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The dark side of soft tissues: Unexpected inorganic carbonate in the invasive slipper limpet Crepidula fornicata and its implications for stable isotope interpretations.

RATIONALE
Stable isotopic analysis is extensively used in trophic ecology. Inorganic carbonates, usually originating from shell fragments, are routinely removed from samples using an acid treatment because they affect δ13 C values. However, acid treatment can also change δ15 N values. For some taxa such as molluscs, the general assumption is that acid pre-treatment is not necessary since their shell is easily dissected from soft tissues and represents the only source of inorganic carbonates. However, other sources of non-dietary carbon (e.g., intracellular inorganic carbon) enriched in13 C thus get overlooked.


METHODS
Soft tissues (foot) of the invasive gastropod Crepidula fornicata of different size classes were analysed for their δ13 C and δ15 N values with and without acid pre-treatment using isotope ratio mass spectrometry. In toto microscopic investigations coupled with acid treatment, scanning electron microscopy and energy-dispersive spectroscopy were used to highlight the presence of inorganic carbonate. A correction model was derived and applied to existing stable isotope data for C. fornicata. We used both seasonal variations in δ13 C signatures and mixing model outputs to assess the error in δ13 C values.


RESULTS
Acid pre-treatment had a significant effect on the stable isotope compositions of C. fornicata foot tissue, especially on δ13 C values: isotopic differences increased with size, up to 3‰ for large females. No effect was detected for small (below ~20 mm) and motile males. In toto microscopic analysis revealed the presence of small spherules of inorganic carbonate, hence explaining the differences in δ13 C values. Mixing model outputs and seasonal variation of δ13 C values showed that untreated samples can lead to large misinterpretations about diet proportions and degree of trophic niche overlap, respectively.


CONCLUSIONS
Spherules of inorganic carbonate in C. fornicata soft tissues are likely to be linked with the motility of this species and their mucus production. We recommend assessing the presence of inorganic carbonate in soft tissue of sessile gastropods.