Summary Determining location and timing of ontogenetic shifts in the habitat use of highly migratory species, along with possible intrapopulation variation in these shifts, is essential for understanding mechanisms driving alternate life histories and assessing overall population trends. Measuring variations in multi-year habitat use patterns is especially difficult for remote oceanic species.To investigate the potential for differential habitat use among migratory marine vertebrates, we measured the naturally occurring stable nitrogen isotope (δ15N) patterns that differentiate distinct ocean regions to create a “regional isotope characterization”, analyzed the δ15N values from annual bone growth layer rings from dead-stranded animals, then combined the bone and regional isotope data to track individual animal movement patterns over multiple years.We used humeri from juvenile North Pacific loggerhead turtles (Caretta caretta), animals that undergo long migrations across the North Pacific Ocean (NPO), using multiple discrete regions as they develop to adulthood. Typical of many migratory marine species, ontogenetic changes in habitat use throughout their decades-long juvenile stage is poorly understood, but each potential habitat has unique foraging opportunities and spatially explicit natural and anthropogenic threats that could affect key life history parameters.We found a bimodal size/age distribution in the timing that juveniles underwent an ontogenetic habitat shift from the oceanic central North Pacific (CNP) to the neritic east Pacific region near the Baja California Peninsula (BCP) (42.7±7.2 vs. 68.3±3.4 cm carapace length, 7.5±2.7 vs. 15.6±1.7 years). Important to the survival of this population, these disparate habitats differ considerably in their food availability, energy requirements, and threats, and these differences can influence life history parameters such as growth, survival, and future fecundity. This is the first evidence of alternative ontogenetic shifts and habitat use patterns for juveniles foraging in the eastern NPO.We combine two techniques, skeletochronology and stable isotope analysis, to reconstruct multi-year habitat use patterns of a remote migratory species, linked to estimated ages and body sizes of individuals, to reveal variable ontogeny during the juvenile life stage that could drive alternate life histories and that has the potential to illuminate the migration patterns for other species with accretionary tissues.

Rationale The sampling of sequential, annually formed bone growth layers for stable carbon (δ13C values) and nitrogen (δ15N values) isotope analysis (SIA) can provide a time series of foraging ecology data. To date, no standard protocol exists for the pre-SIA treatment of cortical samples taken from fresh, modern, bones. Methods Based on the SIA of historical bone, it is assumed that fresh bone samples must be pre-treated with acid prior to SIA. Using an elemental analyzer coupled to an isotope ratio mass spectrometer to measure stable carbon and nitrogen ratios, we tested the need to acidify cortical bone powder with 0.25M HCl prior to SIA to isolate bone collagen for the determination of δ13C and δ15N values. We also examined the need for lipid extraction to remove potential biases related to δ13C analysis, based on a C:N ratio threshold of 3.5. Results It was found that acidification of micromilled cortical bone samples from marine turtles does not affect their δ15N values, and the small effect acidification has on δ13C values can be mathematically corrected for, thus eliminating the need for pre-SIA acidification of cortical bone. The lipid content of the cortical bone samples was low, as measured by their C:N ratios, indicating that lipid extracting cortical bone samples from modern marine turtles is unnecessary. Conclusions We present a standard protocol for testing fresh, modern cortical bone samples prior to SIA, facilitating direct comparison of future studies. Based on the results obtained from marine turtle bones, pre-acidification and lipid removal of cortical bone are not recommended. This is especially useful as there is frequently not enough bone material removed via micromilling of sequential growth layers to accommodate both acid treatment and SIA.