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.

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Understanding the structure and dynamics of food webs requires accurate estimates of energy flow among organisms. Bulk tissue carbon (δ13C) and nitrogen (δ15N) isotope analysis is often used to this end; however, the limitations of this technique can outweigh the benefits. The isotope analysis of individual amino acids is being increasingly employed to trace energy flow and estimate consumer trophic level. Central to this compound‐specific approach are the concepts of essential amino acid (AAESS) δ13C fingerprinting and amino acid (AA) δ15N β‐values, both of which have been understudied and are poorly constrained in terrestrial and freshwater producers. We present AAESS δ13C data for 112 terrestrial and freshwater producers collected from two aridland habitats in the northern Chihuahuan Desert (New Mexico, USA) and AA δ15N data for a subset (n = 28) of these samples. We characterized AAESS δ13C fingerprints by performing linear discriminant analysis on the δ13C values of isoleucine, leucine, lysine, phenylalanine, threonine and valine for four producer groups—C3 plants, C4 plants, CAM plants and filamentous green algae. We explored potential biochemical mechanisms underlying these AAESS δ13C fingerprints by calculating differences between the δ13C values of AAESS products and their AA precursors. This allowed us to estimate and compare isotopic discrimination for specific AAESS synthesis pathways across producer groups. We found a near‐perfect separation of AAESS δ13C fingerprints among producer groups; all groups reclassified with >95% success within our multivariate framework. We also found varied isotopic discrimination for specific AAESS synthesis pathways among producer groups. Contrary to previous studies, we found no differences in β‐values between terrestrial C3 and C4 plants for any trophic‐source AA pairing. Furthermore, we found that Lys δ15N values were less variable and more closely related to bulk tissue δ15N values than Phe δ15N values in terrestrial and freshwater producers. Synthesis. We conclude that AAESS δ13C fingerprints are a higher resolution tracer for freshwater food webs, where instream algae have overlapping bulk tissue δ13C values with terrestrial C3 plants. Additionally, βGlx‐Lys and βPro‐Lys are the best for AA δ15N‐based consumer trophic‐level estimates in freshwater food webs containing both terrestrial and aquatic resources.

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Herbivory is a dominant feeding strategy among animals, yet herbivores are often protein limited. The gut microbiome is hypothesized to help maintain host protein balance by provisioning essential macromolecules, but this has never been tested in wild consumers. Using amino acid carbon (δ13C) and nitrogen (δ15N) isotope analysis, we estimated the proportional contributions of essential amino acids (AAESS) synthesized by gut microbes to five co‐occurring desert rodents representing herbivorous, omnivorous and insectivorous functional groups. We found that herbivorous rodents occupying lower trophic positions (Dipodomys spp.) routed a substantial proportion (~40%–50%) of their AAESS from gut microbes, while higher trophic level omnivores (Peromyscus spp.) and insectivores (Onychomys arenicola) obtained most of their AAESS (~58%) from plant‐based energy channels but still received ~20% of their AAESS from gut microbes. These findings empirically demonstrate that gut microbes play a key functional role in host protein metabolism in wild animals.