The measurement of stable isotopes in ‘bulk’ animal and plant tissues (e.g., muscle or leaf) has become an important tool for studies of functional diversity from organismal to continental scales. In consumers, isotope values reflect their diet, trophic position, physiological state, and geographic location. However, interpretation of bulk tissue isotope values can be confounded by variation in primary producer baseline values and by overlapping values among potential food items. To resolve these issues, biologists increasingly use compound-specific isotope analysis (CSIA), in which the isotope values of monomers that constitute a macromolecule (e.g., amino acids in protein) are measured. In this review, we provide the theoretical underpinnings for CSIA, summarize its methodology and recent applications, and identify future research directions. The key principle is that some monomers are reliably routed directly from the diet into animal tissue, whereas others are biochemically transformed during assimilation. As a result, CSIA of consumer tissue simultaneously provides information about an animal’s nutrient sources (e.g., food items or contributions from gut microbes) and its physiology (e.g., nitrogen excretion mode). In combination, these data clarify many of the confounding issues in bulk analysis and enable novel precision for tracing nutrient and energy flow within and among organisms and ecosystems.

Stable-isotope analysis (SIA) has revolutionized animal ecology by providing time-integrated estimates of the use of resources and/or habitats. SIA is based on the premise that the isotopic composition of a consumer's tissues originates from its food, but is offset by trophic-discrimination (enrichment) factors controlled by metabolic processes associated with the assimilation of nutrients and the biosynthesis of tissues. Laboratory preparation protocols dictate that tissues both of consumers and of their potential prey be lipid-extracted prior to analysis, because (1) lipids have carbon isotope (δ(13)C) values that are lower by approximately 3-8‰ than associated proteins and (2) amino acids in consumers' proteinaceous tissues are assumed to be completely routed from dietary protein. In contrast, models of stable-isotope mixing assume that dietary macromolecules are broken into their elemental constituents from which non-essential amino acids are resynthesized to build tissues. Here, we show that carbon from non-protein dietary macromolecules, namely lipids, was used to synthesize muscle tissue in an omnivorous rodent (Mus musculus). We traced the influence of dietary lipids on the synthesis of consumers' tissues by inversely varying the dietary proportion of C4-based lipids and C3-based protein while keeping carbohydrate content constant in four dietary treatments, and analyzing the δ(13)C values of amino acids in mouse muscle after 4 months of feeding. The influence of dietary lipids on non-essential amino acids varied as function of biosynthetic pathway. The source of carbon in ketogenic amino acids synthesized through the Krebs cycle was highly sensitive to dietary lipid content, with significant increases of approximately 2-4‰ in Glutamate and Aspartate δ(13)C values from the 5% to 15% dietary lipid treatment. Glucogenic amino acids (Glycine and Serine) were less sensitive to dietary lipid, but increased by approximately 3-4‰ from the 25% to 40% lipid diet. As expected, the δ(13)C values of essential amino acids did not vary significantly among diets. Although lipids provide a calorie-rich resource that fuels energy requirements, our results show that they also can be an important elemental source of carbon that contributes to the non-essential amino acids used to build structural tissue like muscle. As such, the calculation of trophic-discrimination factors for animals that consume a lipid-rich diet should consider lipid carbon as a building block for proteinaceous tissues. Careful consideration of the macromolecular composition in the diet of the consumer of interest will help to further refine the use of SIA to study animal ecology and physiology.

Reef‐building corals are mixotrophic organisms that can obtain nutrition from endosymbiotic microalgae (autotrophy) and particle capture (heterotrophy). Heterotrophic nutrition is highly beneficial to many corals, particularly in times of stress. Yet, the extent to which different coral species rely on heterotrophic nutrition remains largely unknown because it is challenging to quantify. We developed a quantitative approach to investigate coral nutrition using carbon isotope (δ¹³C) analysis of six essential amino acids (AA_ESS) in a common Indo‐Pacific coral (Pocillopora meandrina) from the fore reef habitat of Palmyra Atoll. We sampled particulate organic matter (POM) and zooplankton as the dominant heterotrophic food sources in addition to the coral host and endosymbionts. We also measured bulk tissue carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope values of each sample type. Patterns among δ¹³C values of individual AA_ESS provided complete separation between the autotrophic (endosymbionts) and heterotrophic nutritional sources. In contrast, bulk tissue δ¹³C and δ¹⁵N values were highly variable across the putative food sources and among the coral and endosymbiont fractions, preventing accurate estimates of coral nutrition on Palmyra. We used linear discriminant analysis to quantify differences among patterns of AA_ESS δ¹³C values, or ‘fingerprints’, of the food resources available to corals. This allowed for the development of a quantitative continuum of coral nutrition that can identify the relative contribution of autotrophic and heterotopic nutrition to individual colonies. Our approach revealed exceptional variation in conspecific colonies at scales of metres to kilometres. On average, 41% of AA_ESS in P. meandrina on Palmyra are acquired via heterotrophy, but some colonies appear capable of obtaining the majority of AA_ESS from one source or the other. The use of AA_ESS δ¹³C fingerprinting analysis offers a significant improvement on the current methods for quantitatively assessing coral trophic ecology. We anticipate that this approach will facilitate studies of coral nutrition in the field, which are essential for comparing coral trophic ecology across taxa and multiple spatial scales. Such information will be critical for understanding the role of heterotrophic nutrition in coral resistance and/or resilience to ongoing environmental change. A free Plain Language Summary can be found within the Supporting Information of this article.

Compound‐specific stable isotope analysis of individual amino acids (CSIA‐AA) has emerged as a transformative approach to estimate consumer trophic positions (TP_CSIA) that are internally indexed to primary producer nitrogen isotope baselines. Central to accurate TP_CSIA estimation is an understanding of beta (β) values—the differences between trophic and source AA δ¹⁵N values in the primary producers at the base of a consumers’ food web. Growing evidence suggests higher taxonomic and tissue‐specific β value variability than typically appreciated. This meta‐analysis fulfils a pressing need to comprehensively evaluate relevant sources of β value variability and its contribution to TP_CSIA uncertainty. We first synthesized all published primary producer AA δ¹⁵N data to investigate ecologically relevant sources of variability (e.g. taxonomy, tissue type, habitat type, mode of photosynthesis). We then reviewed the biogeochemical mechanisms underpinning AA δ¹⁵N and β value variability. Lastly, we evaluated the sensitivity of TP_CSIA estimates to uncertainty in mean β_Glx‐Phe values and Glx‐Phe trophic discrimination factors (TDF_Glx‐Phe). We show that variation in β_Glx‐Phe values is two times greater than previously considered, with degree of vascularization, not habitat type (terrestrial vs. aquatic), providing the greatest source of variability (vascular autotroph = −6.6 ± 3.4‰; non‐vascular autotroph = +3.3 ± 1.8‰). Within vascular plants, tissue type secondarily contributed to β_Glx‐Phe value variability, but we found no clear distinction among C₃, C₄ and CAM plant β_Glx‐Phe values. Notably, we found that vascular plant β_Glx‐Lys values (+2.5 ± 1.6‰) are considerably less variable than β_Glx‐Phe values, making Lys a useful AA tracer of primary production sources in terrestrial systems. Our multi‐trophic level sensitivity analyses demonstrate that TP_CSIA estimates are highly sensitive to changes in both β_Glx‐Phe and TDF_Glx‐Phe values but that the relative influence of β values dissipates at higher trophic levels. Our results highlight that primary producer β values are integral to accurate trophic position estimation. We outline four key recommendations for identifying, constraining and accounting for β value variability to improve TP_CSIA estimation accuracy and precision moving forward. We must ultimately expand libraries of primary producer AA δ¹⁵N values to better understand the mechanistic drivers of β value variation.