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.

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 δ²H, δ¹⁸O, δ¹³C, δ¹⁵N, δ³⁴S 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 δ²H, δ¹³C, δ¹⁵N, δ¹⁸O and δ³⁴S 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 δ¹³C, δ¹⁵N and δ³⁴S (i.e. in the same run) was accomplished using a Vario Isotope Cube isotope ratio mass spectrometer (Elementar, Germany). The δ²H and δ¹⁸O 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 δ¹³C, Air for δ¹⁵N, V-SMOW (Vienna-Standard Mean Ocean Water) for δ²H and δ¹⁸O, and V-CDT (Vienna – Canyon Diablo Triolite) for δ³⁴S. The isotopic values of δ²H were calculated using the comparative equilibration approach (Wassenaar and Hobson 2003, 2006) based on the keratin standards CBS (δ²H -197 ‰, δ¹⁸O = +3.8 ‰) and KHS (-54.1 ‰, δ¹⁸O = +20.3 ‰). Values of δ¹³C, δ¹⁵N and δ³⁴S 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 δ¹³C, L-glutamic acid USGS 40 (-26.39 ‰ and -4.5 ‰ for δ¹³C and δ¹⁵N), hair USGS 42 (δ¹⁵N = +8.05 ‰ and δ¹³C = -21.09 ‰) and USGS 43 (δ¹⁵N = +8.44 ‰ and δ¹³C = -21.28 ‰) for ¹³C/¹²C and ¹⁵N/¹⁴N; USGS 42 (+7.84 ‰) and USGS 43 (+10.46 ‰) for ³⁴S/³²S. Values of δ¹³C 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 δ¹⁵N were expressed versus Air-N₂ 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 δ³⁴S were expressed relative to VCDT on a scale defined by assigning a δ³⁴S value of -0.3 ‰ to the reference material IAEA-S-1. Method uncertainty (calculated as one standard deviation) was 0.1 ‰ for δ¹³C, 0.2 ‰ for δ¹⁵N, 0.3 ‰ for δ¹⁸O and δ³⁴S and 2 ‰ for δ²H.

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.