A 15 million-year long record of phenotypic evolution in the heavily calcified coccolithophore Helicosphaera and its biogeochemical implications

The biogeochemical performance of coccolithophores is defined by their overall abundance in the oceans, but also by a wide range in cell size, degree of calcification and carbon production rates between different species. Species’ “sensitivity” to environmental forcing has been suggested to relate to their cellular PIC:POC ratio and other physiological constraints. Understanding both the short and longer-term adaptive strategies of different coccolithophore lineages, and how these in turn 15 shape the biogeochemical role of the group, is therefore crucial for modeling the ongoing changes in the global carbon cycle. Here we present data on the phenotypic evolution of a large and heavily-calcified genus Helicosphaera (order Zygodiscales) over the past 15 million years (Ma), at two deep-sea drill sites from the tropical Indian Ocean and temperate South Atlantic. The modern species Helicosphaera carteri, which displays eco-physiological adaptations in modern strains, was used to benchmark the use of its coccolith morphology as a physiological proxy in the fossil record. 20 Our results show that, on the single-genotype level, coccolith morphology has no correlation with physiological traits in H. carteri. However, significant correlations of coccolith morphometric parameters with cell size and physiological rates do emerge once multiple genotypes or closely related lineages are pooled together. Using this insight, we interpret the phenotypic evolution in Helicosphaera as a global, resource limitation-driven selection for smaller cells, which appears to be a common adaptive trait among different coccolithophore lineages, from the warm and high-CO2 world of the middle Miocene to the 25 cooler and low-CO2 conditions of the Pleistocene. However, despite a significant decrease in mean size, Helicosphaera kept relatively stable PIC:POC (as inferred from the coccolith “aspect ratio”) and thus highly conservative biogeochemical output on the cellular level. We argue that this supports its status as an “obligate calcifier”, like other large and heavily-calcified genera such as Calcidiscus and Coccolithus, and that other adaptive strategies, beyond size-adaptation, must support the persistent, albeit less abundant, occurrence of these taxa. This is in stark contrast with the ancestral lineage of Emiliania and 30 Gephyrocapsa, which not only decreased in mean size but also displayed much higher phenotypic plasticity in degree of calcification while becoming globally more dominant in plankton communities. https://doi.org/10.5194/bg-2019-472 Preprint. Discussion started: 22 January 2020 c © Author(s) 2020. CC BY 4.0 License.


Introduction
Coccolithophores (calcifying Haptophyte algae) are a globally abundant group of marine phytoplankton and an important 35 component of the biogeochemical carbon cycle. Their calcification (Monteiro et al., 2016), primary production (Rousseaux and Gregg, 2013) and deep-sea burial of calcite scales (coccoliths) Ziveri et al., 2007) have been crucial for the evolution of past and modern ocean chemistry, and are still alleviating the negative effects of rising atmospheric and oceanic CO2 levels (Ridgwell and Zeebe, 2005). Their net biogeochemical impact, which is commonly summarized as a cellular balance of inorganic (PIC) and organic (POC) carbon production rates (i.e. PIC:POC ratio), is highly susceptible to 40 environmental forcing such as high-temperature stress (Gerecht et al., 2017(Gerecht et al., , 2014Rosas-Navarro et al., 2016), nutrient limitation (Bolton and Stoll, 2013;Gerecht et al., 2015;Müller et al., 2017) and ocean acidification (Ridgwell et al., 2009;Riebesell et al., 2000). Understanding how coccolithophores adapt to environmental changes and how adaptive strategies shape their biogeochemical impact is thus essential for modeling the carbon cycle dynamics in the projected warmer, high-CO2 oceans of the near future (Bopp et al., 2013;Doney et al., 2004;Feely et al., 2004). 45 Deep-sea sediments hold a ~200 million year (Ma) long fossil record of coccoliths (Bown et al., 2004). Some morphometric features of coccoliths, such as length and thickness, correlate with biogeochemically relevant traits: cell size (Henderiks, 2008), growth rates (Gibbs et al., 2013) and the production rates of organic (POC) and inorganic (PIC) carbon (Bolton et al., 2016;McClelland et al., 2016). Combined with morphospecies-diversity and community composition that is readily inferred from the fossil record (Bown et al., 2004;Knappertsbusch, 2000;Suchéras-Marx and Henderiks, 2014), coccolith morphometry 50 could therefore help elucidate evolutionary processes from the cellular-to the community-level. In this context, the transition from the warm and high-CO2 world of the middle Miocene (~15 Ma) to the colder, low-CO2 conditions of the Pleistocene (Super et al., 2018;Zachos et al., 2001) provides an optimal case-study for investigating the long-term evolutionary adaptation of modern coccolithophore lineages to relevant climate-forcing, and understanding the biogeochemical implications of their evolutionary adaptation. This was a period of large-scale shifts in coccolithophore community structure, abundance and 55 biogeochemical impact (Hannisdal et al., 2012;Si and Rosenthal, 2019), as also evidenced by a macroevolutionary decrease in cell size (Suchéras-Marx and Henderiks, 2014) and cellular calcification rates (Bolton et al., 2016).
To date, studies of coccolithophore evolutionary adaptation have mostly focused on members of the Noelaerhabdaceae family, which dominated coccolithophore communities during the middle Miocene and Pleistocene (Henderiks and Pagani, 2007;Suchéras-Marx and Henderiks, 2014). While their most important modern representative Emiliania huxleyi is highly abundant 60 in today's oceans, this lineage represents only a small fraction of the morphological, ecological and physiological diversity of coccolithophores (Young et al., 2005). The Noelaerhabdaceae also have a unique evolutionary history with an early Jurassic divergence (~195 Ma) from the other calcifying haptophytes (Medlin et al., 2008). Clearly, there is a need to investigate evolutionary adaptation across the range of diversity and include other prominent coccolithophore lineages to determine whether the evolutionary patterns observed in the Noelaerhabdaceae are universal for the group as a whole.
This study focuses on the phenotypic evolution of the heavily calcified coccolithophore genus Helicosphaera (order Zygodiscales; Fig. 1). This genus was a constant component of coccolithophore communities during the Cenozoic (Aubry, 1988) and is still today an important contributor to oceanic carbonate fluxes Menschel et al., 2016;Ziveri et al., 2007). The most common modern species within this genus, Helicosphaera carteri (Wallich 1877) Kamptner, 1954, can be grown in culture (Sheward et al., 2017;Šupraha et al., 2015) and has a long fossil record spanning over 20 Ma 70 (Aubry, 1988). Earlier experimental studies have found a range of strain-specific physiological traits in H. carteri, likely related to local eco-physiological adaptation (Šupraha et al., 2015). In addition, this species is considered highly susceptible to ocean acidification due to its heavily calcified coccoliths and high PIC:POC ratio (Gafar et al., 2019). Finally, both modern and fossil Helicosphaera morphospecies are well-defined and identifiable under a light microscope, which allows for long-term tracking of speciation events, community dynamics and phenotypic evolution at the morphospecies-level. The main aims of this study 75 are to (I) benchmark the use of coccolith morphology as a potential physiological proxy in Helicosphaera based on the coccolith morphology of physiologically distinct H. carteri strains in culture, (II) explore the adaptive strategies of the genus and main evolutionary drivers during the past 15 Ma, and (III) compare the evolutionary patterns of Helicosphaera with other major coccolithophore lineages and discuss the biogeochemical implications of their evolutionary adaptation.

Helicosphaera carteri cultures
Culture data, obtained from batch experiments presented in Šupraha et al. (2015), are herein used as a bench-mark for phenotypic plasticity in modern H. carteri. Two strains were isolated from contrasting environments, the eutrophic South Atlantic Ocean (strain RCC1323) and oligotrophic western Mediterranean Sea (strain RCC1334) (Fig. 2a).
These strains exhibited different physiological traits such as cell size, growth and calcification rates (PIC production rates). 85 Strains were acclimated to experimental conditions for 10 generations prior to inoculation. Experiments were run in triplicate at a temperature of 17 °C and an irradiance of ~160 μ mol m −2 s −1 under a 14:10 h light:dark cycle. Further details of the experimental setup are given in Šupraha et al. (2015). All phenotypic and physiological data used in the present study were obtained from the exponentially growing, non-limited control experiments only.

Deep-sea sediment records 90
Fossil time series data were collected from pelagic foraminiferal nannofossil oozes and chalks recovered at two deep-sea drill sites (Fig. 2a). Deep Sea Drilling Project (DSDP) Leg 74 Site 525 is located in the southeastern Atlantic Ocean, at the Walvis Ridge (29°04.24' S, 02°59.12' E; water depth 2467 m) (Moore et al., 1984). Ocean Drilling Program (ODP) Leg 115 Site 707 is located in the tropical western Indian Ocean, at the Mascarene Plateau (07°32.72' S, 59°01.01' E; water depth 1552 m) (Backman et al., 1988). The sites were selected to represent contrasting productivity levels and climatic conditions. Modern-95 day primary production estimates are on average 50-100 g C m -2 yr -1 higher in the South Atlantic than in the equatorial Indian https://doi.org/10.5194/bg-2019-472 Preprint. Discussion started: 22 January 2020 c Author(s) 2020. CC BY 4.0 License.
Ocean (Antoine et al., 1996;Beaufort et al., 1997;Fischer et al., 2000). Consistently higher carbonate mass accumulation rates at Site 525 compared to Site 707 (Suchéras-Marx and Henderiks, 2014) confirm consistent site-to-site carbonate production contrasts during the geological past. Regional differences in mean sea surface temperature extend back at least 10 million years ( Fig. 2b). Age models are based on calcareous nannoplankton biostratigraphy, updated to the most recent geological time scale 100 (Gradstein et al., 2012) (Table S1). Linear sedimentation rates were estimated between age-depth tie-points.

Coccolith morphometry
Helicosphaera carteri coccoliths harvested from triplicate cultures were filtered on cellulose nitrate filters (0.8 μm, Whatman) after dispersing the coccoliths with a Triton-NaOCl treatment (Paasche et al., 1996). Filters were dried at 60°C and mounted with Canada Balsam (Merck, USA) on microscope slides, rendering transparent filters under polarized light. Microscope slides 105 for fossil coccolith morphometry were prepared from fine fraction (<38µm) sediment using the spraying technique (Henderiks and Törner, 2006) for a total of 36 (Site 525) and 38 (Site 707) stratigraphic levels. Sub-samples of freeze-dried bulk sediments were wet-sieved with buffered distilled water into three separate size fractions (<38 μm, 38-63 μm and >63 μm). The fine fraction, dominated by coccoliths, was collected in suspension, concentrated on cellulose acetate membrane filters using a Millipore vacuum pump and oven-dried at 50°C. 110 In total, 331 culture-harvested and 4115 fossil Helicosphaera specimens were analysed using a Leica DM6000B microscope equipped with polarizing filters placed at different angles (Beaufort et al., 2014). Besides size measurements, mean grey level (MGL) was used as a proxy for a change in coccolith thickness based on the birefringence of calcite (Beaufort, 2005). Standard imaging and light settings were ensured during the course of image collection (Intensity=200, Field=10, Aperture=10). Image collection was performed within a short time frame in order to minimize the effect of light bulb aging. For each sample, at 115 least 50 Helicosphaera coccoliths were imaged at 1000x magnification with a SPOT Flex color digital camera (Diagnostic Instruments Inc.; µm-scale calibrated pixel resolution 0.061µm [1.2x C-mount] or 0.074µm [1.0x C-mount]) under three different orientations of the polarizer (0°, 35°, and 45°). Coccoliths were randomly selected and identified to morphospecies level. Images taken with the three orientations of the polarizer were merged in a single 8-bit greyscale image, showing no extinction cross (Beaufort et al., 2014) (Fig. 1). Images from each sample were analyzed as a batch using a custom-made macro 120 in ImageJ software v1.47c. It determined the MGL of the selected area, measured in grey-level values of 0 (black) to 255 (white), and performed size measurements on individual coccoliths (area, perimeter and length and width of the fitted ellipse).
The calculation of coccolith thickness and volume was conducted following the protocol described in Beaufort et al. (2014) of image segmentation, but Helicosphaera coccoliths (also known as "helicoliths") are not entirely birefringent under crossed or circular polarized light due to the crystallographic orientation of calcite elements; the proximal plate and the "blanket" that covers most of the central area are birefringent in analysis, but the "flange" that extends from the proximal plate and underlies 130 the blanket is composed of so-called V-units that are only partially birefringent in the same orientation on a microscope slide ( Fig. 1) (Beaufort et al., 2014;Young et al., 2004). In addition, parts of the central area exceed 1.55µm (or 255 (white) value; Beaufort et al., 2014), showing faint yellow birefringence colors that render grey values (<255) in 8-bit, thus lowering the mean grey-value estimate calculated from all pixels in one specimen). An examination of random specimens from both sites performed in ImageJ using the "Threshold color" function has shown that the yellow signal from the thickest parts of the 135 central area usually accounts for up to 5% of surface area in medium-sized species (e.g. H. carteri and H. sellii) and up to 10%-15% in the largest species H. granulata. Therefore, Helicosphaera volume estimates are not converted to calcite mass (multiplied by the density of calcite, 2.7 pg/µm 3 ). Instead, in analogy with previous studies on Coccolithus pelagicus (Cubillos et al., 2012;Gerecht et al., 2014), we report on changes in "coccolith volume index" (CVI, in ln µm 3 ) that scales with other coccolith size and shape parameters. Statistically significant changes in CVI are meaningful indicators, if not a muted 140 expression, of the true phenotypic changes in both modern and fossil Helicosphaera assemblages.
Finally, we compiled morphometric time series data for Calcidiscus (Knappertsbusch, 2000) and Reticulofenestra (Bolton et al., 2016;Hannisdal et al., 2012) from tropical and mid-latitude locations to compare patterns of phenotypic variation and evolution across major coccolithophore clades. This comparison includes a total of 4907 Reticulofenestra coccoliths measured in 37 samples at Site 525. The latter database is limited to linear size measurements (length) only, because it was collected 145 with a different polarized light microscopy and digital image analysis setup (Henderiks, 2008;Henderiks and Törner, 2006).

PIC:POC and geometric proxies for cell physiology
Cellular production rates of particulate inorganic (PIC) and organic carbon (POC) were determined for H. carteri in the exponentially growing control experiments as a product of growth rate and the corresponding cellular elemental quotas (Šupraha et al., 2015). The molar ratio PIC:POC is widely used to assess the relative balance between calcification and 150 photosynthesis, which in turn informs about the net CO2 uptake or release by coccolithophores (review by Ridgwell et al., 2009) and references therein).
These physiological rates cannot be directly measured in the fossil record. Nevertheless, within a theoretical framework of the relationships between cell geometry, coccolith dimensions and coccolith coverage, cellular levels of organic and inorganic carbon (calcite) quota can still be estimated with a range of approaches (Bolton et al., 2016;Gibbs et al., 2013Gibbs et al., , 2018Henderiks, 155 2008;Jin et al., 2018;McClelland et al., 2016). In this study, we use the approach described by McClelland et al. (2016) who correlated a coccolith shape index, the "aspect ratio" (AR), with PIC:POC ratios measured in modern strains of Emiliania huxleyi and Gephyrocapsa oceanica, deriving the following power-law relationship: Despite the fact that coccolith thickness in Helicosphaera is systematically underestimated, we took a similar approach to investigate relative changes in shape (AR) and cellular degree of calcification (PIC:POC) between mean phenotypes.

Coccolith abundance counts and paleo-fluxes 165
For the quantitative analysis of fossil coccolith assemblages, microscope slides were prepared directly from freeze-dried bulk sediments using the drop technique (Bordiga et al., 2015). The 74 samples were analysed at 1000x magnification under an Olympus BX53 polarized-light microscope. A minimum of 300 coccoliths was counted and at least 20 fields of view (FOV) were analysed in each sample to determine the relative contribution of the major coccolithophore taxa. The absolute abundance of coccoliths (in N/g) was calculated using the formula: 170 Where N is the number of coccoliths counted, A is the area of the coverslip, f is the area of one FOV (0,0367 mm 2 ), n is the number of FOVs counted and W is the weight of the sediment on the coverslip (g).

Statistical analysis
Raw biometry data (µm-scale) were transformed to their natural logarithm (ln µm-scale) before calculating sample means and 185 variances, when investigating allometric relationships and comparing temporal trends within the time series.
One-tailed t-tests were performed in R version 3.2.2. using the "t.test" command from the Stats package. Paired t-tests were used to compare means between culture strains and between the fossil measurements. For the latter, site-to-site comparisons were made at the genus and (morpho)species level. P-values < 0.05 were considered statistically significant. The 95% confidence intervals for relative abundance estimates were calculated in PAST version 3.20 freeware (Hammer et al., 2001;190 Suchéras-Marx et al., 2019).

Helicosphaera carteri allometry
The scaling between coccolith size and thickness estimates of H. carteri shows slightly negative allometry and is highly similar in modern strains and fossil specimens (Fig. 3a,b). Mean phenotype dimensions in modern single genotypes fall within the 195 lower range of the fossil measurements. Differences in mean coccolith traits between the South Atlantic and Mediterranean strains are not statistically significant, or only borderline significant (paired t-tests, p-values= 0.029-0.039; Table S2). The range of shapes (AR) and dimensions (CVI) in cultured H. carteri largely overlap with its fossil counterparts (Fig. 3c,d). The Mediterranean strain (RCC1334), however, produced higher proportions of small and lightly calcified coccoliths ('outliers' from the distribution; Fig. 3d). Likewise, the raw fossil data ranges indicate that the Indian Ocean Site 707 hosted higher 200 proportionas of small H. carteri specimens than South Atlantic Site 525 (Fig. S1).

Mean phenotypic evolution of the Helicosphaera genus
The fossil time series data, now considering all measurements of H.carteri and other fossil Helicosphaera morphospecies, show long-term trends in mean CVI and AR at both sites (Fig. 4a,b). Linear regression of sample means vs. age suggests a 16% decrease in mean size and CVI (ln-scale) from the middle Miocene to late Pleistocene at South Atlantic Site 525 (r=0.849), 205 which corresponds to a 46% decrease in mean volume on the linear scale (µm 3 ). The same trend is less pronounced at the tropical Indian Ocean Site 707, with a 9% decrease in mean size and CVI (27% decrease in volume) over the same time interval (r=0.525). Mean shape (AR) estimates shows an opposite trend with age at both sites, implying a 24% (525; r=0.946) and 18% (707; r=0.815) increase in aspect ratio over the past 15 million years. The AR is mainly driven by changes in size, since the decrease in thickness over time is less pronounced (Fig. S2) suggesting that the relative degree of calcification increased as 210 coccolith dimensions (and thus mass per individual coccolith) decreased at both sites. When comparing all fossil measurements over the past 15 Ma, mean CVI was significantly smaller at Site 707 than at Site 525 (paired t-test; t=20.36, df=3863.8, p-value <0.001). The Indian Ocean record also reveals larger variation in Miocene-Pliocene sample means compared to the more gradual trend observed in the South Atlantic, so that site-to-site offsets in mean CVI varied over time (Fig. 4a). These offsets were largest throughout the middle Miocene, after which mean CVI reached more 215 similar values that persisted during the late Miocene between ~8.5 -5.5 Ma. Site-to-site offsets increased again during the latest Miocene and Pliocene, ~5.5 -3 Ma. From a low in the early Pleistocene (~2.5 Ma) both sites display a near-synchronous increase in CVI that peaked during the middle Pleistocene (~1.2 Ma). A steady decrease in CVI followed during the late Pleistocene at both sites; this trend appears steeper at Site 707 so that mean CVI values diverged again between sites during the past ~1 million years. 220

Phenotypic evolution at the morphospecies level
Both sites reveal distinct temporal patterns in Helicosphaera morphospecies composition ( Fig. 5; Fig. S2 Fig. 1) were the most abundant, with common trends in relative abundances over the ~15 Ma interval (Fig. 5a). 225 Helicosphaera granulata was the most dominant species at the South Atlantic site during the middle  and occasionally within the same period (~14.5 and ~12 Ma) at Site 707. The rest of the interval was dominated by H. carteri, with higher contributions of H. sellii during the Pliocene and Pleistocene (~4.5-1 Ma). Of the three studied species, only H. carteri was present continuously during the past 15 Ma ( Fig. 5b; Fig. S1). Helicosphaera granulata was present until ~2-3 Ma, whereas H. sellii had its first occurrence at ~15 Ma at Site 525 and ~12 Ma at Site 707 and its last occurrence at ~1 Ma at 230 both sites. Interestingly, extinctions of H. sellii and H. granulata were preceded by a decrease in their mean CVI at both sites ( Fig. 5c, d). Similarly, H. carteri has experienced a distinct decline in CVI over the past million years and modern H. carteri strains have lower mean size and volume than most of their fossil sample counterparts ( Fig. 3; Fig. 5b 0.001). Nevertheless, the long-term species-level patterns, as well as site-to-site offsets, were comparable among the three species (Fig. 5b-d), demonstrating that the genus-level coccolith volume trends and offsets between the sites (Fig. 4a) were not merely caused by relative contributions of differently-sized species, but also by phenotypic evolution at the species-level.
Florisphaera profunda-based primary productivity estimates (Fig. 4c) and fluxes of upper-photic zone taxa ("total placolith fluxes" in Fig 4d) confirm that significant site-to-site differences in primary production were maintained throughout the past 15 million years. High %Fp (up to 75-80%) at Site 707 indicates an oligotrophic system with low to moderate phytoplankton 250 production in the upper photic zone (PP average 131±34 g C m -2 yr -1 ) compared to the more productive Site 525 where %Fp was consistently <25% and, therefore, PP >200 g C m -2 yr -1 (average 340±65 g C m -2 yr -1 ; Fig 4c; Fig. S3). The highest nannofossil fluxes were recorded during the late Miocene and earliest Pliocene (~9 and 4 Ma), when lowered %Fp values also corroborate elevated PP levels at both sites. Despite the highly different community structures and productivity, a similar transition from larger Reticulofenestra species that dominated the middle Miocene community to smaller Gephyrocapsa spp. 255 in the late Pliocene and Pleistocene is observed at both sites (Fig. S4).

Coccolith dimensions as a proxy for cell physiology?
Mean coccolith dimensions (CVI, AR or any other parameters tested) in modern Helicosphaera carteri strains did not reflect the significant differences in growth rates, cell size or PIC and POC production rates measured in the same strains (Šupraha et 260 al., 2015). This means that, when analyzed on the cellular and genotype level, coccolith volume cannot be used to indirectly infer cell size, carbon production rates or growth rates in this species. This observation confirms that intracellular coccolith production in Helicosphaera is constrained by genetics of biomineralization and defined by the dimensions of the organic template and coccolith vesicle, not by growth and other physiological rates Young et al., 2004).
Similar to H. carteri, mean CVI has no correlation to physiological rates or cell size in single genotypes of the heavily calcified 265 species Coccolithus pelagicus and Coccolithus braarudii (Gerecht et al., 2014). Nevertheless, a strong positive relationship between coccolith dimensions and cell size of Coccolithus spp. is well established based on fossilized coccospheres, representing multiple genotypes and thousands of generations on geological timescales (Gibbs et al., 2018;Henderiks, 2008).
Likewise, a positive correlation of coccolith size and cell size in Coccolithus pelagicus is evident when different genotypes from field communities and culture experiments are pooled together (Gibbs et al., 2013). 270 Apparently conflicting results have been reported for the members of the Noelaerhabdaceae family. For example, Fritz (1999) found that coccolith size does not correlate with physiological rates or cell size within a single genotype of Emiliania huxleyi, while studies by Bolton et al. (2016) and McClelland et al. (2016) found significant correlation between coccolith thickness, aspect ratio and the PIC:POC ratio within Noelaerhabdaceae, based on multiple strains from two closely related taxa (Emiliania and Gephyrocapsa). These observations support our conclusion that correlations between coccolith morphometry, cell size 275 and physiology are weak or not identifiable within single strains (=single genotypes), and that such relationships can only be established when considered across different genotypes or closely-related species and lineages (Fig. S5).

Phenotypic evolution during the past 15 Ma: Selection for smaller cells
The long-term phenotypic trends in fossil Helicosphaera indicate a selection for smaller cells in the South Atlantic Ocean and the equatorial Indian Ocean over the past 15 Ma. Our dataset supports that this selection for smaller cells occurred on various scales: (I) the (morpho)species level, (II) the community level, as well as (III) the evolutionary level through extinction of larger species and speciation of smaller species. 290 At the (morpho)species level, this includes selection for smaller-celled phenotypes within each of the three most dominant species (H. carteri, H. sellii and H. granulata; Fig. 5). Along with the species-level trends, the selection for smaller cell-size is also expressed in the relative contribution of different-sized species and their speciation patterns. This is evident in the gradual decrease in relative abundance and Pliocene extinction of the largest species H. granulata whose dominance was gradually replaced by the rise of H. carteri and H. sellii. Subsequently, the extinction of H. sellii in the middle Pleistocene was 295 followed by the rise of an even smaller extant species, H. hyalina. The adaptations at the species level, ecological trends on the community level as well as the evolutionary processes (speciation and extinction) on the genus level, all followed the same pattern in favoring smaller-celled genotypes and species within Helicosphaera, a trend that is consistent between the two sites.
This selection for smaller morphotypes appears to be a common long-term adaptive response across multiple coccolithophore coccolithophore lineages indicates that the phenotypic evolution of the group is driven by (a) common macroevolutionary driver(s) that selected for smaller coccolith and thus smaller cell size since the middle Miocene. 305

What drives the phenotypic evolution of Helicosphaera spp.?
The phenotypic expression observed at any point in the fossil record is a mean phenotype of millions of generations of coccolithophore cells sedimented from the photic zone. As such, it represents the genotypes which at the time were selected by their environment. It is a result of selection by global and local environmental drivers (e.g. hydrography, temperature, insolation, nutrients, CO2 levels) as well as evolutionary (extinction, speciation) and ecological processes combined with inter-310 and intra-specific interactions such as competition and predation (Falkowski and Oliver, 2007;Finkel et al., 2007). Keeping in mind this environmental and biological complexity behind the final product -a coccolith with its phenotypic features, what does the selection for smaller cell size tell us about the possible global and regional drivers of phenotypic evolution in Helicosphaera and in coccolithophores in general?
The past 15 million years of Earth's geological history has included prominent environmental and paleoceanographic changes. 315 Most importantly, this period saw the transition from the warm and high-CO2 world of the  Ma) to the cooler, low-CO2 world of the Pleistocene with its alternating ice ages and interglacial periods (Super et al., 2018;Zachos et al., 2001;Zhang et al., 2013). The global decrease in pCO2 since the middle Miocene and associated carbon limitation of photosynthesis and calcification has been suggested as the main global driver of coccolithophore phenotypic evolution (Bolton et al., 2016). Carbon limitation during the late Miocene may have triggered a physiological adaptive response in 320 coccolithophores around 7-5 Ma ago in the form of an active allocation of carbon from calcification to photosynthesis, as inferred from coccolith vital effects (Bolton and Stoll, 2013). A number of studies, spanning beyond the interval addressed here, have shown that carbon limitation especially affects larger, heavily calcified taxa, due to their low surface-to-volume (SA:V) ratio and high calcification-demand for carbon (Hannisdal et al., 2012;Henderiks and Pagani, 2007;McClelland et al., 2016;Pagani et al., 2011). Being among the largest and most calcified species, members of the genus Helicosphaera would 325 therefore be strongly affected by decreasing CO2 levels during the middle to late Miocene, leading to progressive selection for smaller morphotypes with higher SA:V ratio and lower carbon-demand for calcification. If the adaptive carbon allocation mechanism observed in Noelaerhabdaceae and Coccolithaceae has also developed in Helicosphaera spp., it may account for the apparent reversal in global phenotypic trends during the late Pliocene and early Pleistocene found in our dataset. Also, it may indicate that other global environmental drivers and adaptive responses were at play in the most recent time intervals. 330 While the global decrease in coccolith and cell size across coccolithophore lineages is likely related to decreasing pCO2, the site-to-site contrasts observed in this study could be related to other environmental drivers which commonly shape coccolithophore communities such as nutrient availability and temperature. Experiments on H. carteri have shown that this species can strongly reduce its nutrient requirements, arguably as an adaptation to growth in oligotrophic environments (Šupraha et al., 2015). This comes at a price of slower growth rates, on average larger cell size and slower calcification rates, but conserved rates of photosynthesis, which is prioritized in respect to calcification under nutrient limitation. Similar shortterm response patterns, where limited resources are allocated to maintain photosynthesis at the expense of calcification, were observed in semi-continuous cultures of C. pelagicus . On the other hand, E. huxleyi is notably insensitive to phosphorus limitation (Gerecht et al., 2017;Oviedo et al., 2014) due to its small size (and SA:V ratio), low nutrient requirements and remarkable activity of its alkaline phosphatase (Riegman et al., 2000). These experimental observations show 340 that nutrient availability can have a strong effect on coccolithophores and that it likely exerts strong selective pressure in the natural environment, forcing the cells to channel limiting nutrients from calcification to photosynthesis to maintain their division rates or to develop physiological adaptations. Even though physiological low nutrient-adaptation was not reflected in the coccolith phenotype of single strains of H. carteri and C. pelagicus, it is plausible to expect that prolonged nutrient limitation would have strong effects on size-selection over longer, evolutionary time-scales (Falkowski and Oliver, 2007). In 345 this sense, lower nutrient availability in the oligotrophic Indian Ocean as opposed to the more productive South Atlantic (inferred from modern data as well as our %Fp-based PP estimates and nannofossil community composition) can account for the observed general offset in size between the two sites. In other words, lower nutrient concentrations coupled with carbon limitation have likely channeled the Indian Ocean genotypes towards even smaller cell-size compared to their South Atlantic counterparts. This process was likely supported and even exacerbated by consistently higher temperatures in the tropics 350 (27±1°C, long-term average ± 1 s.d.; Herbert et al., 2016) compared to the South Atlantic and higher latitudes (Fig. 2b). Cooler sea surface temperatures in the South Atlantic would have favored both the solubility of CO2 in the ocean and coastal upwelling off SW Africa would have supplied nutrients to the photic zone, thus alleviating the effects of resource limitation and allowing for on average larger cell size at the South Atlantic site compared to the Indian Ocean site.
An important adaptive trait to consider in the context of resource limitation is the heteromorphic, haplo-diploid life-cycle that 355 is prevalent among coccolithophores including Helicosphaera (Young et al., 2005). The haploid life-phase is generally significantly smaller and less calcified than the diploid life-phase, and is considered an adaptation to seasonal resource limitation (D'Amario et al., 2017;Šupraha et al., 2016). In addition, both life-cycle phases of Helicosphaera are flagellated and have a haptonema, which is a strong indication that they are putative mixotrophs (as demonstrated for Coccolithus pelagicus; Houdan et al., 2006) and can relieve nutrient limitation by ingesting bacteria. However, due to their sensitivity to 360 dissolution, holococcoliths of Helicosphaera are scarce or near-absent from the fossil record. It is thus difficult to assess how important the life-cycle dynamics of this genus were during its evolutionary history, but given the prevalence of this adaptive strategy in the modern ocean it is safe to assume that it was an important mechanism for overcoming resource limitation also in the past.

Biogeochemical implications of phenotypic evolution 365
Despite the evolutionary selection for smaller coccolith-and cell size in Helicosphaera since the middle Miocene, the trend in AR was less pronounced, indicating relatively consistent biogeochemical output (i.e. similar PIC:POC ratio; Fig. 7) of the https://doi.org/10.5194/bg-2019-472 Preprint. Discussion started: 22 January 2020 c Author(s) 2020. CC BY 4.0 License.
genus. The observed AR trend suggests a slight increase in PIC:POC over time, which could mean that on the cellular level, the adaptive strategy of decreasing coccolith and cell size compensated for resource limitation and resulted in conserved physiological performance. Moreover, the regional trends in size selection were not reflected in regional patterns of AR, which 370 were largely overlapping between both sites throughout the studied interval. Evidently, any global or regional patterns in phenotypic evolution, speciation or adaptation (either in size or physiology) ultimately led to very similar biogeochemical performance among the populations in different regions. A significant divergence in AR between the sites happened in the late Pleistocene, with an increase in AR (and thus PIC:POC) at Site 707. This coincided with an increase in PP estimate as well as coccolith size (Fig. 4), suggesting that locally weaker resource limitation could have led to increasing calcification rates and 375 some degree of regional trends was possible despite overall globally conserved biogeochemical performance.
However, when considered in a wider phylogenetic context and compared with AR patterns observed in the Noelaerhabdaceae (Fig. 6c), the AR trends observed in Helicosphaera, though statistically significant, appear biologically meaningless. During the same time interval, the AR and thus the biogeochemical output of Noelaerhabdceae exhibits remarkable variability and a wide range of possible PIC:POC ratios (i.e. degrees of calcification) with an overall decrease between the middle Miocene and 380 Pleistocene. This phenotypic variation is likely related to the overall higher taxonomic and morphological diversity of the Noelaerhabdaceae and it points to a high adaptive potential of this group that ensured its evolutionary success and global dominance during the Neogene, not just within coccolithophore communities (Figs S3 and S4) but in marine phytoplankton in general. Our data show that despite the global trends of size decreases across coccolithophore lineages, phenotypic evolution in different groups can lead to highly different biogeochemical outputs (Fig. 6c). A prime example of this is the comparison 385 between the Helicosphaera genus, with its highly conserved morphology, little phenotypic innovation, low abundances and relatively stable biogeochemical performance; and the Noelaerhabdaceae, with the potential for inter-species hybridization (Bendif et al., 2015) and rapid diversification (Bendif et al., 2019), global dominance in marine phytoplankton communities (Suchéras-Marx and Henderiks, 2014;Fig. S3) and a range of possible biogeochemical outputs ( Fig. 7; Fig. S5). Clearly, there are various adaptive strategies to the same global drivers among coccolithophore lineages, and a wide range of biogeochemical 390 consequences of their phenotypic evolution.
One possible explanation for different adaptive strategies can be related to the distinction between obligate calcifiers, including Coccolithus and Helicosphaera, and non-obligate calcifiers such as Emiliania huxleyi (Durak et al., 2016;Walker et al., 2018).
The phenotypic plasticity in obligate calcifiers could be more restricted when it comes to morphological innovation and biogeochemical performance compared to non-obligate calcifiers, who can regulate their calcification rates (and thus PIC:POC 395 ratio) to zero and overcome carbon limitation or other environmental stressors such as ocean acidification (Gafar et al., 2019).
Obligate calcifiers such as Coccolithus, Calcidiscus and Helicosphaera, combined, were relatively more abundant than reticulofenestrids until ~7 Ma when their fluxes decreased significantly at both sites (Fig. S3). This supports that obligate calcifiers have been evolutionary and ecologically less successful compared to non-obligate calcifier lineages during the global https://doi.org/10.5194/bg-2019-472 Preprint. Discussion started: 22 January 2020 c Author(s) 2020. CC BY 4.0 License.
shift to the "icehouse" world of the late Miocene to Pleistocene, each of the groups adapting within the constraints of their 400 phenotype.

Conclusions
Coccolithophore communities are globally linked on evolutionary time scales through speciation and extinction events. On millennial time scales, environmental changes cause selective pressures which result in common signals of phenotypic evolution across different coccolithophore lineages, as shown for Helicosphaera (Zygodiscales order), Calcidiscus 405 (Coccolithales order) and the ancestral lineage of Emiliania and Gephyrocapsa (Isochrysidales order). However, the biogeochemical output of the group is defined by lineage-specific physiology and adaptive strategies, regionally distinct community compositions and local ecophysiological adaptations. Inferring physiological adaptive strategies from coccolith morphometry proves to be challenging if not impossible when considered within single genotypes. Valid correlations between morphology and physiology emerge only when more genotypes or closely related taxa are pooled together. Accordingly, short-410 term physiological responses in e.g. cell size or PIC:POC ratios observed in single-genotype experiments do not necessarily translate into long-term phenotypic evolution observed in the fossil record, which is a result of far more complex and longterm selection with multiple biotic and abiotic drivers. To conclude, the possible scenarios for biogeochemical performance of coccolithophores under ongoing global environmental changes are difficult to predict, as they likely rest on specific adaptive responses and complexities of different lineages that are beyond simple size-adaptation and are shaped by the adaptive potential 415 of different species and groups, and ultimately by the biodiversity and taxonomic structure of coccolithophore communities.

Data availability
The data will be made available on PANGAEA after the manuscript is published.

Acknowledgements
This study was supported by the Royal Swedish Academy of Sciences through a grant from the Alice and Knut Wallenberg 420 foundation (KAW 2009.0287) and funding from the Norwegian Research Council (project 197823/V40) to J.H. We are grateful to Prof. Bente Edvardsen for access to laboratory facilities at the Section for Aquatic Biology and Toxicology (AQUA) at the University of Oslo (UiO) as well as helpful discussions during the PhytoSCALE project. We thank Andrea Gerecht (Centre for Ecological and Evolutionary Synthesis, UiO) for her support in conducting the culture experiments.    (CVI, ln µm 3 ) vs. aspect ratio (AR, unitless). Here, individual specimens are also shown for the modern strains.  Primary productivity estimates (PP, gC m -2 yr -1 ; 95% confidence intervals) based on the relative abundance of Florisphaera profunda (Fp%) cf. Beaufort et al., (1997). Bars to the left show the range in modern primary production estimates for the South Atlantic (blue) and the Eastern Indian Ocean (red) (Antoine et al., 1996;Beaufort et al., 1997;Fischer et al., 2000). (d) Long-term trends in placolith fluxes (or burial rates, N m -2 yr -1 ) of upper-photic zone dwelling species only. Error bars show 460 ±15% reproducibility as determined from repeat analyses (Bordiga et al., 2015).    HC (1323) HC (1334) https://doi.org/10.5194/bg-2019-472 Preprint. Discussion started: 22 January 2020 c Author(s) 2020. CC BY 4.0 License.