Statistical analysis
All statistical analyses were performed in R 4.0.3 (R Core Team, 2017). Linear mixed models (LMMs) were run using the package lmer (Bates, Machler, Bolker, & Walker , 2015), with package lmerTest (Kuznetsova, Brockhoff, & Christensen 2016) to calculate degrees of freedom andp -values. We calculated marginal R2 values for the LMMs using the method described by Nakagawa and Schielzeth (2013). We initially performed a series of linear regression analyses to test for possible effects of body size on telomere length, possible effects of body size on percent DNAm changes and possible effects of temperature on growth rate/body size (Supplementary Analysis 1). We also performed a LMM to examine the effect of baseline telomere length (at day 3) on change in telomere length after adjusting for the effects of regression to the mean (RTM) (Verhulst, Aviv, Benetos, Berenson, & Kark, 2013; Supplementary Analysis 2).
In a first, longitudinal analysis, we used a LMM (Model 1) to explore the effects of temperature, clutch size, change in DNAm and change in tarsus (growth from day 3 to day 11) (all as fixed effects) on the rate of changes in telomere length from day 3 to day 11 (dependent variable). Our preliminary analysis (Supplementary analysis 2c) detected a negative correlation between tarsus length at day 3 and telomere length at day 3. Therefore, we also included tarsus length at day 3 as a fixed effect to account for potential effects of body size on telomere length changes. Nest ID was included as a random effect in this model to control for possible familial/genetic effects, and the repeated sampling of the same individuals within a brood. Only individuals with data for both day 3 and day 11 were included in this longitudinal analysis. Change between day 3 and day 11 was calculated using the equation (change = (value at day 11 – value at day 3)). Models were built on the biological variables of interest.
In our cross-sectional analyses, we performed two LMMs to examine a) Model 2: the effects of temperature, clutch size, DNAm at day 3, and tarsus length at day 3 (fixed effects) on telomere length at day 3 (the dependent variable), and b) Model 3: the effects of temperature, brood size, DNAm at day 11, and tarsus length at day 11 (fixed effects) on telomere length at day 11. We accounted for genetic and/or familial effects by including Nest ID as a random effect.