INTRODUCTION
Identifying mechanisms involved in rapid adaptation to novel
environmental conditions is a central theme in evolutionary biology and
a pressing concern in the context of global changes characterising the
Anthropocene (Malhi, 2017). The vast majority of studies investigating
mechanisms involved in rapid adaptation to new environments have focused
on phenotypic plasticity on the one hand and on genetic responses to
selection on the other hand. At their crossroad, recent work underlines
the potential role of epigenetics in rapid adaptation to new
environments (Liu, 2013). In particular, environmental variations can
induce differences in DNA methylation patterns and hence modulate gene
expression and upper-level phenotypes (Duncan, Gluckman, & Dearden,
2014; Jaenisch & Bird, 2003). Such methylation-linked phenotypic
variation can occur during an individual’s lifetime, especially early on
during the organism’s development (Waterland & Jirtle, 2003; Weaver et
al., 2004). Although methylation changes acquired across an individual’s
lifetime may often be non-heritable (7 ) but see (8,9 ),
epigenetically induced phenotypic shifts may nevertheless enhance
individual fitness in new environments. Moreover, during the course of
evolution, divergent genetic variants regulating epigenetic
modifications may also under selection, hence promoting the evolution of
divergent epigenotypes and epigenetically-linked phenotypic variation
(Richards, Bossdorf, & Pigliucci, 2010). While epigenetic studies
focused on human diseases and medical topics are now abundant, studies
in an ecological context are still rare (Derks et al., 2016). A few
epigenetic studies in natural populations revealed that DNA methylation
shifts might play a determinant role in local adaptation to
environmental variation (Foust et al., 2016). There is hence an urgent
need for further empirical investigations of simultaneously rapid
genetic and epigenetic evolution in response to environmental change
(Danchin et al., 2011).
Urbanization rapidly and irreversibly changes natural habitats into
human-made environments and is considered a major threat to biodiversity
(Brondizio, Settele, & Díaz, 2019). For species who appear to cope with
urbanisation, urban habitats present a myriad of novel environmental
conditions compared to the habitat where they evolved, including high
levels of chemical, light and sound pollution, high proportion of
impervious surfaces, high habitat fragmentation, low vegetation cover
and high human densities (Grimm et al., 2008; Szulkin, Garroway,
Corsini, Kotarba, & Dominoni, 2020). Such extreme environmental changes
compared to natural areas are expected to result in numerous novel
selection pressures for city-dwelling species (Szulkin, Munshi-South, &
Charmantier, 2020). Accordingly, rates of recent phenotypic change,
concerning multiple types of traits related to behaviour, morphology,
phenology and physiology, were found greater in urban areas than in any
other habitat types, including non-urban anthropogenic contexts (Alberti
et al., 2017; K. A. Thompson, Rieseberg, & Schluter, 2018). The
exploration of the molecular mechanisms implicated in urban-driven
phenotypic changes has only begun, with both genetic (Mueller, Partecke,
Hatchwell, Gaston, & Evans, 2013; Perrier et al., 2018; Salmón et al.,
2020), and epigenetic investigations (McNew et al., 2017; Riyahi,
Sánchez-Delgado, Calafell, Monk, & Senar, 2015; Watson, Powell, Salmón,
Jacobs, & Isaksson, 2021). For instance, DNA methylation variations
have been associated in vertebrates with high levels of traffic-related
air pollution (Ding et al., 2017). Yet, epigenetic studies have been
performed at relatively small genomic resolution. In addition, very
little is known about the level of parallelism and hence of the
predictability of genetic and epigenetic evolution in response to
urbanisation in distinct cities (Rivkin et al., 2018; Santangelo,
Rivkin, & Johnson, 2018). So far, a small number of studies have
provided evidence for a range of situations: from local adaptation
despite strong gene flow (e.g. in the red-tailed bumblebee Bombus
lapidaries Theodorou et al., 2018) to restricted gene flow and
independent colonization in different cities by a few founders, followed
by adaptation (e.g. in the burrowing owl Athene cunicularia,Jakob C. Mueller et al., 2018). Providentially, recent genomic tools of
high resolution and the multitude of cities around the globe offer
unique opportunities to compare simultaneously individuals’ genomic and
epigenomic responses in several cities and thereby study the parallelism
and predictability in molecular mechanisms implicated in rapid
adaptation to urbanization (Perrier, Caizergues, & Charmantier, 2020;
Santangelo et al., 2020).
In this study, we used both genome-wide and epigenome-wide sequencing
approaches to compare genetic and epigenetic responses among three pairs
of great tit Parus major populations in urban and forest
habitats. At the European level, population monitoring of Great tits
revealed parallel phenotypic shifts in city birds compared to their
forest conspecifics, with in particular smaller and lighter urban birds
laying earlier and smaller clutches (Biard et al., 2017; Caizergues et
al., 2021; Chamberlain, Hatchwell, & Gaston, 2009; Corsini et al.,
2020). In addition, genomic analyses showed patterns of genome-wide
differentiation between urban and forest birds (Perrier et al., 2018)
while a large scale analysis revealed some parallel footprints of
adaptation to urbanization across nine European cities (Salmón et al.,
2020). At the epigenetic level, a preliminary Great tit study recently
described methylation shifts associated with urbanization (Watson et
al., 2021). However, this analysis focused on a single location (n = 6
urban and 6 forest males) and hence could not test for a potential
parallelism in the urban-related epigenomic response. Moreover, the
search for DMSs (Differentially Methylated Sites) rather than DMRs
(differentially methylated regions) in this recent study most probably
weakened the power to detect significantly differentially methylated
regions of the genome. In order to advance our understanding of the
genome-wide and epigenome-wide responses to urbanization, and its
putative spatial parallelism, we here searched for genomic footprints of
divergent selection and for DMRs between three pairs of urban-forest
populations across Europe. Our results show that despite limited genetic
differentiation and few genomic footprints of divergent selection
between forest and urban populations, urban life was associated with
numerous differentially methylated regions notably associated with
neural development, behaviour and immunity. Hence, this study shows the
potential role of an epigenetic response in rapid adaptation to
urbanization. Importantly, we found little parallelism between cities in
both the genomic and the epigenomic responses to urbanization, possibly
confirming the hypothesis that multiple evolutionary ways exist to
independently cope with similar novel environmental conditions.