Methodological constraints for the stable isotope analysis of different tissues: A comparison between muscle and whole‐body samples in freshwater gammarids

Abstract

Carbon (δ ¹³ C) and nitrogen (δ ¹⁵ N) stable isotope ratios allow the reconstruction of food webs, provided that all trophic levels are properly characterized. In freshwater ecosystems, small‐bodied invertebrates often occupy central positions in food webs, with whole‐body samples commonly used for their stable isotope analysis, due to insufficient sampling material for single‐tissue analysis. However, whole‐body samples include multiple tissues, each with its own metabolic routing and turnover rate, thus potentially having different isotopic values. Using two genetic lineages of the Gammarus fossarum complex (Crustacea: Amphipoda) as models, we compared the δ ¹³ C and δ ¹⁵ N stable isotope values of muscle and whole‐body samples using isotopic niche analysis and diet reconstruction via mixing models. When tested, decarbonation of whole‐body samples through acidification had a significant effect on the δ ¹³ C values due to the inorganic carbonate in crustaceans' exoskeleton. Thus, decarbonated δ ¹³ C values were used for whole‐body samples in the analyses. Whole‐body samples had significantly higher δ ¹⁵ N and a narrower niche than muscle samples. The interspecific niche overlap did not vary among tissues for one of the lineages, but was twice as high using whole‐body samples compared to muscle samples for the other lineage. Mixing models for whole‐body samples performed better than those for muscle samples, due to the absence of muscle tissue‐specific trophic discrimination factors (TDFs) in the literature, highlighting the need for tissue‐specific TDFs in macroinvertebrates. Our results reinforce the pre‐established caution around the use of whole‐body samples and underline biases that can arise from comparing different tissues.