The classification of coralline algae commonly relies on the morphology of cells and reproductive structures, along with thallus organization, observed through Scanning Electron Microscopy (SEM). Nevertheless, species identification based on morphology often leads to uncertainty, due to their general plasticity. Evolutionary and environmental studies featured coralline algae for their ecological significance in both recent and past Oceans and need to rely on robust taxonomy. Research efforts towards new putative diagnostic tools have recently been focused on cell wall ultrastructure. In this work, we explored a new classification tool for coralline algae, using fine-tuning pretrained Convolutional Neural Networks (CNNs) on SEM images paired to morphological categories, including cell wall ultrastructure. We considered four common Mediterranean species, classified at genus and at the species level (Lithothamnion corallioides, Mesophyllum philippii, Lithophyllum racemus, Lithophyllum pseudoracemus). Our model produced promising results in terms of image classification accuracy given the constraint of a limited dataset and was tested for the identification of two ambiguous samples referred to as L. cf. racemus. Overall, explanatory image analyses suggest a high diagnostic value of calcification patterns, which significantly contributed to class predictions. Thus, CNNs proved to be a valid support to the morphological approach to taxonomy in coralline algae.

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.

The $\mathrm{B}/\mathrm{Ca}$ ratio in calcareous marine species is informative of past seawater CO${}_{\mathrm{3}}^{\mathrm{2}-}$ concentrations, but scarce data exist on $\mathrm{B}/\mathrm{Ca}$ in coralline algae. Recent studies suggest influences of temperature and growth rates on $\mathrm{B}/\mathrm{Ca}$ , the effect of which could be critical for the reconstructions of surface ocean pH and atmospheric pCO₂. In this paper, we present the first laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) analyses of Mg, Sr, Li, and B in the coralline alga Lithothamnion corallioides collected from different geographic settings and depths across the Mediterranean Sea and in the Atlantic Ocean. We produced the first data on putative temperature proxies ($\mathrm{Mg}/\mathrm{Ca}$ , $\mathrm{Li}/\mathrm{Ca}$ , $\mathrm{Sr}/\mathrm{Ca}$ , $\mathrm{Mg}/\mathrm{Li}$ ) and $\mathrm{B}/\mathrm{Ca}$ in a coralline algal species grown in different basins from across the photic zone (12, 40, 45, and 66 m depth). We tested the $\mathrm{B}/\mathrm{Ca}$ correlation with temperature proxies and growth rates in order to evaluate their possible effect on B incorporation. Our results suggested a growth rate influence on $\mathrm{B}/\mathrm{Ca}$ , which was evident in the sample with the lowest growth rate of 0.10 mm yr⁻¹ (Pontian Isl., Italy; 66 m depth) and in Elba (Italy; 45 m depth), where the algal growth rate was the highest (0.14 mm yr⁻¹). At these two sites, the measured $\mathrm{B}/\mathrm{Ca}$ was the lowest at 462.8 ± 49.2 µmol mol⁻¹ and the highest at 757.7 ± 75.5 µmol mol⁻¹, respectively. A positive correlation between $\mathrm{B}/\mathrm{Ca}$ and temperature proxies was found only in the shallowest sample from Morlaix (Atlantic coast of France; 12 m depth), where the amplitude of temperature variation (ΔT) was the highest (8.9 ^∘C). Still, fluctuations in $\mathrm{B}/\mathrm{Ca}$ did not mirror yearly seasonal temperature oscillations as for $\mathrm{Mg}/\mathrm{Ca}$ , $\mathrm{Li}/\mathrm{Ca}$ , and $\mathrm{Sr}/\mathrm{Ca}$ . We concluded that growth rates, triggered by the different ΔT and light availability across depth, affect the B incorporation in L. corallioides.

Calcareous red algae are foundation species and ecosystem engineers with a global distribution. The principles governing their calcification pathways are still debated and the morphological characters are frequently unreliable for species segregation, as shown by molecular genetics. The recent description of the new species Lithophyllum pseudoracemus, previously undetected and morphologically confused with Lithophyllum racemus, offered a challenging opportunity to test the effectiveness of microanatomy and ultrastructural calcification traits as tools for the identification of these two species, for integrative taxonomy. High resolution SEM images of molecularly identified samples showed that the different size of the perithallial cells and the features of the asexual conceptacle chambers may contribute to the separation of the two species. The two species share the same crystallite morphology in the primary and secondary cell-wall calcification, as previously described in other species belonging to the same clade. However, the perithallial secondary calcification was significantly thicker in L. racemus than in L. pseudoracemus. We described a granular calcified layer in the innermost part of the cell wall, as a putative precursor phase in the biomineralization and formation of the secondary calcification. The hypothesis of different pathways for the formation of the primary and secondary calcification is supported by the observed cell elongation associated with thicker and higher Mg/Ca primary calcification, the inverse correlation of primary and secondary calcification thickness, and the absence of primary calcification in the newly formed wall cutting off an epithallial cell from the meristem.

The B / Ca ratio in calcareous marine species is informative of past seawater CO₃²⁻ concentrations, but scarce data exist on B / Ca in coralline algae (CA). Recent studies suggest influences of temperature and growth rates on B / Ca, the effect of which could be critical for the reconstructions of surface ocean pH and atmospheric pCO₂. In this paper, we present the first LA-ICP-MS analyses of Mg, Sr, Li and B in the CA Lithothamnion corallioides collected from different geographic settings and depths across the Mediterranean Sea and in the Atlantic Ocean. We produced the first data on temperature proxies (Mg, Li and Sr / Ca) and B / Ca in a CA species grown in different Basins (the Mediterranean Sea and the Atlantic Ocean), from shallow to deep waters (12 m, 40 m, 45 m and 66 m depth). We tested the B / Ca correlation with temperature proxies and growth rates, in order to evaluate their possible effect on B incorporation. Our results showed a growth rate influence on B / Ca, especially in the deepest sample (Pontian Isl., Italy; 66 m) and in the shallowest sample (Morlaix, Atlantic coast of France; 12 m), where the growth rates were respectively 0.11 mm/yr and 0.13 mm/yr and the B / Ca was respectively 462.8 ± 49.2 μmol/mol and 726.9 ± 102.8 μmol/mol. A positive correlation between B / Ca and the temperature proxies was found only in Morlaix, where the seasonal temperature variation (ΔT) was the highest (8.90 °C). These pieces of evidence suggest that growth rates, triggered by the different ΔT and light availability across depth, affect the B incorporation in L. corallioides.