A stable ultrastructural pattern despite variable cell size in Lithothamnion corallioides

Recent advances on the mechanism and pattern of calcification in coralline algae led to contradictory conclusions. The evidence of a biologically controlled calcification process, resulting in distinctive patterns at the scale of family, was observed. However, the coralline calcification process has been also interpreted as biologically induced because of the dependency of its elemental composition on environmental variables. To clarify the matter, five collections of Lithothamnion corallioides from the Atlantic Ocean and the Mediterranean Sea, across a wide depth range (12-66 m), have been analyzed for morphology, anatomy and cell wall crystal patterns in both perithallial and epithallial cells to detect possible ultrastructural changes. L. corallioides shows the alternation of tiers of short-squared and long-ovoid/rectangular cells along the perithallus, forming a typical banding. The perithallial cell length decreases according to water depth and growth rate, whereas the diameter remains constant. Our observations confirm that both epithallial and perithallial cells show primary (PW) and secondary (SW) calcite walls. Rectangular tiles, with the long axis parallel to the cell membrane forming a multi-layered structure, characterize the PW. Flattened squared bricks characterize the SW, with roundish outlines enveloping the cell and showing a zigzag and cross orientation. Long and short cells have different thicknesses of PW and SW, increasing in short cells. Epithallial cells are one to three flared cells with the same shape of the PW and SW crystals. Despite the diverse seafloor environments and the variable L. corallioides growth rate, the cell walls maintain a consistent ultrastructural pattern with unaffected crystal shape and arrangement. A comparison with two congeneric species, L. minervae and L. valens, showed similar ultrastructural patterns in the SW but evident differences in the PW crystal shape. Our observations point to a biologically control rather than an induction of the calcification process in coralline algae and suggest a possible new morphological diagnostic tool for species identification, with relevant importance for paleontological applications. Finally, secondary calcite, in the form of dogtooth crystals that fill the cell lumen, has been observed. It represents a form of early alteration in living collections which can have implications in the reliability of climate and paleoclimate studies based on geochemical techniques.