Telomere extraction and assay
We measured relative telomere length (RTL) from blood samples of sparrows after they fledged, collected from 2000 to 2015. DNA sample concentration was first measured using a Nanodrop 8000 Spectrophotometer (Thermo Fisher) and normalized to 20–30 ng/µl. Next we used a monochrome multiplex quantitative polymerase chain reaction (MMqPCR) method to quantify RTL (Cawthon, 2009) as described in Chik et al. (2023). In brief, MMqPCR quantifies RTL as a ratio of telomeric signals to that of a single-copy reference gene (GAPDH in our study), where amplification of both target sequences occur within a single well to eliminate error from sample loading. Samples were allocated to plates using a slicing approach (van Lieshout et al., 2020), where each ‘slice’ contained samples obtained from the same year, and each plate contained samples from three consecutive slices, to avoid confounding plate and sample year effects. We measured samples in duplicates in adjacent wells. Plates were run using two machines, a QuantStudio 12K Flex Real-Time PCR System (Thermo Fisher Scientific, five plates) and a StepOnePlus (Applied Biosystems, 77 plates), and by two technicians (MEM ran 52 plates and NdR ran 30 plates), but machine identity did not have an effect on RTL (Sibma, 2021). After MMqPCR, we removed samples with Ct values > 25, and a between-duplicate relative difference > 0.2. We averaged the RTL of the remaining duplicates as the final measure for each sample. Mean qPCR amplification efficiencies for telomeres and the reference gene were 90.7% (range: 71 – 109%) and 94.5% (range 76 – 119%) respectively (Sibma, 2021). The inter-plate repeatability was 0.49 (s.e. = 0.07), while the intra-plate repeatability was 0.98 (Sibma, 2021). The final telomere dataset consisted of 2,083 telomere length measurements from 1,225 birds, 476 of which have telomere length measurements at multiple ages.