Stable Isotope Mixing Models Are Biased by the Choice of Sample Preservation and Pre-treatment: Implications for Studies of Aquatic Food Webs

Stable isotope analysis has become one of the most widely used techniques in ecological studies. However, there are still uncertainties about the effects of sample preservation and pre-treatment on the ecological interpretation of stable isotope data and especially on Bayesian stable isotope mixing models. Here, Bayesian mixing models were used to study how three different preservation methods (drying, freezing, formalin) and two pre-treatments (acidification, lipid removal) affect the estimation of the utilized organic matter sources for two benthic invertebrate species (Limecola balthica, Crangon crangon) collected in the Baltic Sea. Furthermore, commonly used mathematical lipid normalization and formalin correction were applied to check if they were able to adjust the model results correctly. Preservation effects were strong on model outcomes for frozen as well as formalin preserved L. balthica samples, but not for C. crangon. Pre-treatment effects varied with species and preservation method and neither lipid normalization nor mathematical formalin correction consistently resulted in the desired model outcomes. Our analysis highlights that particularly small, not significant changes in stable isotope ratios introduced by different preservation and pre-treatments display a so far unrecognized source of error in stable isotope mixing models. We conclude that mathematical correction of benthic invertebrate stable isotopes data should be avoided for Bayesian mixing models and that previously unaddressed effects of sample preservation (especially those arising from preservation by freezing) have potentially biased our understanding of the utilization of organic matter in aquatic food webs.

Sample preservation and pre-treatment in stable isotope analysis: Implications for the study of aquatic food webs

Stable isotope analysis has become one of the most widely used techniques in ecology. However, uncertainties about the effects of sample preservation and pre-treatment on the ecological interpretation of stable isotope data and especially on Bayesian stable isotope mixing models remain. Here, Bayesian mixing models were used to study how three different preservation methods (drying, freezing, formalin) and two pre-treatments (acidification, lipid removal) affect the estimation of diet composition for two benthic invertebrate species (Limecola balthica, Crangon crangon). Furthermore, commonly used mathematical lipid normalization and formalin correction were applied to check if they improve the model results. Preservation effects were strong on model outcomes for frozen as well as formalin preserved L. balthica samples, but not for C. crangon. Pre-treatment effects varied with species and preservation method and neither lipid normalization nor mathematical formalin correction consistently resulted in improved model outcomes. Our analysis highlights that particularly small changes in δ15N introduced by different preservation and pre-treatments display a so far unrecognized source of error in stable isotope studies. We conclude that mathematical correction of stable isotopes data should be avoided for Bayesian mixing models and that previously unaddressed effects of sample preservation (especially those arising from preservation by freezing) have potentially biased our understanding of the utilization of organic matter in aquatic food webs.

Fish and maize: Bayesian mixing models of fourteenth- through seventeenth-century AD ancestral Wendat diets, Ontario, Canada

Freshwater and marine fish have been important components of human diets for millennia. The Great Lakes of North America, their tributaries and smaller regional freshwater bodies are important Native American fisheries. The ethnohistorical record, zooarchaeological remains, and isotopic values on human bone and tooth collagen indicate the importance of fish in fourteenth- through seventeenth-century ancestral Wendat diets in southern Ontario, which is bordered by three of the Great Lakes. Maize (Zea mays ssp. mays) was the primary grain of Native American agricultural systems in the centuries prior to and following sustained European presence. Here we report new Bayesian dietary mixing models using previously published δ13C and δ15N values on ancestral Wendat bone and tooth collagen and tooth enamel. The results confirm previous estimates from δ13C values that ancestral Wendat diets included high proportions of maize but indicate much higher proportions of fish than has previously been recognized. The results also suggest that terrestrial animals contributed less to ancestral Wendat diets than is typically interpreted based on zooarchaeological records.