Methods for sampling sequential annual bone growth layers for stable isotope analysis

Abstract

Stable carbon (δ13C) and nitrogen (δ15N) isotope analysis (SIA) has proven useful in addressing fundamental questions in ecology such as reconstructing trophic interactions, habitat connections and climate regime shifts. The temporal scales over which SIA can be used to address ecological problems vary depending on the protein turnover times of the analysed tissue. Hard, inert tissues, such as teeth, bones and mollusc shells, grow in regular intervals (i.e. daily or annually), and sequential sampling of these growth layers provides a time series of isotopic patterns. As a result, SIA on these tissues is useful for elucidating behaviour and ecology of animals over time, especially those with cryptic life-history stages, such as marine turtles that retain growth layers in their humerus bones. To date, there exists no standard protocol for the sequential sampling of cortical bone samples taken from fresh, modern samples for SIA. We tested two different methods, micromilling untreated bone cross sections and biopsy coring bone cross sections processed for skeletochronology, for sequentially sampling individual growth layers from marine turtle humerus bones. We present a standard protocol for sequential bone growth layer sampling for SIA, facilitating direct comparison of future studies. We recommend using the micromilling sampling technique on untreated bone cross sections, as it facilitated higher precision sampling of growth layers that were not affected by chemical processing, and minimized sample handling, thereby reducing chances for contamination. This is the first study to present a standardized method to sequentially sample annual bone growth layers for stable isotope analysis and facilitates direct comparison among future studies.