Exploring the use of amino acid stable hydrogen isotope (δ2H) analysis to improve geographic assignment of wildlife.

Stable isotopes of hydrogen (δ2H) have long been used to trace the movement and geographic origins of wildlife. δ2H analysis of individual amino acids (AA) offer a multivariate alternative to bulk tissue analysis, but the geographic resolving power of this approach remains underexplored. Here, we analyzed AA δ2H, along with bulk tissue δ2H, δ13C, and δ15N values of 32 feathers collected from Canada geese (Branta canadensis) with known molting locations along a latitudinal gradient in the Midwestern United States (34-48 °N). We evaluated isotopic variation among three latitudinally defined geographic groups across this gradient. The δ2H values of essential AA showed strong latitudinal separation, with isoleucine, leucine, and valine differing among all pairwise comparisons and exhibiting between-group differences exceeding those observed in bulk feather δ2H. In contrast, δ2H values of non-essential AA were far more variable within groups and, apart from proline and glutamic acid, did not exhibit significant differences among geographic localities. A bootstrapped flexible discriminant analysis (FDA) incorporating δ2H values of five essential AA yielded a mean classification accuracy of 82 %, with misclassification concentrated between adjacent North and Central groups. Including bulk tissue δ13C values marginally improved model accuracy (mean 83 %), likely reflecting geographic variation in native or agricultural C4 plant abundance. An FDA model based only on non-essential AA δ2H values performed poorly (mean 59 % accuracy), which contrasts with recent studies on humans in which non-essential AA δ2H values were better predictors of region of origin. We suggest this discrepancy reflects differences in food sourcing; barring carry-over effects, wildlife primarily incorporate locally derived essential AA whereas human (supermarket) diets often integrate protein sources across continental spatial scales. Our results indicate that essential AA exhibit clear latitudinal structure in δ²H values and hence these analyses offer strong potential for geographic assignment of migratory birds.