonas (Dunstan et al., 2005; Chen et al., 2011) and E. huxleyi (Volkman et al., 1981; Riebesell et al., 2000). A few other minor sterols have been reported, but they were not detected or below detection in our study, such as cholesterol, 24-methylcholest-5-en-3β-ol, and cholesta-5,22-dienol in P. tricornutum (Ballantine et al., 1979; Véron et al., 1996; Cvejić and Rohmer, 2000) and cholesterol and 24-methylcholesta-5,7,22-trien-3β-ol in Rhodomonas/Proteomonas sp. (Dunstan et al., 2005) and E. huxleyi (Yamamoto et al., 2000; Sawada and Shiraiwa, 2004; Mausz and Pohnert, 2015).

Alkenones were only observed in E. huxleyi in our study, consistent with previous results showing that these compounds were only synthesized by a few haptophytes including E. huxleyi (Volkman et al., 1980b; Volkman et al., 1998). The alkenone composition of E. huxleyi was characterized by the presence of four pairs of isomers, including eight alkenone compounds (Table S4) (Marlowe et al., 1984; Riebesell et al., 2000; Sachs et al., 2016). Moreover, higher abundance of several alkenone components have been also observed in some E. huxleyi strains under certain culture conditions, e.g., C_37 : 4 Me in the strain 1742 (Eltgroth et al., 2005) or at low temperatures (our study; Prahl and Wakeham, 1987). In addition, two other compounds C_38 : 4 Et and C_38 : 4 Me were also found in one E. huxleyi strain (Marlowe et al., 1984).

Collectively, the results above highlight the similarity of sterol and alkenone composition in algal species in our study with those in conspecifics or congeneric phytoplankters in previous studies. Sterol and alkenone composition can vary between algal strains and can be affected by environmental changes (Conte et al., 1994; Volkman, 2003), which may explain the differences between our findings and previous results. In the following section, specific response patterns of brassicasterol ∕ epi-brassicasterol and alkenones are evaluated and quantified, which are further compared with FA responses under changing temperature, N : P supply ratios and pCO₂.

4.2 Responses of brassicasterol ∕ epi-brassicasterol contents

Increasing temperature caused an overall 12 % increase in carbon-normalized contents of brassicasterol ∕ epi-brassicasterol in E. huxleyi but nonsignificant changes in P. tricornutum and Rhodomonas sp. (Tables 1 and 2). Consistent with our findings, positive correlations between increasing temperature and sterol carbon-normalized contents have been observed in the dinoflagellates Karenia mikimotoi and Prorocentrum minimum (Ding et al., 2019) and the green alga Scenedesmus quadricauda (Piepho et al., 2012). High sterol contents at high temperature could be predicted based on its biochemical function, because increasing levels of sterols can reduce membrane fluidity to enable an organism's functional activity as temperature increases (Ford and Barber, 1983). Enhanced partial CO₂ pressure caused a 21 % decrease in carbon-normalized brassicasterol ∕ epi-brassicasterol contents in Rhodomonas sp. and nonsignificant responses in P. tricornutum and E. huxleyi; however, per-cell contents of brassicasterol ∕ epi-brassicasterol and POC showed nonsignificant changes in all three species (Tables 1 and 2). Minor effects of CO₂ concentration on per-cell contents of sterols have been observed in another strain of E. huxleyi (PML B92/11) (Riebesell et al., 2000) and the Chlorophyceae D. viridis (Gordillo et al., 1998). While the mechanism underlying sterol responses to pCO₂ is still unclear, our results indicate that enhanced pCO₂ did not induce substantial changes in per-cell contents of sterols in phytoplankton due to the role of sterols in membrane composition and functions (Riebesell et al., 2000). Nevertheless, enhanced pCO₂ might change carbon metabolism in phytoplankton (Gordillo et al., 2001), as revealed by variable carbon-normalized contents of brassicasterol ∕ epi-brassicasterol in Rhodomonas sp. in our study.

N and P deficiency caused overall 8 % and 37 % decreases in carbon-normalized brassicasterol ∕ epi-brassicasterol contents, respectively, in E. huxleyi but nonsignificant changes in other two species (Tables 1 and 2). Carbon-normalized or dry-weight contents of sterols in phytoplankton generally reduced in response to N or P deficiency (Breteler et al., 2005; Piepho et al., 2010; Ding et al., 2019). Furthermore, the relatively higher per-cell contents of sterols in response to P deficiency than N deficiency have been also found in the three species in this study and in the freshwater diatom Stephanodiscus minutulus (Lynn et al., 2000). Lipid modifications triggered by nutrient deficiency have been well studied in the plant Arabidopsis and more recently elucidated in typical phytoplankters (Van Mooy et al., 2009; Abida et al., 2015; Shemi et al., 2016). In P. tricornutum, N deficiency exerted more severe stress on membrane glycerolipids than P deficiency, which caused a stepwise adaptive response, resulting in undetectable phospholipids and instead the increase in the synthesis of non-phosphorus lipids (Abida et al., 2015). Also in plants, P deficiency resulted in the replacement of phospholipids by non-phosphorous glycolipids such as glucosylceramide, sterol glucoside and acylated sterol glucoside (Siebers et al., 2015). Consequently, sterols are synthesized and accumulate in the plasma membrane in response to P deficiency. Thus, N deficiency may inhibit the capacity of the cells to synthesize sterols, while upon P deficiency membrane glycerolipid remodeling with the accumulation of non-phosphorous lipids may explain the relatively higher per-cell contents of sterols in response to P deficiency in our study.

In summary, our study shows that temperature, N : P supply ratios and pCO₂ had significant separate effects on per-cell and per-carbon contents of brassicasterol ∕ epi-brassicasterol in certain algal species. Previous studies have shown significant interactions of two environmental factors on carbon-normalized contents of sterols in phytoplankton (Piepho et al., 2012; Chen et al., 2019; Ding et al., 2019). We here found potentially confounding effects of multiple environmental drivers on phytoplankton sterol contents. For example, the responses of E. huxleyi brassicasterol ∕ epi-brassicasterol carbon-normalized contents to increasing temperature varied with N : P supply ratios, i.e., an increase under lower N : P supply ratios, but no clear changes under the highest N : P ratio (Fig. 1e), suggesting that nutrient availability may potentially alter the effects of warming on sterol contents in E. huxleyi.

4.3 Responses of alkenone contents and ratios

Carbon-normalized contents of total alkenones showed nonsignificant responses to the changes in temperature, N : P supply ratios or pCO₂ in E. huxleyi (Table 1), which can be attributed to similar response patterns of per-cell contents of alkenones and POC (Table 2). We observed that per-cell contents of alkenones changed significantly in response to N and P deficiency but showed nonsignificant responses to warming or enhanced pCO₂. In the following, the responses of per-cell contents of alkenones and the ratios of certain alkenone isomers are discussed.

N deficiency in semicontinuous E. huxleyi cultures grown at 20 % of μ_max led to a 35 % decrease in per-cell contents of alkenones in our study (Table 2). However, all published culture studies we could find in which E. huxleyi was grown under N deficiency were performed with batch cultures and reported either an increase in per-cell alkenone contents or a nonsignificant change (Epstein et al., 1998; Prahl et al., 2003; Bakku et al., 2018; Wördenweber et al., 2018). Several possible explanations exist for these contradictory findings. An increase in growth rate has been shown to reduce alkenone concentrations (ng mL⁻¹) in continuous cultures of E. huxleyi (Sachs and Kawka, 2015). Thus, the higher growth rate of E. huxleyi (20 % of μ_max) in semicontinuous cultures in our study is a possible cause of lower alkenone contents compared to the batch culture studies where cells were harvested at or near the stationary phase of growth (i.e., growth rate approaching 0) (Epstein et al., 1998; Prahl et al., 2003; Bakku et al., 2018; Wördenweber et al., 2018). Because growth rate and growth phase strongly affect sterol contents (Sachs and Kawka, 2015; Chen et al., 2019), the conjunctions above-described may change if the maximum growth rates of batch cultures were used for comparison. In addition, phytoplankton in a continuous culture has a constant growth rate under a given dilution rate, while the growth rate at the stationary phase of a batch culture is zero. Thus, energy-availability for sterol remodeling differs between the two culture systems. Contradictory results obtained in batch and semicontinuous cultures could indicate different alkenone contents produced by E. huxleyi during the bloom period and summertime growth of this species, respectively (Lampert and Sommer, 2007). Another possibility is that gene complements within the species of E. huxleyi vary considerably, which may explain different phenotypic variations, including differences in N and P uptake in this species (Read et al., 2013). N deficiency severely impairs the synthesis of nucleotides, amino acids and ultimately all enzymatic machinery, consequently resulting in a decrease of most central metabolites (Wördenweber et al., 2018). Intense lipid turnover with the reduction of most central metabolites have been reported in E. huxleyi under N deficiency based on transcriptomic and metabolomic studies (Rokitta et al., 2014; Wördenweber et al., 2018), which may also result in lower per-cell alkenone contents in our study.

In contrast, P deficiency caused an increase (49 %) in per-cell contents of alkenones in E. huxleyi (Table 2), as well as in other strains and life-cycle stages of E. huxleyi (Wördenweber et al., 2018). Experimental data presented here agree with a metabolic model predicted from transcriptomic data (Rokitta et al., 2016) and the findings in a comprehensive metabolomics study showing a significant accumulation of several key metabolites, especially neutral lipids such as triacylglycerols, alkenones and alkenes in response to P deficiency (Wördenweber et al., 2018). E. huxleyi contains only very small amounts of triacylglycerols and hence alkenones have been suggested to have a storage role (Volkman et al., 1980c; Bell and Pond, 1996; Eltgroth et al., 2005). Our results support this view and suggest that P deficiency can induce the accumulation of alkenones which can serve as storage molecules in E. huxleyi cells. The increased abundance of metabolites in response to P deficiency is likely derived from the arrest of cell-cycling due to decreased nucleic acid synthesis, and the reduction equivalents are preserved by lipogenesis as enzymatic functionality (Wördenweber et al., 2018).

The carbon chain-length distribution of alkenones (C₃₇ ∕ C₃₈ alkenone ratios) showed a 13 %–21 % increase from the cold to warm treatments and in response to N and P deficiency and a slight decrease (6 %) with enhanced pCO₂ (Tables 1 and 2). Previous studies have shown that C₃₇ ∕ C₃₈ alkenone ratios not only varied with temperature and physiological stages (such as growth stage) but also differed between alkenone-producing species (Conte et al., 1998; Pan and Sun, 2011; Nakamura et al., 2014). In agreement with our findings, a slight increase in C₃₇ ∕ C₃₈ alkenone ratios at higher temperatures has been also found in four E. huxleyi strains in exponential phase cultures (Conte et al., 1998). In contrast to our results, it has been reported lower C₃₇ ∕ C₃₈ alkenone ratios occurred under nutrient deficiency at the stationary phase of E. huxleyi in comparison to those at the exponential phase (Conte et al., 1998; Pan and Sun, 2011). As discussed above, different culturing approaches may cause conflicting results in different studies, as the effects of nutrient deficiency and growth rate cannot be well distinguished in the batch approach. The proposed biosynthetic pathways of classical C₃₇–C₄₀ alkenones show that biosynthesis of C₃₇ Me involves chain elongation with malonyl-CoA, while C₃₈ Et compounds are formed by the condensation of methylmalonyl-CoA; C₃₈ Me compounds are produced after the involvement of an additional α-oxidation (Rontani et al., 2006). Our results suggest that warming, N and P deficiency, and enhanced pCO₂ may have independent effects on the synthesis of C₃₇ Me and C₃₈ alkenones, which ultimately result in changes in C₃₇ ∕ C₃₈ ratios.

The relative abundance of C₃₈ homologs (C₃₈ Et ∕ Me ratios) showed an 82 % increase from the cold to warm treatments (Tables 1 and 2). The prominent increase in C₃₈ Et ∕ Me ratios resulted from nonsignificant changes in per-cell contents of C₃₈ Et and the decrease in C₃₈ Me. An increase in C₃₈ Et ∕ Me ratios with increasing temperatures has been found in four E. huxleyi strains in mid-exponential phase of batch cultures (Conte et al., 1998). More importantly, our experimental results agree well with the findings in the sedimentary records back to ∼120.5 Ma (Brassell et al., 2004), showing the absence of all C₃₈ Me but the occurrence of C₃₈ Et in Cretaceous sediments (warm climate, indicating a high C₃₈ Et ∕ Me ratio) but the presence of C₃₈ Me from Cretaceous to Quaternary ages (warm to cold climate, suggesting a potentially declined C₃₈ Et ∕ Me ratio). The strong increases in C₃₈ Et ∕ Me ratios with warming in our study may reflect the differences in biosynthetic pathways between C₃₈ Et and C₃₈ Me (Rontani et al., 2006). Therefore, the distribution of alkenones in sediments over time can be linked to evolutionary adaption of alkenone biosynthesis in response to global climate change (Brassell, 2014).

The results discussed above show significant changes in per-cell contents of alkenones in response to N and P deficiency, indicating the important role of alkenones as storage molecules. Another type of biomolecules, alkenoates, has been identified in E. huxleyi and may biochemically link with alkenones (Marlowe et al., 1984; Conte et al., 1994). However, alkenoates were converted to FAs by saponification in our sample preparation steps and thus not evaluated. There might be interesting variations in alkenone / alkenoate ratios with changing multiple environmental conditions, which can be assessed in future studies.

4.4 Implications for ecology and biogeochemistry

There has been evidence that carbon allocation in algal cells is highly responsive to environmental changes (Palmucci et al., 2011; Halsey and Jones, 2015). Our new study demonstrated that, under future ocean scenarios (warming, N and P deficiency and enhanced pCO₂), carbon-normalized contents of brassicasterol ∕ epi-brassicasterol, alkenones and FAs have differential responses, i.e., significant but nonsystematic changes in sterols and FAs, and nonsignificant changes in alkenones (Table 1). Our results further suggest rearrangements of cellular carbon pools under future ocean scenarios, and such variations would have important impacts on marine ecological functions and biogeochemical cycles.

Our study revealed an overall decrease (∼20 %) in carbon-normalized contents of brassicasterol ∕ epi-brassicasterol in Rhodomonas sp. and E. huxleyi under ocean-related global change scenarios (Table 2). The low availability or absence of dietary sterols has been shown to constrain growth, reproduction and survival in Daphnia (Martin-Creuzburg et al., 2005; Martin-Creuzburg and von Elert, 2009a) and development and egg production in copepods (Hassett, 2004; Klein-Breteler et al., 2005). The potential influence of sterol deficiency on ecosystem functioning is the reduction of carbon transfer efficiency across the autotroph–herbivore interface, leading to a low production of higher trophic levels (von Elert et al., 2003; Martin-Creuzburg and von Elert, 2009b). Brassicasterol ∕ epi-brassicasterol compounds are cholesterol precursors and can be converted to cholesterol by most crustaceans (Martin-Creuzburg and von Elert, 2009b; Kumar et al., 2018) and thus can efficiently support somatic growth of crustacean zooplankton such as Daphnia magna (Martin-Creuzburg et al., 2014). It is therefore possible that reduced brassicasterol ∕ epi-brassicasterol under projected future ocean conditions may have deleterious ecological consequences in plankton communities, particularly where Rhodomonas or E. huxleyi is dominant.

Carbon-normalized contents of PUFAs showed an overall increase (∼65 %) in Rhodomonas sp. and an overall decrease (∼ 10 %–20 %) in P. tricornutum and E. huxleyi (Table 2). FA composition and contents in phytoplankton have shown significant effects on zooplankton production and trophic carbon transfer from phytoplankton to zooplankton (Müller-Navarra et al., 2000; Jónasdóttir et al., 2009; Rossoll et al., 2012; Arndt and Sommer, 2014). An example of this is the positive effect of increased docosahexaenoic acid (22 : 6n-3; DHA) content in the diatom Skeletonema marinoi on egg production rates of the calanoid copepod Acartia tonsa (Amin et al., 2011). These impacts of FA production remodeling have been discussed in detail in our previous studies (Bi et al., 2014, 2018; Bi and Sommer, 2020). The varying PUFA contents observed in the present study may thus potentially influence zooplankton nutrition. For example, an increase in PUFA contents in the diatoms in cold periods may have positive effects on zooplankton production; in contrast, a decrease in PUFA contents in E. huxleyi at enhanced pCO₂ may reduce trophic transfer efficiency at the phytoplankton–zooplankton interface. Moreover, we found that the overall responses of brassicasterol ∕ epi-brassicasterol were opposite to PUFAs in Rhodomonas sp., while both showed an overall decrease in E. huxleyi; only brassicasterol ∕ epi-brassicasterol decreased in P. tricornutum under future ocean scenarios (Table 2). Co-limitation of sterols and PUFAs has been well studied in a freshwater herbivore D. magna, showing a negative effect on the growth of this herbivore (Martin-Creuzburg et al., 2009; Sperfeld et al., 2012; Marzetz et al., 2017). Less is known about how sterols and PUFAs regulate the performance of marine herbivorous zooplankton. The differential responses of sterols and PUFAs we observed may have tremendous implications for the study of marine food webs, especially in habitats where phytoplankton succession is highly dynamic with environmental changes.

Carbon-normalized contents of total alkenones in E. huxleyi showed nonsignificant changes (Table 2). E. huxleyi is mainly grazed by heterotrophic protists in the context of pelagic food webs (Braeckman et al., 2018; Nejstgaard et al., 1997). Also, early studies on copepod feeding clearly showed ingestion of E. huxleyi (Harris, 1994; Nejstgaard et al., 1997; Vermont et al., 2016) and excretion of alkenones (Volkman et al., 1980a), indicating faecal pellet transport of these compounds to sediments (Volkman et al., 1980a). In the present study, we observed nonsignificant changes in carbon-normalized contents of total alkenones in E. huxleyi under variable temperature, N : P ratios and pCO₂, demonstrating quantitative applicability of alkenones as proxies for E. huxleyi biomass in biogeochemical cycles.

From a paleoceanographic perspective, lipid biomarker data have been reported on the basis of both dry sediment weight and total organic carbon (TOC) content (Zimmerman and Canuel, 2002; Zhao et al., 2006; Xing et al., 2016). Dry sediments contain variable organic and inorganic components; thus, biomarker contents normalized to TOC can partially eliminate the influence of sedimentation rates and between-site or temporal changes in the amount of organic carbon deposition or preservation (Zimmerman and Canuel, 2002). The application of sediment biomarker contents for paleoproductivity reconstruction is based on two key assumptions: (1) relatively constant ratios of biomarker per cell or per POC in a specific phytoplankton group and (2) nonsignificant changes in biomarker ∕ POC ratios during post production and deposition degradation. In laboratory culture studies, carbon-normalized and cell-normalized lipid contents provide broadly similar responses to environmental parameter changes in most cases, but there are exceptions (Ding et al., 2019; this study). However, cell-normalization is difficult for sediment reconstruction, as phytoplankton cell counting is often not quantitative (Piepho et al., 2010; Ahmed and Schenk, 2017). In the present study, we focus on the implications of our findings based on particulate organic carbon (POC)-normalized contents of lipid biomarkers. For example, our study revealed an overall 20 % decrease in carbon-normalized contents of brassicasterol ∕ epi-brassicasterol in Rhodomonas sp. and E. huxleyi in ocean-related global change scenarios but not in the diatom P. tricornutum. Because smaller ranges are expected in temperature, N : P supply ratio and pCO₂ in individual locations over time, our results provide additional support for the applicability of using sterols in paleoproductivity reconstruction, especially in diatom-dominated areas. Furthermore, we observed that C₃₇ ∕ C₃₈ alkenone ratios varied significantly with the changes in temperature, N : P supply ratios and pCO₂, indicating that, besides temperature, other environmental factors may also significantly influence C₃₇ ∕ C₃₈ alkenone ratios. In contrast, C₃₈ Et ∕ Me ratios in our study responded significantly only to temperature changes. These results denote the importance to consider the effects of multiple environmental factors on C₃₇ ∕ C₃₈ alkenone ratios, and they further underline the importance of temperature in geological application of alkenone ratios.