Paper I
Šupraha L., Gerecht A. C., Probert I., Henderiks J. (2015) Eco-physiological adaptation shapes the response of calcifying algae to nutrient limitation.
Manuscript submitted to Scientific Reports
The steady increase in global ocean temperature will most likely lead to nutrient limitation in the photic zone. This will impact the physiology of marine algae, including the globally important calcifying coccolithophores.
Understanding their adaptive patterns is essential for modelling carbon production in a low-nutrient ocean. We investigated the physiology of
Helicosphaera carteri , a representative of the abundant but under-investigated flagellated functional group of coccolithophores. Two strains isolated from contrasting nutrient regimes (South Atlantic and Mediterranean Sea) were grown in phosphorus-replete and phosphorus-limited batch cultures. The
Mediterranean strain exhibited 28% lower growth rate, 30% larger coccosphere size and 24% lower particulate inorganic carbon (PIC) production than the Atlantic strain. Under phosphorus limitation, the same strain was capable of reaching a 2.6 times higher cell density due to 2.5-fold lower phosphorus requirements. These results suggest that local physiological adaptation can define the performance of this species under nutrient limitation.

Paper II
Gerecht, A. C., Šupraha, L., Edvardsen, B., Probert, I., & Henderiks, J.
(2014). High temperature decreases the PIC/POC ratio and increases phosphorus requirements in Coccolithus pelagicus (Haptophyta).
Biogeosciences, 11(13): 3531-3545.
Rising ocean temperatures will likely increase stratification of the water column and reduce nutrient input into the photic zone. This will increase the likelihood of nutrient limitation in marine microalgae, leading to changes in the abundance and composition of phytoplankton communities, which in turn will affect global biogeochemical cycles. Calcifying algae, such as coccolithophores, influence the carbon cycle by fixing CO
2
into particulate organic carbon (POC) through photosynthesis and into particulate inorganic carbon (PIC) through calcification. As calcification produces a net release of
CO
2
, the ratio of PIC /POC determines whether coccolithophores act as a source (PIC/POC > 1) or a sink (PIC/POC < 1) of atmospheric CO
2
. We studied the effect of phosphorus (P-) limitation and temperature stress on the physiology and PIC/POC ratios of two subspecies of Coccolithus pelagicus .
This large and heavily calcified species (PIC/POC generally > 1.5) is a major contributor to calcite export from the photic zone into deep-sea reservoirs.
Phosphorus limitation did not influence exponential growth rates in either subspecies, but P-limited cells had significantly lower cellular P-content. A
5 ° C temperature increase did not affect exponential growth rates either, but nearly doubled cellular P-content under both high and low phosphate availability. The PIC/POC ratios did not differ between P-limited and nutrientreplete cultures, but at elevated temperature (from 10 to 15 ° C) PIC /POC ratios decreased by 40–60%. Our results suggest that elevated temperature may intensify P-limitation due to a higher P-requirement to maintain growth and
POC production rates, possibly reducing abundances in a warmer ocean.
Under such a scenario C. pelagicus may decrease its calcification rate relative to photosynthesis, resulting in PIC/POC ratios < 1 and favouring CO
2
sequestration over release. Phosphorus limitation by itself is unlikely to cause changes in the PIC/POC ratio in this species.

Paper III
Šupraha, L., Ljubeši ć , Z., Mihanovi ć , H., Henderiks, J. (2014). Observations on the life cycle and ecology of Acanthoica quattrospina Lohman from a
Mediterranean estuary. Journal of Nannoplankton Research, 34: 49-56
Different life cycle phases of Acanthoica quattrospina life cycle were studied under the scanning electron microscope. Samples were collected during winter
2013 at two stations in the lower reach of the Krka River estuary (Eastern
Adriatic Sea, Croatia). An ecological analysis revealed that the species was tolerant to low salinity and reached high abundance at the halocline of the estuary. Examination of holococcoliths showed great variability in the morphology of the central area, indicating that several specimens previously described as Sphaerocalyptra sp. represent a variety of the Acanthoica quattrospina holococcolith phase. Furthermore, the observed variability of holococcoliths may represent a distinct mechanism of holococcolith production, with structural similarities to other members of the
Sphaerocalyptra genus.

This thesis is based on the following papers:
I Šupraha L.
, Gerecht A. C., Probert I., Henderiks J. (2015) Ecophysiological adaptation shapes the response of calcifying algae to nutrient limitation . Manuscript submitted to Scientific Reports
II Gerecht, A. C., Šupraha, L.
, Edvardsen, B., Probert, I., &
Henderiks, J. (2014). High temperature decreases the PIC/POC ratio and increases phosphorus requirements in Coccolithus pelagicus (Haptophyta). Biogeosciences , 11(13): 1021-1051.
III Šupraha, L.
, Ljubeši ć , Z., Mihanovi ć , H., Henderiks, J.
(2014).
Observations on the life cycle and ecology of Acanthoica quattrospina Lohman from a Mediterranean estuary.
Journal of
Nannoplankton Research , 34: 49-56

Introduction ..................................................................................................... 9
Coccolithophores ........................................................................................ 9
Physiological response of coccolithophores to phosphorus limitation ..... 12
Background .......................................................................................... 12
Previous investigations ........................................................................ 12
This thesis ............................................................................................ 15
Coccolithophore life-cycle ....................................................................... 16
Previous investigations ........................................................................ 16
This thesis ............................................................................................ 17
Future objectives ........................................................................................... 19
References ..................................................................................................... 21

Marine phytoplankton encompasses phylogenetically and functionally diverse algae and cyanobacteria (Simon et al., 2009) that account for nearly half of the global primary production (Field et al., 1998). The bulk of the phytoplankton activity takes place in the photic zone, where carbon and inorganic nutrients are assimilated into organic or inorganic compounds and subsequently recycled within the pelagic food web (Falkowski et al., 2003).
In addition, a significant portion of assimilated organic and inorganic matter is exported to deeper layers and remineralized or deposited in marine sediments (Ducklow et al., 2001). On a global scale, phytoplankton production is strongly regulated by seasonal patterns in solar insolation and nutrient availability (Alvain et al., 2008). Consequently, changes in global climate can have a major impact on production rates, community structure and biogeography of marine phytoplankton (Gregg et al., 2003, Morán et al., 2010,
Winter et al., 2014).
Figure 1.
Coccolithophore species discussed in this thesis. A) Emiliania huxleyi ; B)
Helicosphaera carteri ; C) Coccolithus pelagicus ssp. braarudi ; D) Acanthoica quattrospina. Scale bar=5µm
The role of different phytoplankton groups in the biogeochemical carbon cycle is defined by their life histories. Most of the major groups act mainly as a sink of CO
2
owing to their photosynthetic activity and siliceous (diatoms) or organic (dinoflagellates and cyanobacteria) cell walls (Ragueneau et al.,
2006). Their primary production is commonly referred to as the “biological carbon pump”, and is considered the key regulator of global CO
2
levels
(Longhurst and Harrison, 1989). Apart from diatoms and dinoflagellates, one
9

of the most important functional groups in the biogeochemical carbon cycle is the calcifying phytoplankton, which is dominated by the coccolithophores
(Fig 1).
Molecular systematics place coccolithophores within the division
Haptophyta, a diverse group of nanoplanktonic flagellates with which they share common ultrastructural features (Edvardsen et al., 2000). The modern diversity of ~280 coccolithophore morphospecies (Young et al., 2005) exhibits a remarkable morphological, physiological and ecological variability
(Fig. 1). They show a cosmopolitan distribution, inhabiting almost exclusively marine habitats, and all layers of the photic zone, generally preferring mesotrophic and oligotrophic environments with lower turbulence (Margalef,
1978). Under favourable environmental conditions, coccolithophores are
Figure 2.
Schematic illustration of the role of coccolithophores in the biogeochemical carbon cycle. capable of forming large oceanic blooms which are characterized by complex biogeochemical interactions (Holligan et al., 1993, Robertson et al., 1994).
Unlike most of the other phytoplankton groups, they contribute both to the biological carbon pump through photosynthesis and to the release of CO
2 through calcification (Iglesias-Rodríguez et al., 2002). Moreover, their calcite
10

exoskeletons (coccospheres) and their building blocks (coccoliths) are significant means of sinking export of both organic and inorganic carbon to oceanic sediments (Ziveri et al. 2007, Fig. 2).
The fossil record indicates that calcification, which is the unique feature of coccolithophores, originated in the Late Triassic, ~220Ma ago (Bralower et al., 1991), possibly several times within the calcifying haptophyte lineage (de
Vargas and Probert, 2004). This is mostly in agreement with the molecular data which estimates the origin of the calcifying haptophyte lineage to the
Middle Triassic, ~250Ma ago (Sáez et al., 2004). The group thrived during the
Mesozoic, reaching a peak in morphospecies diversity and dominating the marine phytoplankton (Bown et al., 2004, Falkowski et al., 2004, Bown,
2005). The occurrence of coccolithophores in marine plankton during the middle Mesozoic enhanced the sinking export of carbon from the photic zone and had a crucial role both in the chemical evolution of modern oceans and the regulation of global CO
2
levels (Ridgwell and Zeebe, 2005). Over geological time, coccolithophores contributed to the formation of kilometresthick layers of calcareous oozes, oceanic sediments containing deposited coccoliths and coccospheres (Blackman, 1902, Baumann et al., 2004, Ziveri et al., 2007). The extensive fossil record obtained from these oceanic sediments provides detailed insights into the macroevolution of the group
(Bown, 2005), which was likely driven by changes in climate forcing (Finkel et al., 2007, Hannisdal et al., 2012). Major extinctions at the K/T boundary
(65Ma) and climate shifts during the Cenozoic (65Ma-present) led to the loss of dominance and extinction of larger coccolithophore taxa and an increase in abundance of the smaller forms (Herrmann and Thierstein, 2012). This general size decrease during the Cenozoic had a profound effect on global carbon production and burial (Suchéras-Marx and Henderiks, 2014).
Although the macroevolutionary patterns in morphology and species diversity of the coccolithophores are well documented, the underlying biological mechanisms remain to be fully understood. Climate forcing is a complex process, and each of the environmental drivers has a specific effect on coccolithophore physiology, ecology and biogeography (Gregg and Casey,
2007, Zondervan, 2007). Understanding the adaptive strategies and response patterns of modern coccolithophores to specific environmental changes is therefore important both for interpreting the fossil record and modeling the group’s dynamics under ongoing climate change. This thesis presents the research on coccolithophore responses to environmental changes which was addressed through:
1) Experiments investigating the response of coccolithophores to phosphorus limitation (Paper I, Paper II)
2) Field investigation of coccolithophore life cycles across gradients of potential environmental drivers (Paper III)
11

Inorganic nutrients, notably nitrogen and phosphorus, are one of the main regulators of phytoplankton production in modern oceans (Redfield, 1934,
Platt et al., 1992, Klausmeier et al., 2004). Phosphorus in particular plays a key role in biological systems, where it acts as a structural molecule in nucleic acids, biological membranes and cellular energy carrier ATP (Westheimer,
1987). Due to the high affinity of marine organisms for phosphorus uptake and generally low concentration in surface waters, low phosphorus availability is commonly limiting phytoplankton production in marine environments, notably oceanic gyres and the Mediterranean Sea (Wu et al.,
2000, Thingstad et al., 2005, Elser et al., 2007). Main phosphorus supply mechanisms such as river inflow, atmospheric transport and upwelling are highly constrained by the climate, and are likely to be affected by the ongoing climate change (Delaney, 1998). Most importantly, models predict that the warming of the modern oceans will enhance the stratification of the water column, reducing the upwelling transport of phosphorus to the photic zone
(Sarmiento et al., 2004, Roemmich et al., 2015) and expanding the oceanic areas affected by phosphorus limitation. Coccolithophores are commonly good competitors under reduced nutrient availability, and some species, like
Emiliania huxleyi are even capable of forming blooms in low-phosphorus conditions (Egge and Heimdal, 1994). However, since the climate-driven changes in nutrient supply are considered a major driver of the long-term evolution of phytoplankton (Finkel et al., 2007), climate change will probably have a profound effect on modern coccolithophores. The effects of reduced phosphorus availability affect coccolithophores on many levels, from the species-specific physiological changes (Zondervan, 2007) to the changes in community composition and biogeography. Ultimately, the above-mentioned changes will define the global biogeochemical impact of the group.
Understanding the physiological effects of phosphorus limitation requires a specific methodological approach in which the effects of changes in an individual environmental driver can be observed. While field investigations provide a good insight into coccolithophore dynamics in natural ecosystems, they often do not allow for decoupling of effects of different drivers.
Controlled laboratory experiments have proved to be suitable for this purpose as they allow for controlled modifications of the growth environment and collection of large amount of chemical, physiological and morphological data.
12

Laboratory experiments have been extensively used during the past 20 years to address the physiological response of coccolithophores to phosphorus limitation (Paasche and Brubak, 1994, Paasche, 1998, Riegman et al., 2000,
Borchard et al., 2011, Langer et al., 2012, Langer et al., 2013, Oviedo et al.,
2014). Main focus was put on the immediate effect of phosphorus limitation on basic cellular parameters such as growth rate, cell and coccosphere size, cellular elemental quotas and rates of photosynthesis (particular organic carbon production, POC), calcification (particular inorganic carbon production, PIC) and nutrient acquisition. In addition, a number of experiments aimed at detecting the effects of phosphorus limitation on the coccolith production mechanism, which can result in the production of malformed or incomplete coccoliths.
Figure 3.
An example of the batch culture experiment with nutrient limitation.
Inoculated cells grow exponentially until consuming the nutrients. Nutrient limited cells are sampled once the culture enters the stationary (zero-growth) phase. Control samples are sampled during the non-limited exponential growth.
Three main methodological approaches are used in phosphorus limitation experiments: The batch culture method is the simplest approach, in which cells are inoculated in a defined volume of growth medium with reduced phosphorus concentrations (Fig. 3). Cells grow exponentially until the phosphorus is consumed, and then enter the stationary (zero-growth) phase, when they are harvested and analyzed (Langer et al., 2012, Oviedo et al.,
2014). This approach is suitable for detecting short term, immediate effects of phosphorus limitation on elemental quotas, cell size, growth rate and coccolith morphology. However, the batch culture method does not allow for the accurate measurement of production rates in phosphorus limited cells as the growth rates change over the course of the experiment (Langer et al., 2013).
In a semi-continuous setup, cells are initially inoculated in a defined volume
13

of a growth medium. During the exponential growth phase, part of the culture is transferred to a new container and diluted to the initial inoculation volume
(Paasche, 1998). The aim of dilutions is to prevent the onset of stationary phase and keep cells in the exponential growth phase. The process is repeated in regular intervals until the desired number of cell generations is reached. The semi-continuous approach is suitable for calculating production rates of exponentially growing cells under low-nutrient availability. The chemostat method represents similar approach to the semi-continuous method. However, initially inoculated cultures are not transferred to a new container. Instead, exponentially growing cells are continuously diluted by equal rates of fresh medium inflow and culture outflow, thus keeping cells in an exponential growth phase (Borchard et al., 2011). The dilution rate is set to correspond to the growth rate of the culture, thus keeping the cell number constant throughout the experiment, which is not the case in semi-continuous method.
Early investigations focused on the most abundant coccolithophore species,
Emiliania huxleyi . Pioneer studies by Paasche and Brubak (1994) and Paasche
(1998) revealed the strong effects of phosphorus limitation on coccolith production. Cells grown in phosphorus limited batch, semi-continuous and chemostat cultures exhibited an increase in number of coccoliths produced per cell, and an increase in number of coccolith layers. Phosphorus limitation also triggered the calcification in otherwise non-calcifying strains of E. huxleyi
(termed “N-cells”). Their studies also detected a clear decrease in growth rate of the phosphorus limited cells and an increase in cell size. Reduction in phosphorus production rate and cellular phosphorus quota was recognized as a clear indication of phosphorus limitation. Calcification was shown to be metabolically independent of cell division and photosynthesis, taking place in the dark or during the stationary phase. A study by Riegman et al. (2000) aimed at understanding the exceptional performance of E. huxleyi in lowphosphorus environments. They experimentally confirmed the physiological findings of previous experiments, and demonstrated that the uptake system of
E. huxleyi has the highest affinity for phosphate ever detected for a phytoplankton species. The complexity of coccolithophore responses to changing environment was revealed by experiments simulating combined effects of phosphorus limitation, increased temperature and higher p CO
2
(Borchard et al., 2011). Here, the decrease in carbon production, detected in phosphorus limited cells under high temperature and high pCO
2
was related to a decrease in POC production. On the other hand, an increase in POC production under combined effects of p CO
2
, irradiance and phosphorus limitation was observed by Leonardos and Geider (2005) in a non-calcifying strain of E. huxleyi . The effects of exclusively phosphorus limitation on carbon production therefore remain unclear. An increase in both PIC and POC production was observed in another species, Calcidiscus leptoporus by Langer et al. (2012). However, the authors argued that the observed increase was due to the methodological error arising from variable growth rates in batch
14

cultures. Their argument was supported further by comparison of different experimental methods used on the same strain of E. huxleyi (Langer et al.,
2013), suggesting that carbon production does not increase in coccolithophores under phosphorus limitation. Subsequent study by Oviedo et al. (2014) demonstrated that there is no uniform physiological response to phosphorus limitation in terms of carbon production and carbon release/uptake balance (PIC:POC production) within E. huxleyi . Instead, strain-specific response patterns clearly need to be considered.
The effects of phosphorus limitation on the coccolith production mechanism were investigated both in E. huxleyi (Paasche, 1998, Oviedo et al.,
2014) and C. leptoporus (Langer et al., 2012). No significant increase in the occurrence of malformations was detected, although phosphorus limitation increased the percentage of over-calcified coccoliths in some strains of E. huxleyi (Oviedo et al., 2014). Again, the over-calcification response appeared highly strain specific and not positively correlated to PIC production rates.
Work of Oviedo et al. (2014) raised the question of the origin of strain-specific responses to phosphorus limitation. The response patterns of the strains of E. huxleyi used in their study did not show any grouping on the basis of their isolation sites and local phosphorus concentrations.
The existence of species-specific and strain-specific response patterns, highlighted the need for introduction of additional ecologically and biogeochemically relevant species in the experimental database. It also indicated that the origin of strain-specific response patterns should be investigated to better understand the regional effects of climate change on coccolithophore physiology. The aim of the studies presented in Paper I and
Paper II of this thesis was to test the response to phosphorus limitation in two previously uninvestigated species, Helicosphaera carteri and Coccolithus pelagicus , using the batch culture method (Fig. 3). Both species are common and globally distributed coccolithophores, relevant for carbon production and sinking export from the photic zone (Ziveri et al., 2007, Daniels et al., 2014a).
The study on H. carteri addressed the role of local physiological adaptation in defining the physiological response to phosphorus limitation (Paper I). Two strains, isolated from two contrasting nutrient regimes (nutrient rich South
Atlantic and oligotrophic Western Mediterranean) exhibited significantly different physiology under non-limiting conditions. Strain-specific physiological parameters indicated that the adaptation to lower phosphorus concentrations lead to larger size, slower growth rate and lower calcification rates in the Mediterranean strain. Most importantly, growth under phosphorus limitation revealed that he Mediterranean strain had much lower phosphorus requirements, which allowed it to perform better i.e. reach higher cell concentrations than the Atlantic strain under the same phosphorus
15

concentration. This experiment clearly demonstrated that in H. carteri , strainspecific response patterns originate from the adaptation to local phosphorus availability.
The experiments conducted on two subspecies of Coccolithus pelagicus showed that phosphorus limitation itself does not affect the PIC/POC ratio in this species (Paper II). However, a 5°C increase in temperature had a strong negative effect on the PIC/POC ratio in ssp. pelagicus . This decrease in the
PIC/POC ratio was attributed to a decrease in PIC production, rather than an increase in POC production. Temperature stress also increased the phosphorus requirements of the species, further enhancing the nutrient stress and reducing the species performance in terms of cell abundance reached under Plimitation. Additionally, this study showed for the first time that phosphorus limitation can increase the frequency of malformations in coccolithophores, a phenomenon that was not observed in previous investigations on E. huxleyi
(Oviedo et al., 2014) and C. leptoporus (Langer et al., 2012).
The coccolithophore life-cycle commonly involves the alternation between a heterococcolith-producing diploid phase and a holococcolith-producing haploid phase (Young et al., 2005). Key morphological distinction between the phases results from the phase-specific coccolith production mechanism. In diploid cells, heterococcoliths are produced intracellulary within a Golgiderived vesicle (Paasche, 1962, Klaveness and Paasche, 1979). Two types of alternating crystalline units grow continuously from nuclei mineralized on the organic template (Young et al., 1999). Once complete, coccoliths are exported on the cell membrane by exocytosis (Taylor et al., 2007). On the other hand, holococcoliths are built from the tiny crystallites, extracellularly mineralized on the organic template placed between the cell membrane and a secondary membrane termed “skin” by Klaveness (1973).
The transition between the two phases has been investigated extensively in cultured material, where the transition between the phases occurred spontaneously (Gayral and Fresnel, 1983, Medlin et al., 1996, Houdan et al.,
2004, Parke and Adams, 2009) or under controlled experimental setup (Nöel et al., 2004). In field samples, transition between the phases is observed in the form of combination coccospheres, containing both heterococcoliths and holococcoliths of the same species (Kamptner, 1941, Kleijne, 1991, Cros et al., 2000, Cros and Fortuno, 2002, Geisen et al., 2002, Malinverno et al., 2008,
Frada et al., 2009, Daniels et al., 2014b). Despite well-documented phase transitions in culture and a number of observations in field material, the clear environmental triggers for the phase transition are still unclear.
16

Available field and experimental data suggest that the two life phases represent an adaptation to nutrient forcing. Houdan et al. (2006) showed experimentally that the haploid holococcolith phases of Coccolithus ssp. braarudi and Calcidiscus leptoporus have higher growth rates in nutrient depleted media, as opposed to their diploid heterococcolith phases which prefer nutrient enriched media. The same nutritional preferences of the two life phases were confirmed by investigations of coccolithophore communities in the Mediterranean Sea (Triantaphyllou et al., 2002, Dimiza et al., 2008,
Malinverno et al., 2009, Cros and Estrada, 2013) and North Atlantic (Renaud and Klaas, 2001, Silva et al., 2013), where the holococcolith phase commonly dominated the stratified oligotrophic environments during summer, and the heterococcolith phase abounded in more eutrophic waters. Different nutritional preferences are likely related to cell size, as the haploid phase needs to support half the genetic material and often smaller cell volume compared to diploid phase (Lewis, 1985, Houdan et al., 2004). Furthermore, strong UV backscattering of the holococcolith crystalline structure, revealed by
Quintero-Torres et al. (2006), was interpreted as an adaptation to high irradiance in oligotrophic surface waters. Most coccolithophore species are flagellated in haploid phase and possess haptonema, a haptophyte-specific organelle used for ingesting particulate organic matter through phagocytosis, as confirmed for C. ssp. braarudi (Houdan et al., 2006). The ability to feed on marine bacteria and organic matter could give a competitive advantage to holococcolithophores in low nutrient conditions.
Since combination coccospheres are rarely found in field samples, it is difficult to attribute their occurrence to specific environmental drivers.
Furthermore, the scarcity of observations often leads to incomplete descriptions of coccolithophore life-cycles or unverified descriptions of lifecycle associations which have to be confirmed by future studies (e.g. figures
112-114 in Cros and Fortuno (2002)). Paper III of this thesis presents part of the exceptional field material collected during winter 2013 in the Eastern
Adriatic Coast (Mediterranean Sea). Samples collected along the estuarydriven environmental gradients were rich in combination coccospheres and both life-cycle phases of several coccolithophore species. This allowed for a detailed description of the complete life cycle of Acanthoica quattrospina
Lohmann, a common coccolithophore species first described in its heterococcolith phase (Lohmann, 1903). Life-cycle association with the holococcolithophore Sphaerocalyptra sp., first suggested by Cros et al. (2000) and Cros and Fortuno (2002) was confirmed, joining the two species in a common life cycle (Fig. 4).
We interpret that the species exhibits a haplo-diploid life cycle, typical of coccolithophores (Houdan et al., 2004). The combination coccospheres are
17

formed after the meiotic division of the diploid cell (Fig. 4). Holococcolithbearing haploid cells are then able to divide mitotically within the haploid phase, and the transition to the heterococcolith phase occurs after the syngamy of two haploid cells. The morphological analysis of combination coccospheres and a holococcolith phase revealed a distinct pattern of holococcolith formation, which could be tracked on the incomplete holococcoliths being formed on the cell surface. Furthermore, distribution of life phases and the combination coccospheres along the estuary revealed the ecological preferences of life phases and possible triggers of the phase transition.
Figure 4.
Life cycle of Acanthoica quattrospina , as interpreted from our results
(Paper III). The species exhibits a haplo-diploid life cycle, with mitotic divisions taking place within both life phases. Scale bar=5µm
18

Work presented in this thesis provides a background for future investigations on coccolithophore adaptation to climate change, which will focus on experimental, fossil and field data. The future work on the experimental data will address the effects of phosphorus limitation on coccolith morphology in
Helicosphaera carteri . Preliminary data analysis suggests that the number of malformations increases in this species under phosphorus limitation.
Observations also show that high phenotypic plasticity within the coccolith set is strongly genetically constrained and unaffected by the perturbations in phosphorus availability. Furthermore, the effects of phosphorus limitation will be investigated in Emiliania huxleyi , along with comparison of two distinct experimental setups (the batch and semi-continuous method). Both experimental methods and the strain selection can significantly influence the experimental results (Langer et al., 2013, Oviedo et al., 2014). Therefore, using the same strain with two experimental methods will improve the quality and relevance of the observed response pattern.
Insights into the morphological and phenotypic response of H. carteri to phosphorus limitation will be used to interpret changes in coccolith size and phenotypic expression within the Helicosphaera genus under climate forcing during the past 16 million years. Further analysis of field data from the Eastern
Adriatic Coast (Mediterranean Sea) will aim at understanding the role of the coccolithophore life-cycle in adaptation to changing environment, as well as detecting the direct triggers of the phase transition. Finally, the field material rich in different life-phases of common coccolithophore species will be analyzed taxonomically to obtain more complete morphological descriptions of their life-cycle associations.
19

I am grateful to my supervisor Jorijntje Henderiks for her guidance and mentorship during the first half of my PhD studies. Experimental work was conducted at the University of Oslo (UiO), Norway, where my second supervisor Bente Edvardsen (UiO) provided great support in using culturing and laboratory facilities. Andrea Gerecht (University of Tromsø, Norway) introduced me into culture experiments and helped me to obtain the results I presented in this thesis.
I would also like to thank Zrinka Ljubeši ć (University of Zagreb, Croatia), for taking part in organizing two research cruises in the Adriatic Sea in 2013.
I am thankful to Sissel Brubak (UiO) for her support in the culture lab, and to
Gary Wife (EBC, Uppsala University) and Miloš Bartol (Department of Earth
Sciences, Uppsala University) for their help in the SEM lab. Finally, I would like to acknowledge all of my colleagues at the Paleobiology Program for friendly working environment and all their help during my time in in Uppsala.
This research was funded by the Research Council of Norway
(FRIMEDBIO project 197823) and the Royal Swedish Academy of Sciences grant received from the Knut and Alice Wallenberg Foundation (KAW
2009.0287) through Jorijntje Henderiks. The Paleobiology Program (Uppsala
University) and Anna Maria Lundin's scholarship (Uppsala University) provided funding to attend meetings and conferences.
20

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