Molecular analyses
DNA extraction of the collected blood samples is described in Appendix S1. Relative erythrocyte TLs were measured in all nestlings (n =566, average age 10.9±1.5 days) from 2002-2006 (5 cohorts from two populations, Fig. 2) using the real-time quantitative polymerase chain reaction (qPCR) amplification method as described by Cawthon et al. (2002) and with modifications by Criscuolo et al. (2009). The qPCR method measures the ratio of the telomere repeat copy number amount of telomere sequence to a control single copy gene number (a non-variable “housekeeping” gene; GAPDH) relative to a reference sample. This ratio is referred to as the relative telomere length (TL). DNA samples were diluted with dH2O to yield 1.67 ng/μL (corresponding to 10 ng of DNA per well in the PCR assay) and subsequently stored at -78°C. All samples were randomized and run in triplicates on 96-well plates, each plate including a 2-fold serial dilution (40-2.5 ng/well) of a “standard sample” of DNA from a single individual used to produce standard curves for each plate, a non-target control sample, and the reference sample (all in triplicates). Following Ringsby et al. (2015), PCR assays were prepared using the Absolute blue qPCR SYBR green Low Rox master mix (ThermoFisher scientific) and run using a Stratagene Mx3005p system and analyzed using the MxPro qPCR software (Agilent). Primers and thermal profiles are specified in Appendix S1. Relative TLs were calculated taking plate amplification efficiencies (all within 100±10%, mean telomere assay efficiency was 102.2±3.8%, and 101.9±3.4% for GAPDH assays across 2x21 plates) into account using the Pfaffl (2001) method. All telomere analyses were performed at the University of Glasgow and included 507 individuals measured by MLP (this study) and 60 individuals (a subset of both male and female nestlings from both populations from 2002 and 2005) measured by WB under identical laboratory conditions and using the same reference sample (Ringsby et al., 2015). The average of the reference sample cycle thresholds (Ct) across all plates were 9.42±0.13 SD (CVinterplate=1.39%, CVintraplate=0.72%) for telomere assays and 20.58±0.09 SD (CVinterplate=0.46%, CVintraplate=0.40%) for GAPDH assays.
Molecular sexing and pedigree reconstruction are described in Appendix S1. Each nestling was classified into one of three selection categories (sample sizes are shown in Table 1): Selected (1): Both parents had been subject to the selection regime, i.e. captured during a winter selection event and allowed to stay in the population. Intermediate (0.5): one parent had been subject to artificial selection. Unselected (0): No parents had been artificially selected (i.e. accidentally not captured during any winter selection event) and these individuals are therefore the unselected controls. Unknown genetic parents were assumed not to have been artificially selected (i.e. not captured or immigrated after the selection event and thus not included in the pedigree).