Stable hydrogen isotope measurements of songbird feathers: effects of intra-feather variability and sample processing

Deuterium composition of feathers has been widely applied to establish provenance of birds. Surprisingly, little attention has been paid to the potential sources of unwanted hydrogen isotopic variability that may affect the comparability and replicability of deuterium measurements. Feathers may exhibit hydrogen isotopic differences between distal and proximal sections, as well as between rachis and vane. For this reason, it is recommended to always analyse ground vane from the same section of the feather. However, this protocol may be seriously limiting when working with songbird feathers, which are usually small and light. In this paper, intra-feather hydrogen isotopic variability was studied in four species of songbirds by comparing four sections of the same feather. No difference in hydrogen isotopic composition was found between distal and proximal sections of the vane. This was expected as songbird feathers grow rapidly and individuals avoid geographical movements during moult, favouring a homogeneous source of hydrogen during keratin synthesis. Conversely, as previously demonstrated for other avian species, rachis showed systematically c. 11‰ δ 2 H more negative values than vane. This is probably a result of their biochemical and ultrastructural differences, which seem strongly conserved across taxa. Such differences imply that we need to describe which feather part was used in the methods of our reports to ensure full comparability and replicability among studies. Nevertheless, vane and rachis δ 2 H were strongly correlated, demonstrating that rachis can be also used in isotope analyses because its δ 2 H can be accurately rescaled. This is useful since rachis represents an important fraction of feather mass in songbirds. Values of δ 2 H from ground and non-ground samples from the same feather were also compared. There was no effect of the processing protocol, suggesting that grinding is unnecessary when working with passerine feathers.