Feather sampling

One or more feathers samples were collected from a total of 233 birds over 3 different years in Portugal (79 birds, years 2009, 2013) and Spain (160 birds, 2013/14) from birds caught in nest boxes in breeding colonies. Adult males are easily recognised, juvenile and adult females have indistinguishable plumage. Based on previous knowledge of lesser kestrel moult patterns (Cramp and Simmons 1977), the primary P9 was considered the best feather to characterise the wintering area.

A total of 79 birds were sampled from breeding colonies in the migratory population in Portugal, where no individuals remain during the winter. In 2009 the winter grown feather P9 and the breeding season grown tail feather T3 were sampled from 32 birds, including 6 individuals whose sub-Saharan African wintering location was known from light level geolocators (see Catry et al 2011a). During the first

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batches of isotopic analysis, the T3 feathers were realised to be unreliable as an indicator of the breeding area due to high isotopic variability in carbon values. They were not included in the results. In the 2013 breeding season an additional 47 Portuguese birds were sampled when P9 (winter grown) and P3 (breeding grown) feather samples were collected. In total 28 individuals were identified from plumage as adult males, 51 were females of unknown age.

To isotopically characterise the Iberian breeding area, and to establish the base level for deuterium in non-breeding birds, breast feathers were collected from 25 chicks from the migratory (Portuguese) colonies, in 2013. Analysis of all primary feathers from dead birds (see below) showed that the P3 feather is grown at the end of the breeding season when some birds may already be migrating. This indicated that P4 would in fact be a better feather to characterise the breeding area.

Consequently, during the 2013 winter and the 2014 breeding season, we sampled P4 and P9 feathers from 160 birds in breeding colonies from the partially migratory (Spanish) population known to have both resident and migrant individuals.

Approximately 25-30% of individuals are resident in these colonies, and it is likely that some individuals overwinter in Iberia away from their breeding colonies (Negro et al 1991, author’s unpublished data). Of birds sampled from Spain, 79 were adult males, 13 were known from rings to be adult females and 68 were females of unknown age.

Breeding season P4 feathers were also taken from 40 birds known from plumage to be juvenile males. Feathers were stored individually in labelled sample bags until analysis. All 3 isotopes were analysed from Portuguese samples, whilst for Spanish, every isotope was analysed in a subset of feathers.

Moult sequence and consistency of isotopic variance across the wing were established by sampling all primary feathers (P1:10) from 9 lesser kestrels (n= 6 female, n= 3 male) found dead in 2013, using stable isotopes of δ¹³C, δ¹⁵N and δD.

Three (2 female, 1 male) were from the migratory Portuguese colonies and 5 were from the partially migratory Spanish colonies and included a known first year female.

133 6.2.3. Stable isotope analysis

A sample of 1.5 x 1.5cmof material was clipped consistently from the same distal portion of the feather to avoid any systematic differences in isotopic ratios within the feather that have been noted in some raptor species (Smith et al 2008). They were washed in a 2:1 chloroform:methanol solution to remove surface oils and dirt, then dried under the fume hood for 48 hours. Each sample (excluding rachis) was cut as finely as possible, cutting parallel to the rachis to capture as much isotopic variation as possible in each fragment. 0.5mg of sample were weighed and tightly packed in to tin capsules. To account for continuous exchange with atmospheric moisture, the standards and replicate samples intended for Deuterium analysis were loosely covered and allowed to equilibrate at room temperature with the ambient laboratory air for a minimum of 72 hours before being scrunched closed.

All samples were analysed using a Thermo Finnigan Delta XP continuous-flow isotope ratio mass spectrometry at the Stable Isotope Laboratory (ENVSIL), University of East Anglia, UK. Carbon and nitrogen isotopic composition δ¹³C and δ¹⁵N were simultaneously analysed using a Costech Elemental Analyser on-line with a mass spectrometer. For hydrogen isotopic analysis, a Vecstar vertical furnace with a glass carbon packed pyrolysis column was used on-line with the same mass spectrometer.

Two newly developed international keratin standards from the US Geological Survey (USGS 42 and USGS 43; Coplen and Qi 2012) and an inter-comparison material independently developed by the Doñana Biological Station, Spain (LIE-PA2, Razorbill feather; Alvarez 2012) were analysed at the start and end of batches to ensure consistency between batches. Some samples had repeated analysis in different batches as an additional consistency check. A triplicate of the USGS43 was analysed after every 12 unknown samples to account for instrument drift quantification. Due to faulty carrier gas flow affecting the nitrogen portion of the analysis, some batches of the Spanish feathers had to be remeasured. In some cases there was insufficient sample remaining so carbon data only exists for these feathers.

Assuming the proportion of exchangeable hydrogen in feather keratin was 20%

(Wassenaar and Hobson 2000) an isotopic scale stretch correction was applied to all samples using the known non-exchangeable fractions of USGS 42 (-78.5‰), USGS 43 (-50.3‰) and Razorbill (20.8‰).

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Stable isotope compositions are reported in δ notation in parts per thousand (‰) deviation from the international standards for Carbon (Pee Dee Belemnite (PDB)), nitrogen (Atmospheric Nitrogen (Air)) and for hydrogen (Vienna Standard Mean Ocean Water (VSMOW)). This is defined by the equation:

δsample=([Rsample/Rstandard]-1)x1000 where δsample is either ¹³C, ¹⁵N or ²H respectively, relative to the standard and R is the ratio of the heavy and light isotopes (¹³C/¹²C, ¹⁵N/¹⁴N or ²H/¹H respectively) in both the sample and reference material.

Measurement precision for δ13C and δ15N was estimated to be ≤0.2‰ and for Hydrogen was ≤2.0‰

6.2.4. Assessing the effects of body size and heat stress on feather hydrogen