Stable isotopes in avian research: a step by step protocol to feather sample preparation for stable isotope analysis of carbon (δ13C), nitrogen (δ15N), and hydrogen (δ2H). Version 1.1 v1

GUIDELINES Stable isotopes in avian research: a step by step protocol to feather sample preparation for stable isotope analysis of carbon (δ C), nitrogen (δ N), and hydrogen (δ H). Version 1.1 Brian Chew , Jeff Kelly , Andrea Contina Corix Plains Institute, University of Oklahoma, Five Partners Place, 201 Stephenson Parkway, Norman, OK 73019, USA, Oklahoma Biological Survey, University of Oklahoma, 111 East Chesapeake Street, SC Building 134, Norman, OK 73019, USA, Department of Biology, University of Oklahoma, 730 Van Vleet Oval, Norman, OK 73019, USA, University of Colorado Denver, Department of Integrative Biology, Science Building 2074, Denver, CO Works for me dx.doi. org/10.17504/protocols.io. z2uf8ew Brian Chew Sep 14, 2019 13 15 2 1, 2 1, 2, 3 4 1 2 3 4 Summary Using a 2:1 chloroform methanol solution and Versatm detergent diluted with deionized water we cleaned feather samples of oil/debris for stable isotope analysis of carbon (δ13C), nitrogen (δ15N), and hydrogen (δ2H). Prepared samples were then measured, cut, and packaged in tin or silver capsules for analysis of carbon(δ13C) and nitrogen(δ15N) or hydrogen(δ2H), respectively. We aim to standardize the laboratory procedure across institutions and research groups focused on avian ecology and conservation and to improve result accuracy and reproducibility. Background Stable isotope analysis of relatively abundant elements such as carbon (δ13C), nitrogen (δ15N), and hydrogen (δ2H) have been widely implemented, particularly over the past two decades, in ecology, conservation, and forensic studies (Vander Zanden et al. 2018.). For example, stable isotope analyses of biological tissues can reveal useful information on diet, trophic level interactions, and large scale animal movements (Bridge et al. 2011; Contina et al. 2013; Zenzal et al. 2018). However, laboratory sample preparation for stable isotope studies requires a standardized protocol to ensure accuracy and repeatability of the results (Paritte and Kelly 2009). Corresponding author : andrea.contina@ucdenver.edu Useful references: Zenzal T.J. et al. 2018. Temporal migration patterns between natal locations of ruby-throated hummingbirds (Archilochus colubris) and their Gulf Coast stopover site. Movement ecology, 6(1), p.2. Vander Zanden et al. 2018. Application of isoscapes to determine geographic origin of terrestrial wildlife for conservation and management. Biological conservation, 228, pp.268-280. Contina et al. 2013. Using geologgers to investigate bimodal isotope patterns in Painted Buntings (Passerina ciris). The Auk, 130(2), pp.265-272. Bridge et al. 2011. Causes of bimodal stable isotope signatures in the feathers of a molt-migrant songbird. Canadian Journal of Zoology, 89(10), pp.951-959. Paritte and Kelly 2009. Effect of cleaning regime on stable-isotope ratios of feathers in Japanese Quail (Coturnix japonica). The Auk, 126(1), pp.165-174. Anytime work with samples is being performed, latex or nitrile gloves should be worn to prevent contamination from outside substances. All work should be performed on a flat, cleaned surface. Some steps in this protocol, such as standard weights and data sheets, might be idiosyncratic to our lab and equipment used at the time 1 1 09/14/2019 This is an open access protocol distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited MATERIALS N AM E CATAL OG # VEN DOR Chloroform Ethanol Methanol double distilled water (ddH2O) Versa-Clean Multi-Purpose Cleaner 04-342 Kimwipes 34120 MATERIALS TEXT SAFETY WARNINGS of the analysis. Please contact andrea.contina@ucdenver.edu if you have any questions or comments. Fisher Scientific Kimberly-Clark These are the standards used for Hydrogen analysis in our lab: KHS (Kudu Horn Standard), CBS (Caribou Hoof Standard), BHCO (Brownheaded Cowbird). Standards used for Carbon and Nitrogen analysis in our lab: USGS40, USGS41a, and USGS 42. USGS 42 can be used for the reference curve and QAQC.Note: different standards, capsules, and weights are used for Carbon and Nitrogen (see Table 1 below). Other materia ls: Staples Compressed-gas duster. When combining Chloroform and Methanol, only open containers under a fume hood. Latex or Nitrile gloves should be worn when handling substances. Protective eyewear should also be worn. 1 Under fume hood, pour 100mL chloroform into clean 250 mL beaker. Preparation of Cleaning Solut ion: 2:1 Chloroform Methanol Solut ion 2 09/14/2019 This is an open access protocol distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited 2 Pour 50mL methanol into beaker. 3 Stir contents with a stir rod, or carefully swirl contents of container under fume hood to mix contents. 4 If desired for repeated use, pour 2:1 chloroform methanol solution into at least 250mL container with sealable, non-plastic lid. 5 In clean 250mL beaker, pour 150mL deionized water , and 5mL detergent . 6 Gently swirl content of container. 6.1 Avoid agitating solution in any manner which might generate bubbles. 7 Mark beaker DETERGENT. 8 Into three clean 250mL beakers, pour 200mL deionized water each. 9 Mark Beakers 1, 2, and 3. Preparation of Cleaning Solut ions: 30:1 Deionized Water Detergent Solut ion 3 09/14/2019 This is an open access protocol distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited 10 Place beakers in order DETERGENT, 1, 2, 3. Feathers will be cleaned in this order to avoid final prepared feather being contaminated with detergent.