Energy dispersive X-ray spectroscopy (EDX)
For analysis of the elemental composition, three cleaned radulae (ultrasonic bath for 20 seconds) were attached to glass object slides by double-sided adhesive tape, following our previous protocol (Krings et al., 2022a, 2022c, 2023). Then, each radula was surrounded by a small metallic ring. Afterwrads the ring was filled with epoxy resin (Reckli Epoxy WST, RECKLI GmbH, Herne, Germany) to cover the readula completely. After polymerization, lasting for three days at room temperature, glass object slides and adhesive tape were removed. Samples were polished with sandpapers of different roughness until teeth were on display and smoothened with aluminum oxide polishing powder suspension of 0.3 μm grainsize (PRESI GmbH, Hagen, Germany) on a polishing machine (Minitech 233/333, PRESI GmbH, Hagen, Germany) to receive a plain smooth surface. The embedding and smoothening prevent artefacts such as electron scattering during EDX analysis. Embedded samples were subsequently cleaned in an ultrasonic bath for five minutes, then mounted on SEM sample holders and sputter-coated with platinum (5 nm layer). Elemental composition was determined with the SEM Zeiss LEO 1525 equipped with an Octane Silicon Drift Detector (SDD) (micro analyses system TEAM, EDAX Inc., New Jersey, USA). For each sample, the same settings were used (i.e. an acceleration voltage of 20 kV, working distance, lens opening, etc.). Before analysis, the detector was calibrated with copper.
Small areas (no mapping) were analyzed to receive the data. Following elements were detected and their proportions measured.: H (hydrogen), C (carbon), N (nitrogen), O (oxygen), Pt (platinum), Al (aluminum), Ca (calcium), Cl (chlorine), Cu (copper), Fe (iron), K (potassium), Mg (magnesium), Na (sodium), P (phosphorus), S (sulphur), Si (silicon), and Zn (zinc). Some elements were not discussed as they are either the elemental basis of chitin and proteins (H, C, N, O), the coating (Pt), or the polishing powder (Al, O). For test purposes, we also performed 10 EDX tests on the epoxy to identify putative pollution due to the mechanical application, embedding or polishing. We could not detect Si (which is part of the sandpaper), or any other elements, that we further discuss as Ae (Ca, Cl, Cu, Fe, K, Mg, P+Pt, S, Si, Zn), in the resin. Their presence is therefore considered part of the teeth.
The single peak of P overlaps with one of Pt. Due of this, the software could not discriminate between these two elements and P content could not be reliably determined. Therefore, P and Pt are discussed together (P+Pt). We, however, measured 20 areas of pure epoxy to receive values on their Pt content (mean ± SD; 0.15 ± 0.02 atomic %) to further estimate the proportions of P in the teeth.
We tested the inner tooth structure by EDX and the thin outer layer (“surface”) of the teeth (500–1000 nm thickness), which covers the inner tooth structure. We did not detect high content of elements in the inner structure and no differences in the distribution there. We thus decided to summarize the point measurements of the inner structure. With regard to the surfaces, we could determine variations in the distribution of elements and thus differentiated between the tooth basis, the bulges, the basal region of the stylus (stylus, basis), the terminal region of the stylus (stylus, terminal), the cusps and the sides (see Figures 1 and 2 for nomenclature). 474 point measurements on 180 mature teeth were conducted: 204 on 70 inner laterals (of which 70 on the inner tooth structure and 134 on the surface); 56 (22 on the inner tooth structure and 34 on the surface) on 22 outer laterals A, B, C; 54 (22 on the inner tooth structure and 32 on the surface) on 22 outer laterals D; 48 (22 on the inner tooth structure and 26 on the surface) on 22 outer laterals E.