Global warming will be combined with predicted increases in thermal
variability in the future surface ocean, but how temperature dynamics will
affect phytoplankton biology and biogeochemistry is largely unknown. Here,
we examine the responses of the globally important marine coccolithophore Emiliania huxleyi to thermal
variations at two frequencies (1 d and 2 d) at low (18.5 ∘C)
and high (25.5 ∘C) mean temperatures. Elevated temperature and thermal
variation decreased growth, calcification and physiological rates, both
individually and interactively. The 1 d thermal variation frequencies were
less inhibitory than 2 d variations under high temperatures, indicating
that high-frequency thermal fluctuations may reduce heat-induced mortality
and mitigate some impacts of extreme high-temperature events. Cellular
elemental composition and calcification was significantly affected by both
thermal variation treatments relative to each other and to the constant
temperature controls. The negative effects of thermal variation on E. huxleyi growth
rate and physiology are especially pronounced at high temperatures. These
responses of the key marine calcifier E. huxleyi to warmer, more variable temperature
regimes have potentially large implications for ocean productivity and
marine biogeochemical cycles under a future changing climate.
Introduction
Climate-driven changes such as ocean warming alter the productivity and
composition of marine phytoplankton communities, thereby influencing global
biogeochemical cycles (Boyd et al., 2018; Hutchins and Fu, 2017; Thomas, et
al., 2012). Increasing sea surface temperatures have been linked to global
declines in phytoplankton concentration (Boyce et al., 2010), changes in
spring bloom timing (Friedland et al., 2018) and biogeographic shifts in
harmful algal blooms (Fu et al., 2012; Gobler et al., 2017). Warming and
acidification may drive shifts away from dinoflagellate or diatom dominance
and towards nanophytoplankton (Hare et al., 2007; Keys et al., 2018).
Similarly, Morán et al. (2010) predicted that a gradual shift will occur
towards smaller primary producers in a warmer ocean.
Effects of temperature increases on phytoplankton diversity are uncertain.
Warming and phytoplankton biodiversity were found to be inversely correlated
in a coastal California diatom assemblage, at least on short timescales
(Tatters et al., 2018). In contrast, a 5-year long mesocosm experiment
found that elevated temperature can modulate species coexistence, thus
increasing phytoplankton species richness and productivity (Yvon-Durocher et
al., 2015). Globally, rising temperatures may result in losses of
phytoplankton biodiversity in the tropics but gains in the polar regions
(Thomas et al., 2012). It is thought that ocean warming will lead to a
poleward range expansion of warm-water species at the expense of cold-water
species (Boyd et al., 2010; Gao et al., 2018; Hallegraeff, 2010; Hutchins
and Fu, 2017; Thomas et al., 2012). It is evident that rising ocean
temperatures will benefit some groups, while having detrimental consequences
for others (Boyd et al., 2010, 2015, 2018; Feng, et al., 2017; Fu et al.,
2014). For example, recent decades of satellite observations show a striking
poleward shift in the distribution of blooms of the coccolithophore
Emiliania huxleyi, a species that was previously virtually absent in polar waters (Boyd et
al., 2010; Neukermans et al., 2018).
Coccolithophores are the most successful calcifying phytoplankton in the
ocean and contribute almost half of global marine calcium carbonate
production. They play crucial biogeochemical roles by performing both
photosynthesis and calcification and facilitate carbon export to the deep
ocean through the ballasting effects of their calcium carbonate shells
(Klaas and Archer, 2002; Krumhardt et al., 2017; Monteiro et al., 2016). E. huxleyi
(Lohm.) is the most abundant and cosmopolitan coccolithophore, forming
prolific blooms in many regions (Holligan, et al., 1983, 1993;
Iglesias-Rodríguez et al., 2002; Westbroek et al., 1993).
The responses of E. huxleyi to global change factors have been intensively
investigated. Many E. huxleyi strains are sensitive to ocean acidification, which
negatively affects their growth rates and calcification (Feng et al., 2018;
Hoppe et al., 2011). However, among the many currently changing
environmental drivers, temperature may be among the most important in
regulating coccolithophore physiology (Boyd et al., 2010). Feng et al. (2008) reported that the growth rate of E. huxleyi was improved by elevated
temperature at low irradiance. Furthermore, temperature was the most
important driver controlling both cellular particulate organic and inorganic
carbon content of a Southern Hemisphere E. huxleyi strain (Feng et al., 2018).
Most research about the effects of global warming on E. huxleyi and phytoplankton in
general has focused on predicted increases in mean temperatures. However, in
the natural environment, seawater temperatures fluctuate over timescales
ranging from hours, to days, to months (Bozinovic et al., 2011; Jiang et
al., 2017). Future climate models predict not only in an increase in mean
temperature but also an increase in temperature variability (frequency and
intensity), as well as a higher probability of extreme events (IPCC, 2013).
The impacts of climatic variability and extremes have been best studied in
metazoans, where they may sometimes have a larger effect than increases in
climatic averages alone (Vázquez et al., 2017; Vasseur et al., 2014;
Zander et al., 2017). Variability can promote greater zooplankton species
richness, compared with long-term average conditions (Cáceres 1997;
Shurin et al., 2010). In corals, temperature variability could buffer warming
stress, elevate thermal tolerance and reduce the risk of bleaching (Oliver
and Palumbi, 2011; Safaie et al., 2018).
In comparison, we still lack a thorough understanding of how thermal
variation affects phytoplankton growth and physiology. Unlike zooplankton,
the few available studies suggest increasing thermal variation may decrease
phytoplankton biomass and biodiversity and shift the community towards
small phytoplankton (Burgmer and Hillebrand, 2011; Rasconi et al., 2017).
Two studies have shown that plastic responses play a key role in acclimation
and adaptation to thermal fluctuations in algae (Kremer et al., 2018; Schaum
and Collins, 2014). Population growth rates of phytoplankton in fluctuating
thermal environments have been quantitatively modeled based on data from
thermal response curves obtained under constant temperatures (Bernhardt et
al., 2018).
In view of this relative lack of information on the effects of nonsteady
state temperatures on biogeochemically important phytoplankton, we carried
out a thermal variability study using the Sargasso Sea E. huxleyi isolate CCMP371. Our
experiments combined ocean warming with thermal variations, with a focus on
the increasing frequency of temperature variations under global climate
change. We examined growth rates, photosynthesis, calcification and
elemental composition under constant, 1 d and 2 d temperature
variations. This study is intended to provide insights into how different
frequencies of thermal variation may influence the physiology and
biogeochemistry of this important marine calcifying phytoplankton species
under both current and future sea surface temperatures.
Materials and methods
The marine coccolithophore E. huxleyi (Lohm.) Hay and Mohler strain CCMP371 (isolated
from the Sargasso Sea) was maintained in the laboratory as stock batch
cultures in an Aquil medium (100 µmol L-1NO3-, 10 µmol L-1PO43-), made with 0.2 µM filtered coastal
seawater collected from the California region (Sunda et al., 2005). Cells
were grown at 22 ∘C under 120 µmol photons m-2 s-1 cool
white fluorescent light with a 12 h to 12 h light to dark cycle.
Experimental set-up
An aluminum thermal gradient block with a range of 13 temperatures was used
to perform the thermal response curve and temperature variation experiments.
For the thermal curve experiment, the extreme temperatures of the
thermal block were set to 8.5 and 28.6 ∘C, with intermediate
temperatures of 10.5, 12, 13.5, 15.5, 17.5, 18.5, 21.3, 22.6, 24.5, 26.6 and 27.6 ∘C. The E. huxleyi cells were transferred from the stock
cultures into triplicate 120 mL acid-washed polycarbonate bottles in the
thermal block under a 12 h light and 12 h dark cycle at 180 µmol photons m-2 s-1. For the light intensity measurement, irradiance was
measured individually at each position in the thermal block using a light
meter with a small detector bulb to fit into the round holes drilled to fit
the experimental bottles (LI-250A light meter, LI-COR). During measurements
the detector bulb was positioned identically in each position and, if
necessary, the positions of the fluorescent lights were adjusted nearer or
farther until the light intensity was between 175 and 185 µmol photons m-2 s-1 for every experimental replicate.
Semicontinuous culturing methods were used for all experiments. Cultures
were diluted with an Aquil medium every 2 d to keep them in exponential
growth stage while acclimating to the treatment temperatures for 2 weeks
before starting the variation experiment. Dilution volumes were calculated
to match growth rates of each individual replicate, as measured using in vivo
chlorophyll a (Chl a) fluorescence. Once steady-state growth rates were
recorded for 3–5 consecutive transfers, the cultures were sampled (Zhu et
al., 2017). Due to the decrease in cell numbers during cultivation at 28.6 ∘C (from our preliminary experiment), these cultures were diluted from
22 ∘C stock cultures. They were then sampled as a batch culture
(without dilution) after 4–6 d to estimate the negative growth rates and
elemental stoichiometry at this upper-limit temperature point.
Six treatments were used to determine the responses of E. huxleyi growth,
photosynthesis and calcification to different frequencies of temperature
fluctuation. Temperature fluctuation treatments included: (1) low
temperature, constant (18.5 ∘C); (2) low temperature, 1 d
fluctuation cycle (16–21, mean = 18.5 ∘C); (3) low temperature,
2 d fluctuation cycle (16–21, mean = 18.5 ∘C); (4) high temperature, constant (25.5 ∘C); (5) high temperature, 1 d
fluctuation cycle (23–28, mean = 25.5 ∘C); and (6) high temperature,
2 d fluctuation cycle (23–28, mean = 25.5 ∘C). For the
variation treatment cycles, cultures were incubated at the cool phase (16
and 23 ∘C for low and high temperatures, respectively) for
either 1 or 2 d. They were then switched to the warm phase (21
and 28 ∘C for low and high temperature, respectively) for the
same amount of time. It took about 0.5 h to readjust the thermal block
to the transformed temperature at the beginning of each new treatment cycle.
The experimental E. huxleyi cultures were grown in triplicate in 120 mL acid-washed
polycarbonate bottles using the thermal block under a 12 h light/12 h dark
cycle at 180 µmol photons m-2 s-1.
For the variable temperature experiment, cultures were diluted
semicontinuously with an Aquil medium every 2 d for constant and 1 d
variation treatments and every 4 d for 2 d variation treatments.
To ensure nutrient-replete conditions in the 2 d variation treatments,
Aquil nitrate and phosphate stocks were added at the 2 d midpoint of
every 4 d thermal cycle to make sure that the final nitrate and
phosphate concentrations were not depleted and were always maintained at
> 100 and > 10 µmol L-1,
respectively. Cultures were grown for at least eight dilutions
(∼16 d for constant and 1 d variation treatments and
∼32 d for 2 d variation treatments) to acclimate to
the different experimental conditions before final sampling. All variation
treatments were sampled twice across the thermal variation cycle, once
during the cool phase and once during the warm phase.
Growth rates
In vivo fluorescence was measured daily for the 1 d variation treatment and
every 2 d for the constant and 2 d variation treatments using a
Turner 10AU fluorometer (Turner Designs, CA). In vivo-derived growth rates were
subsequently verified using cell samples counted with a nanoplankton
counting chamber on an Olympus BX51 microscope. Specific growth rates
(d-1) were calculated using the in vivo fluorescence and cell count data as follows:
μ= ln[N(T2) /N(T1)] / (T2-T1). N(T1)
and N(T2) are the in vivo fluorescence values (for thermal curve experiments
and constant treatments) or cell counts (for variation treatments because
of potential changes in cellular in vivo fluorescence during fluctuation) at
T1 and T2.
Some 20 mL culture samples were filtered onto GF/F glass fiber filters
(Whatman, Maidstone, UK) for Chl a analysis. In vitro Chl a was extracted with
90 % aqueous acetone for 24 h at -20 ∘C and then measured using
a Turner 10 AU fluorometer (Turner Design, USA) (Fu et al., 2007).
Elemental analysis
Elemental composition sampling included total particulate carbon (TPC),
particulate organic carbon (POC), particulate organic nitrogen (PON),
particulate inorganic carbon (PIC) and particulate organic phosphorus (POP),
allowing calculation of cellular elemental stoichiometry and calcite/organic
carbon ratios (PIC / POC) (Feng et al., 2008). Culture samples for TPC, POC
and PON were collected onto pre-combusted GF/F glass fiber filters
(Whatman) and dried in a 60 ∘C oven overnight. For POC analysis,
filters were fumed for 24 h with saturated HCl (∼37 %)
to remove all inorganic carbon prior to analysis. TPC, PON and POC were then
measured by a 440 Elemental Analyzer (Costech Inc, CA), according to previous
studies (Hutchins et al., 1998; Feng et al., 2008). PIC was calculated as
the difference between TPC and POC. For POP measurement, culture samples
were filtered onto pre-combusted GF/F filters (Whatman) and analyzed using a
molybdate colorimetric method (Solórzano and Sharp, 1980) with minor
modifications, as in Fu et al. (2007).
Total carbon fixation, photosynthesis and calcification rates, and ratios
Total carbon fixation, photosynthetic carbon fixation and calcification
rates were measured using the 14C incubation technique (Platt et al.,
1980), with slight modifications, as in Feng et al. (2008). Culture
samples from each treatment (60 mL) were spiked with 0.2 µCi NaH14CO3
and then incubated for 4 h under their respective experimental conditions.
After incubation, samples were filtered on two Whatman GF/F filters (30 mL
each) for total carbon fixation and photosynthetic rate separately. The
filters for photosynthetic rate measurement were fumed with saturated HCl
(∼37 %) before adding scintillation fluid. A sample of 30 mL from
each treatment (10 mL from each replicate bottle) was filtered immediately,
after adding equal amounts of NaH14CO3 for procedural filter
blanks. Filters were then placed in 7 mL scintillation vials with 4 mL
scintillation fluid overnight in the dark. To determine the total
radioactivity (TA), 0.2 µCi NaH14CO3, together with 100 µL phenylalanine was placed in scintillation vials with the addition of 4 mL
scintillation solution. All samples were counted on a Perkin Elmer Liquid
Scintillation Counter to measure the radioactivity. Total carbon fixation
and photosynthetic rate were calculated from TA, final radioactivity and
total dissolved inorganic carbon (DIC) values. Calcification rate was then
calculated as the difference between total carbon fixation and
photosynthetic rate for each sample.
Model for thermal responses of <italic>E. huxleyi</italic>
Growth rates measured under constant temperatures in the thermal block were
fitted to the Eppley thermal performance curve (Eppley, 1972; Norberg, 2004;
Thomas et al., 2012). This function quantifies parameters of growth
temperature effects, including the temperature optimum for growth
(Topt) and high and low temperature limits (Tmax and Tmin,
respectively) in our strain of E. huxleyi. To predict growth rates under variable
thermal regimes from our constant temperature thermal curves, we applied a
recently developed model based on the Eppley curve but incorporating
nonlinear averaging in conjunction with consideration of Jensen's
inequality (Bernhardt et al., 2018). This new thermal fluctuation model
takes into account the amount of time that the cells spend at each portion
of their thermal performance curve, as well as incorporating the observation
that growth rates usually increase slowly with temperature at the cooler end
of the curve but then drop off very quickly at the upper, warm end of the
curve (Jensen's inequality). This model is more realistic and skillful under
variable temperatures than previous work assuming a linear relationship
between temperature and growth rates, as nonlinear averaging allows much
more accurate predictions when dealing with skewed thermal curves. Several
recent studies of phytoplankton thermal variability responses have
successfully applied the Bernhardt et al. (2018) model (Qu et al., 2019;
Kling et al., 2019).
Statistical analysis
The mean values of most parameters measured under the variation treatments
were calculated by averaging the values from the cool and warm phases,
including all the elemental content and ratios, as well as photosynthetic and
calcification rates and ratios. Statistical analyses were performed using R
(version 3.5.0). For the response of E. huxleyi to warming, the mean growth rate or
elemental ratios of three replicates at 12 temperature points were used to
fit the growth rate or elemental ratios curves. A one-way ANOVA was applied
to analyze the difference between the average value for the entire
temperature range and the value at each individual temperature for the
elemental ratios. For the response of E. huxleyi to thermal variation, a one-way ANOVA
was performed to test the statistical significance in growth rate, elemental
stoichiometry, and photosynthetic and calcification rates and ratios among
different frequencies (constant, 1 d and 2 d) of temperature
variabilities at cool and warm phases under high and low temperatures. The p values were
calculated based on Student's t test via two functions including
compare_means() and stat_compare_mean() in the ggpubr package, and the figures were
generated via the ggplot package in open-source statistical software R version 3.5.0 (R Foundation).
ResultsResponses of <italic>E. huxleyi</italic> to warming
The growth rates of E. huxleyi at constant temperature increased significantly with
warming from 0.09±0.01 d-1 at 8.5 ∘C to a maximum value of
0.90±0.02 d-1 at 21.3 ∘C. Growth was optimal up to 24.5 ∘C and then decreased rapidly to -0.46±0.05 d-1 at 28.6 ∘C (p<0.05, Fig. 1).
Thermal performance curve showing cell-specific growth rates
(d-1) of Emiliania huxleyi CCMP371 across a temperature range from 8.5 to 28.6∘. Symbols represent means, and error bars are the standard
deviations of three replicates at each temperature, but in many cases the
errors bars are smaller than the symbols.
The elemental ratios of the cells in the different temperature treatments
were compared to the average elemental ratios across the entire temperature
range (Fig. 2). The thermal trends of TPC / PON ratios were generally similar to those of growth rates, in that ratios increased from 8.5 to 17.5 ∘C and then decreased from 24.5 to 27.6 ∘C. The TPC / PON ratios at
8.5, 10.5 and 27.6 ∘C were significantly lower than the average level
of all the temperature points (p<0.05, Fig. 2a). The POC / PON ratios
of most temperature points were very close to the mean value of 6.3, except
at 27.6 ∘C (7.1) and 28.6 ∘C (7.4), which were significantly
higher than the average (p<0.05, Fig. 2b). The highest PIC / POC ratio
was 0.49±0.07 at 22.6 ∘C, and the lowest PIC / POC ratio was
0.05±0.04 at 27.6 ∘C, a value that was almost 90 % lower than
the highest value. The PIC / POC ratios at the lowest temperature tested (10.5 ∘C) and at the high end of the temperature range (26.6 and 27.6 ∘C) were significantly lower than the average level (Fig. 2c). Chl a/ POC ratios were significantly lower at 8.5, 10.5 and 27.6 ∘C than the
mean and at 17.5, 21.3, 22.6 and 24.5 ∘C were significantly higher
than the average (p<0.05, Fig. 2c). The trends of PIC / POC and Chl a/ POC ratio were similar, in that they gradually increased from low
temperature to the highest value at 22.6 ∘C and then dropped rapidly
as temperature increased further (Fig. 2c, d).
Changes in Emiliania huxleyi TPC / PON ratios (a), POC / PON ratios (b), PIC / POC ratios (c)
and Ch a/ POC ratios (d) across a temperature range from 8.5 to 28.6∘C. Dashed lines represent the average ratios for the entire
temperature range. Bars represent means, and error bars are the standard
deviations of three replicates at each temperature. Asterisks (*) represent the
significant difference (p<0.05) between average ratios and the
ratio at each temperature.
Responses of <italic>E. huxleyi</italic> to temperature variationsGrowth rate
In low-temperature experiments, both 1 d and 2 d temperature
variations had a negative effect on growth rate. The mean growth rates of
the 1 d (0.71±0.01 d-1) and 2 d (0.72±0.01 d-1) variation treatments were not significantly different from each
other (p>0.05), but both were lower than that of the constant
18.5 ∘C treatment (0.76±0.01, p<0.05) (Fig. 3a).
Growth rates were low during the cool phase (16 ∘C) of the experiment
(∼0.5–0.6 d-1), but those of the 2 d variation cycle
were not significantly different from the constant control at this
temperature (p>0.05). However, the growth rates during the cool
phase of the 1 d variation cycle were lower than those of the constant
16 ∘C treatment (p<0.05). During the warm phase of the thermal
cycle (21 ∘C), there were no significant differences in the elevated
growth rates (∼0.85–0.9 d-1) of the constant control and
those of either variable treatment (p>0.05, Fig. 3a).
Emiliania huxleyi growth rate responses to constant temperatures and during the warm
and cool phases of the two thermal variation frequencies (1 d and
2 d) under low (a) and high (b) mean temperatures. The thick black line
in the boxplots represents median values for each experimental treatment;
the whiskers on the boxplots indicate 1.5× interquartile range. Listed
p values with their respective brackets are the statistical significance
between two treatments.
In the high-temperature experiments, as in the low-temperature experiments,
both temperature variation frequencies had a negative effect on mean growth
rates. The growth rates in the 2 d variation treatment were (0.20±0.02 d-1), a decrease of ∼74 % compared with the
constant 25.5 ∘C (p<0.05) and ∼62 % of the
1 d variation treatment value (p<0.05, Fig. 3b). During the
cool phase (23 ∘C), the growth rate of the 1 d variation treatment
was slightly lower (p<0.05) than the constant 23 ∘C, but there
were no significant changes between 2 d variations and the constant 23 ∘C treatment (p>0.05, Fig. 3b). During the warm phase (28 ∘C), the constant 28 ∘C and 2 d variation treatment both had
negative growth rates of -0.45±0.05 and -0.45±0.04 d-1, respectively. However, the 1 d variation treatment had a low
but positive warm-phase growth rate at 0.25±0.02 d-1 (Fig. 3b).
There was a time lag of ∼0.5 h to switch to the
transformed temperature for each new growth phase, which should thus have
had only minimal effect on overall growth rates across the 1 d and
2 d thermal variations.
Cellular PIC and POC contents and ratios
In low-temperature experiments, the cellular PIC content of the constant
18.5 ∘C treatment was 3.5±0.3 pg cell-1, and there were no
significant differences with temperature variation treatments (p>0.05, Table 1). However, the cellular POC content of the constant 18.5 ∘C treatment was 8.0±0.6 pg cell-1, which was lower than in the
2 d variation treatment but significantly higher than in the 1 d
variation treatment (p<0.05).
The effect of temperature variation under low and high temperature
on total carbon (pg cell-1), cellular POC (pg cell-1), cellular PIC (pg cell-1),
cellular PON (pg cell-1), cellular POP (pg cell-1) and cellular Chl a (pg cell-1)
of Emiliania huxleyi.
TreatmentTotal carbonCellular PONCellular POPCellular POCCellular PICCellar Chl aLow temperature18.5 ∘C11.5±0.41.8±0.20.17±0.008.0±0.63.5±0.30.14±0.001 d cool point (16)13.0±0.52.2±0.30.18±0.008.9±0.34.1±0.30.15±0.011 d warm point (21)12.0±0.72.1±0.30.19±0.009.3±0.92.7±0.90.19±0.002 d cool point (16)10.1±0.71.3±0.20.16±0.016.0±0.94.0±0.30.12±0.012 d warm point (21)10.4±0.51.5±0.20.17±0.016.6±0.53.8±0.30.15±0.01High temperature25.5 ∘C15.0±0.72.0±0.10.21±0.019.5±0.35.5±0.70.18±0.021 d cool point (23)16.1±1.43.0±0.20.21±0.0012.9±1.53.2±0.20.15±0.011 d warm point (28)19.1±0.84.4±0.30.24±0.0117.0±0.62.1±0.20.20±0.022 d cool point (23)12.4±1.01.9±0.20.19±0.017.5±1.04.8±0.30.13±0.012 d warm point (28)19.4±2.03.9±0.80.25±0.0318.3±3.72.1±0.90.25±0.02
Like POC, the PIC / POC ratio was significantly affected by temperature
variations (Fig. 4a). The lowest PIC / POC ratio was found in the 1 d
variation treatment (0.38±0.07), which was significantly lower than
the 2 d variation treatment value (p<0.05) but close to that
in the constant 18.5 ∘C (p>0.05). A similar trend was
found in both the cool (16 ∘C) and warm phases (21 ∘C) of the two
variation treatments, in that the PIC / POC ratio of the 1 d variation
treatment was lower than of the 2 d variation treatment (p<0.05, Fig. 4a). Both variation treatments had lower PIC / POC ratios during
the warm phase than during the cool phase, although these differences were
not significant (p>0.05).
Responses of Emiliania huxleyi PIC / POC ratios to constant temperatures and during the
warm and cool phases of two thermal variation frequencies (1 d and
2 d) under low (a) and high (b) mean temperatures. LT: low temperature;
HT: high temperature. The thick black line in the boxplots represents median
values for each experimental treatment; the whiskers on the boxplots indicate 1.5× interquartile range. Listed p values with their respective
brackets denote the statistical significance between two treatments.
High-temperature experiments showed particulate carbon trends that were
contrary to those of the low-temperature treatments. The PIC content and
PIC / POC ratios were significantly decreased by temperature variation. The
cellular PIC content of the constant treatment (25.5 ∘C) was 5.5±0.3 pg cell-1, which was ∼200 % higher than that of the
1 d variation and ∼160 % higher than in the 2 d
variation treatments (p<0.05, Table 1). The same trend was found
for PIC / POC ratios in 1 d variation and 2 d variation treatments,
which decreased ∼67 % and 33 % compared with the constant
25.5 ∘C treatment, respectively (p<0.05, Fig. 4b). However,
the POC content of 1 d and 2 d variation treatments was higher than
in the constant 25.5 ∘C treatment (p<0.05, Table 1). During
the cool phase (23 ∘C), the PIC content and PIC / POC ratio of the
1 d variation treatment was significantly lower than in the 2 d
variation treatment but, contrary to PIC content, the POC content of the
1 d variation treatment was significantly higher than that in the
2 d variation treatment. During the warm phase (28 ∘C), there were
no significant differences of PIC content, POC content, or PIC / POC ratio
between the 1 d and 2 d variation treatments (Fig. 4b, Table 1).
Photosynthetic and calcification rates and ratios
In low-temperature treatments, there were no differences between total
carbon fixation rates (photosynthesis plus calcification) for the two
variable treatments relative to the constant control (Fig. 5a). However,
during the cool phase total carbon fixation rates were higher in the 1 d
variation than in the 2 d variation (p<0.05, Fig. 5a), while
this rate was the same in both variation treatments during the warm phase (p>0.05, Fig. 5a). In high-temperature experiments, the total
carbon fixation rates of the 1 d and 2 d variation treatments were
significantly decreased by about ∼20 % and ∼18 % respectively, compared with the constant 25.5 ∘C treatment
(p<0.05, Fig. 5b).
Responses of Emiliania huxleyi photosynthetic carbon fixation and calcification at
constant temperatures and during the warm and cool phases of two thermal
variation frequencies (1 d and 2 d), including total carbon
fixation (photosynthesis + calcification) at low (a) and high (b)
temperatures, photosynthetic carbon fixation at low (c) and high (d)
temperatures, and calcification rates at low (e) and high (f) temperatures.
LT: low temperature; HT: high temperature. The thick black line in the
boxplots represents median values for each experimental treatment; the whiskers
on the boxplots indicate 1.5× interquartile range. Listed p values
with their respective brackets denote the statistical significance between
two treatments.
The photosynthetic and calcification rates of the constant 18.5 ∘C
treatment were 0.04±0.00 pmol C cell-1 h-1 and 0.02±0.00 pmol C cell-1 h-1, respectively, which were not
significantly different from both of the temperature variation treatments (p>0.05, Fig. 5c, e). Photosynthetic rates changed within the
thermal cycle for both 1 d and 2 d variation treatments, with a
decrease of 22 % and 28 % from the warm phase to the cool phase,
respectively (Fig. 5c). However, there were no significant changes in
calcification rates under either variation frequency treatment between the
cool and warm phases of the thermal cycles (p>0.05).
In the mean 25.5 ∘C experiment, photosynthetic rates were not
significantly different between the 2 d variation and constant
treatments (p>0.05), while the photosynthetic rate of the
2 d variation was slightly higher than that of the constant 25.5 ∘C
treatment (p<0.05, Fig. 5d). In contrast, calcification rates of
1 d and 2 d variation treatments at a mean temperature of 25.5 ∘C were significantly decreased by about ∼46 % and
∼51 %, respectively, relative to the constant control
(p<0.05, Fig. 5f). There were no significant differences in total
carbon fixation and photosynthetic and calcification rates between the 1 d
variation and 2 d variation treatments during both the cool (23 ∘C)
and warm (28 ∘C) phases (p>0.05, Fig. 5b, d, f).
In the low-temperature treatments, there were no significant differences in
calcification to photosynthesis (Cal / Photo) ratios between the constant and
the two variable treatments (p>0.05, Fig. 6a). In contrast, in
the high-temperature experiments, the Cal / Photo ratio of the 1 d
variation and 2 d variation treatments were decreased by ∼40 % and 49 %, respectively, compared with the constant 25.5 ∘C
treatment (p<0.05, Fig. 6b). For both low- and high-temperature
experiments, there were no significant differences between the 1 d and
2 d variation treatments in either the cool or warm phases of the
thermal cycle (p>0.05, Fig. 6b). However, in both temperature
treatments the lower photosynthetic rates during the cool phase (Fig. 5c, d)
resulted in an increase in the Cal / Photo ratio during the cool phase for
both the 1 d and 2 d variation treatments (p<0.05, Fig. 6a, b).
Responses of Emiliania huxleyi calcification to photosynthesis ratios (Cal / Photo) to
constant temperatures and during the warm and cool phases of two thermal
variation frequencies (1 and 2 d) under low (a) and high (b) mean
temperatures. LT: low temperature; HT: high temperature. The thick black
line in the boxplots represents median values for each experimental
treatment; the whiskers on the boxplots indicate 1.5× interquartile range.
Listed p values with their respective brackets denote the statistical
significance between two treatments.
Elemental content and stoichiometry
In the low-temperature experiments, the 1 d variation and 2 d
thermal variations had different effects on cellular elemental contents and
ratios, relative to the constant 18.5 ∘C treatment. The 1 d variation
increased most of the cellular elemental and biochemical contents (TPC, PON
and Chl a) but with no significant difference (p>0.05), except
for POP content (p<0.05), compared with the constant 18.5 ∘C
treatment (Table 1). In contrast, the 2 d variation treatment decreased
all the measured cellular elemental and biochemical contents (TPC, PON, POP
and Chl a, p<0.05) in relation to the constant 18.5 ∘C
treatment (Table 1). However, the TPC / PON and Chl a/ POC ratios of the 2 d
variation treatment were higher than those of the 1 d variation and
constant 18.5 ∘C treatments (p<0.05, Fig. 7a, e), while the
PON / POP ratio was lower than in the 1 d variation and constant 18.5 ∘C treatments (p<0.05, Fig. 7c). There were no significant
differences in TPC / PON, PON/POP and Chl a/ POC ratios between the constant
18.5 ∘C and the 1 d variation treatments (p>0.05, Fig. 7a).
Responses of Emiliania huxleyi elemental ratios in two thermal variation frequency
treatments (1 and 2 d) compared to constant temperatures for TPC / PON
(a low and b high temperature), PON/POP (c low and d high temperature)
and Chl a/ POC ratios (e low and f high temperature). LT: low temperature;
HT: high temperature. The thick black line in the boxplots represents median
values for each experimental treatment; the whiskers on the boxplots indicate 1.5× interquartile range. Listed p values with their respective
brackets denote the statistical significance between two treatments.
In high-temperature experiments, the highest cellular TPC, PON and POP
contents were all obtained under the 1 d variation treatment, which was
significantly higher than under constant 25.5 ∘C conditions (p<0.05, Table 1). However, there were no significant differences in cellular
Chl a content between the constant 25.5 ∘C and both variation treatments
(p>0.05, Table 1). The TPC / PON ratio of the constant 25.5 ∘C treatment was ∼22 % and ∼35 %
higher than that of the 2 d variation and 1 d variation treatments,
respectively (p<0.05, Fig. 7b), while the PON/POP ratio was highest
in the 1 d variation, followed by the 2 d variation and finally by
the constant control (Fig. 7d). The Chl a/ POC ratio of the 1 d variation
treatment was significantly lower than that of the constant 25.5 ∘C and
2 d variation treatments (p<0.05), but there were no
significant differences between the constant 25.5 ∘C and 2 d
variation treatments (p>0.05, Fig. 7f).
During the cool phase of the high-temperature experiments (23 ∘C), the
cellular TPC, PON, POP and Chl a content of 2 d variation were all
significantly lower than in the 1 d variation treatment (p<0.05). Similar decreasing trends during the cool phase were observed for the
TPC / PON ratios (Fig. 7b) but not the Chl a/ POC ratio, which was
∼32 % higher than in the 1 d variation treatment
(p<0.05, Fig. 7f). During the warm phase (28 ∘C), there were
no significant differences of cellular TPC, PON and POP contents between
1 d and 2 d variation treatments (p>0.05, Table 1), as
well as the TPC / PON ratio (Fig. 7b). However, the Chl a content of the 1 d
variation treatment was ∼20 % lower than that of the
2 d variation treatment (p<0.05). The Chl a/ POC ratio was not
significantly different between the 1 d and 2 d variation treatments
at the warm phase (p>0.05, Table 1, Fig. 7f).
Experimental constant temperature performance curves and measured and
modeled fluctuating temperature performance curves
The experimentally determined constant condition temperature performance
curves and the predicted fluctuating temperature condition temperature
performance curves based on the Bernhardt et al. (2018) nonlinear averaging
model are shown in Fig. 8 for E. huxleyi. Compared with the measured temperature
performance curve under constant thermal conditions, the modeled curve of
the fluctuating temperature condition showed a leftward shift towards lower
temperatures at optimum temperatures and above. The maximum and optimal
temperature of the modeled fluctuating temperature performance curve were
all lower than those of the measured constant condition curve. In
particular, the optimal temperature for growth decreased from 22 ∘C in
constant conditions to 21 ∘C under fluctuating temperature conditions.
At the same time, the maximum growth rate (μmax) of the
fluctuating temperature condition was 0.8 d-1, which was lower than the
constant condition value of 0.9 d-1. The measured growth rates of
experimental 1 d (0.71 d-1) and 2 d (0.72 d-1) variation
treatments at the relatively low mean temperature of 18.5 ∘C closely
matched the model-predicted fluctuating temperature growth rate at this
temperature (0.74-1, Fig. 8). However, measured and predicted growth
rates did not match as well at the higher mean temperature. At 25.5 ∘C,
the measured growth rate of the 1 d variation was 0.52 d-1, 30 %
higher than the predicted fluctuating temperature growth rate of 0.40 d-1. In contrast, the measured growth rate of the experimental 2 d
variation treatment was 0.20 d-1, a decrease of 50 % compared to the
model-predicted fluctuating temperature growth rate of 0.40 d-1 at
this temperature (Fig. 8).
Thermal performance curves based on specific growth rates (d-1)
of Emiliania huxleyi, including our experimentally determined constant condition temperature
performance curve (black symbols and solid line) and a predicted fluctuating
condition temperature performance curve (dashed line) according to the model
of Bernhardt et al. (2018). Measured growth rates from the two low- and high-temperature experiments are shown for constant thermal conditions (red
symbols), 1 d (green symbols) and 2 d (blue symbols) variation
treatments.
DiscussionEffects of warming on <italic>Emiliania huxleyi</italic> growth rates and elemental ratios
Thermal response curves and optimum growth temperatures describe the
importance of temperature as a control on the distribution of E. huxleyi strains in
the ocean (Buitenhuis et al., 2008; Paasche, 2001). The optimal temperature
range of 21.3–24.5 ∘C found in our study is similar to that of some
other E. huxleyi strains (De Bodt et al., 2010; Feng et al., 2017; Rosas-Navarro et
al., 2016; Zhang et al., 2014). Most studies have focused on the lower part
of the temperature curve where growth rates increase with rising
temperatures (Feng et al., 2017; Matson et al., 2016), with relatively few
examining stressfully warm temperatures where growth is inhibited (Zhang et
al., 2014).
In our study, the descending portion of the upper temperature performance
curve ranged from 24.5 to 28.6 ∘C, at which point growth rates
became negative. We repeated the upper thermal limit part of the curve
several times to rigorously verify that cultures were unable to grow at this
temperature. The magnitude of the negative growth rate is presented here as
it represents an expression of the degree of stress the culture experienced
at this temperature and thus makes a useful comparison with the other
positive growth rate values in the variation experiments. This E. huxleyi strain was
isolated from the Sargasso Sea where the sea surface temperature can reach
29 ∘C in the summer and will undoubtedly be higher in the future with
global warming
(https://seatemperature.info/sargasso-sea-water-temperature.html, last access: 17 January 2019). This
suggests that this strain may be currently living at or very near its upper
thermal limit for part of the year, as are many other tropical and
subtropical phytoplankton (Thomas et al., 2012) and that it may therefore be
vulnerable to further warming.
Calcification is the key biogeochemical functional trait of this species,
and the PIC / POC ratio of E. huxleyi can be influenced by factors that include CO2
concentration, nutrient status, irradiance and temperature (Feng et al.,
2008, 2017; Raven and Crawfurd, 2012). The cellular PIC / POC of E. huxleyi has been
reported to decrease as irradiance and CO2 concentration rise but to
increase under nitrate and phosphate limitation (Feng et al., 2017; Paasche,
1999; Riegman et al., 2000). The effect of temperature on E. huxleyi cellular PIC / POC
ratio is, however, more complex. De Bodt et al. (2010) and Gerecht et al. (2014) observed that higher cellular PIC / POC ratios were obtained at lower
temperatures for both E. huxleyi and Coccolithus pelagicus. Sett et al. (2014), however, found an opposite
trend, whereby the PIC / POC ratio increased with temperature in another
strain of E. huxleyi. Feng et al. (2017) reported that the cellular PIC / POC of E. huxleyi was
increased as the temperature rose from 4 to 11 ∘C but
decreased with warming from 11 to 15 ∘C and remained steady
afterwards.
In our study, the cellular PIC / POC ratio of E. huxleyi was positively correlated to
growth rate (R2=0.73) and increased with warming from 8.5 ∘C to
a maximum at 22.6 ∘C and then decreased with further warming to 27.6 ∘C. In a meta-analysis of studies using different coccolithophore
subgroups, Krumhardt et al. (2017) found that the highest PIC / POC ratios
were observed between 15 and 20 ∘C, in the same
thermal range where the highest growth rates of E. huxleyi are found, as seen here and
in Sett et al. (2014). In contrast, Rosas-Navarro et al. (2016) reported
that the cellular PIC / POC ratio showed a minimum at optimal growth
temperature (between 20 and 25 ∘C) for three strains of E. huxleyi.
However, the E. huxleyi strain used here was isolated from a warmer area (the Sargasso
Sea) compared with isolates from coastal Japan and New Zealand in previous
studies (Rosas-Navarro et al., 2016; Feng et al., 2017). The growth
temperature for our stock cultures was 22–24 ∘C, higher than that of the
other two E. huxleyi strains. Feng et al. (2017) also found that the optimal
temperature for calcification was close to the stock culture maintenance
temperature in their study.
Our results also support suggestions that stressful high temperatures may
lead to decreases in cellular PIC / POC ratios and calcification (De Bodt et
al., 2010; Feng et al., 2017; Gerecht et al., 2014; Krumhardt et al., 2017).
The cellular PIC / POC ratio of E. huxleyi was much more plastic than the other ratios
we measured, including TPC / PON and POC/PON. Indeed, PIC / POC ratios may
change dramatically (> 2-fold) with temperature for some
coccolithophore subgroups (Krumhardt et al., 2017). The plasticity in
PIC / POC ratios of E. huxleyi during temperature changes in our study may have
implications for shifts in the ballasting of coccolith-containing particles
during sinking, thus affecting the ocean carbon cycle.
The cellular Chl a/ POC ratio of E. huxleyi showed a similar pattern with the PIC / POC
ratio, as it was also positively correlated to growth rate. Zhu et al. (2017) reported the cellular Chl a/ POC ratio of a southern California diatom
was also correlated to growth rate across a very similar temperature range.
In contrast, Feng et al. (2017) found that the cellular Chl a/ POC ratio of
E. huxleyi dramatically decreased with warming. However, in our experiments, the
cellular Chl a/ POC ratio was lower at 27.6 ∘C than at 28.6 ∘C,
likely due to the negative growth rates and consequent lack of acclimation
of the cultures maintained at the highest temperature. Traits such as
PIC / POC ratios, Chl a/ POC ratios and TPC / PON ratios also showed some evidence
for possible carryover from the stock cultures (22–24 ∘C) in this 28.6 ∘C treatment, as we were forced to sample before the cells died
completely, after only 2–3 cycles of dilution.
Effect of thermal variation on <italic>Emiliania huxleyi</italic> growth and physiologyConstant vs. variable temperature
Thermal variability in the surface ocean is becoming an increasingly
relevant topic as global warming proceeds. In our study, we found that the
growth rates of a subtropical E. huxleyi strain were quite sensitive to temperature
variation under both low (18.5 ∘C, “winter”) and high (25.5 ∘C,
“summer”) mean temperatures. In both low- and high-temperature experiments,
growth rates always decreased under temperature variation, compared with the
constant mean temperature. This result agrees with previous studies showing
that temperature variation slowed the growth rates of the fresh water green
alga Chlorella pyrenoidosa and the marine diatom Cyclotella meneghiniana, as observed in laboratory work and during
long-term field observations (Zhang et al., 2016).
This growth rate inhibition under temperature variation was more pronounced
at high temperature than at low temperature, indicating that variability at
the warm range boundary will have a stronger negative effect on population
growth rate than variability near the lower thermal limits (Bernhardt et
al., 2018). This trend suggests that acclimation to high temperature
(whether constant or variable) may require greater investment in cellular
repair machinery, such as heat shock proteins, thus potentially diverting
nutrient and energy supplies and thereby reducing growth rates (O'Donnell et
al., 2018). However, following Jensen's inequality model to predict the
thermal performance curve, there should be an inflection point where the
transfer between positive and negative effects of temperature variability
will occur compared with the constant thermal curve. Conversely, for
phytoplankton living in regions of suboptimal temperatures, thermal
variation can enhance growth (Bernhardt et al., 2018). Thus, for some polar
phytoplankton or for temperate species extending their ranges poleward, such
as E. huxleyi (Neukermans et al., 2018), not only warming but also thermal variability
may need to be taken into consideration in order to understand changes in
high-latitude microbial communities and biogeochemistry cycles.
Temperature variation affected the physiology of E. huxleyi differently compared with
constant temperature. Physiological traits that were affected by thermal
fluctuations also differed at low temperature (“winter”) and high
temperature (“summer”), suggesting different response mechanisms. Under
low temperature variations (16–21 ∘C), photosynthesis and calcification
were correlated with temperature, leading to rates similar to those observed
with constant temperature. However, elemental contents and ratios under
thermal variations differed from constant temperature. For instance, the
cellular POC, PON, POP and Chl a contents increased during 1 d variations
but decreased during 2 d variations, compared with constant temperature.
These cellular quota changes were reflected in elemental ratio differences
(PIC / POC, Chl a/ POC and TPC / POC) between the thermal variation treatments
and constant temperature. However, the changes between thermal variation and
constant treatments were not significant under low temperature (“winter”),
indicating that the thermal variation would not significantly influence
biogeochemical cycles under these conditions. Unlike constant temperature
treatments where selection may favor a higher growth rate, the trade-off for
the thermal variation treatments may involve sacrificing increased growth
rate in order to adjust cellular stoichiometry to adapt to the fluctuating
environment.
In contrast, photosynthetic and calcification rates under high-temperature
thermal variations (23–28 ∘C) were significantly different from those
seen under constant temperature (25 ∘C), especially the calcification
rate. Thermal variation treatments transiently but repeatedly experienced
the extreme high temperature point (28 ∘C), leading to extremely low
calcification rates and PIC contents and thus relatively low PIC / POC and
Cal / Photo ratios. Previous E. huxleyi studies agree that high temperature decreases PIC
content, PIC / POC ratios and Cal / Photo ratios (Feng et al., 2017; 2018;
Gerecht et al., 2014). The two different patterns of responses to thermal
variation we observed under low and high temperatures imply a seasonal
pattern in the ways that thermal variations will affect the elemental
stoichiometry of E. huxleyi .
Under other stresses such as nutrient limitation, trade-offs between growth
rates and resource affinities may be necessary to adapt to thermal changes.
For instance, nitrate affinity declines in cultures of the large centric
diatom Coscinodiscus that have acclimated to warmer temperatures (Qu et al., 2018), while warming
decreases cellular requirements for iron in the nitrogen-fixing
cyanobacterium Trichodesmium (Jiang et al., 2018). In nitrogen-limited cultures of the
marine diatom Thalassiosira pseudonana, long-term thermal adaptation acted most strongly on systems
other than those involved in nitrate uptake and utilization (O'Donnell et
al., 2018). Thus, it is possible that our thermal response results with E. huxleyi
might have differed under nutrient-limited growth conditions.
The 1 d variation vs. the 2 d thermal variation
As temperature fluctuations in the surface ocean increase along with climate
change, phytoplankton will be influenced by the frequencies and intensities
of these thermal excursions. We found that the responses of E. huxleyi to 1 d
vs. 2 d temperature variations were different at both low and high
temperatures. For instance, under low temperature the transition from the
warm phase to the cool phase during the thermal variation could be treated
as a low-temperature stress leading to a lag phase in growth. The growth
rate of the 1 d variation treatment at the cool phase was lower than
that of the 2 d variation, suggesting that physiological acclimation is
not rapid enough to accommodate to the shorter variation treatment, while
the 2 d variation allows enough time for growth to recover. However, at
the warm phase (21 ∘C) there was no difference in growth rates between
the 1 d and 2 d variations compared with the constant 21∘
treatment. These results imply that there was a shorter lag phase after
transfer at the optimal temperature point (21 ∘C at the warm phase)
than during low-temperature stress (16 ∘C at the cool phase).
There was no significant difference in photosynthetic rates between the
1 d and 2 d variation during the warm phase (21 ∘C), but both
were higher than during the cool phase, indicating the photosynthetic rate
was correlated to the thermal variation cycle. However, for the
calcification rate there was no significant difference between 1 d and
2 d variations during either the cool or warm phases. These results
suggested that photosynthesis was more responsive to temperature variations
than calcification and thus ultimately determined the growth rate in both
cool and warm phases. Feng et al. (2017) reported a similar relationship
between growth and photosynthetic rates of a Southern Hemisphere E. huxleyi strain
cultured at different temperatures.
Temperature variation frequencies also strongly influenced elemental
composition. In low-temperature experiments, the cellular contents of PON,
POP and POC in the 2 d variation treatment were all higher than under
2 d variations. A notable exception to this trend was the cellular PIC
content, which was not significantly different between 1 d and 2 d
variation treatments. The PIC content was positively correlated to
calcification and relatively stable, indicating that coccolith production
and storage of E. huxleyi was relatively independent of the frequency of thermal
variation.
Unlike the photosynthetic rate, the cellular elemental content of 1 d
and 2 d variations were significantly different but were not changed
during temperature variation when transitioning from the warm phase to the
cool phase or vice versa. The temperature-dependent photosynthetic enzyme
activity likely determined the similar photosynthetic rate of 1 d and
2 d variation treatments at both cool and warm phase in our short-term
experiment, but the divergent responses of cellular stoichiometry in 1 d
and 2 d thermal variations indicated different mechanisms of rapid
acclimation to different thermal fluctuation frequencies. Our results imply
that the responses of E. huxleyi to 1 d and 2 d thermal variations have
different patterns, but both reach stable states during extended periods of
temperature fluctuation. Due to decreasing POC content, the PIC / POC ratio
increased in the 2 d variation compared with the 1 d variation,
suggesting that more rapid thermal fluctuations might lead to a decrease in
calcite ballasting of sinking organic carbon.
Under the high-temperature scenario, thermal variation forces the microalgae
to intermittently deal with a lethal high temperature during the warm phase
(28 ∘C), with potentially irreversible damage to the cells. In the
“summer” experiments, the mean growth rate of the 2 d variation was
much lower than that of the 1 d variation. This mainly resulted from the
negative growth rate of 2 d variation cultures during the warm phase (28 ∘C), whereas the growth rate of the 1 d variation was > 0.20 d-1. This result demonstrates that high-frequency temperature
variations (1 d) can partly mitigate growth inhibition by high
temperatures in E. huxleyi and thus allow tolerance to extreme thermal events relative
to longer exposures. This observation agrees with previous studies of other
marine organisms such as corals (Oliver and Palumbi, 2011; Safaie et al.,
2018). In the case of our experiments, the lag phase and metabolic inertia
would help to maintain the microalgae during short exposures (1 d) to
high temperature when transitioning from the cool phase (23 ∘C) to the
warm phase (28 ∘C).
Likewise, the particulate organic element contents (PON, POP and POC) of E. huxleyi
were more stable in 1 d than in 2 d temperature variation
treatments. The relatively steady status of cellular particulate organic
matter content in the high-frequency temperature variation treatment may
conserve energy, compared to the energy-intensive redistribution of major
cellular components under lower-frequency temperature variations. This
differential energetic cost may help to explain the differences in growth
rates between the two treatments. Adaptation to high temperature may also
require higher investment in repair machinery, such as heat shock proteins,
leading to an increased demand for nitrogen and other nutrients, thus
increasing cellular POC, PON and POP contents (O'Donnell et al., 2018).
Prediction and modeling of <italic>E. huxleyi</italic> responses to thermal variation
Mathematical curves based on population growth rates from laboratory studies
have been used to predict future population abundance, persistence or
fitness in a changing world (Bernhardt et al., 2018; Deutsch et al., 2008;
Jiang et al., 2017). We applied a modified version of the Eppley thermal
performance curve model with the addition of nonlinear averaging (Bernhardt
et al., 2018) to predict the influence of thermal variation on the growth
rate of E. huxleyi (Fig. 8). E. huxleyi growth rates were predicted to be much lower at warmer
temperatures under variable conditions compared to constant conditions, but
there were no significant differences at cooler temperatures. Thus, the
effect of thermal variation on population growth at the upper thermal limit
was predicted to be stronger than that in the lower portion of the thermal
range (Bernhardt et al., 2018; Sunday et al., 2012). This phenomenon has
been widely observed in ectothermic animal taxa (Dell et al., 2011), but
this model for the effect of thermal variation on population growth rate may
lack the ability to predict species responses at the extreme edges of their
ranges (Bernhardt et al., 2018).
Our results showed that the measured effects of a variable thermal regime on
E. huxleyi growth rate fitted well with model-predicted values at a relatively low
temperature (mean = 18.5 ∘C) but differed considerably at high
temperature (mean = 25.5 ∘C). This was especially evident under the
2 d variation conditions at a mean of 25.5 ∘C, where the growth
rate was sharply lower than predicted from the constant temperature
performance curve-based model. This result suggests that transient heat
waves may erode thermal tolerances of E. huxleyi populations already growing near their
upper thermal limits and that the frequency and duration of such extreme
events is critically important in determining the magnitude of this stress.
Qu et al. (2019) reported that the tropical cyanobacterium Trichodesmium erythraeum only showed a
slight decrease in growth rate with thermal variation treatments at high
temperature (average 30 ∘C), compared with constant 30 ∘C
treatments. In contrast, the sensitivity of this E. huxleyi isolate to increasing
thermal variability may reduce its fitness and its ability to compete with
other taxa such as diatoms and cyanobacteria, with implications for
community structure in the future subtropical ocean.
Although thermal variation at high temperature negatively impacted the
growth rate of E. huxleyi in our experiment, our relatively short-term study did not
address the potential for E. huxleyi to evolve under selection by frequent extreme
heat events. Evolutionary change in the thermal optimum and the maximum
growth temperature in response to ocean warming may reduce heat-induced
mortality and mitigate some ecological impacts of global warming (O'Donnell
et al., 2018; Thomas et al., 2012). For example, Schlüter et al. (2014)
found that after 1 year of experimental adaptation to warming
(26.3 ∘C), the marine coccolithophore E. huxleyi evolved a higher growth rate
when assayed at the upper thermal tolerance limit. Similar results were
reported for the marine diatom Thalassiosira pseudonana in recent studies (O'Donnell et al., 2018;
Schaum et al., 2018). Schaum et al. (2018) also found that the evolution of
thermal tolerance in marine diatoms can be particularly rapid in fluctuating
environments. Furthermore, populations originating from more variable
environments are generally more plastic (Schaum and Collins, 2014; Schaum
et al., 2013). Long-term evolutionary experiments with E. huxleyi will be necessary to
determine how the thermal performance curve of this important marine
calcifier may diverge under selection by different frequencies and durations
of extreme thermal variation events.
Understanding the combination of ocean warming and magnified thermal
variability may be a prerequisite to accurately predicting the effects of
climate change on the growth and physiology of the key marine calcifier E. huxleyi. This
information will help to inform biogeochemical models of the marine and
global carbon cycles and ecological models of phytoplankton distributions
and primary productivity. How changing thermal variation frequencies and
heat wave events will affect marine phytoplankton remains a relatively
under-explored topic but one that is likely to become increasingly
important in the changing future ocean.
Data availability
The data are available by request from the corresponding author (DAH) or at
https://www.bco-dmo.org/project/668547 (Hutchins, 2019).
Author contributions
XW, FF and DAH contributed to conceiving and planning the experiments. XW,
FF, PQ, JDK and H-BJ performed the lab experiments. XW, FF, YG and
DAH contributed to the data analysis and to writing the paper. All of the
authors contributed
comments, revisions and editing.
Competing interests
The authors declare that they have no conflict of interest.
Acknowledgements
We would like to
thank the two reviewers for their constructive comments and suggestions.
Financial support
This research has been supported by the US National Science Foundation Biological
Oceanography (grant nos. OCE1538525 and OCE1638804), the National Key
Research and Development Program of China (grant no. 2016YFA0601302), and the China Scholarship Council.
Review statement
This paper was edited by Julia Uitz and reviewed by two anonymous referees.
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