Evidence for mostly non-parallel differentially methylated
regions between urban and forest environments
We identified a total of 224 distinct DMRs between urban and forest
great tits: 80 for Barcelona, 68 for Montpellier and 93 for Warsaw. Only
14 DMRs (6.25%) were found repeatedly in at least two comparisons, and
only 3 were common to the three cities. 7 of these 14 parallel DMRs were
in the same direction of methylation in urban compared to forest areas
(Figure 5, Figure S4A). Barcelona urban birds presented significantly
more hypomethylated than hypermethylated DMRs
(χ2 = 11.25, P < 0.001),
while no difference was found for Montpellier
(χ2 = 0.941, P = 0.332) nor for Warsaw
(χ2 = 0.011, P = 0.917). DMRs were
distributed across all the 32 chromosomes as well as on 37 unplaced
scaffolds. 203 of the 224 different DMRs (91%) overlapped genes or 5kb
flanking regions, among which 52% were directly located in gene bodies,
promoter or TSS sequences (35.3% in gene bodies and 47.3% in
promoters/TSS) suggesting their putative functional role in gene
expression and regulation and/or splicing events.
Following the procedure previously described, GO analyses on the pooled
genes list revealed an overrepresentation of modules associated with the
nervous system (GO:2000300, regulation of synaptic vesicle exocytosis;
GO:0050804 modulation of synaptic transmission), immunity (GO:005728,
negative regulation of inflammatory response; GO:0050852, T cell
receptor signaling pathway), metabolic activity (GO:006816 calcium ion
transport; GO:0055072, iron ion homeostasis, GO:0043087, regulation of
GTPase activity), behaviour (GO:0007626, locomotory behavior) and
endocrine processes (GO:0044060: regulation of endocrine process). All
enriched GO are presented in Table 2 and SI Figure S5.
We also searched for DMRs between sexes, following the same procedure.
We identified 206 DMRs associated with sex, of which 58 for Barcelona,
81 for Montpellier and 99 for Warsaw. Warsaw presented significantly
more hyper than hypomethylated DMRs (χ2=5.878, P=
0.015), but it was not the case for Barcelona (χ2=0,
P=1) nor Montpellier (χ2=1.25, P=0.264). DMRs were
distributed on 29 chromosomes and 35 unplaced scaffolds. On a total of
206 DMRs, 181 (57.3%) were on genes or in a 5kb upstream/downstream
region around genes. GO analyses revealed enrichment of genes involved
in development, growth and morphogenesis, among others (see detailed
enriched GO Table 3, Figure 6 & S6).
Almost twice more sex DMRs were shared between locations (11,7%) than
between habitats (6.25%, see Figure S4 A & B ; z-test: χ²-squared =
3.885, P = 0.049). When taking into account the direction of
methylation difference, 7 sex DMRs were shared between at least two
cities (9.7%) which was three times more than for habitat DMRs (3.1%;
z-test: X-squared = 7.904, P = 0.005).