DOI: 10.5061/dryad.fn2z34tsk
title: Stable isotope profile of autumnal migratory bird feathers on Italian Alps

description: In the study of migration of birds, there are many aspects to be deepened
 to comprehend both ecology of the species and their geographical
 connectivity between breeding and wintering grounds. In the last decades,
 stable isotopes of light elements such as hydrogen, oxygen, nitrogen,
 carbon and sulphur measured in cheratinous tissues such feathers are
 increasingly used in order to delineate origin of migrants and to define
 ecological aspects of birds. Here we present a complete isotopic profile
 of several passerine species migrating through Italian Alps during
 post-breeding season. Stable isotope ratios of hydrogen, oxygen, carbon,
 nitrogen and sulphur were measured in juvenile feathers of 807 individuals
 of 48 different specie ringed in central Italian Alps during post-breeding
 migration in the years 2010, 2011, 2012, and 2013. Birds were ringed in
 two ringing and monitoring stations of Progetto Alpi
 (http://progetto-alpi.muse.it/). No birds were mistreated, and the
 sampling took place in compliance with the national and international laws
 in force at the time of the study.
description: Feathers were sampled from
 migratory birds in two locations on central Italian Alps during the years
 2010, 2011, 2012 and 2013. All feathers are juvenile, hence grown in the
 nest and though carry the natal origin information. The values of
 <i>δ</i><sup>2</sup>H,
 <i>δ</i><sup>18</sup>O,
 <i>δ</i><sup>13</sup>C,
 <i>δ</i><sup>15</sup>N,
 <i>δ</i><sup>34</sup>S were measured for each
 individual feather. It was possible to determine all five isotope ratios
 in the majority of individuals, with few values missing. To determine
 feather <i>δ</i><sup>2</sup>H,
 <i>δ</i><sup>13</sup>C,
 <i>δ</i><sup>15</sup>N,
 <i>δ</i><sup>18</sup>O and
 <i>δ</i><sup>34</sup>S values, feathers were first
 washed in a solvent mixture (diethyl ether-methanol 2:1) and then prepared
 for analysis (Bontempo et al. 2014). Simultaneous determination of
 <i>δ</i><sup>13</sup>C,
 <i>δ</i><sup>15</sup>N and
 <i>δ</i><sup>34</sup>S (i.e. in the same run) was
 accomplished using a Vario Isotope Cube isotope ratio mass spectrometer
 (Elementar, Germany). The <i>δ</i><sup>2</sup>H
 and <i>δ</i><sup>18</sup>O values were determined
 through pyrolysis combustion using a TC/EA (Thermo Finnigan, Bremen,
 Germany) interfaced with a Delta Plus XP (Thermo Finnigan, Bremen,
 Germany) continuous-flow isotope-ratio mass spectrometer. Pyrolysis was
 carried out at 1450°C in a glassy carbon column. The helium carrier flow
 was 110 ml/min, the GC column was a 1.2 m long molecular sieve 5A, at
 110°C. Isotope ratios were expressed in δ-notation against V-PDB
 (Vienna-Pee Dee Belemnite) for
 <i>δ</i><sup>13</sup>C, Air for
 <i>δ</i><sup>15</sup>N, V-SMOW (Vienna-Standard
 Mean Ocean Water) for <i>δ</i><sup>2</sup>H and
 <i>δ</i><sup>18</sup>O, and V-CDT (Vienna – Canyon
 Diablo Triolite) for <i>δ</i><sup>34</sup>S. The
 isotopic values of <i>δ</i><sup>2</sup>H were
 calculated using the comparative equilibration approach (Wassenaar and
 Hobson 2003, 2006) based on the keratin standards CBS
 (<i>δ</i><sup>2</sup>H -197 ‰,
 <i>δ</i><sup>18</sup>O = +3.8 ‰) and KHS (-54.1 ‰,
 <i>δ</i><sup>18</sup>O = +20.3 ‰). Values of
 <i>δ</i><sup>13</sup>C,
 <i>δ</i><sup>15</sup>N and
 <i>δ</i><sup>34</sup>S were calculated against
 working in-house standards (casein and wheat), which were themselves
 calibrated against international reference materials using multi-point
 normalisation: fuel oil NBS-22 (IAEA International Atomic Energy Agency,
 Vienna, Austria; -30.03 ‰) and sugar IAEA-CH-6 (- 10.45 ‰) for
 <i>δ</i><sup>13</sup>C, L-glutamic acid USGS 40
 (-26.39 ‰ and -4.5 ‰ for <i>δ</i><sup>13</sup>C
 and <i>δ</i><sup>15</sup>N), hair USGS 42
 (<i>δ</i><sup>15</sup>N = +8.05 ‰ and
 <i>δ</i><sup>13</sup>C = -21.09 ‰) and USGS 43
 (<i>δ</i><sup>15</sup>N = +8.44 ‰ and
 <i>δ</i><sup>13</sup>C = -21.28 ‰) for
 <sup>13</sup>C/<sup>12</sup>C and
 <sup>15</sup>N/<sup>14</sup>N; USGS 42 (+7.84 ‰)
 and USGS 43 (+10.46 ‰) for
 <sup>34</sup>S/<sup>32</sup>S. Values of
 <i>δ</i><sup>13</sup>C were expressed versus V-PDB
 on a scale normalised against the two reference materials LSVEC (−46.6 ‰)
 and NBS 19 (+1.95 ‰) (Brand et al. 2014). Values of
 <i>δ</i><sup>15</sup>N were expressed versus
 Air-N<sub>2</sub> on a scale normalised using the two
 reference materials IAEA-N-1 and USGS32, with consensus values of +0.4 ‰
 and +180 ‰ (Brand et al. 2014). Values of
 <i>δ</i><sup>34</sup>S were expressed relative to
 VCDT on a scale defined by assigning a
 <i>δ</i><sup>34</sup>S value of -0.3 ‰ to the
 reference material IAEA-S-1. Method uncertainty (calculated as one
 standard deviation) was 0.1 ‰ for δ<sup>13</sup>C, 0.2 ‰ for
 δ<sup>15</sup>N, 0.3 ‰ for
 <i>δ</i><sup>18</sup>O and
 <i>δ</i><sup>34</sup>S and 2 ‰ for
 <i>δ</i><sup>2</sup>H. References Bontempo, L, et al. (2014)
 Comparison of methods for stable isotope ratio (δ13C, δ15N, δ2H, δ18O)
 measurements of feathers. Methods Ecol Evol 5: 363–371. Brand,
 WA, Coplen, TB, Vogl, J, Rosner, M, Prohaska, T (2014) Assessment of
 international reference materials for isotope-ratio analysis (IUPAC
 technical report). Pure Appl Chem 86: 425–467. Wassenaar, LI, Hobson, KA (2003)
 Comparative equilibration and online technique for determination of
 non-exchangeable hydrogen of keratins for use in animal migration studies.
 Isotopes Environ Health Stud 39: 211–217. Wassenaar, LI, Hobson, KA (2006)
 Stable-hydrogen isotope heterogeneity in keratinous material: mass
 spectrometry and migratory wildlife tissue subsampling strategies. Rapid
 Commun Mass Spectrom 20: 2505–2510.