Methylation variation
Similarly to genetic data, we performed an RDA on methylation level of 157,741 CpG sites to describe epigenetic variation among individuals in relation to location, habitat and sex (Figure 2B). The model was significant but explained less than 1% of the total variance (R²  =  0.007, P = 0.001). All variables contributed significantly (location : P = 0.001, habitat: P = 0.03, sex: P = 0.001, Table S5). Partial RDA revealed that location and sex explained a similar proportion of the total variance which was higher than habitat (location: R² = 0.003, P = 0.001; sex: R² = 0.003, P = 0.001; habitat: R² = 0.001, P = 0.3). When removing the Z chromosome from analyses, results remained similar (Table S6), showing that the difference in methylation was not entirely driven by sexual chromosomes.
We then investigated more finely whether individual methylation on CpG cytosines varied across location, habitat (urban vs forest), sex, and location×habitat interaction, using an ANOVA, run on autosomes and Z chromosome separately (Figure 3, Table S7). For autosomes, we detected a significant effect of location (F = 3.319, P = 0.044), with Montpellier individuals showing lower methylation levels that Warsaw ones (Figure 3, Tukey test: P = 0.04) and no other difference between pairs of cities. Also, no significant effect of sex (P = 0.263) or habitat (P = 0.478) was found, suggesting that urbanization did not have an important overall effect on global methylation levels. For the Z chromosome, we found a strong difference between sexes, with homogametic males showing 2.98% more methylated Z than heterogametic females (Figure 3 ; P = 1.45x10-15), while no significant difference between location (P = 0.577) or habitat (P = 0.915) was found.