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

Methods in Ecology and Evolution 2016 doi: 10.1111/2041-210X.12522 Methods for sampling sequential annual bone growth layers for stable isotope analysis Calandra N. Turner Tomaszewicz 1,2 *, Jeffrey A. Seminoff 2 , Larisa Avens 3 and Carolyn M. Kurle 1 Division of Biological Sciences, Ecology, Behavior and Evolution Section, University of California, San Diego, La Jolla, CA 92093-0116, USA; 2 Southwest Fisheries Science Center, NOAA, National Marine Fisheries Service, La Jolla, CA 92037, USA; and 3 Southeast Fisheries Science Center, NOAA, National Marine Fisheries Service, Beaufort, NC 28516, USA Summary 1. Stable carbon (d 13 C) and nitrogen (d 15 N) 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. 2. We tested two diﬀerent methods, micromilling untreated bone cross sections and biopsy coring bone cross sec- tions processed for skeletochronology, for sequentially sampling individual growth layers from marine turtle humerus bones. 3. We present a standard protocol for sequential bone growth layer sampling for SIA, facilitating direct compar- ison of future studies. We recommend using the micromilling sampling technique on untreated bone cross sec- tions, 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. 4. 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. Key-words: bone, collagen, marine turtles, sequential sampling, skeletochronology, stable isotope analysis Introduction Stable carbon (d 13 C) and nitrogen (d 15 N) isotope analysis (SIA) of organic matter is a powerful tool used in ecological studies to elucidate diet, trophic level, habitat use and migra- tion of a wide variety of taxa in both marine (e.g.Vander Zan- den & Rasmussen 2001; Michener & Lajtha 2007; Graham et al. 2010; Newsome, Clementz & Koch 2010) and terrestrial (e.g. Koch, Fogel & Tuross 1994; Hobson, Barnett-Johnson & Cerling 2010) systems. Examination of both d 13 C and d 15 N values from animal tissues allows for reconstruction of animal movement patterns due to spatial variation in these values that reﬂect diﬀerential carbon and nitrogen processing at the base of terrestrial and marine food webs (DeNiro & Epstein 1978; Rau et al. 1983; Clementz & Koch 2001; McMahon, Hamady & Thorrold 2013). Diﬀerent tissues incorporate and retain stable isotopes from the diet at varying rates, allowing researchers to investigate for- aging ecology over multiple time-scales by sampling-speciﬁc *Correspondence author. E-mail: cturnert@ucsd.edu tissues (Hobson 1999; Dalerum & Angerbj€ orn 2005; Reich, Bjorndal & Martinez del Rio 2008; Kurle 2009). Many hard tissues, such as bone, teeth, otoliths, corals and bivalve shells, do not have regular cellular turnover; instead, subsequent lay- ers formed during growth are retained. These inert layers pre- serve their original chemical composition, thereby reﬂecting the stable isotope values of the environment and the prey con- sumed during the formation of a particular growth layer (e.g. Elorriaga-Verplancken et al. 2013). This creates a time series of data reﬂecting an animal’s diet and location when layers are formed at regular time intervals (e.g. days for otolith rings, or years for bone, tooth, coral and tree rings). Sequential SIA of growth layers has been conducted on tis- sues such as otolith and teeth (e.g. Schwarcz et al. 1998; Hob- son 1999; Newsome et al. 2006; Elorriaga-Verplancken et al. 2013) with promising results for reconstructing habitat use pat- terns for migratory megafauna. For some marine turtle spe- cies, humerus bone tissue is deposited in annual layers (e.g. Snover et al. 2011) and, recently, sequential SIA of marine tur- tle bone growth layers identiﬁed by skeletochronology has been successful, generating a time series reﬂecting the diet and © 2015 The Authors. Methods in Ecology and Evolution © 2015 British Ecological Society