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Evidence of high Sr/Ca in a Middle Jurassic murolith coccolith species

Paleoceanographical reconstructions are often based on microfossil geochemical analyses. Coccoliths are the most ancient, abundant and continuous record of pelagic photic zone calcite producer organisms. Hence, they could be valuable substrates for geochemically based paleoenvironmental reconstructions but only Sr/Ca is exploited even if it remained partially understood. For example, some murolith coccoliths species have very high Sr/Ca compared to the common 1-4 mmol/mol recorded in placolith coccoliths. In this study, we analyzed the elemental composition of the Middle Jurassic murolith Crepidolithus crassus by synchrotron-based nanoXRF (X-ray Fluorescence Spectroscopy) mapping focusing on Sr/Ca and compared the record to two placolith species, namely Watznaueria contracta and Discorhabdus striatus. In C. crassus, Sr/Ca is more than ten times higher than in both placoliths and seems higher in the proximal cycle. By comparison with the placoliths analyzed in the same analytical set-up and from the same sample, we exclude the impact of the diagenesis and seawater Sr/Ca to explain the high Sr/Ca in C. crassus. Based on comparisons to Pontosphaera discopora and Scyphosphaera apsteinii which also have high Sr/Ca, it seems more likely that high Sr/Ca in C. crassus is either due to the vertical elongation of the R-units of the proximal cycle or related to the action of the special polysaccharide controlling the growth of those vertically elongated R-units that may have affinities to Sr. In order to apply the Sr/Ca proxy to muroliths, further investigations are needed on culture experiments.


Introduction
Paleoceanography partly relies on the application of geochemical proxies -i.e. chemical changes of fossils or sediments composition induced by chemical, physical or biological parameters changing through time. Many geochemical proxies are based on elemental ratios in planktic or benthic foraminifera e.g. composition is the Sr/Ca ratio used as a paleoproductivity proxy (Stoll and Schrag, 2000). to Ullmann et al., 2013); ii) the calcium carbonate polymorph; iii) the species specific fractionation of the Sr/Ca ratio; iv) metabolic controls on this fractionation factor; and v) water temperature. The Sr/Ca ratio in calcareous nannofossils is a relative proxy which has no quantitative calibration. The process underlying the positive relation between Sr/Ca and productivity stands on the observation that more Sr is incorporated in calcite with high calcification rates which positively correlates with high cell physiological rates. This proxy is also species-dependent or, at least, group-dependent (Stoll and Ziveri, 2004). The latter is discussed, the species-dependency may be related to different calcification physiologies (Payne et al., 2008;Suchéras-Marx et al., 2016a) or to different partitioning coefficients in relation to different ultrastructure organizations and more precisely between V-and R-crystals in coccoliths (Young et al., 1992) theoretically possible (Paquette and Reeder, 1995) but still not observed (Stoll and Ziveri, 2004).

The most important factors influencing Sr/Ca ratios in biogenic carbonates is outlined in
Recently, analyses on the cultured murolith Scyphosphaera apsteinii showed Sr/Ca ∼22 mmol/mol, an order of magnitude higher than the common ∼1-4 mmol/mol measured in placoliths such as Gephyrocapsa oceanica, Gephyrocapsa huxleyi, Calcidiscus leptoporus, Coccolithus pelagicus or Helicosphaera carteri (Stoll et al., 2007;Hermoso et al., 2017). Hermoso et al. (2017) also highlight observations of high Sr/Ca in fossil muroliths Pontosphaera (Pliocene) and Crepidolithus (Lower Jurassic) but points out that Scyphosphaera, Pontosphaera and Crepidolithus have different crystal organizations and growth directions and thus crystal organization cannot explain murolith high Sr/Ca. The present study aims to map Sr and Ca in Crepidolithus crassus and to compare the calculated Sr/Ca with the crystal growth directions in order to explain the murolith Sr anomaly. This study will discuss then the range of applicability and limitation of the Sr/Ca proxy in coccoliths.

Material
The coccolith analyzed in this study comes from sample CM35, a marlstone from the lower Bajocian In the three coccoliths, membrane contribution in Ca, Fe, Cu and Zn and in Sr in C. crassus is observed. However, it is always two order of magnitude below the concentration in the coccolith thus membrane contribution represents less than 1% in those elements.

Sample preparation
The three coccoliths were picked using Suchéras-Marx et al. (2016b) protocol which consists in a handpicking procedure. Rock powder is smeared on a cover slide in order to isolate particles from each other.
Coccoliths are then observed with an optical microscope Leica DM750P with a x200 magnification (x20 dry objective) under cross-polarized light. Coccoliths are identified with a x400 magnification (x40 dry objective) and a λ/4 gypsum filter may be used if further optical criteria of recognition are needed. Once picked, each coccolith is mounted on 500 nm-thick silicon nitride (Si 3 N 4 ) TEM windows (Silson Ltd. Southam, UK) using a drop of ethanol to detach the coccolith from the needle. No other chemical or physical treatment were made.

NanoXRF 17 keV mapping at ID22NI
The XRF analyses were performed at beamline ID22NI (currently ID16b) at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. All three coccoliths were analyzed at an incident X-ray beam energy of 17 keV to ensure the measure of Sr (Kα = 14.165 keV). The beam spot was focused by an ESRF

XRF spectrum
The three spectra shown in Figure 1 represent the mean spectrum for each coccolith. Because XRF analysis is penetrative, the spectra also record the membrane holding the coccolith and thus the spectrum of zones with only the membrane is also presented. The fit presented for each spectrum is the modeled reconstruction used for the calculation and the map reconstruction (cps and mmol/mol). In D. striatus and W. contracta, 14 elements are recorded namely: S, Cl, Ar, K, Ca, Mn, Fe, Cu, Zn, Br, Kr, Rb, Sr and Pb whereas in C. crassus only 12 are recorded (same elements except Kr and Rb) (Fig. 1)  For the same species, the Mn is scarce in the central area and the tube structures but more abundant in the shields.

Element and ratio maps
Conversely to the placoliths, in C. crassus, the Ca and Sr maps are slightly different. In Sr maps, the central area is easily visible. Around the central area, an inner concentric rim of more abundant Sr is observed. A concentric outer rim up to the lateral border is observed with Sr concentrated in radial rays.
In the Ca map, the outer rim is also observed in radial structures but the inner rim is distinguished from DOI: 10.31233/osf.io/dcfuq | Peer-reviewed by Peer Community in Paleontology 5 of 13

Figure 3. Sr/Ca and Mn/Ca maps (mmol/mol) of C. crassus, D. striatus and W. contracta.
the outer only by a discontinuity. Finally, another discontinuity separates the central area from the inner rim.
For all species, the Sr/Ca maps have the same patterns as Sr and similarly, the Mn/Ca maps have the same patterns as Mn (Fig. 3). In C. crassus then, the Sr/Ca is higher in a ring around the central area and in radially oriented crystals in the outer ring. In the most concentrated area, the Sr/Ca ranges between 10 and 100 mmol/mol and tends to be two orders of magnitude higher than in W. contracta and D. striatus.
Finally, the area with higher Sr/Ca is neither enriched nor depleted in Mn/Ca. and are low in diagenetic fluids (Veizer, 1974; James and Jones, 2015).
The Sr anomaly in C. crassus is then related to either: i) an ion pump concentrating the Sr 2+ inside the cell to higher concentrations than other species; ii) the use of a polysaccharide that tends to increase Sr/Ca in comparison to other species; or iii) the crystal organization and growth directions which are controlled by the cell but the high concentration in Sr would then be a by-product of the coccolith construction. Obviously, C. crassus being a fossil occurring only during the Jurassic, the cell biology of the species cannot be explored. Nevertheless, this species is a large murolith like the extant Pontosphaera discopora and has a similar shape to the extant Scyphosphaera apsteinii's lopadolith, both having also higher Sr/Ca than the common placolith signature of 1-4 mmol/mol (Hermoso et al., 2017).
The first two hypotheses (i.e. Sr 2+ ion pump or special polysaccharide) could be that high Sr/Ca is inherited in descendant species from C. crassus. Both Pontosphaera and Scyphosphaera are very closely related in the coccolithophore phylogeny (family Pontosphaeraceae; De Vargas et al., 2007). Crepidolithus is within the family Chiastozygaceae, whose position is unknown, but could be the ancestor of the family Zygodiscaceae and its descendants, the families Pontosphaeraceae and Helicosphaeraceae (Bown and Young, 1998). Thus, the high Sr/Ca in both Pontosphaera and Scyphosphaera could be an ancestral character inherited from Crepidolithus. This hypothesis may be challenged by Helicosphaera carterii which has a Sr/Ca ratio around 2-3 mmol/mol (Stoll and Ziveri, 2004) and is from the family Helicosphaeraceae, the sister group of the family Pontosphaeraceae (Bown and Young, 1998  V-unit) from a primary ring of crystals called protococcolith (Young et al., 1992). In C. crassus, the V-units are more developed than the R-units (Fig. 4), whereas in P. discopora and S. apsteinii V-units represent only the narrow outer part of the rim wall (Young and Bown, 1997; Fig. 4). According to Hermoso et al.
(2017), the growth of calcite in the same direction as the c-axis should favor Sr 2+ incorporation but the R-units in P. discopora and S. apsteinii actually grow longer in the vertical axis hence grow orthogonally to the c-axis. In the case of C. crassus, the Sr/Ca is higher around the central area which corresponds to the proximal cycle formed by R-units. Counterintuitively for these three species with thick vertical walls, the Sr is concentrated in the R-units. Surprisingly, C. crassus R-units are, like P. discopora and S. apsteinii, more elongated vertically than radially, thus the growth seems orthogonal to the c-axis (Fig. 4). Then, either the orthogonal to c-axis growth actually favors Sr 2+ contradicting Hermoso et al. (2017) or the biomolecule acting in this growth direction has more affinities with Sr 2+ than the other polysaccharides.

Conclusion
Despite the very high and continuous fossil record of coccoliths since the Late Triassic, the use of geochemical analyses on these calcite platelets for paleoenvironmental reconstructions remain rare. So far, coccolith Sr/Ca is the only coccolith-based geochemical proxy commonly used in paleoceanography. Yet, there are many unknown features about this productivity proxy such as the very high Sr/Ca (i.e. above ∼10 mmol/mol) in some species namely S. apsteinii, P. discopora and C. crassus. In this study, we compared Sr/Ca of C. crassus to contemporaneous placoliths using synchrotron-based nanoXRF and observed that: • C. crassus has higher Sr/Ca than contemporaneous placoliths; • C. crassus Sr/Ca map is different than the Mn/Ca map; • Sr/Ca in C. crassus is higher in the proximal cycle than in the distal cycle.
Hence, high C. crassus Sr/Ca, is not linked to diagenetic overgrowth or seawater Sr/Ca but may be related to vertical elongation of R-units and/or affinity to Sr 2+ of the polysaccharide responsible for the growth of those peculiar R-units. The use of murolith's Sr/Ca in the future may be interesting due to their high values, easier to measure and to the large size of these coccoliths, easier to separate from the bulk placoliths. Nevertheless, in order to apply the Sr/Ca productivity proxy to muroliths in the future, DOI: 10.31233/osf.io/dcfuq | Peer-reviewed by Peer Community in Paleontology 8 of 13

High Sr/Ca in a Middle Jurassic murolith coccolith
new culture studies should test the relation between Sr incorporation and growth rate. The study of Pontosphaera or Scyphosphaera with 3D nanoXRF will directly link Sr/Ca relative concentration with coccolith crystal organization and thus would clearly improve the understanding of the Sr anomaly in those coccoliths.