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.