The use of stable isotopes of sulfur (δ³⁴S) to infer avian diets, foraging habitats, and movements is relatively uncommon, resulting in a lack of information on patterns of δ³⁴S incorporation in avian tissue. In a controlled study of Gentoo Penguins (Pygoscelis papua), we found that diet-tissue isotopic discrimination factors (Δ³⁴S_diet-tissue) differed among egg components and feathers synthesized from a common diet, ranging from -0.4 to -1.7 ‰. We also found that methodical choices such as lipid extraction and prey tissue selection influenced calculated Δ³⁴S_diet-tissue values. Specifically, Δ³⁴S_diet-tissue values were lower (i.e., more negative) when calculated using whole fish relative to fish muscle and lipid-extraction biased egg yolk, but not fish tissue, δ³⁴S values. Δ³⁴S_diet-tissue values obtained for Gentoo Penguins fed a marine fish diet were generally lower than those reported for freshwater-fish-consuming Double-crested Cormorants (Phalacrocorax auritus), the only other bird species in which Δ³⁴S_diet-tissue has been quantified. We found support for the hypothesis that tissue Δ³⁴S_diet-tissue values are inversely related to dietary δ³⁴S values in birds, similar to what has been observed in mammals. Given this relationship, the discrimination factors reported here for Gentoo Penguins may be broadly applicable to other avian species with a similar marine diet. Finally, we provide recommendations for future studies seeking to quantify Δ³⁴S_diet-tissue in avian tissues and guidance to allow for greater application of sulfur stable isotope analysis in ornithological research.

Data was collected from a captive breeding population of Gentoo Penguins (Pygoscelis papua) at the Henry Doorly Zoo in Omaha, Nebraska, USA. Penguins were hand-fed ad libitum a consistent diet of Atlantic Herring (Clupea harengus) for eight and ten months prior to the start of egg tissue collection and feather molt, respectively.  Samples of herring (whole fish and muscle tissue), body feathers, eggshell membrane, albumen and yolk were analyzed for sulfur (S) elemental concentration (%S) and stable isotope values (δ³⁴S) using an elemental analyzer system coupled to a continuous-flow stable isotope ratio mass spectrometer. A sub-sample of whole fish, fish muscle, and yolk samples were lipid extracted using a Soxhlet apparatus with a 1:1 Petroleum–Ether: Ethyl–Ether prior to analyses. We calculated Δ³⁴S_diet-tissue by subtracting the δ³⁴S values of each individual egg tissue and feather sample from the mean isotopic values of the diets (whole herring and herring muscle) consumed prior to tissue synthesis.