Calcification and latitudinal distribution of extant coccolithophores across the Drake Passage during late austral summer 2016

: 15 Coccolithophores are globally distributed microscopic marine algae that exert a major influence on the global carbon cycle through calcification and primary productivity. There is recent interest in coccolithophore polar communities, however field observations regarding their biogeographic distribution are scarce for the Southern Ocean. This study documents the latitudinal, as well as in depth, variability in the coccolithophore assemblage composition and the coccolith mass variation of the ecologically dominant Emiliania huxleyi across the Drake Passage. Ninety-six water samples were taken between 10 and 150 m water depth from 18 20 stations during POLARSTERN Expedition PS97 (February-April, 2016). A minimum of 200 coccospheres per sample were identified in scanning electron microscope and coccolith mass was estimated with light microscopy. We find that coccolithophore abundance, diversity and maximum depth habitat decrease southwards marking different oceanographic fronts as ecological boundaries. We characterize three zones: (1) the Chilean margin, where during SEM analyses. coccoliths of


Introduction
The carbon chemistry of the ocean has a fundamental impact on marine life. The current influx of anthropogenic CO2 into the 5 surface ocean is causing a substantial perturbation to marine chemistry, as exhibited by variations in alkalinity, carbonate ion, saturation state or pH (e.g., Gattuso et al., 2011). Many organisms use dissolved inorganic carbon for photosynthesis and/or the production of calcium carbonate biominerals. Open ocean phytoplankton includes coccolithophore algae, a unicellular organism belonging to the phylum Haptophyta (Young and Bown, 1997;Young et al., 2003). Within a single coccolithophore cell there are dual pathways of carbon utilizationfor photosynthesis and for biomineralization of calcium carbonate platelets, called coccoliths. 10 Coccolithophores produce up to ~40% of open ocean calcium carbonate (Poulton et al., 2013) and are responsible for ~20% of global net marine primary production (Malone et al., 2017). Therefore, how coccolithophore respond to changing oceanic conditions is of upmost importance for marine ecology and carbon cycling. The ratio between particulate organic carbon formed during photosynthesis and particulate inorganic carbon produced via calcification varies depending on species or even morphotypes within species (Blanco-Ameijeiras et al., 2016), but can also be highly influenced by environmental conditions, such as seawater 15 CO2 concentration, total alkalinity, and phosphate concentration (Findlay et al., 2011). As climate varies, it is expected that these key conditions will change, and it is predicted that upper oceans may experience increased stratification and decreased nutrient availability in the upper photic zone (Cabré et al., 2015). How exactly coccolithophores will respond to these changes is subject to debate.

20
Ocean acidification combined with the increase in sea-surface temperature due to global warming are major concerns in polar and subpolar regions (e.g., Wassmann et al., 2011;Post et al., 2013;Freeman and Lovenduski, 2015), triggering an increasing interest in coccolithophore ecology at high latitudes (e.g., Harada et al., 2012;Dylmer et al., 2015;Balch et al., 2016;Charalampopoulou et al., 2016;Giraudeau et al., 2016;Saruwatari et al., 2016;Nissen et al., 2018;Rigual Hernández et al., 2018;Krumhardt et al., 2019). Questions remain about how coccolithophore populations will adapt to predicted changes in their environment, if at all. 25 There is growing concern that increasing levels of CO2 in the atmosphere and the subsequent acidification of the ocean may disrupt the production of coccoliths. As more of the water column becomes undersaturated in CaCO3 in the future (Fabry et al., 2009), carbonate dissolution will be favored over precipitation and coccolithophores may be less successful in exporting carbon to the deep ocean (Fabry et al., 2008). Additionally, any change in the global distribution and abundance of coccolithophore species relative to non-calcifying groups of phytoplankton (e.g., naked Haptophyceae cells, diatoms, etc) will have important effects on 30 the biogeochemical cycling of carbon and climatic feedbacks. A known positive correlation exists over long timescales between surface-ocean carbonate ion concentrations [CO3 2-] and the mean coccolith mass of the associated Noëlaerhabdaceae assemblage, a family of coccolithophores, which includes the extant species Emiliania huxleyi (Beaufort et al., 2011). This correlation is driven by the replacement of more-by less-heavily calcified morphotypes or species with declining [CO3 2-]. Although the physiological driver for this strong ecological selective pressure is not known (Beaufort et al., 2011), it may determine Noëlaerhabdaceae 35 biogeography, particularly in high latitudes, both in the past and future (Cubillos et al., 2007).
Geographical shifts in the occurrence or abundance of coccolithophores and assemblage compositions have been recently observed (e.g., Rivero-Calle et al., 2015;Krumhardt et al., 2016). Repeated sampling in the Australian sector of the Southern Ocean (SO) over the past four decades has shown a dramatic range expansion of E. huxleyi south of 60ºS (Cubillos et al., 2007), where any ocean acidification effect appears outweighed by surface-ocean warming. Other authors also recorded a southward expansion of the habitat of E. huxleyi in the SO during the last two decades (Winter et al., 2014), although the actual cause of this latitudinal 5 expanse is still under debate (e.g., Patil et al., 2014;Malinverno et al., 2015). Even with a temperature-driven range expansion of coccolithophores in the SO, surface ocean carbonate chemistry is now capable of exerting a first-order control on the composition of coccolithophore assemblages as well as on overall coccolithophore calcification (Cubillos et al., 2007;Mohan et al., 2008;Beaufort et al., 2011;Freeman and Lovenduski, 2015). 10 With significant changes in marine species distributions already occurring, it is crucial to understand the ecosystem structure as well as the potential impact of environmental change on the provision of essential ecosystem services (O'Brien et al., 2016). In this work, we assess the potential relationship between environmental parameters and the community composition, biogeography and calcification mode of modern high latitude coccolithophore communities across the Drake Passage. Accordingly, we calculated extant coccolithophore species numbers at different stations between 10 and 150 m of the water column, evaluated the coccolith 15 mass variations of E. huxleyi. We compared these observations with in situ conductivity-temperature-depth (CTD) measurements, carbonate chemistry parameters, as well as to previously published Southern Ocean coccolithophore and calcification datasets.

Sample preparation for scanning electron microscope analyses and coccolithophore taxonomical considerations
Ninety-six water samples were taken at 18 stations located in the southern Chilean continental margin and across the western end 20 of the Drake Passage ( Fig. 1) from February to April 2016 during Expedition PS97 (Lamy, 2016). Seawater samples were obtained at different depths using a rosette sampler with 24 × 12 L Niskin bottles (Ocean Test Equipment Inc.) attached to a CTD Seabird SBE911 plus device (Lamy, 2016). The bottles were fired by a SBE32 carousel. For the study of coccolithophore assemblages, 4 to 7 samples per station, between 10 and 150 m water depth, were chosen. Two litres of water were filtered onto 0.45 µm pore size Polycarbonte Track-Etch Membrane, air-dried and stored over silica gel. A small part of the filter was cut out, fixed on an aluminum 25 Scanning Electron Microscope (SEM) stub and sputtered with gold/palladium. A specific area of the center of the filter was analysed with Zeiss DSM 940A SEM at the University of Bremen, to determine quantitative cell counts for all morphotypes, species and total coccolithophore abundance at magnifications of 1000x, 2000x and 5000x when required. A minimum of 200 whole coccospheres per sample were counted and classified following Young et al. (2003), the revised classification of Jordan et al. (2004) and the electronic guide to the biodiversity and taxonomy of coccolithophores Nannotax 3 30 (ina.tmsoc.org/Nannotax3/index.html) by Young et al. (2019).
Initially, seven different morphotypes of Emiliania huxleyi were distinguished in the study area belonging to two main groups, types A and B (for further details see Nannotax). These are type A (huxleyi), type A overcalcified, type B (pujosiae), type B/C, type C (kleijneae), type R and type O (which included specimens with an opened central area and specimens with the central area 35 covered by a thin plate) ( Table 1, Plate 1). Additionally, the degree of calcification was visually assessed while counting, that is why the terms "normal", "calcified" and "heavily calcified" are used in this work to denote some of the most robust E. huxleyi placoliths regardless the morphotype (see Plate 1). Semiquantitative estimates of preservation were based on SEM observations on the coccolithophore assemblage. "Good" preservation implied little or no evidence of carbonate dissolution. Coccoliths with the main morphological characteristics partially altered but still identifiable at species level were tagged as "moderate" (e.g., Telements within the taxon E. huxleyi were present). Specimens affected by strong dissolution or high fragmentation were regarded as "poor" (e.g., T-elements within the taxon E. huxleyi were dissolved). 5
Phosphate, nitrate and silicate contents were retrieved from the World Ocean Atlas 2013 (WOA13) 1°x1° grid austral summer collection (December to February) (Garcia et al., 2014). Total carbon dioxide (TCO2) and total alkalinity (TALK) values were 15 obtained from the Ocean Data View (ODV) global alkalinity and total dissolved carbon 1°x1° grid collection from the uppermost 150 m of the water column during austral summer (December to February) (Goyet et al., 2000). Since carbonate chemistry parameters were not measured in situ and data availability in the Drake Passage/Southern Ocean is limited, bicarbonate ion (HCO3 -), carbonate ion (CO3 2-), saturation state (ΩCa) and pH were calculated (as derived variables) with ODV software version 4.6.3 (Schlitzer, 2015) using the uppermost 150 m of the GLODAPv2 collection (Key et al., 2015) and excluding measurements done 20 before 1980. HCO3 -, CO3 2-, ΩCa and pH were also calculated (for comparison purposes) using the CO2SYS.XLS program for 10-20 m depth (Pierrot et al., 2006) and considering the interpolated values of TALK, TCO2 and nutrients as well as the in situ measurement of temperature, salinity and pressure.
The values of each of these oceanographic parameters were estimated at the location of every CTD station by interpolating the 25 available data points. The interpolation used a triangulation-based linear method for CTD stations located within the boundaries of the available data, and a nearest neighbour extrapolation method for any CTD stations located outside of the data boundaries.
Latitude/longitude coordinates were projected to Universal Transverse Mercator (Zone 19E, World Geodetic System 1984) before interpolation in order to minimize the distance distortion inherent in geographic coordinates. The calculations were done in MATLAB TM using a custom function that is provided as supplementary material. 30

Sample preparation and coccolith calcite estimates in light microscope
Fifteen samples were selected in a latitudinal transect for coccolith calcite estimates, between 10 and 20 m water depth. Sample preparation was designed and carried out at the University of Salamanca (Spain). A part of the filter (ca. 1/4 of the original Polycarbonte Track-Etch Membrane) was cut out and carefully placed into small plastic bags. Buffered water (pH = 9) was prepared with 0.075g/L of Na2CO3, 0.1 g/L and 0.04g/L of unflavored gelatin ("Gold Gelatin"). Six hundred µL of buffered water 35 were added, and the bags were sealed consistently in a triangular shape. After 30 minutes, the bags were shaken in a lab vortex for 3 minutes to ensure that coccoliths were fully detached from the filter and re-suspended. Holding each bag on top of a cover slide placed on a hot plate (ca. 60°C), an incision was made with a scalpel in one of the bag angles, allowing the solution to slowly drop onto the cover slide. Once the water was evaporated, the cover slip was mounted with mixture of 50% Canada balsam and 50% Xylene and left in an oven (40° C) for at least 24h. This technique ensured that coccoliths were in the same plane of focus for polarized light microscopy. The dried filter was checked for presence/absence of coccoliths later on in the SEM of the University of Bremen (see supplementary material).

5
In this study, a Nikon Eclipse LV100 POL polarized light microscope with a 100x H/N2 objective set-up with circular polarization was used at the University of Salamanca. In order to determine coccolith mass and thickness, between 20 and 53 random fields of view were imaged using the Nikon DS-Fi1 digital 8-bit colour camera and the NisElements software, keeping the light level of the microscope and aperture settings constant. The images were processed with the C-Calcita software (for further details see Fuertes et al., 2014). Calibration was done with images of a well preserved and in-focus calcareous spine from the sample PS97/033-1 at 10 10 m (see supplementary material). In total, 796 coccoliths were analysed, with a minimum of 34 coccoliths (up to 94) measured per sample.

Diversity indices
The Shannon index (H), the Simpson diversity index (1-D) and the Fisher's alpha index were calculated with Paleontological 15 Statistics (PAST™) software version 3.22 (Hammer et al., 2001) using the raw coccolithophore counts. H was determined with the following equation (1): where ni is the number of individuals in taxon i, and n is the total number of all individuals. This index takes into account the number of individuals as well as the number of taxa. H ranges from 0 for communities with just one taxon to higher values for 20 communities with many taxa, each with few individuals (Harper, 1999). Dominance, D, was determined with the equation (2): The Simpson index 1-D varies from 0 to 1 and measures evenness of the community. Fisher's alpha was calculated with the following equation (3): where S is number of taxa, n is number of individuals and a is the Fisher's alpha.

Statistics
A principal component analysis (PCA) was performed on the coccolithophore relative abundance data using PAST™ software version 3.22 (Hammer et al., 2001). The objective of the PCA is to find hypothetical variables, called components, that capture the 30 maximum proportion of the variance in the multivariate dataset as possible (Davis, 1986;Harper, 1999). These new variables are linear combinations of the original variables (Hammer et al., 2001). The first principal component has the largest variance possible, and each subsequent component explains the next greatest variance possible. Samples in which less than 50 coccospheres were counted were excluded from the original database, therefore just 74 samples were considered for the PCA. In order to avoid skewness, the relative abundances of the different coccolithophore taxa (x) were log-transformed prior to the PCA using the 35 formula: y=log(x+1). This transformation enhanced the importance of rare taxa, and minimized the dominance of few abundant taxa (Mix et al., 1999), in this case of E. huxleyi types B/C and C.
Seventeen coccolithophore taxa were considered for the PCA (see the taxonomical groups in Table 2). Due to the similar ecological preferences observed, Papposphaera sp. and Pappomonas spp. were lumped together in one taxonomical group, in the same way as holococcolithophores, the Syracosphaera and the Ophiaster species (Table 2). In contrast, E. huxleyi morphotypes were regarded as different groups. A correlation matrix between the principal component scores and the environmental variables (i.e., SST, SSS, 5 fluorescence, oxygen and density measured in situ, as well as nitrate, phosphate and silicate contents interpolated from the WOA13) was performed in order to identify potential relationships between the environmental parameters and the coccolithophore components.

Coccolithophore distribution
The analysis of 96 water samples shows in general higher cell concentrations in the uppermost 100 m of the water column in the SAZ and at shallower depths (ca. 60 m) in the PFZ (Fig. 3 a). The highest coccolithophore number, 214.6*10 3 cells/L, is reached at station PS97/034-2 (60 m) in SAZ, but high cell concentrations are generally observed north of the SAF (Fig. 3 a). The uppermost 150 m average of coccolithophore concentrations drastically drop at the oceanographic fronts, from 119.9*10 3 to 56.4*10 3 cells/L 15 (PS97/036-1, /037-1) at the SAF, and from 32.2*10 3 to 0.1*10 3 cells/L (PS97/040-1, /041-1) at the PF. Stations south of the PF show very low cell numbers or are devoid of coccolithophores, but detached coccoliths are occasionally observed at the southernmost locations, even at station PS97/50-2. Coccolithophore preservation is in general moderate to good north of the PF, with optimum values at 10-20 m, but becomes notably poorer in the AZ (Fig. 3 c).

Coccolithophore community composition
Twenty-three different coccolithophore taxa (including morphotypes) are observed in this transect across the Drake Passage (Table   2). The most dominant species is E. huxleyi, although less abundant taxa also dwell in these (sub-) polar waters. In the following lines, we will comment on the main species composing the coccolithophore community, from the dominant taxa to the rare ones.

Emiliania huxleyi 25
Emiliania huxleyi dominates the coccolithophore assemblage, reaching values up to 212.5*10 3 cells/L at PS97/034-2 (60 m). This Neither malformed E. huxleyi, morphotype D, nor var. corona are present in the studied samples. However, a variation in the 30 degree of coccolithophore calcification is observed; i.e., heavily calcified specimens as well as weakly calcified specimens are present in this transect (Table 1, Plate 1).
Emiliania huxleyi A group is less abundant than group B and includes type A, type A overcalcified and type R, all of them present in coastal waters (Fig. 4 a). Type R (Plate 1 a) is the most uncommon E. huxleyi morphotype, and it just dwells offshore Chile and at station PS97/038-1 (20 m), where it reaches a maximum of 0.9*10 3 cells/L (Fig. 4 c). A similar distribution pattern is shown by 35 E. huxleyi type A overcalcified ( Fig. 4 d, Plate 1 b, c), with concentrations up to 1.8*10 3 cells/L at PS97/018-1 (100 m). Emiliania huxleyi type A (normal form or moderately calcified, Plate 1 d-f) is restricted to the continental margin, where it records numbers of up to1.8*10 3 cells/L (Fig. 4 b).
Emiliania huxleyi B group specimens dwell north of the PF, showing a broader distribution than A group (

Other taxa
On top of E. huxleyi, less abundant taxa are observed. Ophiaster spp., including O. hydroideus (Plate 2) and sporadically O. reductus, is present primarily at the Chilean margin to a maximum 150 m water depth, but unexpectedly reaches up 10.1*10 3 cells/L at PS97/038-1 at a depth of 10 m north of the PF (Fig. 6 a). Calciopappus caudatus is found in the uppermost 60 m of the SAZ 20 and PFZ with maximum abundances of 5.3*10 3 cells/L also at PS97/038-1, 10 m ( Fig. 6 b). Four species belonging to the genus Syracosphaera are recorded in the Drake Passage (i.e., S. dilatata, S. corolla, S. marginaporata and S. pulchra). Syracosphaera spp. dominates among the rare coccolithophore assemblage in the SAZ, except at coastal stations, and its highest numbers (up to 5.3*10 3 cells/L PS97/038-1) are recorded between 10 and 60 m (Fig. 6 c). In contrast, Calcidiscus s.l., mainly Calcidiscus leptoporus, displays moderate numbers at the coastal stations offshore Chile (from 10 to 150 m), but reaches higher numbers (up 25 to 1.4*10 3 cells/L at PS97/038-1, 10 m) southwards in a rather patchy, but shallow distribution (Fig. 6 d). Papposphaera sp. and Rare coccolithophore taxa (maximum below 0.8 *10 3 cells/L) are also recorded in the Drake Passage. Gephyrocapsa muellerae is 30 restricted to the northernmost stations offshore of Chile, while Chrysotila sp. is occasionally observed in the SAZ and Calciosolenia murrayi in the PFZ (Fig. 7 c-e). Acanthoica quattrospina and Wigwamma antarctica display a broader distribution, even south of the PF, in the case of the latter (Fig. 7 a, b). Additionally, non-coccolithophores haptophytes belonging to the genera Petasaria and Chrysochromulina are generally present in the SAZ, PFZ and AZ, reaching maximum concentrations of 7.8 *10 3 cells/L at PS97/037-1 at 60 m water depth (Fig. 7 f). 35 In order to investigate the relationship between the coccolithophore composition and the environmental variables in the study area, a PCA, was performed using the data from 74 sampling points (Fig. 8). Based on the broken stick method, the PCA indicates the existence of three main principal components (PC) explaining 77.4% of the total variance (see supplementary material). PC1 explains 45.3% of the variance and it is positively related to the abundance of E. huxleyi type O and negatively related to E. huxleyi type C. PC2, connected to E. huxleyi type B/C, in a lesser extent to type B, and negatively correlated to E. huxleyi type A, accounts for 17.3% of the variance. PC1 seems associated to a marked temperature gradient. Positive PC1-values are linked to warm/moderately warm water taxa, which dwell north of the SAF, and negative PC1-values are connected to taxa that live in 5 colder waters at higher latitudes. PC1 is correlated to SST and anticorrelated to density, oxygen content, macronutrients (phosphate, nitrate) and silicate (Table 3). PC2 is related to salinity variations (Table 3). Negative values of PC2 are related to extant coccolithophore taxa observed in the low salinity waters offshore Chile or to taxa living in the PFZ. On the contrary, positi ve values of PC2 are linked to taxa dwelling in the SAZ, where the SSS values are the highest of the studied transect. The coccolithophore assemblage composition separates out three different clusters in the PCA (Fig. 8) corresponding to three different 10 oceanographic areas. The SAZ is characterized by positive values PC1 and PC2, the PFZ by negative PC1 and PC2 values, and the Chilean coastal environment (Chl) by positive PC1 and negative PC2.

Diversity
Coccolithophore diversity indices (Shannon index H, Simpson index 1-D, Fisher's alpha) as well as the number of taxa are highest offshore Chile (0-150 m) and they became restricted to the uppermost 60 m in the PFZ (Fig. 9). The number of taxa is maximal at 15 station PS97/038-1 (10 m) and drastically drops south of the PF. The Shannon and Simpson 1-D indices display a similar pattern ( Fig. 9 b, d), are highly correlated (r=0.95, supplementary material) and show that coccolithophore diversity decreases southwards.
The number of taxa and diversity indices are strongly related to latitude ( Fig. 9 c, f), with station PS97/038-1 (10-20 m) being an outlier due to the high diversity estimates recorded there.

Coccolith calcification -E. huxleyi-20
Emiliania huxleyi specimens were classified into non-standardized sub-categories (e.g., regarding level of coccolith calcification) while counting in the SEM. Specimens of type A calcified/heavily calcified and overcalcified (Fig. 10 a) are present at the same locations as the common E. huxleyi type A (Fig. 4). Calcification in the group B implied in most of the cases a thicker central tube in the coccolith central area and thicker T-elements (Plate 1). Although type B calcified is recorded with rather low numbers, it shows a similar distribution to type B/C calcified ( Fig. 10 b, c); both are restricted to the uppermost 150 m of the water column at 25 stations PS97/029-1, /030-1 and /031-1. Type C calcified is occasionally recorded where types B and B/C are present, but shows a much broader and patchy distribution north of the PF (Fig. 10 d).
Coccolith mass measured with the software C-Calcita ranges from 19.8 to 0.8 pg, and median values (per station) vary from 7.3 to 2.4 pg. Relatively high E. huxleyi masses are recorded in the SAZ (Fig. 11), but not at the stations with the highest cell concentrations (such as PS97/034-2). Measurements of the coccolith mass allowed us to compare to the identified morphotypes. 30 In general, coccolith mass decreases southwards across the Drake Passage (Fig. 11). While high coccolith masses are reached offshore Chile (i.e., stations PS97/017-1, /018-1) where E. huxleyi types R and A (overcalcified) are present, low coccolith masses are reached in the PFZ where types B/C and C dominate (i.e., stations PS97/037-1, /040-1). The gradual latitudinal mass decrease is occasionally interrupted by sudden drops in the mass estimates. These decreases in mass estimates appeared to be controlled by the predominance of a specific morphotype, for instance the low E. huxleyi mass values recorded at PS97/016-1 coincide with a 35 sudden increase in the relative abundance of E. huxleyi type C (Fig. 11).

Latitudinal variations in the coccolithophore abundance, distribution and diversity.
The observed maximum coccolithophore abundance recorded (up to 214.6*10 3 cells/L) is in agreement with previous studies carried out in different sectors of the Southern Ocean, which estimated maximum numbers between 130 and 640*10 3 cells/L (e.g., Eynaud et al., 1999;Findlay and Giraudeau, 2000;Cubillos et al., 2007;Gravalosa et al., 2008;Mohan et al., 2008;Hinz et al., 5 2012;Saavedra-Pellitero et al., 2014;Malinverno et al., 2015;Balch et al., 2016;Charalampopoulou et al., 2016). The coccolithophore abundance, diversity and maximum depth habitat drastically drop from North to South and portray the oceanographic fronts as ecological boundaries. Marked shifts in the coccolithophore numbers, community composition and diversity occurring at the SAF and PF observed here, were also previously noted by other authors in different sectors of the Southern Ocean (e.g., Eynaud et al., 1999;Gravalosa et al., 2008;Saavedra-Pellitero et al., 2014;Malinverno et al., 2015;Balch 10 et al., 2016;Charalampopoulou et al., 2016). Although the aforementioned studies reported increases in the abundance of coccolithophores at the SAF and PF, we only observe an increase in the number of cells/L in the PF at shallow depths (< 60 m, Fig. 3), which is not so evident in the SAF. The increase in coccolithophore abundance recorded in the PF could be linked to the high biological productivity occurring at the Antarctic Circumpolar Current fronts (e.g., Murphy, 1995;Pollard et al., 2002;Patil et al., 2013) due to the frontal dynamics itself (e.g., Laubscher et al., 1993) or to the physical accumulation of particulate matter 15 and nutrients at these convergence zones (e.g., Franks, 1992;Eynaud et al., 1999;Gravalosa et al., 2008;Balch et al., 2016).
The southernmost extent of E. huxleyi has been extensively discussed (e.g., Winter et al., 2014;Malinverno et al., 2015). The PF constitutes a natural sharp barrier which marks a drop in coccolithophore diversity and number of coccospheres (Saavedra-Pellitero et al., 2014;Saavedra-Pellitero and Baumann, 2015). Several studies observed the absence of E. huxleyi south of the PF (e.g., 20 Verbeek, 1989;Charalampopoulou et al., 2016). However, specimens of E. huxleyi and W. antarctica are sporadically recorded south of the PF in the studied transect with numbers of <3*10 3 cell/L in the uppermost 80 m of the water column at stations PS97/043-3 and /047-1. Emiliania huxleyi is observed in low numbers at temperatures between 1.7 and -0.7º C (Fig. 2), below the 2º C isotherm limit that McIntyre and Bé (1967) originally established for the Atlantic Southern Ocean. Although it is unusual, few authors occasionally found E. huxleyi also dwelling in cold waters < 2º C (see Table 1 in Holligan et al., 2010). Monospecific 25 assemblages of E. huxleyi have been also recorded south of the PF by other authors in the Pacific sector (e.g., Gravalosa et al., 2008;Saavedra-Pellitero et al., 2014), Australian sector (e.g., Nishida, 1986;Findlay and Giraudeau, 2000;Cubillos et al., 2007;Malinverno et al., 2015), in the Atlantic sector (e.g., Eynaud et al., 1999;Holligan et al., 2010) and Indian sector (e.g., Mohan et al., 2008;Patil et al., 2014). We speculate that the free detached coccoliths of E. huxleyi observed in our study area, down to 61.7ºS, and showing a broader distribution than coccospheres, are not in situ and could have been transported. In any case the southernmost 30 extent of coccolithophores is also influenced by the clear dominance of diatoms south of the PF, as suggested by the high diatom concentration (valves/g dry sediment) and biogenic opal content recorded in surface sediment samples from the AZ of the Drake Passage (Cárdenas et al., 2018) and from Pacific Southern Ocean extant plankton studies (e.g., Saavedra-Pellitero et al., 2014;Malinverno et al., 2016).

35
The number of taxa and coccolithophore diversity decreases southwards (Fig. 9) in agreement with other studies performed in the Drake Passage, the Australian and Pacific sectors of the Southern Ocean (e.g., Findlay and Giraudeau, 2000;Gravalosa et al., 2008;Saavedra-Pellitero et al., 2014;Charalampopoulou et al., 2016). Coccolithophore diversity is related to the temperature gradient, as shown by the correlation between Shannon index and SST (r=0.8, see supplementary material). Contrary to Saavedra-Pellitero et al. (2014), the highest coccolithophore diversity values do not always occur at stations that showed the highest coccolithophore 40 abundances (Figs. 3, 9). Few studies offshore Chile (ca. 33ºS, 36ºS) and in the Drake Passage showed low coastal coccolithophore diversity increasing towards open ocean regions (Charalampopoulou et al., 2016;Menschel et al., 2016;von Dassow et al., 2018) which contrasts with the high number of taxa recorded in this work at uppermost 100 m of the water column at the Chilean margin ( Fig. 9). Amongst different environmental factors, temperature could be one of the main variables favouring high coccolithophore diversity at the coastal stations. 5 The unexpected high diversity and number of taxa recorded at 10-20 m at station PS97/038-1 (previously labelled as "outlier"), coincident with relatively high density of coccospheres, does not seem to have been promoted by high SSTs, but rather by an occasional variation in the nutrient availability. So far, there are no nutrient measurements in situ available for this transect, but the interpolated values from the WOA13 austral summer suggest that nutrients are generally available this part of the SAZ, and 10 shallow (10, 20 m) nitrate or phosphate concentrations (Fig. 12 h, i) do not abruptly change at PS97/038-1. Therefore this could be due to mesoscale eddies which could have advected nutrients (Frenger et al., 2018). We speculate that an increase in the fluorescence values (Fig. 2), reflecting higher chlorophyll-ɑ concentrations, could be attributed to a higher availability of nutrients, which could have favored coccolithophores. The available fluorescence data (Fig. 2) seem to primarily reflect diatom concentration, south of the PF, followed by the non-coccolithophore haptophytes (Petasaria and Chrysochromulina spp.) in the 15 SAZ and PFZ (up to 100 m water depth) superimposed to the coccolithophore distribution. Future quantitative analyses of extant diatoms and nutrients performed at the same stations and depths are envisaged and will be required for better understanding of the phytoplankton community interactions and ecological patterns across the Drake Passage.

2. Community composition across the Drake Passage. 20
Based on the PCA (Fig. 8) it was possible to distinguish three main different oceanographic areas, characterized by different coccolithophore assemblages in the study area.
(1) The Chilean margin. Coccolithophores dwell up to 150 m water depth in this zone (Fig. 3). Emiliania huxleyi type A is present 25 in the stations closest to the Chilean coast (i.e., PS97/018-1 and /017-1), which recorded the highest SST and lowest SSS in the study area. This morphotype of E. huxleyi has been also observed in low abundances north of the SAF in different Pacific/Australian Southern Ocean (e.g., Cubillos et al., 2007;Saavedra-Pellitero et al., 2014;Malinverno et al., 2015), although was not observed by others authors (e.g., Gravalosa et al., 2008;Charalampopoulou et al., 2016), probably due to the high latitudes of those transects. Specimens of type A with different degree of calcification were present ranging from normal to overcalcified 30 (Plate 1), the latter being more abundant. In the Northern Hemisphere, E. huxleyi type A dominates the coccolithophore assemblage in the North Atlantic and in Norwegian coastal waters (e.g., van Bleijswijk et al., 1991;Holligan et al., 1993), but not in the Southern Ocean (e.g., Cook et al., 2011;Hagino et al., 2011). Emiliania huxleyi type R was observed for first time in the Drake Passage, although it has been previously observed off New Zealand (Langer et al., 2009; and in the Eastern South Pacific (Beaufort et al., 2008;Beaufort et al., 2011;von Dassow et al., 2018). 35 Other minor taxa present in the Chilean margin are Calcidiscus s.l., Ophiaster spp and G. muellerae. The first has already been found by other authors in the SAZ (e.g., Saavedra-Pellitero et al., 2014;Malinverno et al., 2015), but its patchy distribution north of the PF is in agreement with observations by Charalampopoulou et al. (2016). Ophiaster spp. shows uneven distribution in the SAZ and PFZ, but it is also present in the Chilean continental margin. Ophiaster spp. is recorded offshore Chile with low number living up to 100 m water depth, which contrast the high numbers observed in the SAZ offshore New Zealand always above 60 m (Saavedra-Pellitero et al., 2014). Occasional low numbers of extant G. muellerae have also been observed in the Southern Ocean by Saavedra-Pellitero et al. (2014) and Findlay and Giraudeau (2000).

5
(2) The SAZ. This oceanographic zone is bounded in the south by the SAF. Salinity values are relatively constant at about 34 psu, but SST gradually decreases, down to ca. 6º C, while the nitrate and phosphate contents progressively increase (Figs. 2, 12). The SAZ is characterized by the dominance of E. huxleyi types C, B/C, O and B. The maximum coccolithophore depth habitat progressively decreases southwards from 150 m in the transitional zone south of the chilean margin to 100 m in the open ocean (Fig. 3). The shift in occurrence from type A group to type B group has been recorded by some authors at the STF in the Australian 10 sector (e.g., Hiramatsu and De Deckker, 1996;Findlay and Giraudeau, 2000;Malinverno et al., 2015).  (Hagino et al., 2011). Emiliania huxleyi type O (including the opened and lamella forms) is abundant in the SAZ, which is in agreement with Malinverno et al. 20 (2015). Emiliania huxleyi type B/C and C (see Table 1) are the dominant taxa in the SAZ of the Southern Ocean (e.g., Findlay and Giraudeau, 2000;Cubillos et al., 2007;Gravalosa et al., 2008;Mohan et al., 2008;Saavedra-Pellitero et al., 2014;Saavedra-Pellitero and Baumann, 2015).
Among minor taxa, Syracosphaera spp., Calcidiscus sp., A. quattrospina as well as holococcolithophores are found in the SAZ, in 25 agreement with the assemblage observed by Charalampopoulou et al., (2016) north of the PF. Four species belonging to the genus Syracosphaera (plus S. strigilis HOL) are recorded in the study area, as previously observed (Gravalosa et al., 2008;Charalampopoulou et al., 2016). Acanthoica quattrospina, a species tolerant to low salinity (Supraha et al., 2014) is present in southern high latitudes (Eynaud et al., 1999;Findlay and Giraudeau, 2000;Malinverno et al., 2015), but has not been recorded in other polar transects (e.g., Mohan et al., 2008). 30 (3) The PFZ. This oceanographic zone is bounded by the SAF and the PF. Salinity and SST gradually decrease with respect to the SAZ, and nutrient contents continue to progressively increase poleward (Figs. 2, 12). The maximum coccolithophore depth habitat in this zone is restricted to 60 m. Emiliania huxleyi types B/C and C dominate the PFZ and reach relatively high numbers north of the PF, although E. huxleyi type O is still present in low abundance (Fig. 11), as also observed by Malinverno et al. (2015). The 35 high coccolithophore numbers observed in the SAZ and at the shallowest depths of the PFZ are part of the Great Calcite Belt, a region of high surface reflectance in the Southern Ocean due to the increased seasonal concentrations of coccolithophore and particulate inorganic carbon (Balch et al., 2011;Balch et al., 2016). We suggest that the uneven distribution of some of the coccolithophore taxa is driven by the physical processes of the ACC; i.e., it is primarily linked to the positions of frontal boundaries but also affected by the dynamics of mesoscale eddies, as mentioned by Holligan et al. (2010). 40 Minor taxa present in the PFZ, and broadly north of the PF include species of the family Papposphaeraceae (i.e., Papposphaera sp., Pappomonas spp. and W. Antarctica). They have small-sized and lightly calcified coccoliths, which makes them easily overlooked even under SEM. Specimens from the genera Papposphaera and Pappomonas have been observed at polar waters in the North Hemisphere (Thomsen, 1981;Samtleben and Schröder, 1992;Charalampopoulou et al., 2011;Thomsen and Østergaard, 5 2014) and also in the Southern Ocean (e.g., Gravalosa et al., 2008;Saavedra-Pellitero et al., 2014;Charalampopoulou et al., 2016).
Apart from the fact that the lightly calcified polar coccolithophores are non-photosynthetic heterotrophs, which gives them a strong competitive advantage to dwell in the darkness for months every year, very little is known about them (Thomsen and Østergaard, 2013). However, because they are weakly calcified, they will be one of the first polar taxa to be threatened by ocean acidification (Thomsen and Østergaard, 2013). 10

Emiliania huxleyi mass variations across the Drake Passage.
In this study, the coccolith mass of E. huxleyi was measured across the Drake Passage up to the PF at depths ranging between 10 and 20 m water depth. The general southwards decreasing trend in E. huxleyi mass (Fig. 11) is in agreement with trends observed 15 by Charalampopoulou et al. (2016) across the Southern Ocean (Fig. 13). Differences in the estimated masses can be attributed to the distinct taxonomical considerations, to the methodologies used in both studies, and mainly to the different oceanographic conditions during the sampling periods (2009,2016). The mean coccolith mass is related to strong latitudinal gradients in temperature (r=0.75), also observed by Charalampopoulou et al. (2016), total alkalinity (r=-0.89), total CO2 (r=-0.86), HCO3 -(r=-0.81 or -0.68 depending on the method used to calculate it) in agreement with Beaufort et al., (2011) and nutrient content (nitrate: 20 r=-0.75, phosphate=-0.71) noted by Charalampopoulou et al., (2016) (Table 4, Fig. 12). In contrast, the coccolith mass relationship to salinity, fluorescence, silicate content, carbonate ion, calcite saturation is not significant (Table 4). Still it could be argued that the poleward decrease in coccolith mass roughly coincides with a reduction in ΩCa (Fig. 12) and CO3 2− (Table 4) in agreement with different studies that found depressed coccolith calcification at low ΩCa and CO3 2− values (e.g., Riebesell et al., 2000;Beaufort et al., 2011). Although the pH variation is rather reduced, the anticorrrelation between coccolith mass and pH becomes significant 25 (r=-0.7) depending on the method used (Table 4, Fig. 12), in agreement with the biogeochemistry and optics South Pacific experiment (BIOSOPE) data from Beaufort et al (2011) (r=-0.52). The negative correlation of the present data contrasts with the global and well established relationship between coccolith mass and pH (r=0.75) (Beaufort et al., 2011). However, the relationship between coccolith mass and the carbonate chemistry parameters should be considered carefully. TALK, TCO2, ΩCa, pH and HCO3have been calculated from the GLODAP-v2 database (in which the majority of the datapoints are scattered and samples were 30 measured just in August 2005 and in February 2009) (Key et al., 2015) or from the global alkalinity and total dissolved carbon collection (Goyet et al., 2000) which shows an austral summer average using the CO2SYS.XLS program (Pierrot et al., 2006).
The observed decreasing trend of the coccolith mass can be linked to the latitudinal succession from type A group to type B group (Fig 12 b), in agreement with other authors who observed a latitudinal trend from E. huxleyi more calcified to weakly calcified 35 morphotypes (e.g., Cubillos et al., 2007;Mohan et al., 2008). In the Chilean margin, the highest coccolith masses recorded are related to the presence of E. huxleyi type A (including normal, calcified and overcalcified specimens) and type R (Fig. 11), observed also by other authors at lower latitudes offshore Chile (Beaufort et al., 2008;Beaufort et al., 2011;von Dassow et al., 2018).
Although it is uncommon, heavily calcified E. huxleyi morphotypes have been recorded in reduced pH and ΩCa conditions in other parts of the globe (e.g., Smith et al., 2012;Triantaphyllou et al., 2018). The presence of calcified specimens of E. huleyi type B/C, and in a lesser extent of type C, in the transitional zone from the chilean margin to the open ocean reflects an increase in the coccolith masses (Figs. 10, 11). In contrast, a higher relative abundance of E. huxleyi type C correponds to smaller coccolith masses in the SAZ. Striking are the relatively low coccolith mass values in the open ocean of the SAZ that coincide with maxima in the density of coccolithophores (Fig. 12). 5 The dataset presented here constitutes an important contribution to the coccolithophore ecology sparsely studied at high latitudes.
This work is also relevant for future climate and ocean model simulations in the context of global warming and ocean acidification threatening calcifying plankton. Taking into account the existing relationships between the physico-chemical parameters and the coccolithophore components, changes in the composition and calcification modes of E. huxleyi morphotypes are expected to occur 10 in the Drake Passage with the ongoing climate change. However, our study does not provide enough evidence to infer how coccolithophores will cope with a forthcoming changing ocean. We speculate that future sea surface warming and stratification (Boyd et al., 2008), concomitant with a southward migration of the Antarctic Circumpolar fronts, will lead to a increase in the numbers of E. huxleyi at higher latitudes and to a potential higher calcification poleward in agreement with the model from Krumhardt et al. (2017). At the same time, pH, CO3 2− and ΩCa are predicted to decrease (e.g., Hauri et al., 2015), which will also 15 affect coccolithophores. Based on our limited data, conflicting conclusions can be drawn from the carbonate parameters. We could hypothesize that E. huxleyi will calcify more in a future Southern Ocean scenario at lower pH, consistent with few culture experiments (e.g., Iglesias-Rodriguez et al., 2008) but also that this species will reduce its calcification rate at lower CO3 2− , ΩCa and higher HCO3 − , in agreement with the negative effects of ocean acidification suggested by several authors (e.g., Riebesell et al., 2000). Bearing in mind that culture experiments in different strains of E. huxleyi have already shown different responses to 20 changing carbonate chemistry (Langer et al., 2009), it seems necessary to consider also the degree of adaptive potential of coccolithophores in future studies to predict their upcoming performance in the Polar realm.

Conclusions
This study documents the latitudinal and the depth variability in the coccolithophore assemblage composition and calcification of 25 Emiliania huxleyi, the dominant species, across the Drake Passage, driven by physical, chemical and biological parameters in the surface ocean. Coccolithophore abundance, diversity and maximum depth habitat decrease southwards portraying the oceanographic fronts as ecological boundaries. Marked shifts in the coccolithophore numbers, community composition and diversity occur at the Subantarctic Front (SAF) and Polar Front (PF). Three main different oceanographic areas are characterized, based on the coccolithophore composition: 30 (1) The Chilean margin. Emiliania huxleyi type A (normal and overcalcified) and type R are present in the stations closest to the Chilean coast, which record the highest SST and lowest SSS in the study area. Rare taxa present offshore Chile are Calcidiscus s.l., Ophiaster spp. and Gephyrocapsa muellerae. (including holococcolithophores from this genus), Calcidiscus sp. and Acanthoica quattrospina.
(3) The Polar Front Zone (PFZ). It is bounded by the SAF in the north and the PF in the south. Salinity and SST progressively decreases with respect to the SAZ, and nutrient contents continue to increase poleward. Emiliania huxleyi types B/C and C dominate the PFZ and reach relatively high numbers north of the PF, although E. huxleyi type O is still present. Minor taxa present in the PFZ, and broadly north of the PF include species of the family Papposphaeraceae (i.e., Papposphaera sp, Pappomonas spp. and Wigwamma antarctica). Specimens of E. huxleyi and W. antarctica are sporadically recorded south of the PF with numbers of 5 <3*10 3 cell/L and dwelling a at temperatures <2° C.
The general decreasing trend in E. huxleyi coccolith mass can be linked to the latitudinal succession from type A group (in the Chilean margin) to type B group (in the PFZ). Coccolith mass and coccolithophore diversity are related to the strong latitudinal gradient in temperature. Coccolith mass also shows anticorrelation to total alkalinity, total CO2, bicarbonate ion (HCO3 -), pH and 10 nutrient content, which contrasts with the global and well established positive relationship between coccolith mass and pH as well as total alkalinity. However, the relationship between coccolith mass and the carbonate chemistry parameters should be considered carefully, since in situ measurements are not available. The existing relationships between the physico-chemical parameters and the coccolithophore components in the Drake Passage suggest that assemblage composition and calcification modes of E. huxleyi will be strongly affected by the ongoing climate change. 15