DOI: 10.5061/dryad.qbzkh18x7
title: Convergent evolution of failure- and wear-prevention in radulae of rock-scraping land snails (Chondrinidae, Gastropoda)

description: This study investigates the morphology, material, and mechanical
 properties of radular teeth in six lineages of Chondrinidae, a family of
 terrestrial gastropods. We examined three species that forage on biofilms
 from mixed substrates (Abida secale, Granopupa granum, Granaria frumentum)
 and three that specialised in scraping lichen off rock faces (Chondrina
 arcadica, Rupestrella rhodia, Solatopupa similis). Using scanning electron
 microscopy (SEM), nanoindentation, and energy-dispersive X-ray
 spectroscopy, we analysed tooth morphology, hardness H, Young’s modulus E,
 and elemental composition. Our findings confirm that the central radular
 region reflects substrate preferences, with rock-scraping species
 exhibiting reduced tooth denticles and significantly higher E and H values
 compared to mixed substrate feeders. Correlations between mechanical
 properties and elemental composition (increased calcium or silicon
 content) suggest convergently evolved strengthening mechanisms in
 rock-scraping species. The results support hypotheses of parallel
 evolution in radular adaptations, highlighting Chondrinidae as a valuable
 model for studying the interplay of morphology, function, ecology, and
 evolution.
description: <em>Energy dispersive X-ray
 spectroscopy</em> To analyse the elemental
 composition of the teeth using energy-dispersive X-ray spectroscopy (EDX,
 EDS), the same radulae were used that were previously documented in SEM.
 In total, 1495 small areas (1 x 1 µm) of three different radular regions
 (central, lateral, and marginal regions; see Supplementary Figure 2 for
 radular regions) were successfully analysed (for the exact quantity of
 measurements per region and species, see Supplementary Table 2).
 Following established procedure [37,51], radulae were attached to
 glass slides with double-sided adhesive carbon tape, commonly used in SEM
 analysis. The adhesive was used because it appeared not to infiltrate the
 teeth. Each radula was surrounded by a small metallic ring, which was
 filled with epoxy resin (Reckli Epoxy WST, RECKLI GmbH, Herne, Germany) to
 fully encase the radula. After the resin polymerized for three days at
 room temperature, we removed the glass slides and adhesive tape. The
 samples were then polished using sandpapers of varying roughness until the
 cross-sections of the teeth became visible, followed by further smoothing
 with a 0.3 μm aluminium oxide polishing powder on a polishing machine
 (Minitech 233/333, PRESI GmbH, Hagen, Germany) to achieve a uniformly
 smooth surface. This embedding and smoothing process was essential to
 ensure a flat sample surface, minimizing electron scattering during
 subsequent EDX analysis. The embedded samples were
 cleaned in an ultrasonic bath for five minutes, mounted on SEM sample
 holders, and sputter-coated with a 5 nm layer of platinum. Elemental
 composition analysis was carried out using the SEM Zeiss LEO 1525,
 equipped with an Octane Silicon Drift Detector (SDD) (microanalysis system
 TEAM, EDAX Inc., Mahwah, USA). Consistent settings were applied across all
 tests (e.g., 20 kV acceleration voltage, same working distance, and lens
 opening). The detector was calibrated using copper prior to
 analysis. We performed point analyses (without mapping)
 on small areas on the cross-section of cusps (for region see Figure 1A) to
 identify the presence of various elements, including Al (aluminium), C
 (carbon), Ca (calcium), Cl (chlorine), Cu (copper), F (fluorine), Fe
 (iron), K (potassium), Mg (magnesium), N (nitrogen), Na (sodium), O
 (oxygen), P (phosphorus), Pt (platinum), S (sulphur), Si (silicon), and Zn
 (zinc) (for representative EDX spectra, see Supplementary Figures 3 and
 4). Elements like C, N, Na, and O were not discussed in detail as they are
 integral to chitin and proteins, while Al and O could come from the
 polishing powder. Additionally, C, N, and O are light elements; they
 cannot be reliably quantified. Quantitative analyses and statistical
 evaluations were restricted to heavier elements, which can be robustly
 measured with this technique. For quality control, we
 conducted 10 additional EDX tests on the epoxy to check for contamination
 from mechanical application, embedding, or polishing. No Si (from the
 sandpaper), Al (from the polishing powder), or any of the discussed
 elements (Ca, Cl, Cu, Fe, K, Mg, P, S, Si, Zn) associated with the resin
 composition were detected, confirming that the elements found in the teeth
 were not due to contamination. Due to the overlap of
 phosphorus (P) and platinum (Pt) peaks, the software could not distinguish
 between these two elements reliably. Therefore, P content was discussed
 together with Pt (P+Pt). To estimate the proportion of P in the teeth, we
 measured the Pt content in 20 areas of pure epoxy, yielding a mean value
 of 0.14 ± 0.02 atomic %. The samples were then used for
 nanoindentation of the teeth.
 <em>Nanoindentation</em> For
 estimating the mechanical properties of the cusps (for the region see
 Figure 1A), nanoindentation was carried out on the embedded and polished
 samples previously analysed by EDX. We followed previous protocols
 [51,57]. A nanoindenter SA2 (MTS Nano Instruments, Oak Ridge, USA),
 equipped with a Berkovich indenter tip and a dynamic contact module (DCM)
 head, was used for the measurements. Both effective hardness
 (<em>H</em>) and Young’s modulus (<em>E</em>) were
 determined from force-distance curves obtained using the continuous
 stiffness mode (CSM). <em>E</em> was calculated using the
 Oliver–Pharr method [68]. The Poisson’s ratio was set to 0.3, and the
 allowable drift rate was set to 0.1 nm/s. All tests were conducted under
 normal room conditions (relative humidity 28–30%, temperature 22–24 °C),
 with each indent and corresponding curve manually monitored. Due to the Pt
 sputter coating, effective <em>E</em> and
 <em>H</em> were determined at penetration depths ranging from
 600 to 800 nm. The metallic coatings may influence nanoindentation if the
 coating thickness is comparable to the indentation depth. In our case, the
 sputter coating was ~5 nm thick, while indentation depths were 600–800 nm
 (&gt;100× thicker). Given that the Oliver-Pharr analysis relies on
 contact depth at these scales, the thin Pt layer should have had no
 measurable influence on hardness or modulus. Approximately 40 values were
 obtained at this indentation depth for each site, and the average was used
 to calculate one mean <em>H</em> and one mean
 <em>E</em> value per indent. In total, nanoindentation was
 successfully performed on 1495 cross-sections of the cusps.
 <em>Statistical analyses</em> All
 statistical analyses were performed using JMP Pro, Version 14 (SAS
 Institute Inc., Cary, USA, 1989–2007). The elemental composition data
 obtained via EDX were recognized as compositional, given that the
 measurements represent proportions that sum to a constant total (100%). To
 address the inherent closure constraint and avoid spurious correlations
 associated with standard statistical methods, we processed the data using
 compositional data analysis. This was not done for the mechanical property
 data, Young’s modulus, and hardness. For the elemental data, we replaced
 components with zero values using a small imputation value (0.0001) to
 allow for log-ratio transformation. Then, we calculated the geometric mean
 g(x) of all components of each measurement and applied the centred
 log-ratio (clr) transformation:
 clr(x<sub>i</sub>)=ln(x<sub>i</sub>/g(x)). The
 conducted statistical analyses were performed with the clr-transformed
 data. The Shapiro-Wilk test was used to evaluate normality. As the data
 were non-normally distributed, a Kruskal-Wallis test was applied. Pairwise
 comparisons were then conducted using the Wilcoxon method. Additionally,
 correlation coefficients and relationships between parameters were
 determined using JMP.
description: # Convergent evolution of failure- and wear-prevention in radulae of
 rock-scraping land snails (Chondrinidae, Gastropoda) Dataset DOI:
 [10.5061/dryad.qbzkh18x7](10.5061/dryad.qbzkh18x7) ## Description of the
 data and file structure Results from elemental analysis and
 nanoindentation. ### Files and variables #### File:
 Rodaten_zum_Hochladen.xlsx **Description:** Elements are presented in
 atomic % and the Hardness H, as well as the Young's modulus E, both
 in GPa ##### Variables * Column A: Species * Column B: feeding substrate
 information * Column C: Tested radular region * Column D-U: Results from
 EDX analysis. Elemental proportions in atomic % * Column V: Sum of P and
 Pt content, given in atomic% * Column W-X: Results from nanoindentation
 (hardness and Young's modulus, both given in GPa) * Column Y: g(A) *
 Column Z-AP: log-transformed elemental content. ## Code/software Excel ##
 Access information NA