The influence of diet on the distribution of nitrogen isotopes in animals was investigated by analyzing animals grown in the laboratory on diets of constant nitrogen isotopic composition. 
The isotopic composition of the nitrogen in an animal reflects the nitrogen isotopic composition of its diet. The δ^(15)N values of the whole bodies of animals are usually more positive than those of their diets. Different individuals of a species raised on the same diet can have significantly different δ^(15)N values. The variability of the relationship between the δ^(15)N values of animals and their diets is greater for different species raised on the same diet than for the same species raised on different diets. Different tissues of mice are also enriched in ^(15)N relative to the diet, with the difference between the δ^(15)N values of a tissue and the diet depending on both the kind of tissue and the diet involved. The δ^(15)N values of collagen and chitin, biochemical components that are often preserved in fossil animal remains, are also related to the δ^(15)N value of the diet. 
The dependence of the δ^(15)N values of whole animals and their tissues and biochemical components on the δ^(15)N value of diet indicates that the isotopic composition of animal nitrogen can be used to obtain information about an animal's diet if its potential food sources had different δ^(15)N values. The nitrogen isotopic method of dietary analysis probably can be used to estimate the relative use of legumes vs non-legumes or of aquatic vs terrestrial organisms as food sources for extant and fossil animals. However, the method probably will not be applicable in those modern ecosystems in which the use of chemical fertilizers has influenced the distribution of nitrogen isotopes in food sources. 
The isotopic method of dietary analysis was used to reconstruct changes in the diet of the human population that occupied the Tehuacan Valley of Mexico over a 7000 yr span. Variations in the δ^(15)C and δ^(15)N values of bone collagen suggest that C_4 and/or CAM plants (presumably mostly corn) and legumes (presumably mostly beans) were introduced into the diet much earlier than suggested by conventional archaeological analysis.

Influence of Diet On the Distribtion of Nitrogen Isotopes in Animals

Within an organism, lipids are depleted in (13)C relative to proteins and carbohydrates (more negative delta(13)C), and variation in lipid content among organisms or among tissue types has the potential to introduce considerable bias into stable isotope analyses that use delta(13)C. Despite the potential for introduced error, there is no consensus on the need to account for lipids in stable isotope analyses. Here we address two questions: (1) If and when is it important to account for the effects of variation in lipid content on delta(13)C? (2) If it is important, which method(s) are reliable and robust for dealing with lipid variation? We evaluated the reliability of direct chemical extraction, which physically removes lipids from samples, and mathematical normalization, which uses the carbon-to-nitrogen (C:N) ratio of a sample to normalize delta(13)C after analysis by measuring the lipid content, the C:N ratio, and the effect of lipid content on delta(13)C (Deltadelta(13)C) of plants and animals with a wide range of lipid contents. For animals, we found strong relationships between C:N and lipid content, between lipid content and Deltadelta(13)C, and between C:N and Deltadelta(13)C. For plants, C:N was not a good predictor of lipid content or Deltadelta(13)C, but we found a strong relationship between carbon content and lipid content, lipid content and Deltadelta(13)C, and between and carbon content and Deltadelta(13)C. Our results indicate that lipid extraction or normalization is most important when lipid content is variable among consumers of interest or between consumers and end members, and when differences in delta(13)C between end members is <10-12 per thousand. The vast majority of studies using natural variation in delta(13)C fall within these criteria. Both direct lipid extraction and mathematical normalization reduce biases in delta(13)C, but mathematical normalization simplifies sample preparation and better preserves the integrity of samples for delta(15)N analysis.

/pdf/getting-to-the-fat-of-the-matter-models-methods-and-1styosvrid.pdf

Getting to the fat of the matter: models, methods and assumptions for dealing with lipids in stable isotope analyses

Summary
1The use of stable isotopic techniques to study animal diets and trophic levels requires a priori estimates of discrimination factors (Δ13C and Δ15N, also called fractionation factors), which are the differences in isotopic composition between an animal and its diet. Previous studies have shown that these parameters depend on several sources of variation (e.g. taxon, environment, tissue) but diet as a source of variation still needs assessment.
2We conducted an extensive review of the literature (66 publications) concerning estimates of animal-diet Δ13C (n = 290) and Δ15N (n = 268). We analysed this data set to test the effect of diet isotopic ratio on the discrimination factor, taking into account taxa, tissues, environments and lipid extraction treatments. Our results showed differences among taxonomic classes for Δ13C, but not for Δ15N, and significant differences among tissues for both Δ13C and Δ15N. We found a significant negative relationship between both, Δ13C and Δ15N, with their corresponding diet isotopic ratios. This relationship was found also within taxonomic classes for mammals (Δ13C and Δ15N), birds (Δ13C), fishes (Δ13C and Δ15N) and invertebrates (Δ13C and Δ15N). From these relationships, we propose a method to calculate discrimination factors based on data on diet isotope ratios (termed the ‘Diet-Dependent Discrimination Factor’, DDDF).
3To investigate current practice in the use of discrimination factors, we reviewed studies that used multi-resource isotopic models. More than 60% of models used a discrimination factor coming from a different species or tissues, and in more than 70% of models, only one Δ13C or Δ15N was used for all resources, even if resources had very different isotopic ratios. Also, we estimated DDDFs for the studies that used isotopic models. More than 40% used Δ15N values and more than 33% used Δ13C values differing > 2‰ from estimated DDDFs.
4Synthesis and applications. Over the last decade, applied ecologists have discovered the potential of stable isotopes for animal diet reconstruction, but the successful adoption of the method relies on a good estimation of discrimination factors. We draw attention to the high variability in discrimination factors, advise caution in the use of single discrimination factors in isotopic models, and point to a method for obtaining adequate values for this parameter when discrimination factors cannot be measured experimentally. Future studies should focus on understanding why discrimination factors vary as a function of the isotopic value of the diet.

https://besjournals.onlinelibrary.wiley.com/doi/epdf/10.1111/j.1365-2664.2009.01620.x

Variation in discrimination factors (Δ15N and Δ13C): the effect of diet isotopic values and applications for diet reconstruction

Summary 1. The use of stable isotopic techniques to study animal diets and trophic levels requires a priori estimates of discrimination factors ( Δ 13 C and Δ 15 N, also called fractionation factors), which are the differences in isotopic composition between an animal and its diet. Previous studies have shown that these parameters depend on several sources of variation (e.g. taxon, environment, tissue) but diet as a source of variation still needs assessment. 2. We conducted an extensive review of the literature (66 publications) concerning estimates of animal-diet Δ 13 C ( n = 290) and Δ 15 N ( n = 268). We analysed this data set to test the effect of diet isotopic ratio on the discrimination factor, taking into account taxa, tissues, environments and lipid extraction treatments. Our results showed differences among taxonomic classes for Δ 13 C, but not for Δ 15 N, and significant differences among tissues for both Δ 13 C and Δ 15 N. We found a significant negative relationship between both, Δ 13 C and Δ 15 N, with their corresponding diet isotopic ratios. This relationship was found also within taxonomic classes for mammals ( Δ 13 C and Δ 15 N), birds ( Δ 13 C), fishes ( Δ 13 C and Δ 15 N) and invertebrates ( Δ 13 C and Δ 15 N). From these relationships, we propose a method to calculate discrimination factors based on data on diet isotope ratios (termed the ‘Diet-Dependent Discrimination Factor’, DDDF). 3. To investigate current practice in the use of discrimination factors, we reviewed studies that used multi-resource isotopic models. More than 60% of models used a discrimination factor coming from a different species or tissues, and in more than 70% of models, only one Δ 13 C or Δ 15 N was used for all resources, even if resources had very different isotopic ratios. Also, we estimated DDDFs for the studies that used isotopic models. More than 40% used Δ 15 N values and more than 33% used Δ 13 C values differing > 2‰ from estimated DDDFs. 4. Synthesis and applications . Over the last decade, applied ecologists have discovered the potential of stable isotopes for animal diet reconstruction, but the successful adoption of the method relies on a good estimation of discrimination factors. We draw attention to the high variability in discrimination factors, advise caution in the use of single discrimination factors in isotopic models, and point to a method for obtaining adequate values for this parameter when discrimination factors cannot be measured experimentally. Future studies should focus on understanding why discrimination factors vary as a function of the isotopic value of the diet.

/pdf/the-effect-of-diet-isotopic-values-and-applications-for-diet-1ota7f4if3.pdf

the effect of diet isotopic values and applications for diet reconstruction

Fifty years ago, GE Hutchinson defined the ecological niche as a hypervolume in n-dimensional space with environmental variables as axes. Ecologists have recently developed renewed interest in the concept, and technological advances now allow us to use stable isotope analyses to quantify these niche dimensions. Analogously, we define the isotopic niche as an area (in δ-space) with isotopic values (δ-values) as coordinates. To make isotopic measurements comparable to other niche formulations, we propose transforming δ-space to p-space, where axes represent relative proportions of isotopically distinct resources incorporated into an animal's tissues. We illustrate the isotopic niche with two examples: the application of historic ecology to conservation biology and ontogenetic niche shifts. Sustaining renewed interest in the niche requires novel methods to measure the variables that define it. Stable isotope analyses are a natural, perhaps crucial, tool in contemporary studies of the ecological niche.

A niche for isotopic ecology

Use of stable isotope ratios to trace pathways of organic matter among consumers requires knowledge of the isotopic shift between diet and consumer. Variation in trophic shift among consumers can be substantial. For data from the published literature and supplementary original data (excluding fluid-feeding consumers), the mean isotopic shift for C was + 0.5 + 0.13%o rather than 0.0%o, as commonly assumed. The shift for C was higher for consumers analyzed as muscle (+ 1.3 + 0.30%o) than for consumers analyzed whole (+ 0.3 +0.14%o). Among consumers analyzed whole, the trophic shift for C was lower for consumers acidified prior to analysis (-0.2 + 0.21%o) than for unacidified samples ( +0.5 + 0.17%o). For N, trophic shift was lower for consumers raised on invertebrate diets (+ 1.4 + 0.21%o) than for consumers raised on other high-protein diets (+3.3 +0.26%o) and was intermediate for consumers raised on plant and algal diets (+2.2 +0.30%o). The trophic shift for S differed between high-protein (+ 2.0 + 0.65%o) and low-protein diets (-0.5 + 0.56%o). Thus, methods of analysis and dietary differences can affect trophic shift for consumers; the utility of stable isotope methods can be improved if this information is incorporated into studies of trophic relationships. Although few studies of stable isotope ratios have considered variation in the trophic shift, such variation is important because small errors in estimates of trophic shift can result in large errors in estimates of the contribution of sources to consumers or in estimates of trophic position.

Claire C. McGrath

Papers

Variation in trophic shift for stable isotope ratios of carbon, nitrogen, and sulfur