Advancing Amino Acid Isotope‐Inferred Trophic Ecology in an Apex Predator: Insights From a Data‐Rich Killer Whale Population

Summary

Amino acid‐specific nitrogen isotope analysis (δ 15 N‐AA) is increasingly used to infer trophic ecology, yet remains poorly validated in marine predators. We analyzed bulk tissue δ 15 N ( 15 N/ 14 N) and δ 13 C ( 13 C/ 12 C), and δ 15 N‐AA in 105 skin samples collected from individually identified Norwegian killer whales between 2017 and 2023. Forty‐two individuals with best documented diets in 2010–2023, based on field observations and corroborated by mercury concentrations and bulk tissue δ 15 N values in skin, provided reference diet groups against which δ 15 N‐AA‐derived hypotheses were tested: Fish‐only (100% fish prey; n  = 15), Intermediate mixed diet (~10% mammalian prey in addition to fish; n  = 14), and Top mixed diet (~50% mammals, 50% fish prey; n  = 13). Univariate and multivariate approaches identified diet as the primary driver of δ 15 N variation across individual amino acids and multivariate isotopic space, whereas sex and life stage had little effect. However, substantial inter‐individual variation remained unexplained and appeared to reflect metabolic processes. Metrics δ 15 N ΣTrophic—ΣSource (δ 15 N difference between the sum of trophic amino acids and the sum of source amino acids) and isotopic difference between glutamic acid/glutamine (Glu) and phenylalanine (Phe) (δ 15 N Glu–Phe ) successfully identified Top mixed diet whales but lacked the sensitivity to identify Intermediate mixed diet individuals, whilst δ 15 N values of Threonine showed no relationship with trophic position. Proline (Pro) and Phe used as trophic‐source pair provided the first plausible δ 15 N‐AA‐derived trophic position estimates for killer whales and performed better than bulk isotope‐derived trophic position when a priori knowledge was not available. Multivariate clustering of δ 15 N‐AA data complemented trophic position estimates by resolving finer‐scale dietary variation occurring within trophic levels.