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Compound-specific nitrogen stable isotope analysis provides an approach for estimating animal trophic position that may overcome key issues associated with stable isotope analysis of bulk tissue. Yet compound-specific trophic discrimination factors have not been estimated for a broad range of habitats, taxa, and diets. We conducted a controlled-feeding experiment to characterize the variation in compound-specific (TDFAA) and bulk (TDFBulk) trophic discrimination factors of four freshwater fish species fed on three distinct diets. We also compared TDFAA of fish muscle and scale to evaluate the viability of scales for making food web inferences. Mean ± 1 SD TDFBulk was 2.2‰ ± 0.9‰, and there were significant effects of species and diet trophic position on TDFBulk. Mean ± 1 SD TDFAA was 6.9‰ ± 0.8‰. Although there was no effect of species on TDFAA, there were significant differences in TDFAA across the three diets. TDFAA from fish scales were not significantly different from those of muscle. Our study illustrates the advantages of estimating trophic position using compound-specific stable isotopes and the need for continued investigation of factors resulting in variation in TDF values.

Over the last 100 yr, anthropogenic stressors have decimated the assemblage of deepwater coregonines that once underpinned the food webs of the Laurentian Great Lakes. As a part of ongoing restoration efforts, fisheries managers are interested in reintroducing deepwater coregonines from remnant populations to reestablish historical food web connections. However, little is known about historical trophic position and niche partitioning among deepwater coregonines in the Great Lakes. We used nitrogen stable isotope analysis of amino acids to compare trophic position of museum‐preserved (1920s) and present‐day forage fishes in Lakes Michigan and Superior. In the 1920s, deepwater coregonines exhibited clear trophic niche partitioning, with trophic positions spanning a full trophic level. Additionally, species trophic positions were tightly conserved between lakes. In Lake Superior, trophic niche partitioning has been maintained over the last 100 yr, but trophic position has shifted downward by ~0.5 trophic level. The more dramatic species loss in Lake Michigan corresponds with a sharp reduction in trophic niche breadth over time. Our study reveals remarkable trophic niche breadth among deepwater coregonines prior to the major anthropogenic impacts on the Laurentian Great Lakes and provides a food web benchmark for restoring the historical trophic diversity of this iconic species flock.