Introduction
The fossil remains of biomineralised plankton provide comprehensive records
of their biogeography, ecology, diversity and evolution that have
significance for our understanding of past ocean and climate systems and
their influence on these microscopic organisms. Despite their small size (2
to 200 µm for nanno- and
microplankton), the vast numbers of photosynthesising plankton in the ocean
drive many regional- to global-scale biogeochemical processes and comprise
the biomass that sustains the ocean ecosystem (e.g. Menden-Deuer and
Kiørboe, 2016). Investigating the biological response of plankton species
to environmental variability is therefore a crucial step in understanding the
potential consequences of future climate change on marine systems.
Coccolithophores are a major group of calcifying marine algae that first
evolved more than 200 million years ago (Ma) during the Late Triassic
(Janofske, 1992; Bown et al., 2004). The remains of their calcite cell
coverings contribute to the export of biogenic carbonate to deep-sea
sediments (Broecker and Clark, 2009), forming a geographically and temporally
extensive fossil record that is mostly in the form of individual calcite
plates called coccoliths. Spatial and temporal analysis of coccoliths reveals
the evolution, biogeography and ecology of past species (e.g. Haq and
Lohmann, 1976; Knappertsbusch, 2000; Ziveri et al., 2004; Gibbs et al., 2006;
Baumann et al., 2016) and the response of species and communities to
palaeoceanographic and palaeoclimatic variability (e.g. Bollmann et al.,
2002, 2009; Bown, 2005; Bown and Pearson, 2009).
Valuable new insights into past coccolithophore communities can also be
provided by the study of intact fossil coccospheres that have not
disarticulated into their component coccoliths, providing intriguing
snapshots of individual cell growth in geological time (Gibbs et al., 2013;
Bown et al., 2014; O'Dea et al., 2014). Whilst the preservation of intact
coccospheres in sediments is generally uncommon, recent investigations
showcase a large diversity of coccospheres from a range of ages, ocean
basins and latitudes in numbers suitable for robust quantitative analyses
(Gibbs et al., 2013; Bown et al., 2014). The discovery of relatively abundant
fossil coccospheres in exceptionally well-preserved sedimentary deposits,
inspired Gibbs et al. (2013) to first explore the quantitative links between
coccosphere geometry (coccosphere size, coccolith length and coccolith
number) and population growth. Their laboratory experiments using the modern
species Coccolithus braarudii and Emiliania
huxleyi identified that cells undergoing rapid cell division (termed
“exponential-phase” growth) were smaller and had fewer coccoliths per
coccosphere compared to cells dividing slowly, or not at all
(“stationary-phase” growth). This initial evidence of a relationship
between growth phase and coccosphere geometry was then used to reconstruct
the response of fossil taxa (Coccolithus and Toweius)
through an interval of rapid warming ∼ 56 Ma called the
Palaeocene–Eocene Thermal Maximum, PETM (Gibbs et al., 2013; O'Dea et al., 2014). As
growth phases describe “states” of rapid or slowed growth rates, these
findings hint that coccosphere geometry could provide opportunities for new
insights into the ecological “fitness” and subsequent evolutionary success
of coccolithophore populations where growth rates (or other physiological
measures) cannot be measured directly.
The development of coccosphere geometry as an indicator, or even proxy, of
growth phase in the fossil record requires further evidence that phases of
rapid and slowed growth produce quantifiably distinct differences in
coccosphere geometry, which can be regarded as a “universal” feature of
coccolithophores rather than just a species-specific attribute. Even across
the diversity of modern species, we observe substantial variability in cell
size, coccolith length and numbers of coccoliths per cell. Given this
observation, can we reasonably hypothesise that the growth–geometry
relationship reported by Gibbs et al. (2013) for two modern species is
similar across coccolithophores in general? If this is the case, then
coccosphere geometry could prove to be a valuable proxy for growth phase and
provide new insights into important fitness-related traits where growth rates
cannot be measured directly. One potential concern is that coccolithophores
show pronounced species-specific and even strain-specific physiological
responses to a variety of environmental manipulations such as carbonate
chemistry and nutrient availability in culture experiments (Langer et al.,
2006, 2009; Krug et al., 2011), which may extend to coccosphere variability.
We therefore require coccosphere geometry data from multiple modern species
experiencing different growth phases in order to further investigate the
relationship between coccosphere geometry and growth.
Here, we aim to determine the relationships (if any) between growth phase and
coccosphere geometry in three modern coccolithophore species –
Calcidiscus leptoporus, Calcidiscus quadriperforatus and
Helicosphaera carteri – and to integrate these new data
with those previously determined by Gibbs et al. (2013) for
Coccolithus and Emiliania. Calcidiscus and
Helicosphaera are particularly pertinent study taxa, as they have
widespread modern and geological occurrences and are important components of
mid- to low-latitude coccolithophore communities, preferring warmer temperate
to tropical waters (Ziveri et al., 2004). These species are also three of the
largest and most heavily calcified of all the modern species, along with
Coccolithus pelagicus in the high latitudes and
Coccolithus braarudii in the mid- to high latitudes (Ziveri
et al., 2004). They are therefore important contributors to the production
(Daniels et al., 2014, 2016) and export of inorganic carbon to the deep ocean
(Ziveri et al., 2007). Variability in coccosphere geometry in these species,
particularly the number of coccoliths per cell, could therefore substantially
alter cellular calcite with significant consequences for calcite production
and export. The well-documented fossil records of these genera extend back to
the first occurrence of Calcidiscus ∼ 57 Ma (Bown et al.,
2007) and Helicosphaera ∼ 54 Ma (Perch-Nielsen, 1985).
Alongside Coccolithus, they have been significant components of
coccolithophore communities over much of the last ∼ 55 Ma
(Perch-Nielsen, 1985; Bown et al., 2007).
Helicosphaera and Calcidiscus also have distinct
evolutionary and physiological differences that may highlight restriction of
the growth–geometry relationship to specific lineages. Species within the
Helicosphaeraceae (Order Zygodiscales) have evolved in a lineage quite
separate to the Coccolithaceae and Calcidiscaceae (Order Coccolithales), with
the two orders diverging very early in coccolithophore evolutionary history
during the Jurassic, ∼ 150–200 Ma (de Vargas et al., 2007).
Helicosphaera carteri is also physiologically distinct from both
Coccolithus and Calcidiscus species as it is motile in the
diploid (heterococcolith-bearing) life-cycle phase. As Coccolithus
and Calcidiscus are considered to be relatively closely related,
though still classified into separate families, we would predict that the
growth-phase diagnostic features of coccosphere geometry in
Calcidiscus species might be more like those reported for
Coccolithus by Gibbs et al. (2013). To our knowledge, the
experiments undertaken for this study have produced the most extensive
data set of modern coccosphere geometry yet to be presented, comprising a
total of more than 13 300 measurements of coccosphere and cell size,
coccolith length and coccoliths per cell from 2850 individual cells.
Methods
Experiment design
Monoclonal cultures of South Atlantic Ocean Calcidiscus
quadriperforatus strain RCC 1135, Calcidiscus leptoporus
strain RCC 1130 and Helicosphaera carteri strain RCC 1323 were
obtained from the Roscoff Culture Collection (RCC) and maintained at an
incubation temperature of 19 ∘C at the National Oceanography Centre,
Southampton. Cultures were acclimated to new experimental temperature and
light conditions for a minimum of 2 weeks (> 10 generations)
prior to the start of each experiment. The light regime remained consistent
across all experiments, at irradiance levels of 75 to
90 µmol photons m-2 s-1 (equivalent to a daily photon
flux of ∼ 3.5 mol photons m-2 d-1) with a 12 h light and
12 h dark irradiance cycle. To achieve a range of cell division rates,
experiments were undertaken at 16, 18, 20 and 22 ∘C, which is well
within the natural temperature range experienced by field populations of
these three species (Ziveri et al., 2004).
For each temperature experiment, all three species were cultured
simultaneously and in duplicate following a “batch culture” procedure,
where an initially low number of cells mL-1 are left to increase in
density, using up nutrients, until initial nutrient levels are completely
depleted and population growth ceases. This approach enables coccosphere
geometry data to be collected from both nutrient-replete rapid cell division
days and nutrient-deplete slowed cell division days towards the end of the
experiment, as used successfully in the experiments of Gibbs et al. (2013)
for Coccolithus. The initial starting density of cells for each
experiment was ∼ 300 cells mL-1 (taken from acclimated cultures)
added to 350 mL of sterilised and filtered natural seawater enriched with
28.8 µM nitrate and 1.8 µM phosphate (lower-nutrient K/20
medium, modified from Keller et al., 1987, following Langer et al., 2006 and
Daniels et al., 2014). The effect of increasing cell density on the carbonate
chemistry of the media over the duration of the experiment was not directly
quantified, but it is likely that there was dissolved inorganic carbon (DIC) consumption throughout
the course of each experiment. However, our aim was to minimise the effect of
cell growth on carbonate chemistry by using low-nutrient media to ensure that
cultures reached nutrient-limiting conditions relatively quickly and at
relatively low final cell concentrations, using 650 mL polycarbonate flasks
(Thermo Fisher Scientific) with vented lids to allow faster diffusive gas
exchange between the culture media and the atmosphere outside the flask, and
aerating and mixing each flask daily under sterile conditions to further
encourage gas exchange. After initial inoculation of the media, experiment
cultures increased in cell number rapidly, termed the exponential growth
phase, and were allowed to grow into the stationary phase, at which point
increasing nutrient limitation reduces growth rates such that the day-to-day
increase in cells mL-1 decreases towards zero. The typical experiment
duration between initial inoculation and the onset of stationary-phase growth
was between 14 and 21 days.
Growth rate calculation
Daily cell abundance was determined from triplicate counts of
cells mL-1 using a Sedgwick Rafter Cell (Pyser-SGI; following Langer et
al., 2006) on a transmitted light microscope at ×100 magnification. As
H. carteri is a motile species, 40 µL per mL (4 %
final volume) of 10 % formaldehyde was added to H. carteri
samples prior to counting to inhibit movement and ensure counting accuracy.
Daily growth rates were calculated as the natural log of the difference
in cell density between the census day and the day before (Langer et al.,
2006). The duration of the exponential-growth phase was then determined by
visual examination of these daily growth rates and plots of cell abundance
over time. Mean exponential growth rates (μ) for each temperature
experiment were calculated from daily cell abundances, where
μ(d-1)= [ln(N1) - ln(N0)/d], and N0 and N1
are the cell concentrations at the beginning and end of the exponential
phase, respectively, and d is the duration of the exponential phase in
days.
Coccosphere geometry
Samples for light microscope (LM) analysis were taken daily using 2–5 mL of
each culture replicate, filtered onto cellulose nitrate filters (pore size
0.8 µm; Sartorius Stedim Biotech) and dried overnight at
50 ∘C. One half of each filter was then fixed between a glass
microscope slide and a cover slip using Norland Optical Adhesive 74 (Norland
Products Inc.) and cured under UV light exposure. All LM analysis was
performed using a cross-polarised light microscope (Olympus BX51) with a
colour camera attached (Olympus DP71). Coccosphere geometry data were
obtained through LM following the same techniques applied by Gibbs et
al. (2013) and Daniels et al. (2014) and described in detail here. Random
transects across the widest section of the filter hemisphere were performed
until 30 individual coccospheres per slide were located from slides
corresponding to alternate day or, in some instances, daily samples. First,
the number of coccoliths around each cell (CN) was counted by
finely adjusting focal depth. Then, in-focus images of the upper coccosphere
surface and maximum cell cross-section were captured, from which biometric
measurements (Fig. 1) of coccolith length (CL), coccosphere size
(∅; size including calcite covering) and cell size (Θ;
size excluding calcite covering, which is assumed to be equivalent to cell
diameter) were taken (CellD software, Olympus). Unlike the spherical
coccospheres of Coccolithus and Calcidiscus species, H.
carteri coccospheres are prolate spheroids (Fig. 4), so here we
report cell and coccosphere sizes for this species as equivalent spherical
diameters. Prolate spheroid volume is calculated as V=(π/6)d2h,
where d is the short-axis cell or coccosphere diameter,
and h is the long-axis cell or coccosphere height
(Sun and Liu, 2003). This volume is used to calculate the equivalent
spherical radius. This coccosphere geometry data set is available from
https://doi.pangaea.de/doi:10.1594/PANGAEA.865403.
Light microscope image of a C. quadriperforatus
coccosphere,
illustrating the coccosphere geometry terminology used in this study and the
size measurements made on each individual coccosphere. After counting the
number of coccoliths per cell (CN), images are taken of
(a) an in-focus, representative coccolith on either the top or
bottom surface of the coccosphere from which coccolith length
(CL) is measured, and (b) a cross-sectional view from
which the coccosphere diameter (∅) and internal coccosphere
diameter, assumed to represent cell diameter (Θ), are measured.
The full range of coccosphere geometry in H. carteri, C.
quadriperforatus and C. leptoporus. (a–d)
Histograms of coccosphere diameter (∅) calculated for frequency
bins of 1 µm size. Note the different frequency scale in plot
d. (e–h) Number of coccoliths per cell (CN)
against ∅, showing a strong and statistically significant
(p<0.0001) positive relationship. The reduced major axis
regression lines show slopes of H. carteri – 2.10 (bootstrapped,
95 % confidence interval, CI
[2.03, 2.17]); C. quadriperforatus – 2.11 (95 % CI [2.02,
2.20]); C. leptoporus – 3.01 (95 % CI [2.87, 3.14]); C. braarudii Gibbs et al., 2013 – 1.35 (95 % CI [1.27, 1.43]); C. braarudii Daniels et al., 2014 – 1.17 (95 % CI [1.09, 1.25]); and
C. pelagicus Daniels et al., 2014 – 1.50 (95 % CI [1.31,
1.67]). (i–j) Coccolith length (CL) with ∅.
(m–p) CL and ∅ with data points coloured by
CN. For comparison purposes, we include data for C.
braarudii and C. pelagicus that can be found in Gibbs et
al. (2013) and Sheward et al. (2014) accompanying Daniels et al. (2014).
Cellular calcite calculation
Particulate inorganic carbon (PIC) per cell was calculated for each
individual coccosphere following Young and Ziveri (2000):
CellularPICpmolCcell-1=CN×CL3×ks×2.7100,
where CN is number of coccoliths per cell, CL is
coccolith length (µm), ks is a shape factor that
numerically describes species-specific coccolith morphology, and
2.7 is the density of calcite (pg µm-3). Division by 100
calculates cellular PIC in pmol C cell-1 from pg cell-1. We use
the shape factors of ks= 0.08 for Calcidiscus spp.,
ks= 0.05 for H. carteri and ks= 0.06
for Coccolithus spp. from Young and Ziveri (2000). Mean, 25th and
75th percentiles, and the range of cellular PIC were calculated from the
22 ∘C experiment data of each species using coccosphere geometry
data from selected mid-exponential-phase days (C. leptoporus = days 7, 9 and 11; C. quadriperforatus = days 3,
5 and 7; and H. carteri = days 6, 7 and 8) and all non-exponential-phase
days. Mean exponential- and non-exponential-phase calcite production rates at
22 ∘C were calculated based on these mean cellular calcite values
multiplied by mean exponential and non-exponential growth rates,
respectively, for the same temperature experiment. The minimum to maximum
range in growth rates was based on growth rates observed across all
temperature experiments.
Additional experimental results from Coccolithus
This study reports the new experimental results for Calcidiscus and
Helicosphaera, alongside coccosphere geometry and growth data for
Coccolithus from two previous studies that used identical LM methods
to collect coccosphere geometry data. Gibbs et al. (2013) obtained
coccosphere geometry data from a comparable batch culture experiment at a
single temperature in Coccolithus braarudii strain RCC
1197. These data are presented for direct comparison with the three new species
of this study, as much of the Gibbs et al. (2013) data was originally
presented as Supplementary Information to accompany that short-format paper.
We also present results from a previously unanalysed data set of
exponential-phase coccosphere geometry in C. braarudii strain RCC
1198 and C. pelagicus strain RCC 4092, originally published as a
data report by Sheward et al. (2014) and available from
http://www.pangaea.de (10.1594/PANGAEA.836841). For that study,
batch culture experiments were undertaken at multiple temperatures
(6–12 ∘C in C. pelagicus and 12–19 ∘C in C.
braarudii), and samples for coccosphere geometry analysis were collected
on a single mid-exponential-phase experiment day (further details in Daniels
et al., 2014).
Statistical analyses
The relationships of ∅ with CL and CN in
each species were tested by Model II reduced major axis (RMA) linear
regression analysis. Confidence intervals (95 %) for the regression slope
were calculated by bootstrapping over 1999 iterations using the freeware
Paleontological Statistics (PAST;
v. 3.13; Hammer et al., 2001). We compare species-specific mean ∅
and mean CN between growth phases using a t test in GraphPad
Prism (version 7.0a for Mac OS X; GraphPad Software, Inc., USA). The
differences in mean ∅ or CN between exponential-phase
growth and non-exponential-phase growth were considered significant at p<0.05.
Results
Growth rates
The four temperature experiments resulted in a modest range of mean
exponential growth rates (μ) across Helicosphaera and
Calcidiscus species. The highest mean exponential growth rate for
C. quadriperforatus was achieved at 22 ∘C (μ= 0.44 d-1), for C. leptoporus at 20 ∘C (μ= 0.44 d-1) and for H. carteri at 20 ∘C (μ= 0.45 d-1). Mean exponential growth rates for C. braarudii
at 15 ∘C were 0.68 d-1. These values are well within the
ranges reported in other studies carried out at similar temperatures for
Calcidiscus (Langer et al., 2006; Buitenhuis et al., 2008; Fiorini
et al., 2010, 2011; Langer et al., 2012; Candelier et al., 2013; Müller
et al., 2014) and H. carteri (Stoll et al., 2002; Šupraha et
al., 2015). Exponential growth rates of 0.4–0.5 d-1 signify that
roughly half of the culture population undergoes cell division each day.
Maximum cell density was ∼ 100 000 cells mL-1 in C. leptoporus cultures, 60–100 000 cells mL-1 for C. quadriperforatus, ∼ 30 000 cells mL-1 for H. carteri
and ∼ 25 000 cells mL-1 for C. braarudii.
Within-species range in coccosphere geometry
Coccosphere (∅) and cell size (Θ), coccolith length
(CL) and number of coccoliths per cell (CN) show
clear species-specific differences (Fig. 2, Table 1). A considerable range in
∅ is seen in all species: 13.8 to 24.4 µm in C. quadriperforatus, 9.4 to 20.9 µm in H. carteri and 10.0
to 19.7 µm in C. leptoporus. This is a comparable
∅ range to C. pelagicus (11.7 to 20.8 µm) but
slightly less than the ∅ range observed in C. braarudii
(13.7 to 29.7 µm). Cell size exhibited a similarly large range of
6.5 to 18.0 µm in H. carteri, 6.4 to 16.5 µm in
C. leptoporus, 8.6 to 18.8 µm in C. quadriperforatus, 7.9 to 18.1 µm in C. pelagicus and 9.9
to 15.8 µm in C. braarudii (Table 1).
Summary statistics of species-specific coccosphere geometry data,
PIC (particulate inorganic carbon) and CN (number of coccoliths
per cell) thresholds for classifying the proportion of recently divided and
ready-to-divide cells in a population, based on the complete coccosphere
geometry data set from all experiment days. Summary statistics for both growth
phases are shown in Table S1. The full data set of experimental conditions,
daily growth rates and coccosphere geometry measurements from each individual
coccosphere is available as Sheward et al. (2016) at
https://doi.pangaea.de/doi:10.1594/PANGAEA.865403.
Parameter
Helicosphaera
Calcidiscus
Calcidiscus
Coccolithus
Coccolithus
carteri
leptoporus
quadriperforatus
pelagicus
braarudii
Number ofvalues
990
1020
840
180
880
Coccosphere
Min
9.35
10.02
13.84
11.74
13.66
diameter,
Mean
15.01
13.28
18.56
16.12
20.49
∅ µm
Max
20.90
19.72
24.39
20.80
29.68
Cell diameter,
Min
6.53
6.39
8.64
7.94
9.92
Θ µm
Mean
12.01
9.90
13.72
12.89
16.36
Max
17.99
16.54
18.81
18.11
25.83
Coccolith
Min
6.70
5.02
5.67
5.68
7.87
length,
Mean
8.89
6.72
9.10
8.95
12.21
CL µm
Max
11.22
8.76
11.67
11.59
17.32
Coccoliths
Min
6
10
8
7
5
per cell,
Mean
16
19
18
14
12
CN
Max
30
45
29
23
20
PIC, pmol
Min
7.79
3.27
6.09
4.15
6.97
C cell-1
Mean
24.48
13.00
30.69
17.28
35.89
Max
54.95
36.58
80.19
42.08
147.80
Recentlydivided cells, CN≤
CN≤ 12
CN≤ 14
CN≤ 14
CN≤ 11
CN≤ 8
Ready-to-divide cells, CN≥
CN≥ 21
CN≥ 23
CN≥ 25
CN≥ 18
CN≥ 16
Reference
This study
This study
This study
Sheward et al. (2014)
Gibbs et al. (2013),
Sheward et al. (2014)
Calcidiscus spp. and H. carteri show a much greater range
in CN compared to Coccolithus spp. (Fig. 2e–h). The
most frequently observed CN is 16 in H. carteri cells,
18 in C. quadriperforatus cells and 19 in C. leptoporus
cells, with a maximum number of ∼ 30 coccoliths in all of these
species. In one C. leptoporus cell, the coccosphere was formed from
45 coccoliths (Fig. 4c). In contrast, Coccolithus cells more
typically have 11 to 14 coccoliths per cell, up to a maximum of 20
coccoliths. The relationship between CN and ∅
subsequently shows a steeper gradient in Helicosphaera and
Calcidiscus (greater CN increase per µm
∅) compared to Coccolithus (Fig. 2). The similar
coccosphere sizes but significantly greater number of coccoliths per
coccosphere of C. quadriperforatus compared to C. braarudii, and C. leptoporus compared to C. pelagicus,
indicate that Calcidiscus species achieve a greater degree of
coccolith overlapping compared with Coccolithus species of a similar
coccolith size. This is likely the result of the circular shape and narrower
central tube structure in Calcidiscus coccoliths, which therefore
pack more tightly around the cell with increasing CN, moderating
a corresponding increase in ∅. The minimum CN in
H. carteri is similar to Coccolithus (CN= 6
and CN= 5–7, respectively). The smallest H. carteri
cells, with just 6 coccoliths, formed cuboid coccospheres (Fig. 4a) and are
most likely recently divided cells. Cubiform coccospheres have also been
reported in Bown et al. (2014) for the extinct Palaeogene taxa
Toweius pertusus and Umbilicosphaera bramlettei, and
“boxy” coccospheres are also seen in several Chiasmolithus
species, which are probably also related to small cell sizes soon after cell
division.
Although coccosphere geometry is similar in the two Calcidiscus
species (Fig. 2f, g), it is not identical, with C. leptoporus
producing coccospheres with a slightly greater CN on average than
C. quadriperforatus (slopes of 3.01 and 2.11, respectively). In
contrast, the two species of Coccolithus are more closely
comparable, with the linear regression gradient between ∅ and
CN being 1.50 and 1.35 in C. pelagicus and C. braarudii, although the gradients are offset from each other (y-intercepts
of -10.17 and -17.33, respectively; Fig. 2h). Until recently, these two
Calcidiscus species were considered to be intraspecific morphotypes
(Knappertsbusch et al., 1997; Knappertsbusch, 2000) or subspecies (Geisen et
al., 2002) but have since been shown to be genetically distinct, which is
also the case for C. pelagicus and C. braarudii (Sáez
et al., 2003; de Vargas et al., 2004). The considerable overlap
in CL, ∅ and CN in Calcidiscus
species makes species differentiation based solely on any one of these
parameters difficult. However, the species-specific coccosphere geometry
identified here lends further support to the genetic distinction between
these species, alongside previously identified morphological and ecological
differences (Knappertsbusch et al., 1997; Knappertsbusch, 2000; Geisen et
al., 2002, 2004; Renaud et al., 2002; Sáez et al., 2003; Baumann et al.,
2016).
Coccolith length varies between cells by up to 4.5 µm in H. carteri, 6.0 µm in C. quadriperforatus and
3.7 µm in C. leptoporus, which is similar
to CL ranges of 3.0 to 8.5 µm reported in selected
studies on sediment samples (e.g. Baumann, 2004; Henderiks and Törner,
2006; Herrmann et al., 2012; Baumann et al., 2016). Unfortunately, no
culturing experiments on Calcidiscus or Helicosphaera
report CL measurements for comparison. In contrast
to CN, CL shows no relationship with ∅
within these clonal populations (Fig. 2i–l), and superimposing
CN onto plots of ∅ against CL
(Fig. 2m–p) clearly demonstrates the strong covariance of ∅
and CN. In our clonal populations, cells have relatively
restricted ranges in ∅ and CL that have no
statistically significant relationship (Fig. 2i–l). A weak relationship
between ∅ and CL appears to exist in
Coccolithus when data for C. pelagicus are combined with
data from two strains of C. braarudii (Fig. 2l, p). This
CL–∅ relationship only occurs in these culture
experiments when data from several growth-synchronised populations are mixed.
This effect is also seen in the culture and field data of Gibbs et al. (2013)
and is greatly amplified in fossil assemblages, which typically integrate the
remains of surface populations over longer time spans (Gibbs et al., 2013,
their Fig. 3a). In our single-clone culture populations, however, the
principal coccosphere geometry relationship is between CN
and ∅ rather than CL and ∅.
Frequency of coccosphere diameter (∅) and number of
coccoliths per cell (CN) for experiment days in exponential
growth (solid line) and experiment days no longer in exponential growth
(dashed line), averaged across all temperature treatments.
(a–f) H. carteri, C. quadriperforatus and
C. leptoporus data from this study. (g–h) A reproduction
of C. braarudii experiment data from Gibbs et al. (2013) SI
Fig. 1e and f for comparison purposes. The lines drawn on CN
plots indicate cells that are recently divided and
ready-to-divide or non-dividing, based on the
10th and 90th percentiles of the complete species CN data shown
in Fig. 2.
Light microscopy images illustrating the full range of cell geometry
observed across all experiment days within cultures of (a) H.
carteri, (b) C. quadriperforatus and
(c) C. leptoporus at 16–22 ∘C. The upper image
of each pair shows the cross-sectional view of the cell, from which
coccosphere diameter and cell diameter are measured. The lower image of each
pair shows a coccolith-focused view of the cell, from which coccolith length
is measured. Number of coccoliths per cell (CN) and coccosphere
diameter (∅) are given for each cell. End-member geometries
illustrating recently divided and ready-to-divide cells are shown, based on
their CN and ∅. Both exponential-phase and
non-exponential-phase cultures will contain some recently divided and some
ready-to-divide cells, but the proportion (%) of each will differ
depending on growth phase, as shown in Figs. 3 and 6. A reference code for
the experiment day that the image was taken from is also given. For example,
22D7 would be a cell from day 7 of the 22 ∘C experiment. All images
are to the same scale.
Coccosphere geometry as a function of growth
This study demonstrates that coccosphere size in all the species studied is
statistically smaller during days of rapid, nutrient-replete,
exponential-phase growth than during days of slowed, nutrient-depleted,
non-exponential-phase growth (Fig. 3). Mean ∅ across all four
temperature experiments during exponential-phase growth is 14.8 µm
in H. carteri, 18.4 µm in C. quadriperforatus,
13.1 µm in C. leptoporus and 20.5 µm in C.
braarudii. Mean coccosphere diameter during non-exponential growth
is modestly but statistically (unpaired t test) larger than during
exponential-phase growth, with mean ∅ being 0.55 µm larger in
C. quadriperforatus (t=3.324, df=839, p<0.001),
0.64 µm larger in H. carteri (t=4.659, df=990,
p<0.0001) and 0.90 µm larger in C.
leptoporus (t=5.669, df=1020, p<0.0001). Mean
∅ in C. braarudii (Gibbs et al., 2013) shows a larger
increase of 1.34 µm (t=9.216, df=548, p<0.0001)
between exponential- and non-exponential-phase growth. An increase in cell
size has also previously been observed in response to nutrient limitation in
Coccolithus and Helicosphaera (Gerecht et al., 2014, 2015;
Šupraha et al., 2015).
In addition to size differences, coccospheres also typically consist of fewer
coccoliths during exponential-phase growth and a greater number of coccoliths
during non-exponential-phase growth (Fig. 3). This is shown by an increased
frequency of cells in higher CN classes and an increased
mean CN during non-exponential-phase growth in each species.
Cells no longer able to maintain exponential rates of growth have an average
of 1 to 2 extra coccoliths per cell in H. carteri (t=5.067,
df=990, p<0.0001) and C. quadriperforatus
(t=5.451, df=840, p<0.0001), 2 to 3 extra coccoliths per cell
in C. leptoporus (t=6.312, df=1020, p<0.0001) and 3
to 4 extra coccoliths per cell in C. braarudii (t=14.24,
df=548, p<0.0001). The frequency distribution of CN
for each species (Fig. 3) can be used as a quantitative indicator of whether
cells are in a recently divided state (close to the minimum number of
coccoliths per cell observed, CN≤ 10th percentile of the
data) or are in a ready-to-divide state (close to the maximum number of
coccoliths per cell observed, CN≥ 90th percentile of the
data). These CN “thresholds” for recently divided and
ready-to-divide cells for each species are shown in Fig. 3 and Table 1. Based
on the species-specific geometries observed, recently divided cells typically
have CN≤ 12 in H. carteri and CN≤ 14 in Calcidiscus spp., whilst cells that are ready-to-divide
have CN≥ 21 in H. carteri, CN≥ 23
in C. quadriperforatus and CN≥ 25 in C. leptoporus (Fig. 3). During exponential growth, the mean and frequency
distribution of population CN is skewed towards the minimum
observed CN and, therefore, the population has a higher
percentage of “recently divided” coccosphere geometries. Populations
exhibiting slowed growth are more likely to have an increased percentage of
cells in a “ready-to-divide” state. However, there are always some recently
divided cells and some ready-to-divide cells in both exponential- and
non-exponential-phase populations due to ongoing cell division, albeit at
different rates. There is therefore a large overlap in ∅ and
CN size range between exponential- and non-exponential-phase
populations (Fig. 3), with a
negligible change in the maximum ∅ and CN of each
(Table S1).
Cellular particulate inorganic carbon
PIC can be calculated directly from the extensive data set of coccosphere
geometry collated for this study by multiplying CN by individual
coccolith calcite (following Eq. 1, Sect. 2.4; Young and Ziveri, 2000). Mean
exponential-phase cellular PIC (calculated at mid-exponential-phase for each
temperature experiment) was 10.7 to 12.6 pmol C cell-1 in C. leptoporus and 21.3 to 25.8 pmol C cell-1 in H. carteri, but
higher in C. quadriperforatus, 21.5 to 30.0 pmol C cell-1,
and C. braarudii, 27.9 pmol C cell-1 (Table 1). At
22 ∘C, mean PIC during non-exponential experiment days was 9 to
45 % higher compared to mid-exponential-phase across all species due to
an increase in median CN of 2 to 4 coccoliths (Table S1). The
25th and 75th percentiles are also clearly shifted towards higher cellular
PIC in cells no longer growing exponentially (Fig. 5a.). The 25th percentile
increases 50 to 60 % in Calcidiscus, with C.
braarudii and H. carteri increasing by 20 to 25 %.
The increase in the 75th percentile is not as large, but it is still
considerable in C. leptoporus and C. braarudii at 36 and
24 %, respectively, with C. quadriperforatus and H.
carteri showing more modest increases of 6 and 11 %.
Calcification rates in Coccolithus, Calcidiscus
and Helicosphaera at 22 ∘C. (a) Mean and 25th to
75th percentile of cellular calcite for cultures dividing exponentially
(mid-exponential-phase days, see Table S1; filled circles) and cultures no
longer maintaining exponential growth (unfilled circles). (b) Range
in cellular calcite, daily growth rates and calcite production observed
across the experiment. (c) Percentage decrease in mean calcite
production when cultures can no longer divide exponentially. The black box in
b and c represents typical calcite production rates
(∼ 0.2–0.8 pmol C cell-1 d-1) for E. huxleyi for
comparison (Balch et al., 1996; Poulton et al., 2010).
Discussion
Physiological insights into coccosphere geometry
Within these experiments, coccosphere size (∅) and the number of
coccoliths per cell (CN) varied depending on whether the culture
population was increasing in cell numbers each day at a rapid rate
(exponential-growth phase) or a slowed rate (non-exponential-growth phase).
Across all four species investigated, the transition from exponential into
non-exponential-phase growth was clearly associated with a shift towards
cells with a greater CN (mean CN increased by 1–3
coccoliths per cell) and larger coccosphere sizes (mean ∅
increased by 0.6 µm in H. carteri and C. quadriperforatus, 0.9 µm in C. leptoporus and
1.3 µm in C. braarudii; Fig. 3). This represents a
significant increase of 4 to 7 % on exponential-phase mean ∅
and an increase of 10 to 27 % on exponential-phase mean CN
(t test, p<0.0001). CN is not a frequently recorded
variable, but ∅
and CN in both nutrient-replete and nutrient-deplete cultures can
sometimes be inferred from supplementary information (Balch et al., 1993; Paasche,
1998; Gerecht et al., 2014, 2015; Šupraha et al., 2015). These are
consistent with the extensive observations from our experiments for
Calcidiscus and H. carteri and those of Gibbs et al. (2013)
for C. braarudii.
The relationship between growth phase, ∅ and CN can
be understood by considering the process of cell division and how it is
affected by the nutrient depletion that instigates non-exponential-phase
growth. Both ∅ and CN vary as each cell progresses
through the cell division cycle (unpublished observations; Taylor et al.,
2007; Müller et al., 2008). Recently divided cells are small, with
approximately the minimum number of coccoliths required to form a
complete cell covering (unpublished observations; Fig. 4). After division,
cells recommence coccolith production and increase CN until
the cell has sufficient coccoliths to cover two newly divided cells.
Coccosphere diameter correspondingly increases alongside
increasing CN as the cell synthesises organic cellular
components such as proteins, lipids and carbohydrates. Cultures that are able
to maintain exponential rates of cell division subsequently have a lower mean
∅, Θ and CN, as the majority of cells are in a
recently divided state (Figs. 3, 4). When cells are no longer able to
maintain exponential rates of cell division, in this instance due to
decreasing nutrient availability, they divide less frequently on average.
This is observed in the later days of each experiment as an increase in the
mean ∅, Θ and CN, an interpretation that is
consistent with the findings of Gibbs et al. (2013).
An increase in cell size, Θ, under decreasing nutrient availability
may seem counterintuitive, as nutrients are essential for phytoplankton
growth. Nitrate and phosphate are the two key nutrients required by most
phytoplankton (Arrigo, 2005; Moore et al., 2013), and they fulfil different
purposes within the cell. Phosphate limitation primarily impedes production
of the RNA, phospholipids and DNA that are essential for cell replication, and
phosphate is a key component of cellular energy carriers (Zhao et al., 2015).
Nitrate limitation particularly impacts the synthesis of proteins and
pigments used in photosynthesis (Zhao et al., 2015). However, despite the
suppression of cell division and photosynthetic activity by phosphate and
nitrate limitation, respectively, the cell is still able to synthesise
non-essential lipids and carbohydrates. Cell size and particulate organic
carbon content (POC) are therefore able to increase under nutrient limited
conditions (e.g. Müller et al., 2008). A similar increase in POC could
also reflect DIC limitation, which sometimes results from DIC drawdown as
cell numbers rise to high concentrations in non-exponential-phase growth. An
increase in POC under DIC limitation was previously shown in C. braarudii (Rickaby et al., 2010).
The greater CN of coccospheres during non-exponential-phase
growth (Fig. 3) includes the occurrence of some large coccospheres with very
high CN (Fig. 4) and more than enough coccoliths to cover two
daughter cells. This is evidence that cellular calcification (coccolith
production) can proceed uninterrupted despite decreasing nutrient
availability, and it indicates that the calcification process has a lower
nutrient “cost” compared to cell division processes (Paasche, 1998;
Monteiro et al., 2016). This is also illustrated by the dramatic
overproduction of coccoliths in E. huxleyi under nutrient
limitation (Balch et al., 1993; Paasche, 1998) and supported by the
CN evidence from Calcidiscus and Helicosphaera
in this study and from Coccolithus in Gibbs et al. (2013). An
alternative possibility is that the continued production of coccoliths by
cells in stationary phase leaves them poised and ready-to-divide should
nutrients become newly available. In support of this, the recommencement
of cell division in stationary-phase cultures after the addition of
nutrient-replete seawater has been observed in E. huxleyi cultures
(personal observations; J. Young, personal communication, 2007).
Contrasting growth phase and growth rate
The clear relationship we observe between growth phase, ∅
and CN is interpreted to be the result of cellular physiology
(calcification, biomass production and the synthesis of molecules involved
in cell division) responding to shifts in nutrient availability over the
course of the experiments, with stationary-phase nutrient depletion
decreasing growth rates to zero once levels became inhibiting to cell
division. Exponential-phase growth rates, the proportion of the culture
undergoing cell division between two consecutive days (daily growth rates) or
averaged across multiple days (mean exponential growth rates), are instead
affected by temperature (which determines the rate of nutrient uptake and the
rate of metabolic cell processes) and irradiance (which affects
photosynthetic rates, i.e. the rate at which the cell can produce energy).
Our manipulation of experiment temperature (16–22 ∘C) aimed to
achieve a range of exponential-phase growth rates that might reveal any
correlation between growth rate and coccosphere geometry. However, no clear
relationship between ∅, Θ, CL
or CN and exponential growth rate (daily or mean) was observed in
our experiments. One explanation for this might be that mean exponential
growth rates (μexp) were not sensitive enough to the temperature
range we applied (C. quadriperforatus μexp= 0.30–0.44 d-1, C. leptoporus
μexp= 0.31–0.44 d-1 and H. carteri μexp= 0.28–0.45 d-1). In addition, growth rates would not
necessarily be expected to influence coccosphere geometry in the same way as
a shift in growth phase caused by nutrient depletion, as temperature and
light primarily affect physiological rates (e.g. Eppley, 1972; Falkowski et
al., 1985), whilst nutrient limitation primarily impedes molecule synthesis
(e.g. Zhao et al., 2015). Calcification, for example, is contingent on both
the rate at which nutrients can be supplied to the cell
(temperature-dependent) and processed into energy (light-dependent) and can
proceed under nutrient limitation, as shown in our experiments, but may be
less efficient under suboptimal temperature or light conditions. As yet, no
studies have investigated the response of cell size and/or coccosphere
geometry under a range of optimum vs. limiting temperature or light
conditions in coccolithophores. As growth phase describes two different
physiological states, one of which manifests as slowed to zero daily growth
rates caused by depleted nutrient availability, the ability to identify
coccolithophore populations with coccosphere geometries characteristic of
each growth phase is an important advancement in interpreting
growth information directly from the coccosphere. Valuable additional
perspectives on the specific role of growth rate on coccosphere geometry
would be gained from future work using semi-continuous or continuous
culturing techniques to achieve a range of steady-state exponential growth
rates under different nutrient, temperature or light conditions.
Coccosphere geometry as a proxy for growth phase in the fossil
record
A notable finding of this study is that coccosphere geometry (coccosphere
size, coccolith length and coccoliths per cell) is species-specific, but
∅ and CN respond identically to growth-phase changes
across four different species of Calcidiscus, Coccolithus
and Helicosphaera. This strongly suggests that coccosphere geometry
within the major coccolithophore families Calcidiscaceae and
Helicosphaeraceae responds to nutrient-driven changes in growth phase, and
therefore cell physiology, in the same way as species within the families
Coccolithaceae (Gibbs et al., 2013) and Noelaerhabdaceae (Balch et al., 1993;
Paasche, 1998; Gibbs et al., 2013). This is compelling evidence that, as a
group, coccolithophores express a common physiological response to shifts
from exponential to non-exponential (stationary) growth phase, seen as a
modest but significant increase in the average CN and
∅ of a population (Figs. 3, 4). This specifically results from
the ability of the cell to maintain calcification processes even when rates
of cell division are suppressed by nutrient limitation.
One of the aims of this study was to further develop the proxy application of
fossil coccosphere geometry first proposed by Gibbs et al. (2013) for
Coccolithus and Toweius. Culture experiments on
Coccolithus and Emiliania huxleyi showed that ∅
and CN responded to growth phase as described above, and Gibbs et
al. (2013) applied this to coccosphere records of fossil Coccolithus
and Toweius (an ancestor of E. huxleyi) across the
Palaeocene–Eocene Thermal Maximum climate change event (56 Ma). Given the
tendency of coccolithophores to show strong species- and strain-specific
responses to external factors, extending this application to other fossil
species might be seen as highly speculative based on data from only two
modern species. The new experimental data presented here for
Calcidiscus and Helicosphaera, in combination with previous
results for Coccolithus and Emiliania (Balch et al., 1993;
Paasche, 1998; Gibbs et al., 2013; Gerecht et al., 2014, 2015), provide
validity that coccosphere geometry persistently responds to growth phase in a
common manner, regardless of species, and notably that mean
population CN increases under slowed growth in
Calcidiscus, Helicosphaera, Coccolithus and
Emiliania.
To further develop this proxy, we need to establish threshold values of
CN that distinguish recently divided cells and cells with
theoretically sufficient coccoliths to undergo cell division. As an
exponential-growth phase population is undergoing cell division at a rapid
rate, it has a greater percentage of recently divided cells (CN≤ lower threshold, Table 1). In contrast, a slowly dividing population
in stationary-phase growth has a greater percentage of ready-to-divide cells
(CN≥ upper threshold; Table 1) but fewer recently divided
cells. Here, we report these CN threshold values (Table 1) for
Calcidiscus and Helicosphaera (Fig. 3) and add them to
those identified by Gibbs et al. (2013) for
Coccolithus. CN is relatively easy to measure in both
fossil and modern coccospheres using light microscopy, and so this
potentially provides a robust method for identifying populations that are
growing rapidly (exponential populations where ≳ 15 % population
is characterised by cells with CN typical of recently divided
cells) compared to populations that are growing slowly (non-exponential
populations where ≳ 15 % population is characterised by cells
with CN typical of ready-to-divide cells). This is illustrated in
Fig. 6 and these specific CN threshold values can be used to
approximate the growth state of any fossil or modern population of
Coccolithus, Helicosphaera or Calcidiscus species.
In reality, the mixing of populations of different growth states in the
fossil record (and open ocean) will frequently result in percentages of
recently divided and ready-to-divide cells lying between the two end members
shown in Fig. 6 (Gibbs et al., 2013). However, where time series of
coccosphere geometry data are available, intervals of changing growth states
can be identified as substantial temporal shifts in the proportional
percentage of recently divided to ready-to-divide cells indicative of less or
more favourable growth conditions (Gibbs et al., 2013; O'Dea et al., 2014).
Whilst in these experiments non-exponential-growth phase is initiated by
nutrient depletion, this would be an overly simplistic interpretation for
modern field, sediment trap or fossil populations. It is more reasonable to
interpret shifts in population coccosphere geometry as a response to less or
more favourable growth environments, incorporating a combination of nutrient,
temperature, light and other environmental factors that may influence
population growth.
Contrasting exponential and non-exponential-phase culture
populations based on the percentage of recently divided and ready-to-divide
cells within the population, as characterised by CN thresholds
specific to each species (Fig. 3; Table 1). Mean percentages for exponential
days are shown as filled data points, and the mean non-exponential experiment
day percentages are shown as unfilled data points. Also indicated (grey
squares) are the characteristic percentages of three Coccolithus
field population data sets presented in Gibbs et al. (2013) – Field
(a) is Scotland, Field (b) is Iceland non-bloom (both
experiencing slowed growth) and Field (c) is Iceland bloom
experiencing rapid growth.
For fossil taxa that have no direct modern counterpart, the general
characteristics of rapidly growing populations, consisting of an increased
proportion of smaller cells with fewer coccoliths relative to slowly dividing
populations, can be used as a qualitative indicator of changes in growth
phase through time. Based on the species studied here, the
typical CN of recently divided and ready-to-divide cells can be
tentatively proposed for any species (living or extinct) based on the 10th
and 90th percentiles of the CN histogram produced from a compiled
taxon-specific data set of coccosphere geometry. Relative changes in the
distribution of CN through time within any species can then
provide a valuable indication of intervals where species may be experiencing
nutrient conditions that are more (shift towards lower CN,
recently divided geometry) or less (shift towards higher CN,
ready-to-divide geometry) favourable for growth. This can be achieved for any
taxon by first compiling a data set of coccosphere geometry for the focal
species and then calculating the 10th and 90th percentiles to estimate
taxon-specific CN thresholds. Growth phase can then be estimated
by calculating the percentage of each sample with coccospheres of
ready-to-divide and recently divided CN before plotting as
Fig. 6. We would caution users to be mindful that the full range of
coccosphere sizes in a species may not be represented in any particular
sample and that, as fossil species are typically morphospecies concepts, the
range in CN observed is likely to incorporate multiple
intraspecies morphotypes or ecotypes with similar but subtly varied
coccosphere geometries. We therefore recommend that as many samples as is
feasible are considered in the full data set before
calculating CN thresholds and that the minimum to maximum range
in coccosphere geometry parameters (Table 1) within modern species are heeded
as an indication of how variable coccosphere geometry can be within even a
single clone. The coccolithophore fossil record is also vulnerable to
size-related preservational biases (Young et al., 2005) that may affect the
abundance of very small or very large coccospheres within a sample and may
not be consistent through time at the same site. The overall quality of
preservation should therefore be considered when interpreting changes in
coccosphere geometry through time, and caution should be exercised if there
is a suspected strong bias against the preservation of particular taxa, very
small coccoliths, or very small or
very large coccospheres within a sample.
Thus far, relatively common fossil coccospheres have been documented from at
least 24 localities (Burns, 1975; Covington, 1985; Lambert, 1987; Young and
Bown, 1991; Mai, 1997; Mai et al., 1998; Henderiks, 2008; Ciurej, 2010; Bown
et al., 2014) representing low to high latitudes, the North and South
Atlantic oceans, the North Pacific Ocean, the Indian Ocean and Southern Ocean, and
ranging from Kimmeridgian (Late Jurassic) to Pleistocene. Coccosphere
geometry analysis is therefore likely to prove applicable at a range of
localities and time intervals, but reasoned selection of sampling sections
is likely to be important for retrieving sufficient coccospheres for robust
data analysis. Hemipelagic sediments, particularly those with less intense
bioturbation, are perhaps more likely to contain coccospheres than deep-sea
oozes (Bown et al., 2014).
Whilst we conclude that coccosphere geometry can be used with confidence as a
proxy of growth phase in the fossil record or modern ocean, we must be clear
that the environmental and growth signal recorded in field populations is
always more complex than any laboratory experiment result. Populations may
only experience a specific nutrient state for a few weeks or less before
conditions change, and the coccosphere geometry response of any individual
cell is likely to be further complicated by temperature and light conditions
that are also essential for growth. At present, there are few to no
experimental data to demonstrate the response of coccosphere geometry to
temperature or irradiance, or how changes in growth rate specifically (rather
than growth phase) may manifest in coccosphere geometry. The fossil record of
coccolithophores further compounds these considerations, as fossil
assemblages are typically temporal integrations of many thousands of very
short-lived population states. The coccosphere geometry signal of species
populations transitioning between rapid and slowed growth phases clearly
becomes obscured and diluted by the mixing of population remains and subtle
shifts in species morphotypes and ecotypes as environmental conditions vary,
as illustrated by Gibbs et al. (2013).
Nevertheless, the use of coccosphere geometry as a proxy for growth phase is
valid across coccolithophores generally, and not just specific species. As
exponential- and stationary-growth phases describe two distinct physiological
states, the former with rapid growth rates experiencing optimal nutrient
supply and the latter with slowed growth rates suffering nutrient limitation,
coccosphere geometry provides a unique link to the physiology of individual
cells and may contribute towards our understanding of population fitness
(measured as growth rate or fitness-related traits such as cell size and
calcification) and ultimately the long-term success of species responding to
varying nutrient conditions. As such, the coccosphere geometry–growth phase proxy is a highly valuable tool, for
the first time allowing direct considerations of growth phase in evolutionary
and palaeoceanographic studies.
Implications of growth-driven cellular PIC and POC for calcite
production
Coccolithus, Calcidiscus and Helicosphaera are
potentially major regional calcite producers in both the modern (Daniels et
al., 2014, 2016) and past ocean (Ziveri et al., 2007), as they are some of
the largest, most heavily calcified modern species with distributions
throughout subpolar (C. pelagicus), temperate (C. braarudii) and subtropical (Calcidiscus and
Helicosphaera) oceans (Ziveri et al., 2004). The process of biogenic
calcification is thought to be responsive to climate and particularly
sensitive to changes in ocean carbonate chemistry (for reviews see Riebesell
and Tortell, 2011; Bach et al., 2015; Meyer and Riebesell, 2015). Our
experiments show that calcite per cell can also change significantly with
growth phase, as the number of coccoliths in the coccosphere varies in
response to changing nutrient availability. This was not previously known for
any species other than E. huxleyi, which produces
high CN, multilayered coccospheres under nutrient limitation.
E. huxleyi additionally sheds excess coccoliths into the surrounding
waters (e.g. Balch et al., 1993), potentially amplifying the biogeochemical
impact of increased coccolith production under low-nutrient conditions,
although, to our knowledge, this species is unique in this respect. Calcite
production is a function of cellular calcite (particulate inorganic carbon)
and growth rate, and it could therefore change considerably with
environmental conditions through time, with implications for the
biogeochemical cycling of carbon in the ocean.
PIC can be calculated directly from coccosphere geometry by multiplying
CN by individual coccolith calcite (following Eq. 1, Sect. 2.4;
Young and Ziveri, 2000). Using the 22 ∘C experiment as an example,
mean exponential-phase cellular calcite ranged from 10.9 pmol C cell-1
in C. leptoporus to 19.3 pmol C cell-1 in C.
quadriperforatus. In the non-exponential phase, cellular calcite
increased by 4.9 pmol C cell-1 (45 %) in C. leptoporus,
2.6 pmol C cell-1 (9 %) in C. quadriperforatus, 2.9 pmol
C cell-1 (11 %) in H. carteri and 5.9 pmol C cell-1
(21 %) in C. braarudii (Fig. 5; Table S1 in the Supplement) due
to the higher CN proportion of each population with
greater CN (Fig. 3). Calcite production per cell per day
(pmol C cell-1 d-1) can be calculated by multiplying cellular
calcite (pmol C cell-1) by growth rate (d-1) (e.g. Daniels et al.,
2014, 2016). Calcite production in these four species is 6 to 20 times higher
than in E. huxleyi at a comparable growth rate (Fig. 5; Balch et
al., 1996; Poulton et al., 2010), and, hence, these heavily calcified species
(e.g. the calcite of one C. braarudii cell is equivalent to
∼ 78 cells of E. huxleyi) do not necessarily need to be
abundant or maintain comparative growth rates to still dominate calcite
production (Daniels et al., 2014, 2016). A dramatic difference in calcite
production can be seen between populations growing exponentially and those no
longer growing exponentially, with reductions in calcite production of 77 to
88 % in all species due to the order of magnitude decrease in growth
rates (based on mean exponential and non-exponential growth rates for the
22 ∘C experiment; Fig. 5c). In field populations, growth rates can
reach as low as < 0.2 d-1 (Poulton et al., 2014), similar to
the culture populations in slowed growth shown in Fig. 5, and therefore these
shifts to such low calcite production per cell per day are approximate
minimum calcite production values for these species. However, it is clear
that rates of calcite production can be altered by up to 50 % for even a
moderate change of growth rate of 0.1 to 0.2 d-1, for example where
coccolithophore populations experience changes in nutrient supply,
temperature or light availability that no longer support optimal rates
of cell division (Poulton et al., 2010, 2014).
The majority of studies attribute environmentally driven changes in calcite
production to variation in calcite per coccolith through coccolith size,
thickness or malformation (e.g. Beaufort et al., 2011; Horigome et al.,
2014). However, CL would need to increase by roughly 5 to
20 % to achieve the same change in cellular calcite as that produced by
an increase of just 2 to 4 coccoliths per cell based on our data. O'Dea et
al. (2014) similarly found that changes in coccolith calcite mass of
∼ 5 to 11 % and ∼ 6 to 16 % for Toweius
pertusus and Coccolithus pelagicus during the PETM, were
dwarfed by up to 500 % changes in cellular calcite resulting from
combined changes in CL, ∅ and CN across
the same time interval of Palaeogene climate change. Change in CN
with growth phase is, therefore, key when considering the impact of environmental
parameters such as nutrient availability on cellular PIC and calcite
production rates. The dominant control of growth rates on calcite production,
as demonstrated recently by Gerecht et al. (2015) for C. pelagicus,
is an important consideration that is often overlooked when investigating the
impact of climate on long-term calcite production, carbon export and
sequestration and should be accounted for alongside-growth phase changes in
calcite.