Morphological changes in coccoliths, tiny calcite
platelets covering the outer surface of coccolithophores, can be induced by
physiological responses to environmental changes. Coccoliths recovered from
sedimentary successions may therefore provide information on
paleo-environmental conditions prevailing at the time when the
coccolithophores were alive. To calibrate the biomineralization responses of
ancient coccolithophore to environmental changes, studies often compared the
biological responses of living coccolithophore species with paleo-data from
calcareous nannofossils. However, there is uncertainty whether the
morphological responses of living coccolithophores are representative of
those of the fossilized ancestors. To investigate this, we exposed four
living coccolithophore species (Emiliania huxleyi, Gephyrocapsa oceanica, Coccolithus pelagicus subsp. braarudii, and Pleurochrysis carterae) that have been evolutionarily
distinct for hundreds of thousands to millions of years, to a range of
environmental conditions (i.e., changing light intensity, Mg/Ca ratio,
nutrient availability, temperature, and carbonate chemistry) and evaluated
their responses in coccolith morphology (i.e., size, length, width,
malformation). The motivation for this study was to test if there is a
consistent morphological response of the four species to changes in any of
the tested abiotic environmental factors. If this was the case, then this
could suggest that coccolith morphology can serve as a paleo-proxy for that
specific factor because this response is conserved across species that have
been evolutionary distinct over geological timescales. However, we found
that the four species responded differently to changing light intensity,
Mg/Ca ratio, nutrient availability, and temperature in terms of coccolith
morphology. The lack of a common response reveals the difficulties in using
coccolith morphology as a paleo-proxy for these environmental drivers.
However, a common response was observed under changing seawater carbonate
chemistry (i.e., rising CO2), which consistently induced malformations.
This commonality provides some confidence that malformations found in the
sedimentary record could be indicative of adverse carbonate chemistry
conditions.
Introduction
Coccolithophores are calcifying marine phytoplankton and are among the most
important calcifiers in the ocean (Tyrrell and Young, 2009). They produce
single calcitic platelets named coccoliths and nannoliths. Due to their
ability to calcify, coccolithophores played an important role in
rock-formation during the Jurassic and Cretaceous as well as through the
Cenozoic (e.g., Erba, 2006). They are directly affected by environmental
drivers such as temperature, salinity, nutrient concentration, light, and
carbonate chemistry that can modify physiological rates and morphology of
certain taxa (e.g., Paasche, 1998; Riebesell et al., 2000; Langer et al.,
2006; Trimborn et al., 2007; Zondervan et al., 2007; Rosas-Navarro et al.,
2016). Due to their sensitivities to environmental changes fossil remains of
coccolithophores (coccoliths and nannoliths) have often been used as
paleo-proxies to reconstruct past physical and chemical conditions in the
surface ocean of local or global significance (e.g., Erba, 1994; Lees et al.,
2005; Tiraboschi et al., 2009; Erba et al., 2010, 2019; Lübke and Mutterlose,
2016; Faucher et al., 2017a; S1 in the Supplement). To
calibrate the biomineralization responses of ancient coccolithophorid algae
to climatic changes, studies often compared the biological responses of
living coccolithophore species with paleo-data from calcareous nannofossils
(Table 1).
Compilation of papers documenting morphological
(malformation) or morphometrical variations in calcareous nannofossils. We
considered papers that compared shape and size variations in calcareous
nannoplankton in the fossil record with biological responses of living
coccolithophores from culture experiments. Papers are organized in
alphabetic order; the following information is given: authors, analyzed
species, morphological and morphometrical variations, environmental
parameter/s considered for the detected alteration, age, and investigated
cores/sections.
PaperAnalyzed genera or speciesmorphological variationsimplicated environmentalparameterAgeInvestigated sections or coresMalformationsAgnini et al. (2006)calcareous nannoplankton assemblage referred to as the Calcareous Nannoplankton Excursion Taxa (CNET)malformations of CNEThigh CO2 and transient chemical modifications of the world's oceansPaleocene–Eocene Thermal Maximum(PETM)Possagno section, ItalyAgnini et al. (2007)calcareous nannoplankton assemblage referred to as the Calcareous Nannoplankton Excursion Taxa (CNET)high asymmetry of CNEThigh CO2, low pH, and change in temperature structurePETMForada section, ItalyBralower and SelfTrail (2016)Discoasterirregularity of individual rays or whole Discoasterhigh CO2PETMBass River, Wilson Lake, South Dover Bridge cores, USAErba et al. (2010)Biscutum constans, Discorhabdus rotatorius, Zeugrabdotus erectus, Watznaueria barnesiaemalformations of Watznaueria barnesiaehigh CO2 and low pHAptian OceanicAnoxic Event (OAE) 1aCismon core, Italy; DSDP site 463Jiang and Wise (2006)Discoastermalformed Discoaster araneus and Discoaster anartios;low pHPETMODP Site 1259Mutterlose et al.(2007)Discoasteraraneusasymmetrical, aberrant Discoaster araneushigh CO2 and/or low pHand/or warming, increasednutrientsPETMODP Site 1260BRaffi and De Bernardi (2008)Fasciculithus, Discoaster nobilis group, Discoaster mediosus, and Discoaster multiradiatusmalformed Discoaster nobilis, Discoaster falcatus and Discoaster mediosus and weakly calcified fasciculith specimens (F. thomasii morphotype)high CO2PETMODP Site 1263Size variationsBornemann et al.(2003)Watznaueria spp., Conusphaera mexicana, Polycostella beckmannii and Nannoconus spp.small Watznaueriachanges in climate and the circulation patternlatest Tithonian and earliest BerriasianDSDP Sites 105, 534A, 367Bornemann and Mutterlose (2006)Biscutum constans, Watznaueria barnesiaereduced Biscutum constans sizecooler water conditionsLate AlbianCol de Palluel section, FranceErba et al. (2010)Biscutum constans, Discorhabdus rotatorius, Zeugrabdotus erectus, Watznaueria barnesiaedwarf Biscutum constans, Discorhabdus rotatorius and Zeugrabdotus erectushigh CO2 and low pHOAE 1aCismon core, Italy; DSDP site 463Ferreira et al. (2017)seven morphospecies of Lotharingiussmaller morphotypesunstable environmental conditions: seawater temperature fluctuations, water trophic variations, and expansion/contraction of the photic zone and nutriclineToarcian – early AalenianRabaçal, Brenha, Cabo Mondego sections, Portugal; Truc-de-Balduc section, FranceFaucher et al. (2017)Biscutum constans, Discorhabdus rotatorius, Zeugrabdotus erectus, Watznaueria barnesiaesize reduction of Biscutum constanshigh CO2 and high trace metal concentrationsLate Cenomanian and latest CenomanianOAE 2Clot Chevalier section, France; Eastbourne section, United Kingdom; Novara di Sicilia section, Italy; Cuba and Pueblo sections, USA
Continued.
PaperAnalyzed genera or speciesmorphological variationsimplicated environmental parameterAgeInvestigated sections or coresSize variationsFraguas and Young(2011)genus Lotharingius“dwarfing” of Lotharingius hauffii, Lotharingius sigillatus and Lotharingius crucicentralisincreased temperature and perturbation of the carbon cycleEarly ToarcianWest Rodiles section, SpainGiraud et al. (2006)Watznaueria britannicabigger Watznaueria britannicaoligotrophic conditions and warmer climateLate Oxfordian-early KimmeridgianBalingen–Tieringen section, GermanyLinnert and Mutterlose (2012)genera Biscutum, Broinsonia, Prediscosphaera, Retecapsa and Watznaueriareduction in size of Biscutum and Broinsonia spp.reduced nutrient availability (Biscutum), increase in sea-surface temperature (Broinsonia spp.)Cenomanian–TuronianGoban Spur cores Site 549, Site 551, GermanyLübke et al. (2015)Biscutum constans, Zeugrabdotus erectus and Watznaueria barnesiaesmall Biscutum constanslow seawater temperatures, low light availability, high nutrient levelOAE 1aNorth Jens-1, Adda-2, Alstätte1 cores, Germany; Cismon core, Italy; DSDP Leg 62;Mattioli et al. (2004a)genera Biscutum and Similiscutumbigger Biscutum and Similiscutumhigh seawater temperature and/or high nutrient concentrationPliensbachian-ToarcianDSDP 547B; Monte Genuardo, Somma section, Italy; Dotternhausen, section GermanyMattioli et al. (2004b)Schizosphaerellasmall Schizosphaerella and undercalcified coccolithsincreased CO2Early Toarcian OAEPozzale and Somma sections, Italy; Dotternhausen section, Germany; Brown Moor Borehole, UKMattioli et al. (2009)Schizosphaerellasmall Schizosphaerellaincreased CO2 or temperature rise or less saline marine surface watersEarly Toarcian OAEDotternhausen section, Germany; Somma section, Italy; Peniche section Portugal; HTM-102 borehole, Saint Paul de Fonts section, FranceO'Dea et al. (2014)Coccolithus pelagicusthinning of Coccolithus pelagicus coccolithsocean acidificationPETMBass River, Lodo Gulch, USA; Tanzania Drilling Project Site 14, TanzaniaSuan et al. (2008)Schizosphaerellasmall Schizosphaerellaincreased CO2 and high seawater temperatureEarly Toarcian OAEPeniche section, PortugalSuan et al. (2010)Schizosphaerellasmall Schizosphaerellahigh nutrient concentrations (less oligotrophic conditions) and/or increased temperatureEarly ToarcianPeniche section, PortugalSuchéras-Marx et al.(2010)Crepidolithus crassusvariation in pseudo-cryptic species dominance, alternation of “small” and “big” Crepidolithus crassusfluctuation of the nutricline and photic zone depth under the control of the orbital cycles of eccentricity and precessionEarly PliensbachianPeniche section, PortugalTremolada et al. (2008)Discoaster multiradiatusmigration of allochthonous specimens of larger Discoaster multiradiatusincreased seawater temperature, stratification of water masses, and establishment of a well-defined thermoclinePETMODP Sites 690 and 1209Wulff et al. (2020)Biscutum constans, Rhagodiscus asper and Watznaueria barnesiaesmall Biscutum constansoligotrophic surface water conditionsBarremian“Frielingen” 9 core, road cut “A39” motorway, Braunschweig, Germany
The primary goal of our study was to understand if physiological experiments
with contemporary species are a valid tool to predict responses of ancient
coccolithophores to environmental change in the geological record. The
assumption that modern species respond identically to environmental change as ancient species did is implicit in many studies (e.g., Giraud et al., 2006;
Erba et al., 2010; Faucher et al., 2017a, Table 1) but, to the best of our
knowledge, has not been explored in depth. To test this assumption, we did a
series of identical stress test experiments with four selected modern
species that have been evolutionarily distinct for hundreds of thousands to
millions of years (Fig. 1). Our hypothesis was as follows: in the case that coccolith
morphology responses to a changing environmental driver are similar in the
four species, this could be indicative of a response pattern that was
physiologically conserved over geological timescales because the species
were evolutionarily separated for so long. In other words, if species
conserve a similar response to certain types of environmental change for
geological timescales, despite very different evolutionary trajectories,
then this would strengthen our confidence that responses recorded for modern
species also apply for the geological past.
Phylogeny and divergence times of the Haptophytes, modified from
Liu et al. (2010). Time is indicated in billion years. The species selected
for this study are shown in red. The nodes represent following divergence
episodes. The number in green represents specific nodes: node 47:
Exanthemachrysis gayraliae and Helicosphaera carteri; node 57: Coccolithus pelagicus and H. carteri; node 62: C. pelagicus and Umbilicosphaera hulburtiana; node 63: Calcidiscus leptoporus and Umbilicosphaera foliosa; node 77: Coronosphaera mediterranea and
Scyphosphaera apsteinii; and node 79: H. carteri and S. apsteinii. Numbers are related to calculated divergence
times. For further information see Liu et al. (2010).
Indeed, there is considerable uncertainty when trying to reconstruct
paleo-environmental conditions based on coccolith morphology. This in itself
is not surprising considering that there are millions of years of evolution
between the time when the fossil coccolithophores lived and when the
physiological experiments were done (Bown, 2005; De Vargas et al., 2007).
Moreover, for the fossil record, it is extremely difficult to disentangle the
individual factor(s) that drove changes in coccolith morphology. Therefore,
it is unsurprising that studies occasionally come to different conclusions
about what environmental factor drove a morphological change in the
paleo-record. For example, Erba et al. (2010), detected the reduction in
size and variation in shape of some nannofossil species during a time of
excess volcanogenic CO2 emissions. They explained their trend with
detrimental carbonate chemistry conditions based on physiological incubation
studies by Riebesell et al. (2000) who found decreasing calcification rates
under increasing CO2. Conversely, Bornemann and Mutterlose (2006)
explained decreasing coccolith size with decreasing sea surface temperature,
a conclusion that was also based on incubation experiments with living
coccolithophore species (Renaud and Klaas, 2001; Renaud et al., 2002).
In order to investigate our hypothesis outlined above, we selected four
different coccolithophore species: Emiliania huxleyi (morphotype R), Gephyrocapsa oceanica, Coccolithus pelagicus subsp. braarudii, and Pleurochrysis carterae. According to
“molecular-clock-data”, they are evolutionarily distinct since the
Triassic or the Jurassic (with the exception of G. oceanica and E. huxleyi, which diverged
∼290 Kya; Liu et al., 2010; Bendif et al., 2014). We present data
on how coccolith size and morphology change in response to a suite of
different environmental drivers and explore whether there is a common
response to any of these drivers among the different species. Afterwards, we
discuss if morphological features of coccoliths have the potential to serve
as paleo-proxies.
Material and methodsExperimental setup
Five experiments are presented in this study with a similar design. Every
experiment tested the influence of one abiotic parameter on four different
coccolithophore species which were cultured individually (i.e., in separate
bottles). The tested abiotic factors were as follows: light intensity, nutrient
limitations (N or P limitations), Mg/Ca ratio, temperature, and carbonate
chemistry. Monospecific cultures of the coccolithophores Emiliania huxleyi (strain RCC 1216,
from the Tasmanian sea), Gephyrocapsa oceanica (strain RCC 1303, from the French coast of the
Atlantic Ocean), Coccolithus pelagicus subsp. braarudii (strain PLY182G; it will be called hereafter C. braarudii, from
the English Channel, Atlantic Ocean), and Pleurochrysis carterae (unknown strain number, coastal
species) were grown in artificial seawater (Kester et al., 1967) under
dilute batch culture conditions (LaRoche et al., 2010). The artificial
seawater medium was enriched with 64 µmol kg-1 nitrate,
4 µmol kg-1 phosphate to avoid nutrient limitations with the
exception of the nutrient limitation experiment (see Sect. 2.1.3). In all
experiments we added f/8 concentrations of vitamins and trace metals
(Guillard and Ryther, 1962), 10 nmol kg-1 of SeO2 (Danbara and
Shiraiwa, 1999), and 2 mL kg-1 of natural North Sea water to provide
potential nutrients which were not added with the nutrient cocktail (Bach et
al., 2011). The medium was sterile-filtered (0.2 µm). The carbonate
chemistry was adjusted with aeration for 24 h using a controlled CO2
gas mixing system reaching the treatment levels of fCO2 400 µatm (total alkalinity,
TA, 2302 µmol kg-1) with the exception of the carbonate chemistry
experiment (see Sect. 2.1.5).
The medium was then transferred into 0.5 L Nalgene™ bottles. Cultures were
incubated in a thermo-constant climate chamber (Rubarth Apparate GmbH) at a
constant temperature of 15 ∘C, (with the exception of the
temperature experiment; see Sect. 2.1.4), with a 16 : 8 (hour : hour)
light / dark cycle, at a photon flux density of 150 µmol photons m-2 s-1 (with the exception of the light experiment; see Sect. 2.1.1). Before the beginning of the experiments, coccolithophore cultures
were acclimated for about 7–10 generations to each of the experimental
conditions. Cultures were in the exponential growth phase at the initiation
of the experiments (also in the nutrient limitation experiment; see Sect. 2.1.3). All culture bottles were manually and carefully rotated three times
a day, each time with 20 rotations in order to reduce sedimentation bias.
Final samples were taken when cells were exponentially growing (except for
nutrient limitation experiments; see Sect. 2.1.3) but cell numbers were
still low enough to limit their influence on the chemical conditions of the
growth medium. Sampling was conducted at the same time for every experiment
to avoid changes in cell diameter/volume, which develop in light–dark cycles
due to the synchrony of the cell cycle (Müller et al., 2012; Sheward et
al., 2017).
Specifics in the light experiment
The light setup was adjusted to test the response of the four species to a
gradient of photon flux densities (PFD). Because light intensities are
difficult to replicate we chose a gradient design in this experiment at the
expense of replication (Cottingham et al., 2005). Therefore, the light was
set to the highest possible intensity in the light chamber, and the bottles
were placed at different positions so that 12 different PFDs were
established (50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,
600 µmol photons m-2 s-1). Light intensities were measured at every
treatment position in the light chamber, using a Li-250A light meter
(Li-Cor, Heinz Walz GmbH, Effeltrich).
Specifics in the Mg/Ca experiment
This experiment was designed to test the physiological response of
coccolithophore algae to changing [Ca2+] while keeping [Mg2+]
constant at the modern seawater value. In the control, the Mg/Ca ratio was
set to simulate the modern ocean values (Mg/Ca=5.2) with [Ca2+]=9.8 mmol L-1 and [Mg2+]=50 mmol L-1. The low-Mg/Ca
treatments were set by increasing [Ca2+] to 25 and 50 mmol L-1,
respectively. The control and both treatments were replicated three times.
Specifics in the nutrient experiment
Batch cultures were grown under N or P limitations. For N limitation, all
cultures were run into N limitation during the acclimation phase, but care
was taken that this occurred at low cell densities so that the chemical
conditions in the seawater (apart from nutrients) remained largely
unaffected. During the main experiment, cell concentrations were counted
every other day, and 0.14 pmol N per cell (as NaNO3) was added to the
medium when cultures reached the stationary phase (i.e., they stopped
dividing). The same was done in P limitation experiments except that
0.01 pmol P per cell (as NaH2PO4) was added when reaching the
stationary phase. As control, we used exponentially growing cells which were
replete in both N and P. Nutrient concentrations were not measured, but
limitations were assured by measuring and comparing growth rates which were
much lower than in the nutrient replete controls. Controls and both
treatments were replicated three times.
Specifics in the temperature experiment
The experiments were carried out in two temperature-controlled light
chambers in order to test the response of the coccolithophores to increased
temperature. Batch cultures were grown at 15 and 22.5 ∘C. Both temperature treatments were replicated three times.
Specifics in the carbonate chemistry experiment
In the ocean acidification (OA) treatment, TA was kept constant (2348 µmol kg-1), whereas fCO2 was increased to 1020.5 µatm. In
the Cretaceous scenario1 (CS1) treatment, fCO2 was kept constant at
1020.5 µatm, while TA was increased to 3729 µmol kg-1. In
the Cretaceous scenario2 (CS2) treatment, fCO2 was increased up to 3061 µatm and TA up to 4978 µmol kg-1. Carbonate chemistry
parameters (pHf – free scale, HCO3-, CO32-, CO2) were calculated using the program CO2SYS (Pierrot et al., 2006) from
measured TA and calculated estimated dissolved inorganic carbon (DIC), temperature, salinity, and
[PO4], and the dissociation constants determined by Roy et al. (1993).
In the OA, CS1, and CS2 treatments, DIC and TA levels were adjusted by adding
calculated amounts of Na2CO3 (Merck, Suprapur quality and dried
for 12 h at 500 ∘C) and hydrochloric acid (3.571 mol L-1,
certified by Merck) following Gattuso et al. (2010).
Samples for pH and TA analyses were taken at the beginning and at the end of
the experiments. Samples were filtered (0.7 µm) and stored at 4 ∘C until measurements that were performed within 2 d for pH
measurements and 14 d for TA. pH was measured spectrophotometrically with
Varian Cary 100 in 10 cm cuvette at 25 ∘C as described in Dickson
et al. (2007) and then recalculated to in situ temperature (15 ∘C) using CO2SYS as is described by Schulz et al. (2017). Every sample was
measured three times. Samples for TA were measured in duplicate with a Metrohm 862
Compact Titrosampler device following Dickson (2003). TA data were
accuracy controlled with certified reference material (A. Dickson, La Jolla,
CA).
Cell abundance, coccosphere, and cell size
Samples for cell abundance were taken at the end of the experiment with the
exception of the nutrient experiments where samples were taken every second
day. Incubation bottles were turned to resuspend all cells and to obtain a
homogenous suspension of the cells before sampling. Cell numbers were
immediately measured three times without addition of preservatives using a
Beckman Multisizer Coulter counter. After the abundance measurements, samples were
acidified with 0.1 mmol L-1 HCl to dissolve all free and attached
coccoliths and subsequently measured another three times each in order to obtain
cell diameters and volumes (Müller et al., 2012).
Scanning electron microscopy (SEM)
Samples for SEM analysis were filtered by gravity onto polycarbonate filters
(0.2 µm pore size). For every sample, 5–10 mL of water was used.
Filters were subsequently dried at 60 ∘C for 2 d. Samples
were sputtered with gold–palladium. SEM analysis was performed at the Earth
Sciences department of the University of Milan with SEM Cambridge
Stereoscan 360. All pictures were taken with the same magnification (5000×),
and the scale bar given on SEM pictures was used for calibration. For every
experiment, in all treatments and replicates, 50 specimens for each species
were analyzed. For every coccolith, the length (DSL) and the width (DSW) of
the coccolith distal shield were manually measured using the public domain
program Fiji distributed by ImageJ software (Schindelin et al., 2012). For
E. huxleyi, the inner tube thickness, the number of distal shield elements, and the
distal shield elements thickness were also measured. For G. oceanica the tube thickness
and the bridge orientations were measured. Moreover, the presence of
malformations was quantified by visual inspection (Fig. 2): morphologies
were grouped following Langer et al. (2006, 2010) categories.
Examples of different morphological categories: normal, malformed,
incomplete, and incomplete/malformed for E. huxleyi, G. oceanica and C. braarudii.
Statistics
Data were tested for normality and homogeneity of variances (Bartlett and
Fligner–Killeen tests). To test the null hypothesis that differences in
growth rates and sizes among treatments are the same, the average values of
parameters from triplicate cultures were compared between treatments. A
one-way analysis of variance was used to determine the statistical
significance of the main effect of the different parameters tested on the
variables. A Tukey post-hoc test was used to assess whether differences
between treatments or control were statistically significant. Statistical
treatments of data were performed using R software. Statistical significance
was accepted for p<0.05. For the light experiment, a nonlinear
regression was used to explore the relationship between light and
coccolithophore parameters (growth and sizes).
ResultsLight
In the four species selected, coccolithophore, cell, and coccolith sizes
did not show any distinct trend with variable light intensity. Data are
reported in Table 2. Emiliania huxleyi coccoliths were less elliptical with light intensities
above 400 µmol photons m-2 s-1 and characterized by a higher
number of distal shield elements with light intensities above 400 µmol photons m-2 s-1. Gephyrocapsa oceanica and C. braarudii coccolith size and shape did not change
with light intensity. Finally, P. carterae coccoliths were less elliptical only at
irradiances of 350 µmol photons m-2 s-1. (Fig. 3; Plate S1). Malformed coccoliths increased in percentage only
in E. huxleyi at 500 µmol photons m-2 s-1 and in G. oceanica at 200 µmol photons m-2 s-1 (Fig. 4).
Light experiments data. Growth rate (μ, in cells d-1);
coccosphere and cell diameters (in µm); coccolith morphometric analyses
were performed on 50 specimens for every treatment for the following: average of coccolith
distal shield length (DSL; in µm) and coccolith distal shield width
(DSW; in µm); ellipticity (DSL/DSW); average E. huxleyi distal shield elements number
(SE) and average distal shield elements width (in µm; SEW); average E. huxleyi inner
tube thickness (in µm; tube thick.); G. oceanica tube thickness (in µm; tube
thick.); G. oceanica bridge angle (angle ∘). For G. oceanica and P. carterae, data from 150 and
200 µmol photons m-2 s-1 are missing due to errors in light
intensity inside the light cabinet; SD: standard deviation. SDl and SDw refer to DSL and DSW standard deviation.
Box plots of coccolith length from the different experiments. In (a)E. huxleyi; (b)G. oceanica; (c)C. braarudii; (d)P. carterae. C: control treatment for every experiment. Light:
experiment with 12 different light intensities from 50 to 600 µm photons m-2 s-1; Ca: calcium manipulation experiment; 25,
[Ca2+]=25 mmol L-1; 50, [Ca2+]=50 mmol L-1. N: nutrient limitation experiment, N: nitrogen-limited condition; P: phosphate-limited condition. T: temperature experiment; H is for 22.5 ∘C; CC: carbonate chemistry experiment; theoretical CO2
values: C: 400 ppm; OA, ocean acidification: 1000 ppm; C1, Cretaceous
scenario1: 1000 ppm; C2 Cretaceous scenario2: 3000 ppm (for further
information see Sects. 2.1.5 and 3.5). The tops and bottoms of each
“box” are the 25th and 75th percentiles of the samples respectively. The
red line in the middle of each box is the median. The whiskers, extending
above and below each box, represent the furthest observations. Observations
beyond the whisker length are marked as outliers (red cross). For the light
experiment, 50 specimens were considered for every treatment. For Mg/Ca
experiment (Ca), nutrient experiment (N), temperature (T), and carbonate
chemistry manipulations (CC) experiments, every box plot represents 150
measurements in total (50 measurements for each replicate). The Light
experiment was performed in December 2013; the Ca experiment was performed
in June 2014; the N experiment was performed in December 2017; the T
experiment was performed in October 2017; the CC experiment was performed in
August 2014.
Percentage of normal, malformed, incomplete, and
incomplete/malformed coccoliths versus experiments. The experiments
displayed represent Mg/Ca, nutrient limitation, carbonate chemistry,
temperature, and light intensity manipulations. C. braarudii did not survive at high
temperature (22.5 ∘C), and no malformations were observed under
the different light intensities tested; therefore, percentage of
malformations are not represented for these experiments for this species.
Furthermore, no malformation was observed for P. carterae, and percentages are not shown.
C: control treatment. Mg/Ca: calcium manipulation experiment; 25,
[Ca2+]=25 mmol L-1; 50, [Ca2+]=50 mmol L-1.
Nutrient limited: nutrient limitation experiment; N: nitrogen-limited
condition; P: phosphate-limited condition. Temperature experiment; H is for 22.5 ∘C; carbonate chemistry experiment; theoretical CO2
values: C: 400 ppm; OA, ocean acidification: 1000 ppm; C1, Cretaceous
scenario1: 1000 ppm; C2 Cretaceous scenario2: 3000 ppm. Light: 12
different light intensities from 50 to 600 µm photons m-2 s-1. For every treatment and for every replicate 100 specimens were
considered.
Mg/Ca
Emiliania huxleyi coccosphere and cell sizes were influenced by changes in seawater
[Ca2+]. Elevating seawater [Ca2+] to ≈25
and 50 mmol L-1 resulted in a significant increase in the coccosphere
and cell diameters (p<0.05). Increased [Ca2+] concentrations
impacted G. oceanica, C. braarudii, and P. carterae cell sizes with a reduction in size in comparison to
[Ca2+] of 9.8 mmol L-1, when seawater [Ca2+] was
elevated to ≈25 and 50 mmol L-1. Gephyrocapsa oceanica and P. carterae
coccosphere diameters were unaffected, while the C. braarudii coccosphere was smaller when
grown under [Ca2+] of 50 mmol L-1 (Table 3). Emiliania huxleyi, G. oceanica, and C. braarudii coccolith sizes
were not affected by changing [Ca2+]. Pleurochrysis carterae coccoliths were smaller at the
highest [Ca2+] concentrations than in the control (Fig. 3; Table 3). Emiliania huxleyi produced
a higher percentage of malformed and/or incomplete coccoliths with
increasing calcium concentrations (Fig. 4; Plate S2), while no
increased malformation was observed in the other species.
Mg/Ca experiment data. Data presented are the average of three
replicates. Growth rate (μ; in cells d-1); coccosphere and cell
diameters (in µm); coccolith morphometric analyses were performed on 50
specimens for every treatment and for every replicate. Data represent the
average of three replicates: average of coccolith distal shield length (DSL; in µm) and coccolith distal shield width (DSW; in µm); ellipticity
(DSL/DSW) diameter; average E. huxleyi distal shield elements number (SE) and average
distal shield elements width (in µm; SEW); average E. huxleyi inner tube thickness
(in µm; tube thick.) and G. oceanica bridge angle (angle ∘); SD: standard deviation. SDl and SDw refer to DSL and DSW standard deviation.
Emiliania huxleyi and C. braarudii coccospheres were larger under P limitation than under N limitation and
the control. Gephyrocapsa oceanica coccospheres were larger under N limitation than under
P limitation and the control. Pleurochrysis carterae coccospheres were larger under N limitation
compared to the control. Cell size remained unaffected in E. huxleyi by nutrient
limitation. Gephyrocapsa oceanica cells and C. braarudii cells were larger under P limitation compared to the
control and N limitation. Pleurochrysis carterae cells were larger under N limitation compared to
the control (Table 4).
Nutrient-limited condition experiment data. Data presented are the
average of three replicates. Growth rate (μ; in cells d-1);
coccosphere and cell diameters (in µm); coccolith morphometric analyses
were performed on 50 specimens for every treatment and for every replicate.
Data represent the average of three replicates: average of coccolith distal
shield length (DSL; in µm) and coccolith distal shield width (DSW;
in µm); ellipticity (DSL/DSW) diameter; average E. huxleyi distal shield elements
number (SE) and average distal shield elements width (in µm; SEW);
average E. huxleyi inner tube thickness (in µm; tube thick.) and G. oceanica bridge angle
(angle ∘); SD: standard deviation. SDl and SDw refer to DSL and DSW standard deviation.
Emiliania huxleyi and G. oceanica coccoliths were larger under P limitation, while there was no
significant difference between N limitation and the control. Emiliania huxleyi coccoliths had
a higher number of distal shield elements under P limitations, while the
inner tube was thinner in N- and P-limited treatments compared to the
control. Gephyrocapsa oceanica produced thicker inner tubes under N and P limitation. Coccolithus braarudii was less
elliptical under P limitation, and P. carterae was less elliptical under N limitation.
Furthermore, E. huxleyi and G. oceanica produced relatively more malformed coccoliths under P limitation (Fig. 4). Coccolithus braarudii and P. carterae coccolith sizes remained unaffected with no sign of
malformations by nutrient limitation (Fig. 3; Plate S3).
Temperature
Emiliania huxleyi and G. oceanica coccospheres and cell sizes were smaller at 22.5 ∘C. Pleurochrysis carterae
coccosphere and cell sizes remained unaffected (Table 5). Emiliania huxleyi coccoliths were
smaller at high temperatures. Furthermore, E. huxleyi had less distal shield elements
and a thinner inner tube when grown at 22.5 ∘C. Gephyrocapsa oceanica and P. carterae coccolith size remained
largely unaffected by changing temperature, but G. oceanica produced thicker inner tubes
under high temperature. Pleurochrysis carterae coccoliths were less elliptical when grown at
22.5 ∘C (Fig. 3; Table 5; Plate S3). Coccolithus braarudii did not
survive at conditions of 22.5 ∘C.
Temperature experiment data. Data presented are the average of three
replicates. Growth rate (μ; in cells d-1); coccosphere and cell
diameters (in µm); coccolith morphometric analyses were performed on 50
specimens for every treatment and for every replicate. Data represent the
average of three replicates: average of coccolith distal shield length (DSL;
in µm) and coccolith distal shield width (DSW; in µm); ellipticity
(DSL/DSW) diameter; average E. huxleyi distal shield elements number (SE) and average
distal shield elements width (in µm; SEW); average E. huxleyi inner tube thickness
(in µm; tube thick.) and G. oceanica bridge angle (angle ∘). C. braarudii did not grow at
22.5 ∘C, and therefore, any data are presented; SD: standard
deviation. SDl and SDw refer to DSL and DSW standard deviation.
E. huxleyiμSDCoccosphereSDCellSDDSLSDlDSWSDwEllipticitySESEWTube thick.Control0.940.014.940.074.280.033.180.312.610.281.22320.120.48High1.470.013.700.103.180.032.820.342.330.291.21290.110.27G. oceanicaμSDCoccosphereSDCellSDDSLSDlDSWSDwEllipticityTube thick.Angle ∘Control0.630.078.010.146.290.095.470.544.660.481.181.4864.58High1.110.087.210.065.510.025.550.664.740.621.171.5967.65P. carteraeμSDCoccosphereSDCellSDDSLSDlDSWSDwEllipticityControl0.440.0411.380.258.940.642.040.141.270.101.60High0.310.0411.820.239.050.231.990.131.370.101.45Carbonate chemistry parameters
Emiliania huxleyi coccospheres and cells were the largest in the OA treatment and smallest in
the CS2 treatment. Gephyrocapsa oceanica and C. braarudii coccospheres were the largest in the control and
smallest in CS2 treatment. Gephyrocapsa oceanica cell size was lower in the CS2 treatment than in
the control, as well as the OA and CS1 treatments. The cell size of C. braarudii was
smaller in the OA, CS1, and CS2 treatments compared to the control.
Pleurochrysis carterae coccosphere and cell size were unaffected by changing carbonate chemistry (Table 6).
Carbonate chemistry experiment data. Data presented are the average
of three replicates. Growth rate (μ; in cells d-1); coccosphere and
cell diameters (in µm); coccolith morphometric analyses were performed
on 50 specimens for every treatment and for every replicate. The following data represent
the average of three replicates: average of coccolith distal shield length
(DSL; in µm) and coccolith distal shield width (DSW; in µm);
ellipticity (DSL/DSW) diameter; average E. huxleyi distal shield elements number (SE) and
average distal shield elements width (in µm; SEW); average E. huxleyi inner tube
thickness (in µm; tube thick.) and G. oceanica bridge angle (angle ∘).
Carbon chemistry speciation calculated as the mean of start and end values
of measured pH and TA are given. SD: standard
deviation. SDl and SDw refer to DSL and DSW standard deviation.
Emiliania huxleyi formed significantly bigger coccoliths in the OA treatment compared to the
control and the CS2 treatment (Fig. 3; Table 6). Furthermore, the inner
tubes were thicker in the OA and CS1 treatments compared to the control and
the CS2 treatments. Malformations were 20 % more frequent in the OA, CS1,
and CS2 treatments than in the control (Fig. 4; Plate S4).
Gephyrocapsa oceanica generated a high number of malformed coccoliths in the OA and CS2
treatments. For G. oceanica, under OA and CS2 conditions, morphometric analyses were
not performed because a large majority of the coccoliths were extremely
malformed, and it was not possible to measure the shape of the specimens (Fig. 2). In the CS1 treatment, coccoliths were slightly smaller compared to the
control with a thinner inner tube. Coccolithus braarudii coccoliths were smaller in the OA and CS2
treatments compared to the control and the CS1. In the OA and CS1
treatments, 40 % of the C. braarudii coccoliths were malformed, and ∼10 % were incomplete. In the CS2 treatment, 97% of coccoliths were
malformed or incomplete. Pleurochrysis carterae coccolith size remained unaffected by carbonate
chemistry variations, but coccoliths are less elliptical under OA, CS1, and
CS2 compared to the control.
Discussion
Coccolithophores started to calcify in the late Triassic, and this biological
innovation appeared in a period of strong climatic and biotic pressure (De
Vargas et al., 2007). The earliest coccoliths had very simple morphologies
and small sizes (2–3 µm; Bown et al., 2004). Calcareous nannoplankton
underwent a major diversification in the Mesozoic and Paleocene where many
new morphologies occurred. The appearance of new coccolith shapes followed
the main geological events, at the Cretaceous/Paleogene (K/Pg) boundary, and the Paleocene/Eocene (P/E) boundary, and
these big reorganizations suggest that certain kinds of morphologies might
have been no longer advantageous for coccolithophore algae under the new
ecological circumstances. The evolution of calcareous nannoplankton through
∼220 Ma documents a remarkable morphological diversity within
the group, and in the last 30 Ma there has been a loss of species that
produced large and heavily calcified coccoliths but an increase in the
modern community of coccolith architectures (Bown et al., 2004). The cause
of this impressive number of structures is unknown, but there might be a
reason connected to the function of coccoliths for the different species to
produce such different shapes ranging from protection against excess sun
light and/or against grazing (Monteiro et al., 2016). Accordingly, coccolith
morphologies are likely only indirectly linked to physical or chemical
conditions such as temperature or CO2 but may rather reflect their
adaptation to a specific, yet-unknown ecological function (Aloisi, 2015). If
morphological changes in coccoliths are the result of a physiological
response to environmental variations (e.g., CO2, nutrient, temperature),
coccoliths recovered from marine sediments could potentially conserve
paleo-environmental information prevailing when the coccolithophore was
alive (Aloisi, 2015). Indeed, many studies of geological records calibrated
biomineralization responses of ancient species to environmental drivers with
experiments with modern species (e.g., Bornemann et al., 2006; Erba et al.,
2010; Suchéras-Marx et al., 2010; Linnert and Mutterlose, 2012; O'Dea et
al., 2014; Lübke et al., 2015; Gibbs et al., 2016; Faucher et al.,
2017a, b; Table 1). Calibrating paleo-responses with
observations from living species depends on the assumption that
coccolithophores conserved a certain response to certain environmental
parameters over geological timescales. However, fossils and living
coccolithophores diverged a long time ago and have a different genetic
background, and therefore, calcareous nannoplankton in the past and nowadays
did and do not necessarily act in the same way to external stress.
Furthermore, morphology may not only depend on abiotic environmental
conditions but could perhaps also be the result of evolutionary development
induced through ecological interactions. For example, if a coccolithophore
genotype which forms larger coccoliths is better suited to protect a cell
against prevalent grazers, then these genotypes will likely proliferate,
whereas related genotypes forming smaller variants could eventually go
extinct. The geological record would not easily allow us to distinguish if
morphological changes are caused by physiological or ecological drivers as
it is difficult enough to reconstruct abiotic paleo-environmental conditions
but almost impossible to unravel relevant processes in the food web of the
geologic past. Therefore, the fundamental question we asked ourselves was
whether morphological features observed in living coccolithophores under
specific environmental parameters could help to build reliable proxies for
abiotic paleo-environmental conditions.
Overall, none of the five tested variables induced a consistent response of
coccolith size and shape across all four species. For example, under high
CO2, E. huxleyi formed larger coccoliths, while C. braarudii formed smaller coccoliths.
Interestingly, our observation of inconsistent responses among species to
various environmental drivers is in line with observations from the fossil
record. There are several observations where just some of the prevailing
species showed changes in morphology during intervals characterized by
extreme climatic conditions even though all species were exposed to
environmental stress. For example, dwarf specimens were recorded for Biscutum constans in all Mesozoic episodes characterized by abnormal conditions, during
intervals of extreme volcanic activity (e.g., during Oceanic Anoxic Events 1a, 1b, and 2; Bornemann et al., 2006; Erba et al., 2010, 2019;
Lübcke et al., 2015; Faucher et al., 2017a). In
conclusion, the inconsistency of morphological responses to changing
environmental drivers observed in both our experiments and the geological
record suggests that morphological responses of living species cannot be
used as analogues for morphological changes in extinct species.
The exception in our dataset is the observed responses in malformation to
changes in carbonate chemistry where some consistency was noted among the
four tested species. Malformations are generally considered as an evidence
of errors during intracellular coccolith formation so that a disturbance of
coccolithogenisis conserved in a malformation could be the consequence of a
direct (i.e., physiological) impact. Indeed, malformations are unlikely to be
the consequence of an evolutionary (i.e., ecological) adaptation to
environmental stress because there seems to be no obvious ecological
advantage of producing malformed coccoliths. The high degree of malformation
when coccolithophores were grown under high CO2 concentrations provides
some evidence that at least this response variable could be used as
paleo-proxy for episodes of acute carbonate chemistry perturbations.
In the fossil record, there are several examples of intervals characterized
by high abundances of malformed specimens, linked to the low calcite
saturation state of the ocean (Jiang and Wise, 2006; Raffi and De Bernardi,
2008; Agnini et al., 2007; Erba et al., 2010; Bralower and Self Trail,
2016). Different authors argued for high CO2 influence on causing these
malformations during the Mesozoic OAEs, Paleocene-Eocene Thermal Maximum
(PETM) and Eocene Thermal Maximum 2. All these intervals were characterized
by excess CO2 concentrations and/or slightly reduced pH. Malformations
were expressed in different ways: they were represented by variation in
ellipticity of coccoliths (Erba et al., 2010), asymmetry (Agnini et al.,
2007), irregular arrangement and length of their rays, and diminished
calcification in some nannoliths (Jiang and Wise, 2006; Mutterlose et al.,
2007; Raffi and De Bernardi, 2008; Bralower and Self Trail, 2016). The short
stratigraphic ranges where these malformations occurred, during the core of
major ocean perturbations, indicated that pH played a role in inducing the
production of these aberrant specimens (Mutterlose et al., 2007; Erba et
al., 2010). There is still not a clear explanation of why only some species
of calcareous nannoplankton were producing aberrant specimens, and there is
not a general consensus on the role of carbonate chemistry in
coccolithophore biomineralization (Gibbs et al., 2010, 2016).
However, a recent work provides a plausible explanation of what might have
happened during the PETM. Here, only some species moved and inhabited the
deep part of the photic zone, to possibly refuge from stressful warm and
eutrophic conditions of the surface water, but had to deal with lower
saturation conditions that induced malformations in these taxa (Bralower and
Self Trail, 2016).
The increase in the percentage of malformed coccoliths observed in our
experiments could suggest a more global occurrence of malformation in modern
coccolithophore species under low pH. However, it is important to bear in
mind that in the geological record critical intervals characterized by
excess CO2 concentrations lasted for some tens or hundreds of thousands of
years, whereas our experiments lasted a few generations (days). Thus,
environmental stress on geological timescales may still be long enough for
coccolithophores to adapt, which can occur within months to years (Lohbeck et
al., 2012; Bach et al., 2018). It also needs to be kept in mind that even if
the four coccolithophore species tested here showed similar morphological
responses to changing environmental drivers, it cannot be excluded that this
resulted from convergent evolution. Indeed, restriction on biological
conditions and adaptation to particular habitats can produce widespread
convergence as convergent evolution is often a consequence of adaptation to
a similar niche (Arbuckle et al., 2014). Therefore, we want to point out
that convergent morphological developments could represent similar
adaptations of different species to abiotic parameters that occurred
multiple times separate from each other.
Conclusions
According to the data provided in this study we report the following
results: (1) sizes and morphologies of the four tested species change
differently in response to temperature, light, nutrient, and Mg/Ca
variations. In some cases, there were opposing reactions among species under
the same abiotic stress; (2) a high number of malformations were detected
when coccolithophores were grown under excess CO2, and this response
occurred in all species tested here.
Overall, there is no support for the suitability of coccolith morphometry to
serve as proxy for temperature, light, nutrient, and Mg/Ca conditions of the
past. However, coccolith malformations could perhaps be useful indicators
for carbonate chemistry stress. Indeed, it will be crucial to evaluate
whether malformations remain over a long time period or if coccolithophores
have and had an adaptive potential towards extreme carbonate chemistry
conditions that might rapidly eliminate malformation in some generations.
Data availability
Data presented in the paper are available in the Supplement. Further information can be accessed by contacting the corresponding author (giulia.faucher@unimi.it).
The supplement related to this article is available online at: https://doi.org/10.5194/cp-16-1007-2020-supplement.
Author contributions
GF, LTB, and UR developed the paper concept. GF and LTB conceived and designed the experiments. GF performed the experiments and analyzed the data. All authors contributed to the writing and discussion of the paper.
Competing interests
The authors declare that they have no conflict of interest.
Acknowledgements
We are indebted to the editor, Erin McClymont, and Mariem Saavedra-Pellitero
and an anonymous reviewer for their fruitful comments that greatly improved
the quality of this paper. A special note of thank goes to Elisabetta
Erba for sharing ideas and huge support. We acknowledge Agostino Rizzi for
assistance during the never-ending SEM analyses.
Financial support
This research was funded
through MIUR-PRIN 2011 (Ministero dell'Istruzione, dell'Università e
della Ricerca–Progetti di Ricerca di Interesse Nazionale) for Elisabetta
Erba (grant no. PRIN 2017RX9XXXY) and through SIR-2014 (Ministero dell'Istruzione, dell'Università e
della Ricerca–Scientific Independence of young researchers) for Cinzia
Bottini (grant no. SIR-2014 RBSI14UU81).
Review statement
This paper was edited by Erin McClymont and reviewed by Mariem Saavedra-Pellitero and one anonymous referee.
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