Despite considerable interest in using stable-hydrogen isotope ratio (δD) measurements in ecological research, it was previously unknown whether hydrogen derived from drinking water, in addition to that derived from diet, contributed to the nonexchangeable hydrogen in animal tissues. We raised four experimental groups of quail ( Coturnix coturnix japonica ) from hatch on two isotopically distinct diets (mean nonexchangeable δD: −146 and −60‰, Vienna Standard Mean Ocean Water Standard) and drinking waters (mean δD: −130 and +196‰, Vienna Standard Mean Ocean Water Standard). Here we show that both dietary and drinking water hydrogen are incorporated into nonexchangeable hydrogen in both metabolically active (i.e., muscle, liver, blood, fat) and inactive (i.e., feather, nail) tissues. Approximately 20% of hydrogen in metabolically active quail tissues and 26–32% of feathers and nail was derived from drinking water. Our findings suggest environmental interpretations of δD values from modern and fossil animal tissues may need to account for potentially large isotopic differences between drinking water and food and require a good understanding of the physiological ecology of study organisms.

Tracking small migrant organisms worldwide has been hampered by technological and recovery limitations and sampling bias inherent in exogenous markers. Naturally occurring stable isotopes of H (δ²H) in feathers provide an alternative intrinsic marker of animal origin due to the predictable spatial linkage to underlying hydrologically driven flow of H isotopes into foodwebs. This approach can assess the likelihood that a migrant animal originated from a given location(s) within a continent but requires a robust algorithm linking H isotopes in tissues of interest to an appropriate hydrological isotopic spatio-temporal pattern, such as weighted-annual rainfall. However, a number of factors contribute to or alter expected isotopic patterns in animals. We present results of an extensive investigation into taxonomic and environmental factors influencing feather δ²H patterns across North America. Stable isotope data were measured from 544 feathers from 40 species and 140 known locations. For δ²H, the most parsimonious model explaining 83% of the isotopic variance was found with amount-weighted growing-season precipitation δ²H, foraging substrate and migratory strategy. This extensive H isotopic analysis of known-origin feathers of songbirds in North America and elsewhere reconfirmed the strong coupling between tissue δ²H and global hydrologic δ²H patterns, and accounting for variance associated with foraging substrate and migratory strategy, can be used in conservation and research for the purpose of assigning birds and other species to their approximate origin.