Statistical analyses
All analyses were conducted using R programming environment (R Core Team 2017). The effect of egg type (mimetic vs. non=mimetic) on corticosterone levels was analyzed using a linear model. Corticosterone levels were natural log-transformed to conform to the expectations of normal distribution (log-transformed corticosterone: Shapiro-Wilk W=0.966, p = 0.238). In addition to the egg type, we included the date of the manipulation as a fixed factor in the analyses, because maternal hormone levels often show strong seasonality (Tyrrell & Cree 1998; Jawor et al. 2006; Hauber et al. 2020a) and in this study corticosterone showed a significant decrease over the breeding season (see below). We then used a linear model with date as a covariate to test if corticosterone levels differed between females who rejected or accepted a non-mimetic egg at the two-hour mark.
For RNAseq analysis, all paired-end reads were mapped to the Swainson’s thrush genome (Accession GCF_009819885.1) as it was the closest available full genome assembly at the time of this analysis. Paired-end mapping was completed using rsem (Li & Dewey 2011) and bowtie 1.0.0 (Langmead et al. 2009) with default parameters. To exclude unexpressed genes and genes not present in robins, only genes with at least 10 identified read counts in over one-third of samples were included for analysis. Differential expression was analyzed using DESeq (Anders & Huber 2010) for differences between treatments and differences between individuals who rejected and those that did not reject the mimic egg. For all analyses, the threshold for significance was set at a false-discovery-rate of 5%.
We calculated instantaneous heart rate each minute, for the first 10 minutes, after the bird arrived at the nest (first audible detection). The average instantaneous heart rates over this 10-minute period were then log-transformed to satisfy assumptions of normality (log-minute averages: Shapiro-Wilk W = 0.992 p-value = 0.058). We then used linear mixed models using R package nlme (Pinheiro et al. 2015) to analyze the effect of model egg color on average heartrate, adding time since arrival, treatment date, and treatment order as covariates, and bird ID as a random factor. Time since arrival was included because robin heart rate was typically high immediately after the arrival at the nest (presumably due to the metabolic demands of flight) following which the heart rate rapidly decreased. To test if individuals differed in the rate of which their heart rate declined following the arrival we asked if addition or random slope terms to the model resulted in a better model fit. However, random slope models had a higher AIC, therefore the final models did not include the random slope term. Only one female rejected the non-mimetic beige egg within the 10 min heart rate recording window and we, therefore, did not include the rejection as a covariate in the model. However, we ran a separate model asking if females that rejected the non-mimetic beige egg within two hours had a higher heart rate in response to the beige eggs compared to females that did not reject these eggs within this time period.