Amorphous calcium carbonate loaded multilamellar vesicles within Amphistegina (Rotaliida) foraminifera

Foraminifera play a crucial role in the global carbon cycle largely due to their prolific biocalcification. Mineralization of shells in Rotaliid Foraminifera have been interpreted to follow two contrasting pathways. The first model assumes in situ mineralization of extracellular organic matrix by attracting calcium and bicarbonate ions that are taken up directly via trans-membrane transport from seawater and/or from the cytosol. The second model assumes that the foraminiferal biocalcification is associated with the production of intracellular amorphous calcium carbonate (ACC), a precursor phase for the formation of calcite shells. However, foraminiferal ACC has not been directly documented. Here, we show evidence of intracellular ACC-bearing vesicles in Rotaliid Foraminifera with a combination of in vivo fluorescence studies, Raman spectroscopy applied to fresh and untouched intracellular vesicles, laboratory synthesis experiments, and chemical/mineralogical analysis of FIB-lamellae from fixed vesicles. We show that foraminiferal ACC vesicles are a natural example of multilamellar vesicles, with a SiO 2 -core and several layers with differing chemical compositions, likely enabling rapid and efficient ACC production and stabilization. Moreover, we tested and described in detail the first non-destructive autofluorescence method enabling in vivo ACC imaging. This might be applicable to various organisms and increase our understanding of biocalcification throughout the tree of life. Recognition of Foraminifera calcification process is a precondition to correctly read geochemical parameters archived in biocalcite that are commonly used as reliable proxies for past environmental and climatic conditions, but also to understand carbonate sedimentation that drives the global C cycle. This work presents the key facts to support foraminifera calcification mechanism by true high-Mg amorphous calcium carbonate (Mg-ACC) production within intracellular vesicles. This is a strong argument against commonly existing extracellular foraminiferal (Rotaliida) biomineralisation theory which states that Foraminifera secrete calcium carbonate on an outer organic membrane by attracting Ca 2+ and bicarbonate ions that are taken up directly via trans-membrane transport.