# The city and forest bird flock together in a common garden: genetic and environmental effects drive urban phenotypic divergence

Megan Thompson, Denis Réale, Baptiste Chenet, Ségolène Delaitre, Amélie

Fargevieille, Marc Romans, Samuel Caro, Anne Charmantier

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Megan Thompson, Denis Réale, Baptiste Chenet, Ségolène Delaitre, Amélie Fargevieille, et al. The city and forest bird flock together in a common garden: genetic and environmental effects drive urban phenotypic divergence. Evolution - International Journal of Organic Evolution, 2025, 79 (5), pp.800-822. 10.1093/evolut/qpaf028. hal-04952645

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Submitted on 22 Nov 2025

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# The city and forest bird flock together in a common garden: genetic and environmental effects drive urban phenotypic divergence

Megan J. Thompson<fnref n="1" />,<fnref n="2" />, ID, Denis Réale<fnref n="1" />, ID, Baptiste Chenet<fnref n="3" />, Ségolène Delaitre<fnref n="2" />, Amélie Fargevieille<fnref n="2" />, ID, Marc Romans<fnref n="3" />, Samuel P. Caro<fnref n="2" />,†, ID, Anne Charmantier<fnref n="2" />,†, ID

## Abstract

Urban phenotypic divergences are documented across diverse taxa, but the underlying genetic and environmental drivers behind these phenotypic changes are unknown in most wild urban systems. We conduct a common garden (CG) experiment using great tit (Parus major) eggs collected along an urbanization gradient to: (1) determine whether documented morphological, physiological, and behavioral shifts in wild urban great tits are maintained in birds from urban and forest origins reared in a CG (N = 73) and (2) evaluate how different sources of genetic, early maternal investment, and later environmental variation contributed to trait variation in the experiment. In line with the phenotypic divergence in the wild, CG birds from urban origins had faster breath rates (i.e., higher stress response) and were smaller than birds from forest origins, while wild differences in aggression and exploration were not maintained in the experiment. Differences between individuals (genetic and environmentally induced) explained the most trait variation, while variation among foster nests and captive social groups was limited. Our results provide trait-specific evidence of evolution in an urban species where genetic change likely underlies urban differences in morphology and stress physiology but that urban behavioral divergences are more strongly driven by plasticity.

**Keywords:** urbanization, urban gradient, common garden experiment, plasticity, genetic differentiation, great tits

## Introduction

Various evolutionary and ecological processes shape the phenotypic diversity we observe in nature and lead to phenotypic divergences between populations (Hendry, 2017; Mitchell-Olds et al., 2007). For instance, local selection pressures on heritable traits can lead to divergent adaptation to local environmental conditions (Kawecki & Ebert, 2004). Developmental or reversible phenotypically plastic responses to local environments can also drive phenotypic adjustments (Ghalambor et al., 2007). Determining how local adaptation and plasticity interact to shape phenotypes is crucial as these processes can have different impacts on demographic and evolutionary trajectories of wild populations (Ghalambor et al., 2007; Nicolaus & Edelaar, 2018; Snell-Rood, 2013).

Some of the most striking examples of phenotypic diversity occur along urbanization gradients, such as urban shifts in multiple taxa towards smaller body sizes (Merckx et al., 2018). Such phenotypic shifts in urban populations are frequently documented across diverse taxa and traits (Diamond & Martin, 2021; Szulkin et al., 2020), through changes in both phenotypic means (Lambert et al., 2021; Miranda et al., 2013) and more recently in phenotypic variation (Capilla-Lasheras et al., 2022; Thompson et al., 2022). Urban

phenotypic divergences are commonly assumed to be driven by genetic change via selection, but there is still a lack of evidence that urban organisms are adapting to these novel urban conditions (Lambert et al., 2021) and plasticity could play a major role in urban phenotypic change (Hendry et al., 2008; Yeh & Price, 2004). Determining the mechanisms behind phenotypic changes in urban organisms could importantly inform on whether urban populations will continue to adjust in pace with further environmental change. For these reasons, there have been several calls for research that disentangle the genetic and plastic contributions on urban phenotypes and, more specifically, calls for urban common garden (CG) experiments (Lambert et al., 2021; Rivkin et al., 2019; Sanderson et al., 2023).

CG experiments are a useful approach for exploring the genetic basis of phenotypic differences between populations. These manipulations rear individuals from different populations under the same environmental conditions from very early life stages and ideally across several generations. As individuals develop and mature under common conditions, phenotypic differences that persist in this context should reflect underlying genetic differentiations rather than plastic responses to environmental conditions (Lambert et al., 2021).

<footnote n="1">¹Département des sciences biologiques, Université du Québec à Montréal, Montréal, QC, Canada</footnote>

<footnote n="2">²Centre d'Ecologie Fonctionnelle et Evolutive, Univ Montpellier, CNRS, EPHE, IRD, Montpellier, France</footnote>

<footnote n="3">³Parc de Lunaret, Montpellier, France</footnote>

<footnote>Corresponding author: Département des sciences biologiques, Université du Québec à Montréal, 141 Av. du Président-Kennedy, Montréal, QC H2X 1Y4, Canada. Email: thompsonjoymegan@gmail.com</footnote>

<footnote n="1">¹Caro and Charmantier should be considered joint last authors.</footnote>
Thus, CGs can help determine whether evolutionary (i.e., genetic) change drives documented phenotypic divergences in wild populations and can be used to explore interactions between genetic and plastic changes acting in these systems (Conover et al., 2009). For these reasons, CG approaches are needed in urban evolution research to explore the potential processes acting in these contexts (Alberti et al., 2017; Sanderson et al., 2023).

We censused 77 CG studies with urban populations in the literature; an impressive number despite the effort and resources these experiments require (see synthesis in Table 1). Most of these studies have been published within the last ten years (83%, N = 64) and many support genetic divergence underlying shifts in urban phenotypes (86%, N = 66), which could indicate local adaptation to urban conditions via evolution. Fewer studies document plasticity to environmental conditions as a driver of phenotypic change (58%, N = 45), but this conclusion is especially common in multitreatment CGs where individuals are reared under multiple environmental treatments (e.g., temperature treatments). Experiments so far tend to use invertebrate or plant models (75%, N = 58), likely as these organisms are more easily reared, reproduced, and manipulated in captive environments. Many studies have focused on physiological phenotypes associated with tolerance to temperature as the urban heat island effect is known to increase temperature and heat stress in urban environments (Mohajerani et al., 2017). For example, urban damselflies (Coenagrion puella), water fleas (Daphnia magna), wood louse (Oniscus asellus), and acorn ants (Temnothorax curvispinosus) have higher heat tolerance compared to non-urban conspecifics when reared under common conditions, providing evidence that urban invertebrates have adapted to urban heat islands (Brans et al., 2017a; Diamond et al., 2018a; Tüzün & Stoks, 2021; Yilmaz et al., 2021).

While CG studies provide support in favor of urban evolution in some well-studied invertebrate and plant taxa, whether genetic change drives phenotypic shifts in other urban taxa is not as well known (e.g., Table 1: 10% or N = 8 CG studies in birds). For instance, birds are one of the most studied taxa in urban ecology and evolution, and there are growing generalizations on how urbanization impacts the traits of birds globally (e.g., earlier lay dates or smaller body sizes, Capillal-Lasheras et al., 2022; Hahs et al., 2023). Great tits (Parus major), specifically, have become a model species for studying urban evolution across Europe and Asia and, thus, research on this species is now contributing to large collaborative research efforts that evaluate trends across replicated urban gradients in continent-wide analyses (Salmón et al., 2021; Thompson et al., 2022; Vaugoyeau et al., 2016). Despite these exciting efforts, a fundamental gap exists about whether urban phenotypic shifts in this model species are driven by evolutionary change between populations or by plastic responses to urban conditions. CG experiments in urban dark-eyed juncos (Junco hymemalis) and European blackbirds (Turdus merula) suggest that genetic change could at least partially play a role in phenotypic differences across morphological, physiological, and behavioral traits (Table 1; Atwell et al., 2012; Miranda et al., 2013; Reichard et al., 2020), but it remains to be seen whether this holds for other urban bird species and, specifically, the great tit where urban phenotypic shifts have been well documented in the wild.

This study uses a CG experiment to disentangle the mechanisms that shape urban phenotypes in populations of great

tits in and around Montpellier, France. In this system, we have documented several phenotypic differences between urban and forest populations in life history, morphology, physiology, and behavior (Caizergues et al., 2018, 2022; Charmantier et al., 2017); trends that tend to be consistent across other European populations (Biard et al., 2017; Corsini et al., 2021; Thompson et al., 2022; Vaugoyeau et al., 2016). In the wild, urban great tits are smaller, have faster breath rates under constraint, show higher aggressiveness when handled, and are faster explorers than their forest counterparts, although estimates of selection gradients suggest that these urban phenotypic shifts are not favored by natural selection (Caizergues et al., 2018, 2022). Genomic studies have revealed that, despite evident gene flow in this system, a small but significant proportion of genetic variation is explained by urbanization. This result suggests some genetic divergence between the urban and forest populations (i.e., significant $F_{ST} = 0.006–0.009$ between urban and forest comparisons; Perrier et al., 2018). A CG approach is the next logical step in deciphering the genetic and plastic influences on the phenotypic divergences documented in this urban system.

We reared great tits from eggs collected from urban and forest sites around Montpellier under common conditions to evaluate whether documented morphological, physiological, and behavioral differences persist under the same environment. We had two major aims. The first was to compare the phenotypes of birds from urban and forest origins reared in a CG. We hypothesized stronger genetic change for highly heritable traits (e.g., morphology, physiology) than for lowly heritable ones (e.g., behavior; Kinnison & Hendry, 2001; Stirling et al., 2002) and, therefore, that morphological and physiological differences would be more likely to persist under common conditions. More specifically, we predicted that birds from urban origins would be smaller and more stressed (phenotypic difference persists) but not more aggressive or exploratory (phenotypic difference does not persist) than birds from forest origins. Our second aim was to evaluate how different sources of variation (i.e., genetic and environmental variation) shaped phenotypes in the experimental context. We present phenotypic estimates from wild populations alongside those from the CG for comparison.

## Methods

### Study system and quantifying urbanization

Populations of urban and forest great tits have been monitored at nest boxes in and around the city of Montpellier, France, as a part of a long-term study (Charmantier et al., 2017). The forest population has been monitored since 1991 in La Rouvière forest located 20 km north of Montpellier, where the number of nest boxes of 32 mm diameter entrance ranged from 37 to 119 because of theft/replacement. The urban population has been monitored since 2011 throughout the city of Montpellier at study sites that differ in their degree of urbanization (163–208 urban nest boxes across 8 study areas, see Figure 1 in Caizergues et al., 2024). During each spring, nest boxes are visited once per week to follow the reproduction of breeding pairs. We catch adults at nest boxes when nestlings are around 12 days old, ring them with a unique metal band, age them based on plumage as either yearling (born previous year) or adult (born at least the year before last), take a blood sample, and measure several phenotypes (see section “phenotyping”; Caizergues et al., 2018, 2022). We quantified the
<table><caption>**Table 1.** Synthesis of studies (N = 77) that have used a common garden approach to compare the phenotypes of urban and nonurban populations (e.g., rural, forest, mountain, agricultural) across a variety of different groups (amphibians, birds, invertebrates, fish, plants, reptiles) and traits (physiology, behavior, morphology, life history, phenology).</caption><thead><tr><th>Group</th><th>Urban vs.</th><th>Traits</th><th>Approach<sup>a</sup></th><th>Urban results<sup>b</sup></th><th>Conclusion<sup>c</sup></th><th>Notes</th><th>Reference</th></tr></thead><tbody><tr><td><strong>AMPHIBIANS</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Agile frog<br>(Rana dalmatina)</td><td>Woodland</td><td>Physiology</td><td>Eggs</td><td>Lower heat tolerance</td><td>E (&amp; G)</td><td>Difference in common garden opposite to wild divergence</td><td>(Bökony et al., 2024)</td></tr><tr><td>Common toads<br>(Bufo bufo)</td><td>Agricultural &amp; natural</td><td>Physiology</td><td>F1 generation</td><td>No difference in toxicity and potency (or in opposite directions)</td><td>E</td><td>Urban and agricultural higher toxicity and potency in wild</td><td>(Bökony et al., 2019)</td></tr><tr><td>Guttural Toad<br>(Sclerophrys gutturalis)</td><td>Natural</td><td>Behaviour</td><td>F1 generation</td><td>No difference in boldness</td><td>E</td><td>Urban and agricultural higher stress response in wild</td><td>(Bökony et al., 2021)</td></tr><tr><td><strong>BIRDS</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Dark-eyed juncos<br>(Junco hymemalis)</td><td>Mountain</td><td>Behaviour<br>Physiology<br>Morphology</td><td>Fledglings<br>Nestlings</td><td>Higher boldness and lower stress response<br>Urban birds have shorter wing and tail lengths, and whiter tails</td><td>G<br>G</td><td>Same results in wild<br>Same results in wild</td><td>(Atwell et al., 2012)<br>(Rasner et al., 2004; Yeh, 2004)</td></tr><tr><td>European blackbirds<br>(Turdus merula)</td><td>Rural<br>Forest</td><td>Physiology</td><td>Fledglings<br>Nestlings</td><td>No difference in testosterone<br>Higher minimum frequency, no difference in peak or max frequency<br>Higher gonadal sensitivity<br>Attenuated stress responses</td><td>E<br>G<br>G</td><td>Lower urban peak testosterone<br>Same results in wild<br>Advanced reproduction in wild<br>Higher urban repeatability</td><td>(Atwell et al., 2014)<br>(Reichard et al., 2020)<br>(Fudickar et al., 2017)<br>(Miranda et al., 2013)<br>(Partecke et al., 2006)</td></tr><tr><td><strong>INVERTEBRATES</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Water fleas<br>(Daphnia magna)</td><td>Rural</td><td>Physiology</td><td>F2 generation<br>2 temperature treatments</td><td>Higher thermal tolerance, hemoglobin, and smaller body size</td><td>G &amp; E</td><td>Higher thermal tolerance at warmer rearing temperature</td><td>(Brans et al., 2017a)</td></tr><tr><td>Morphology</td><td>Morphology</td><td>F2 generation<br>2 temperature treatments</td><td>Smaller body size</td><td>Higher energy reserves and lower enzyme activity</td><td>G &amp; E</td><td>Smaller body sizes at warmer rearing temperatures</td><td>(Brans et al., 2017b)</td></tr><tr><td>Physiology<br>Life history</td><td>Physiology<br>Life history</td><td>F3 generation<br>2 temperature treatments</td><td>Higher energy reserves and lower enzyme activity</td><td>Rearing temperature affected some traits<br>Pace of life syndrome in urban fleas</td><td>G &amp; E</td><td>Rearing temperature affected some traits<br>Pace of life syndrome in urban fleas</td><td>(Brans et al., 2018)</td></tr></tbody></table>
<table><caption>**Table 1. Continued**</caption><thead><tr><th>Group</th><th>Urban vs.</th><th>Traits</th><th>Approach<sup>a</sup></th><th>Urban results<sup>b</sup></th><th>Conclusion<sup>c</sup></th><th>Notes</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="2">Water fleas<br>(Ceriodaphnia cornuta)<br>Mosquito<br>(Anopheles gambiae)</td><td>River/<br>Lake</td><td>Life history<br>Morphology</td><td>F2 generation<br>2 temperature<br>treatments</td><td>Faster maturation, smaller, increased<br>fecundity, early release of progeny</td><td>G & E</td><td>Trait changes in response<br>to rearing temperature<br>in same direction<br>Faster pace of life in urban<br>fleas<br>Some evidence of G × E for<br>fecundity</td><td>(Brans & De<br>Meester,<br>2018)</td></tr><tr><td>Rural</td><td>Life history<br>Morphology<br>Physiology</td><td>F2 generation<br>2 temperature<br>treatments<br>Toxicity treatment<br>Food treatments<br>Larvae</td><td>Higher survival<br>Physiology</td><td>G</td><td>No differential impacts of<br>temperature</td><td>(Brans et al.,<br>2021)</td></tr><tr><td rowspan="2">Asian tigermosquito<br>(Aedes albopictus)<br>Blowfly<br>(Calliphora vicina)</td><td>Rural</td><td>Life history<br>Morphology</td><td>F2 generation<br>Food treatments<br>Larvae</td><td>Reduced dietary tolerance to cyano-<br>bacteria<br>Agricultural sites higher tolerance to<br>carbamates</td><td>G × E</td><td>Genomic differentiation<br>between populations</td><td>(Zhang et al.,<br>2022)</td></tr><tr><td>Rural</td><td>Life history<br>Morphology</td><td>F1 generation<br>Light and dark<br>treatment<br>F2 and F3 gener-<br>ation<br>4 temperature<br>treatments</td><td>Higher tolerance to ammonia<br>Agricultural sites higher tolerance to<br>DDT<br>No difference in diapause incidence</td><td>G</td><td>Diapause lower and more<br>variable under light<br>treatment</td><td>(Antonio-<br>Nkondjio et<br>al., 2011)<br>(Tene Fossog<br>et al., 2013a)<br>(Tene Fossog et<br>al., 2013b)</td></tr><tr><td>Wormlion fly<br>(Vermileonidae sp.)<br>Damselflies<br>(Coenagrion puella)</td><td>Natural</td><td>Life history<br>Physiology</td><td>Larvae<br>Larvae with<br>immune challenge<br>Control and heat<br>wave treatments</td><td>Higher foraging investment and more<br>selective habitat choice<br>No difference in survival, growth rates,<br>immune response, or bioenergetics.<br>No reduction in energy budgets when<br>exposed to a simulated heat wave.</td><td>G</td><td>Sex difference across tem-<br>peratures</td><td>(Westby &<br>Medley,<br>2020)<br>(Hwang &<br>Turner,<br>2009)</td></tr><tr><td>Behaviour</td><td>Rural</td><td>Life history<br>Physiology</td><td>Larvae<br>5 temperature<br>treatments</td><td>Lower growth rates and higher survival</td><td>G</td><td>No large difference across<br>temperatures<br>Some evidence for counter-<br>gradient variation</td><td>(Tüzün et al.,<br>2017a)</td></tr><tr><td>Behaviour</td><td>Rural</td><td>Life history<br>Physiology</td><td>Larvae<br>Control and pesti-<br>cide treatments<br>Larvae<br>2 temperature<br>treatments<br>Control and pesti-<br>cide treatments</td><td>Less active, bolder, and have slower<br>growth rates when exposed to<br>pesticides<br>More active when exposed to pesti-<br>cides, but no difference in explora-<br>tion or food intake</td><td>G × E</td><td>Heat wave experiment<br>significantly influenced<br>all traits measured</td><td>(Tüzün et al.,<br>2017b)</td></tr></tbody></table>
<table><caption>**Table 1. Continued**</caption><thead><tr><th>Group</th><th>Urban vs. Traits</th><th>Approach<sup>a</sup></th><th>Urban results<sup>b</sup></th><th>Conclusion<sup>c</sup></th><th>Notes</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="3">(Ischnura elegans)</td><td>Life history</td><td>F1 generation<br>3 temperature<br>treatments<br>2 predator treatments</td><td>Lighter and slower growth rate</td><td>G × E</td><td>Central and high latitude<br>origin populations<br>Different responses across<br>latitudes</td><td>(Palomar et al.,<br>2023)</td></tr><tr><td>Behaviour</td><td>F1 generation<br>2 temperature<br>treatments</td><td>Higher encounter and predation rates,<br>but only on prey from rural origins</td><td>G × E</td><td>Daphnia prey also reared<br>in common garden</td><td>(Brans et al.,<br>2022)</td></tr><tr><td>Pristine</td><td>Larvae</td><td>Urban moths less attracted to light</td><td>G</td><td>Males more likely to be<br>attracted to light than<br>females</td><td>(Altermatt &<br>Ebert, 2016)</td></tr><tr><td rowspan="2">Spindle ermine moths (Ypo-<br>nomenta cagnagella)</td><td>Morphology</td><td>Larvae</td><td>Smaller wings and less attracted to light</td><td>G</td><td></td><td>(Van de Schoot<br>et al., 2024)</td></tr><tr><td>Rural</td><td>F2 and F3 gener-<br>ation</td><td>Higher heat tolerance in adults</td><td>G</td><td>Parallel patterns across<br>three countries<br>Divergence not evidence in<br>larval stage</td><td>(Merckx et al.,<br>2024)</td></tr><tr><td>Latticed heath moth<br>(Chiasmia clathrate)</td><td>Physiology</td><td>F3 generation<br>Light treatments</td><td>Reduced diapause induction</td><td>G & E</td><td>Light increases direct<br>development and<br>reduces development<br>time</td><td>(Merckx et al.,<br>2023)</td></tr><tr><td rowspan="2">& green-veined white butterfly<br>(Pieris napi)</td><td>Life history</td><td>F1 generation<br>Photoperiod treat-<br>ments</td><td>Lower day length threshold for direct<br>development and reduced diapause<br>induction</td><td>G × E</td><td>2 replicate cities<br>Longer and later urban<br>flight period in the wild</td><td>(Merckx et al.,<br>2021)</td></tr><tr><td>Rural</td><td>Eggs</td><td>No difference in personality</td><td>E</td><td>Urban males who were<br>more active as larvae<br>were more exploratory<br>as adults</td><td>(Kaiser et al.,<br>2018)</td></tr><tr><td>Speckled wood<br>(Pararge aegeria)</td><td>Behaviour</td><td>F1 generation</td><td>No difference in boldness or activity</td><td>E</td><td>Covariation between bold-<br>ness and activity existed<br>in urban butterflies</td><td>(Kaiser et al.,<br>2020)</td></tr><tr><td rowspan="2">Acorn ants<br>(Temnothorax curvispinosus)</td><td>Agricultural &<br>woodland</td><td>F1 generation</td><td>Urban earlier reproduction</td><td>G & E</td><td>Earlier reproduction in<br>colonies from lower<br>latitudes and those<br>reared under warmer<br>temperatures</td><td>(Chick et al.,<br>2019)</td></tr><tr><td>Rural</td><td>F1 generation<br>5 temperature<br>treatments</td><td></td><td></td><td>Higher fitness under<br>treatment like source<br>environment<br>Higher heat tolerance<br>under warmer rearing<br>temperatures</td><td>(Diamond et<br>al., 2018b)</td></tr><tr><td></td><td>Physiology</td><td>F1 generations—3<br>cities<br>5 temperature<br>treatments</td><td>Higher heat tolerance (2 out of 3 cities)</td><td>G & E</td><td></td><td></td></tr></tbody></table>
<table><caption>**Table 1. Continued**</caption><thead><tr><th>Group</th><th>Urban vs. Traits</th><th>Approach<sup>a</sup></th><th>Urban results<sup>b</sup></th><th>Conclusion<sup>c</sup></th><th>Notes</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="10">(Temnothorax nylanderi)</td><td>Physiology</td><td>F1 generation<br>2 temperature<br>treatments</td><td>Higher plasticity in heat tolerance when<br>reared under warmer temperature</td><td>G × E</td><td></td><td>(Diamond et<br>al., 2018a)</td></tr><tr><td>Physiology</td><td>F1 generation<br>2 temperature<br>treatments</td><td>Higher heat tolerance and lower cold<br>tolerance</td><td>G × E</td><td></td><td>(Diamond et<br>al., 2017)</td></tr><tr><td>Physiology</td><td>F1 and F2 gener-<br>tions</td><td>Higher heat tolerance and lower cold<br>tolerance</td><td>G</td><td>Maintained over F1 and<br>F2 so likely not trans-<br>generational plasticity</td><td>(Martin et al.,<br>2019)</td></tr><tr><td>Physiology</td><td>F1 generation</td><td>Higher metabolism, and faster running<br>speed at higher temperatures</td><td>G × E</td><td></td><td>(Chick et al.,<br>2021)</td></tr><tr><td>Behaviour</td><td>Life history<br>Morphology</td><td>Higher cadmium resistance</td><td>G & E</td><td>Difference not observed<br>in field</td><td>(Jacquier et al.,<br>2021a)</td></tr><tr><td>Life history</td><td>Cadmium treatment</td><td>Higher emergence rate and larger<br>worker size when exposed to cad-<br>mium</td><td>G & E</td><td>Cadmium negatively<br>affected life history traits</td><td>(Jacquier et al.,<br>2021b)</td></tr><tr><td>Morphology</td><td>Cadmium treatment</td><td>Higher heat tolerance, but no difference<br>in cold or desiccation tolerance</td><td>G & E</td><td>Tolerance varies between<br>temperature treatments</td><td>(Yilmaz et al.,<br>2021)</td></tr><tr><td>Rural</td><td>Physiology</td><td>F1 generation<br>2 temperature<br>treatments</td><td>G × E</td><td>Reduced running speed<br>when reared at higher<br>temperatures</td><td>(Yilmaz et al.,<br>2021)</td></tr><tr><td>Behaviour</td><td>F1 generation<br>2 temperature<br>treatments</td><td>Higher running speed</td><td>G × E</td><td>Rearing temperature<br>affected measured<br>traits, but generally sex<br>dependent</td><td>(y Gomez &<br>Van Dyck,<br>2012)</td></tr><tr><td>Rural</td><td>Morphology</td><td>F1 generation<br>2 temperature<br>treatments</td><td>Longer femur and wing lengths<br>Higher body mass and growth rate in<br>females only</td><td>G & E</td><td>Higher frequency signals<br>increased syllable to pause ratio</td><td>(Lampe et al.,<br>2014)</td></tr><tr><td rowspan="2">Grasshopper<br>(Chorthippus brumeus)</td><td>Nonroad-<br>side</td><td>Behaviour</td><td>Nymphs<br>2 noise treatments</td><td>G & E</td><td>Higher frequency vocal signals and<br>increased syllable to pause ratio</td><td>(Ichikawa &<br>Kuriwada,<br>2023)</td></tr><tr><td>Rural</td><td>Life history</td><td>F2 generation<br>Light and noise<br>treatments</td><td>G & E</td><td>Higher survival and smaller body size,<br>but no other difference</td><td>(Kuriwada,<br>2023)</td></tr><tr><td rowspan="2">(Chorthippus biguttulus)</td><td>Rural</td><td>Behaviour</td><td>F1 generation</td><td>G</td><td>Higher frequency and shorter chirp<br>vocal signal</td><td>(Kuriwada,<br>2023)</td></tr><tr><td>Rural</td><td>Behaviour</td><td>Egg-sacs</td><td>G</td><td>Lower avoidance of light</td><td>(Czaczkes et<br>al., 2018)</td></tr><tr><td>Bend-legged ground cricket<br>(Dianemobius nigro/ascia-<br>tus)</td><td>Rural</td><td>Behaviour</td><td>F1 generation</td><td>G</td><td>Higher frequency and shorter chirp<br>vocal signal</td><td>(Diamon<br>et<br>al.,<br>2022)</td></tr><tr><td>Synanthropic spider (Steatoda<br>triangulosa)</td><td>Rural</td><td>Behaviour</td><td>Egg-sacs</td><td>G</td><td>Higher heat tolerance and lower cold<br>tolerance</td><td>(Sex difference<br>Compare mid and low<br>urbanization)</td></tr><tr><td>Fruit flies<br>(Drosophila tripunctata)</td><td>Rural</td><td>Physiology</td><td>F2 + generation</td><td>G</td><td>Lower thermal limit tolerance, but no<br>difference in heat tolerance</td><td>(Difference not found in<br>wild)</td></tr><tr><td>(Drosophila suzukii)</td><td>Rural</td><td>Physiology</td><td>F3 generation</td><td>G</td><td>Higher tolerance to pollutants</td><td>(Sato & Taka-<br>hashi, 2022)</td></tr><tr><td>FISH</td><td>Clean</td><td>Physiology</td><td>F2 generation</td><td>G</td><td>Transcriptional mechanism<br>underflies divergence</td><td>(Whitehead et<br>al., 2012)</td></tr><tr><td>Atlantic killfish<br>(Fundulus heteroclitus)</td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>
<table><caption>**Table 1. Continued**</caption><thead><tr><th>Group</th><th>Urban vs. Rural</th><th>Traits</th><th>Approach<sup>a</sup></th><th>Urban results<sup>b</sup></th><th>Conclusion<sup>c</sup></th><th>Notes</th><th>Reference</th></tr></thead><tbody><tr><td>Creek chub (Semotilus atromaculatus)</td><td>Rural</td><td>Behaviour</td><td>Fry<br>2 water velocity treatments</td><td>Higher swimming efficiency</td><td>G & E</td><td>Same results in wild<br>Marginal support for G × E for one urban location</td><td>(Kern & Langerhans, 2019)</td></tr><tr><td></td><td></td><td>Morphology</td><td>Juvenile<br>2 water velocity treatments</td><td>More streamlined body shape</td><td>G</td><td>Marginal support for G × E for one urban location</td><td>(Kern & Langerhans, 2018)</td></tr><tr><td>MAMMALS</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Egyptian fruitbat (Rousettus aegyptiacus)</td><td>Rural</td><td>Behaviour</td><td>Pups</td><td>Bolder, faster learners, and less exploratory</td><td>E</td><td>Cross-fostering revealed that maternal rather than genetic effects may drive difference</td><td>(Harten et al., 2021)</td></tr><tr><td>PLANTS</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Holy hawksbeard (Crepis sancta)</td><td>Rural</td><td>Life history</td><td>Seeds</td><td>Higher number of nondispersing seeds</td><td>G</td><td>Same results in wild</td><td>(Cheptou et al., 2008)</td></tr><tr><td></td><td></td><td>Phenology</td><td>Seeds</td><td>Later phenology, larger, and higher water-use efficiency and photosynthesis</td><td>G</td><td>Most patterns same direction as selection in urban field site</td><td>(Lambrecht et al., 2016)</td></tr><tr><td>Common ragweed (Ambrosia artemisiifolia)</td><td>Rural</td><td>Phenology</td><td>Seeds</td><td>Earlier phenology and lower fitness</td><td>G & E</td><td>Reciprocal transplant Trend for G × E for life history traits, but not phenology</td><td>(Gorton et al., 2018)</td></tr><tr><td></td><td></td><td>Life history</td><td>Urban and rural treatments</td><td></td><td></td><td>Other traits, including flowering time, showed no difference</td><td>(Kostanecki et al., 2021)</td></tr><tr><td>Common milkweed (Asclepias syriaca)</td><td>Rural</td><td>Phenology</td><td>Seeds</td><td>Higher percent germination, smaller height, and higher leaf desiccation index</td><td>G & E</td><td>Heritable genetic variation and weak phenotypic divergences in wild</td><td>(Breitbart et al., 2023)</td></tr><tr><td></td><td></td><td>Morphology</td><td>Germination experiment</td><td>Limited support for difference</td><td>E</td><td>Some differences may be driven by pollinators along urban gradient</td><td>(Santangelo et al., 2020; Thompson et al., 2016)</td></tr><tr><td>White clover (Trifolium repens)</td><td>Nonurban</td><td>Phenology</td><td>F1 generation</td><td>Later phenology, larger flowers and biomass, thinner stolons, reduced cyanogenesis, and greater seed set</td><td>G</td><td>City populations more genetically related</td><td>(Yakub & Tiffin, 2017)</td></tr><tr><td>Virginia pepperweed (Lepidium virginicum)</td><td>Rural</td><td>Phenology</td><td>Seeds</td><td>Earlier phenology, taller, and higher fecundity</td><td>G</td><td>Evidence for cogradian or countergradient variation in growth rate</td><td>(McLean et al., 2005)</td></tr><tr><td></td><td></td><td>Morphology</td><td>5 cities</td><td></td><td></td><td></td><td></td></tr><tr><td>Chitinolytic fungi (Chrysosporium pannorum, Trichoderma koningii, Torulmocyes lagena, Penicillium bilaii)</td><td>Rural</td><td>Physiology</td><td>F1 generation<br>5 temperature treatments</td><td>Higher growth rate at higher temperatures</td><td>G × E</td><td></td><td></td></tr><tr><td>Southern crabgrass (digitaria ciliaris)</td><td>Farmland</td><td>Life history</td><td>Seeds<br>2 competition treatments</td><td>More shoots, shorter, and higher growth rates in high-competition treatment</td><td>G × E</td><td></td><td>(Fukano et al., 2020)</td></tr></tbody></table>
<table><caption>**Table 1. Continued**</caption><thead><tr><th>Group</th><th>Urban vs.</th><th>Traits</th><th>Approach<sup>a</sup></th><th>Urban results<sup>b</sup></th><th>Conclusion<sup>c</sup></th><th>Notes</th><th>Reference</th></tr></thead><tbody><tr><td>Yellow jessamine (Gelsemium sempervirens)</td><td>Wild</td><td>Morphology</td><td>F1 generation</td><td>Larger floral traits</td><td>G</td><td>Patterns in same and opposite direction to those observed in wild</td><td>(Irwin et al., 2014)</td></tr><tr><td>Asian dayflower (Commelina communis)</td><td>Rural</td><td>Morphology</td><td>Seedlings</td><td>Lower leaf number and specific leaf area, no difference in other traits</td><td>G & E</td><td>Different patterns observed in the wild</td><td>(Taichi & Ushimaru, 2024)</td></tr><tr><td>Pineappleweed (matricaria discoidea)</td><td>Rural</td><td>Life history</td><td>Seeds</td><td>Limited support for differences</td><td>E</td><td>Reciprocal common garden as treatments mimicked urban conditions</td><td>(Géron et al., 2022)</td></tr><tr><td>Common dandelion (taraxacum officinale)</td><td>Suburban</td><td>Morphology</td><td>Temperature treatments</td><td>Reduced early seed production following herbivory</td><td>G × E</td><td>Increased herbivory resistance after exposure to herbivores</td><td>(Pisman et al., 2020)</td></tr><tr><td>Thale cress (Arabidopsis thaliana)</td><td>Rural</td><td>Morphology</td><td>Soil treatments</td><td>Larger and higher tolerance to caterpillars, but not aphids</td><td>G</td><td>No wild size difference and higher urban aphid herbivory</td><td>(Qu et al., 2022)</td></tr><tr><td>Phenology</td><td>Phenology</td><td>Morphology</td><td>Soil treatments</td><td>Several differences including earlier flowering time</td><td>G</td><td>Genomics identifies set of clonal lineages</td><td>(Schmitz et al., 2024)</td></tr><tr><td>Life history</td><td>Physiology</td><td>Morphology</td><td>Soil treatments</td><td>Greater stress tolerance characteristics</td><td>G × E</td><td>Some traits adaptive in treatments</td><td>(Weston et al., 2021)</td></tr><tr><td>Phenology</td><td>Phenology</td><td>Morphology</td><td>Soil treatments</td><td>Higher boldness, but no difference in exploration or neophilia</td><td>G</td><td>Boldness only repeatable trait</td><td>(Baxter-Gilbert et al., 2019)</td></tr><tr><td>Life history</td><td>Physiology</td><td>Morphology</td><td>Soil treatments</td><td>No difference in heat tolerance</td><td>E</td><td>Higher urban heat tolerance in wild</td><td>(Campbell-Staton et al., 2021)</td></tr><tr><td>Phenology</td><td>Phenology</td><td>Morphology</td><td>Soil treatments</td><td>Higher urban gene expression change after exposed to heat challenge</td><td>E</td><td>Higher urban gene expression change after exposed to heat challenge</td><td>(Winchell et al., 2016)</td></tr><tr><td>Natural</td><td>Natural</td><td>Behaviour</td><td>F1 generation</td><td>Longer limbs and more lamellae</td><td>G</td><td>Same results in wild</td><td>(Winchell et al., 2016)</td></tr><tr><td>Phenology</td><td>Natural</td><td>Morphology</td><td>F1 generation</td><td>Longer limbs and more lamellae</td><td>G</td><td>Same results in wild</td><td>(Winchell et al., 2016)</td></tr></tbody></table>

Note. Information concerning the traits measured, approach taken, results, conclusions, and other notes are shown for each study. Studies were collated from Lambert et al. (2021) (Table 1) and from a literature search using Google Scholar for articles since 2020 that included both “common garden” and “urban” (conducted March 22, 2024).

aGeneration measured in the common garden (F1-F3) and, in cases where the F0 generation was used, the life stage individuals were brought to common conditions is shown. In the case of multitreatment common garden approaches, details on the rearing treatments are also shown.

bComparative results using the urban group as the reference category. In the study listed last, urban anoles were found to have longer limbs and more lamellae than natural anoles.

cConclusion of underlying mechanisms responsible for phenotypic difference between populations recorded as either G = genetic differentiation between populations, E = plasticity to environment, G & E = both genetic differentiation and plasticity, and G × E = genetic differentiation in plastic responses to environment.
proportion of impervious surface area (ISA; sealed nonnatural surfaces) at each nest box to generate a continuous urbanization metric to characterize the territory of breeding wild birds (captured at nest boxes) and the territory of origin for the birds raised during the CG experiment (see Supplementary Methods). We also categorized territories as urban or forest habitat types since phenotypic changes may not be consistent across continuous and categorical urbanization, which can provide additional insights. For example, in cases where a phenotypic change differs between habitat types but does not clearly change along the gradient, this could suggest that (i) urban effects other than ISA explain phenotypic change or (ii) phenotypic changes may be nonlinear.

## CG manipulation

### Egg transfer to wild foster parents

Between April 5 and April 22, 2022, we collected eggs from urban (N = 50 eggs from 4 sites) and forest (N = 40 eggs from 1 site) populations (Figure 1A; Supplementary Table S1). We collected three to four unincubated eggs (cold to the touch) from each origin nest box. We ensured eggs were unincubated by collecting eggs from nest boxes where we were confident that females had initiated laying within 3 to 4 days before collection and the collected eggs were still covered by nest material. We replaced collected eggs with false eggs to encourage the origin female to continue its reproduction, and we moved collected eggs into foster nest boxes at our Montpellier Zoo study site where wild females had just commenced incubation (Figure 1B). The Montpellier Zoo is an intermediate site along our urban gradient because it is natural in its vegetative characteristics but is exposed to humans and related stimuli (Demeyrier et al., 2016). We transferred eggs from their

origin to foster nests within 6 hours. In one case, we transferred eggs two days later and we kept these clutches in a dark room and rotated eggs every 12 hr until their transfer (foster ZOO46; Supplementary Table S1). On average, the collected urban eggs were significantly lighter than the collected forest eggs (urban: N = 34 eggs weighed, mean = 1.56 g, variance = 0.02; forest: N = 40 eggs weighed, mean = 1.70 g, variance = 0.0075; Welch's t-test: -4.87, df: 52.88, p < 0.001; Supplementary Table S1).

Foster nest boxes contained eggs from two origin broods (N = 6–8 eggs total; Supplementary Table S1). We did not mix urban and forest eggs in the same foster broods to ensure that we could confidently assign all nestlings a habitat of origin, even if biological parents abandoned or were not captured. Urban lay dates in our system were earlier than forest lay dates (urban origin nests laid on average 7.5 days earlier), but there was still overlap in reproductive phenology between habitats (Supplementary Table S1). The percentage of unhatched eggs was similar across habitat of origin (18% urban and 20% forest). Unhatched forest eggs were all from one abandoned foster nest (invaded by hornets), whereas unhatched urban eggs were distributed across successful foster clutches (Supplementary Table S1). Of the 90 eggs transferred, we had N = 73 nestlings hatch (41 urban and 32 forest; Supplementary Table S1). We did not have mortality events so these sample sizes are representative of the number of individuals phenotyped after rearing (Supplementary Table S2).

### Captive rearing

Once nestlings could thermoregulate on their own at 10 days of age (Mertens, 1977), we transferred nestlings to the Montpellier Zoo nursery between April 29 and May 16, 2022

<figure><img src="image_2.png" /><figcaption>**Figure 1.** Procedure of common garden experiment including (A) egg collection, (B) transfer of eggs to wild foster parents, (C) transfer of nestlings to nursery for hand rearing under common conditions, and (D) phenotyping of common garden birds across (i) handling aggression, (ii) breath rate index, (iii) exploration in a novel environment, (iv) tarsus length, and (v) body mass traits.</figcaption></figure>
(Figure 1C; Supplementary Table S1). Due to advanced urban lay dates in our system, urban nestlings entered captivity on average 6 days before forest nestlings (urban mean = 126 Julian days, urban range = 119–135; forest mean = 132 Julian days, forest range = 128–136; Supplementary Table S1). Upon arrival, we ringed and weighed nestlings before placing them into artificial nests with their foster broods. We kept them in incubators that mimicked a dim cavity and kept chicks in a quiet environment (1–3 broods per incubator; Figure 1C.i). At this stage, we hand-raised nestlings by feeding individuals every 30 min between 7:00 and 21:00 (see Supplementary Methods for “captive diet”). Individuals began to “fledge” their brood nests at the age of 18 days. We transferred these individuals in groups of 2–3 birds into small wire cages in the order of when they fledged (irrespective of sex, foster brood, or habitat of origin), where we trained them to feed by themselves (Figure 1C.ii). At this stage, we still fed individuals every 30 min. Once birds were approximately 23 days old, we transferred them to larger cages (0.8 × 0.35 × 0.4 m) that allowed more movement (hops and flights) in groups of 2 individuals. At this stage their feeding schedule became less frequent, and birds were considered independent at an age of approximately 35 days when we transferred individuals to large outdoor aviaries (N = 8; size = 2.2 × 4.4–5.5 m; Figure 1C.iii). Individuals were randomly organized into aviary groups blind of habitat of origin and sex (N = 6–10 individuals per group). All individuals were hand-reared by the same caretakers during the experiment who were blind to the origin of the birds and birds from both origins were mixed through all stages of the rearing protocol.

## Blood sampling and genotyping

We took blood samples from individuals the day before birds were transferred to outdoor aviaries to determine (i) sex and nest of origin to control for genetic relatedness and (ii) assign each bird with an ISA of origin. For each foster brood, nestlings had two possible nests of origin from which eggs were collected so parents of nests of origin were also blood sampled and genotyped to assign nest of origin for each CG bird (see Supplementary Methods).

## Phenotypic measurements

Here, we examine five phenotypic traits: handling aggression, breath rate index, exploration in a novel environment, tarsus length, and body mass. Phenotypic measurements of CG birds (Figure 1D) followed the same protocols used to phenotype wild birds (Caizergues et al., 2018, 2022). We took all phenotypic measures of CG birds indoors at the nursery, so all individuals were phenotyped under similar conditions (i.e., constant temperature, noise, and light levels). We took repeated measures between June 6, 2022 and January 31, 2023 and all observers were blind to habitat of origin while phenotyping individuals during four separate phenotyping sessions. On average, we measured birds 1.8 times in the wild (range = 1–8) and 3.4 times in the CG (range = 1–4; summary by trait Supplementary Table S2). Following our phenotyping protocol in the wild, where birds are mainly measured annually during the breeding season (April 2 to July 16), we measured phenotypes at the same time in the following order:

### Handling aggression

We measured handling aggression immediately following capture (from nest box, cage, or aviary) by provoking the

bird while holding it (Figure 1D.i). We scored their aggressive response between 0 (no reaction) to 3 (tail and wings extended, pecking, and vocalization) on a scale that increased in increments of 0.5 (see Dubuc-Messier et al., 2018; Caizergues et al., 2022 for further details).

### Breath rate index

We placed the bird in a cloth bag and allowed a 5-min standardized period of rest. Once removing the bird from the bag and properly holding the bird (Figure 1D.ii), we recorded the time it took for a bird to take 30 breaths (i.e., movements of the breast). We took this measurement twice in immediate succession and took the average between these measures to represent an individual's breath rate index.

### Exploration

We placed birds into a small compartment next to a novel environment arena where they had a standardized 2-min rest period. We then initiated the novel environment exploration trial by coaxing birds into the novel arena. We recorded their behaviors in this arena for 4 minutes on video then an observer later counted the number of hops and flights birds took while exploring this novel environment (Figure 1D.iii). Only one observer scored videos from the CG (MJT), while multiple observers have scored videos from the wild populations (inter-observer reliability rho > 0.95, including MJT).

### Tarsus length and body mass

Finally, we measured tarsus length (millimeters) with pliers to determine the length between the intertarsal notch and the end of the bent foot (i.e., Svensson's alternative method; Svensson, 1992; Figure 1D.iv) and body mass (grams) using an electronic scale (Caizergues et al., 2021; Figure 1D.v).

### Statistical analyses

We examined wild and common garden (CG) data using separate Bayesian mixed-effect models since model structures between contexts accounted for different fixed and random effects, while examining a similar main effect of interest (i.e., habitat type; aim 1). All models included habitat type (urban vs. forest) and sex (male and female) and their interaction, as fixed effects in the model to evaluate how phenotypic differences vary across habitats and sexes. If the interaction between habitat and sex was weak and largely overlapped zero, we dropped this effect and refitted the model to evaluate the phenotypic differences between habitats and sexes independently. In subsequent models, we replaced the habitat type effect with proportion ISA to further evaluate changes in the wild and CG phenotypes along a gradient of urbanization.

We have already published results on the phenotypic differences between wild urban and forest populations (Caizergues et al., 2018, 2022; Charmantier et al., 2017), but here we report estimates from wild populations that (i) include more years of data (3 additional years, year range: 2011–2022) and (ii) use data only from the study sites used in our CG experiment (i.e., 1 forest and 4 urban study sites). All wild models included random effects that accounted for differences between individuals, study sites, and years, and we additionally accounted for differences between observers for handling aggression, breath rate index, and tarsus length (Table 2). In addition to examining habitat and sex differences in these models, we also accounted for fixed effects such as time of day, date of measurement, and protocol type
<table><caption>**Table 2.** Summary of (1) wild and (2) common garden model structures that account for different fixed and random effects.</caption><thead><tr><th></th><th>(A) Aggression<br>Range: 0-3 score</th><th>(B) Breath rate<br>Range: 7.7-21.7 s</th><th>(C) Exploration<br>Range: 0-320 hops</th><th>(D) Tarsus length<br>Range: 17.32-21.70 mm</th><th>(E) Body mass<br>Range: 14-20.4 g</th></tr></thead><tbody><tr><td colspan="6"><strong>(1) Wild Models</strong></td></tr><tr><td colspan="6"><strong>Fixed effects</strong></td></tr><tr><td>Habitat</td><td>forest vs. urban</td><td>forest vs. urban</td><td>forest vs. urban</td><td>forest vs. urban</td><td>forest vs. urban</td></tr><tr><td>Sex</td><td>female vs. male</td><td>female vs. male</td><td>female vs. male</td><td>female vs. male</td><td>female vs. male</td></tr><tr><td>Age</td><td>adult vs. yearling</td><td>adult vs. yearling</td><td>adult vs. yearling</td><td>-</td><td>adult vs. yearling</td></tr><tr><td>Time of day</td><td>Range: 7.16-17.13</td><td>-</td><td>Range: 6.47-16.67</td><td>-</td><td>Range: 7.17-17.13</td></tr><tr><td>Date of measure</td><td>Range: 101-196</td><td>Range: 108-190</td><td>Range: 109-190</td><td>-</td><td>Range: 101-197</td></tr><tr><td>Protocol</td><td>-</td><td>new vs. old</td><td>new vs. old</td><td>-</td><td>-</td></tr><tr><td>Temperature</td><td>-</td><td>Range: 8-33.7</td><td>-</td><td>-</td><td>-</td></tr><tr><td colspan="6"><strong>Random effects</strong></td></tr><tr><td>Individual ID (V<sub>IND</sub>)</td><td>N = 772</td><td>N = 531</td><td>N = 472</td><td>N = 837</td><td>N = 810</td></tr><tr><td>Site ID (V<sub>SITE</sub>)</td><td>N = 5</td><td>N = 5</td><td>N = 5</td><td>N = 5</td><td>N = 5</td></tr><tr><td>Year ID (V<sub>YEAR</sub>)</td><td>N = 12</td><td>N = 9</td><td>N = 9</td><td>N = 12</td><td>N = 12</td></tr><tr><td>Observer ID (V<sub>OBS</sub>)</td><td>N = 29</td><td>N = 12</td><td>-</td><td>N = 27</td><td>-</td></tr><tr><td colspan="6"><strong>(2) Common Garden Models</strong></td></tr><tr><td colspan="6"><strong>Fixed effects</strong></td></tr><tr><td>Habitat</td><td>forest vs. urban</td><td>forest vs. urban</td><td>forest vs. urban</td><td>forest vs. urban</td><td>forest vs. urban</td></tr><tr><td>Sex</td><td>female vs. male</td><td>female vs. male</td><td>female vs. male</td><td>female vs. male</td><td>female vs. male</td></tr><tr><td>Time of day</td><td>Range: 7.42-11.67</td><td>Range: 7.60-11.97</td><td>Range: 7.67-12.02</td><td>-</td><td>Range: 8.22-11.53</td></tr><tr><td>Assay number</td><td>1-4 (categorical)</td><td>1-4 (categorical)</td><td>1-3 (categorical)</td><td>-</td><td>1-3 (categorical)</td></tr><tr><td>Observer ID</td><td>-</td><td>-</td><td>-</td><td>1-2 (categorical)</td><td>-</td></tr><tr><td colspan="6"><strong>Random effects</strong></td></tr><tr><td>Individual ID (V<sub>ID</sub>)</td><td>N = 73</td><td>N = 73</td><td>N = 70</td><td>N = 72</td><td>N = 71</td></tr><tr><td>Origin nest ID (V<sub>ON</sub>)</td><td>N = 23</td><td>N = 23</td><td>N = 23</td><td>N = 23</td><td>N = 23</td></tr><tr><td>Foster nest ID (V<sub>NF</sub>)</td><td>N = 11</td><td>N = 11</td><td>N = 11</td><td>N = 11</td><td>N = 11</td></tr><tr><td>Aviary ID (V<sub>AV</sub>)</td><td>N = 8</td><td>N = 8</td><td>N = 8</td><td>N = 8</td><td>N = 8</td></tr></tbody></table>

Note. Ranges for continuous fixed effects and number (N) of random effect levels for each trait are shown. Note that urbanization was also examined as a continuous effect in additional models (see model results in Supplementary Table S4).

following previously established model structures for these traits (Table 2; Caizergues et al., 2018, 2022).

Besides examining habitat of origin effects in CG models (aim 1), we also explored how different components of genetic and environmental variation shaped traits in the CG (aim 2). All CG models included the same random effects (Table 2): Individual ID accounted for variance among individuals (V<sub>ID</sub>), nest of origin ID accounted for variance among origin nests (V<sub>NO</sub>), and foster nest ID accounted for variance among foster nests (V<sub>NF</sub>). We also included aviary ID as a random effect that accounted for variation among social groups in the experiment (V<sub>AV</sub>) for the behavioral and physiological traits considered (i.e., aggression, exploration, breath rate). Since individuals in the CG experiment were genotyped, we conducted a complementary analysis of the CG data using mixed-effect animal models (Charmantier et al., 2014) by fitting a genetic relatedness matrix between individuals in our CG context (de Villemereuil et al., 2018a). Since we collected unincubated eggs for the experiment, the nest of origin random effect (V<sub>NO</sub>) if fitted alone, may capture both genetic differences between individuals (i.e., whether they are siblings)

and early environmental differences such as early maternal investment in the eggs. Therefore, the genetic relatedness matrix approach allowed us to further evaluate how the variation of each trait was partitioned when accounting for individual genetic variation and nest of origin variation (V<sub>A</sub> and V<sub>NO</sub>, respectively) at the same time (see Supplementary Methods for model description).

We computed the repeatability (R) of each trait in the CG experiment as:

$$R_{CG} = \frac{V_I}{V_P} \qquad (1)$$

$$\text{where } V_I = V_{ID} + V_{NO} + V_{NF} + V_{AV} + V_{SEX} + V_{HAB} \quad (2)$$

$$\text{where } V_P = V_I + V_R \qquad (3)$$

where V<sub>I</sub> is the among-individual variance that comprises effects that vary consistently among individuals and drive biological differences between individuals, including variance across individuals (V<sub>ID</sub>), nests of origin (V<sub>NO</sub>), foster nests (V<sub>NF</sub>), aviaries or social groups (V<sub>AV</sub>), and fixed effect variance
among sexes and habitat types ($V_{SEX}$ and $V_{HAB}$). We only chose to include fixed effect variance generated by biological effects in the model (i.e., habitat and sex) rather than experimental effects (e.g., time of day or observer) to quantify repeatability using natural sources of variation that may improve comparability to other studies (Wilson, 2018; de Villemereuil et al., 2018b). $V_p$ is the total phenotypic variance and includes sources of among-individual variance ($V_i$) and residual variance ($V_R$). For comparison, we computed $R$ in wild birds as:

$$R_{WILD} = \frac{V_{IND}}{V_p} \qquad (4)$$

$$V_{IND} = V_{ID} + V_{SITE} + V_{SEX} + V_{HAB} \qquad (5)$$

$$\text{where } V_p = V_{IND} + V_{YEAR} + V_{AGE} + V_R \qquad (6)$$

where $V_{IND}$ is the among-individual variance and comprises variance across individuals ($V_{ID}$), study sites or local habitats ($V_{SITE}$), and sexes and habitat types (fixed effect variance: $V_{SEX}$ and $V_{HAB}$). $V_p$ is the total phenotypic variance and includes among-individual variance ($V_{IND}$) and residual variance ($V_R$). In the wild context, we also incorporated biological effects in $V_p$ that vary within individuals, including variance among years ($V_{YEAR}$) and fixed effect variance among ages ($V_{AGE}$). For Poisson models (i.e., exploration) that use a log link transformation, we used the QCglmm package (de Villemereuil et al., 2016) to convert the variance components and repeatability estimate from the latent scale to the data scale.

We conducted all analyses in R v4.3.3 (R Core Team, 2024) using Bayesian mixed-effect models in the MCMCglmm package (Hadfield, 2010) using a Gaussian error structure for all traits except exploration (number of hops) where we used a Poisson error structure. See Supplementary Methods and Table 2 for description of other fixed effects included for each model. We used weakly informative inverse-Gamma priors ($V = 1, nu = 0.002$) for fixed and random effects. We ran all models for 1,000,000 iterations, with a thinning of 500 and a burn-in period of 10,000, which achieved effective sample sizes > 1000 across all estimates. We verified model fit by visually inspecting histograms and QQPlots of model residuals, and the relationship between the residuals and fitted values. We confirmed convergence of models by visually inspecting trace plots, verifying low autocorrelation, and by using Heidelberg stationary tests (de Villemereuil, 2018).

# Results

## Aggression in hand

We found clear evidence that wild urban males were more aggressive than wild forest males, but no habitat difference for females (habitat* sex effect, Table 3.1A; Figure 2A). Results in the wild were qualitatively similar when examining how phenotypes changed along the urban gradient; there was clear evidence that wild males in habitats with higher proportion ISA had increased aggression (Supplementary Table S4; Figure 3A). There was no clear evidence that this phenotypic difference was maintained in the CG (weak habitat* sex or habitat effects overlapping zero; Table 3.2A; Supplementary Table S5; Figure 2A), and no clear evidence that handling aggression increased with proportion ISA of the origin habitat of CG birds (credible interval [CI] overlaps zero; Supplementary Table S4). Individual ID explained 23% of the variation in aggression in the CG, nest of origin explained 2%, foster nest

explained 2%, and housing aviary explained 2% of the variation (Table 3.2A).

## Breath rate index

We found weak evidence that wild urban birds had faster breath rates than wild forest birds (6% posterior distribution crossing zero, Table 3.1B; Figure 2B), but clear evidence that the amount of time to take 30 breaths decreased across the urbanization gradient in the wild populations (i.e., faster breath rates in more urbanized habitats, CI excludes zero, Supplementary Table S4; Figure 3B). We found clear evidence that this phenotypic difference was maintained in CG birds where birds from urban origins had faster breath rates than birds from forest origins (Table 3.2B; Figure 2B), but there was no clear change in breath rate across the urbanization gradient in the CG (CI overlaps zero; Supplementary Table S4; Figure 3B). Individual ID explained 40% of the variation in breath rates in the CG, while origin nest (11%), foster nest (4%), and housing aviary (4%) explained less variation in this trait (Table 3.2B).

## Exploration

We found clear evidence that exploration in the wild was higher in urban compared to forest birds (Table 3.1C; Figure 2C) and less clear evidence that exploration increased with increasing urbanization (5% posterior distribution crossing zero; Supplementary Table S4; Figure 3C). There was no clear evidence that these differences were maintained in the CG (credible overlapping zero; Table 3.2C; Figure 2C), and exploration of CG birds did not increase clearly with proportion ISA of the origin habitat (Supplementary Table S4; Figure 3C). Individual ID explained 17% of the variation in CG exploration behaviors, while origin nest (7%), foster nest (10%), and housing aviary (2%) explained less variation in this trait (Table 3.2C).

## Tarsus length

We found clear evidence that tarsus length was significantly shorter in wild urban birds than wild forest conspecifics (Table 3.1D; Figure 2D) and with increasing urbanization in the wild (Supplementary Table S4; Figure 3D). In the CG experiment, this phenotypic divergence was in the same direction and had similar effect sizes as in the wild, with birds from urban origins tending to have shorter tarsi than birds from forest origins (CG: $\beta_{habitat} = -0.22$ vs. wild: $\beta_{habitat} = -0.29$; Table 3D) and tarsus length decreasing with increasing ISA (CG: $\beta_{ISA} = -0.29$ vs. wild: $\beta_{ISA} = -0.20$, Supplementary Table S4). The large CIs in the CG context however reduced certainty and suggests the smaller CG sample size may limit the ability to detect statistical differences for this trait (CI overlapping zero, but note 91% of ISA CI excludes 0). Out of the random effects considered, individual ID explained 46% of the variation in tarsus in the CG, followed by origin nest and foster nest, which explained 25% and 7% of the variation, respectively (Table 3.2D).

## Body mass

We found weak evidence that wild urban birds were lighter than wild forest birds (7% posterior crossing zero; Table 3.1E; Figure 2E), but clear evidence that body mass decreased with increasing urbanization (CI excluded zero, Supplementary Table S4; Figure 3E). We found clear evidence that CG birds from urban origins were lighter than birds from forest origins
<table><caption>**Table 3.** Fixed and random effect estimates and credible intervals (CI) for (1) wild and (2) common garden contexts across phenotypic traits (A: handling aggression, B: breath rate index, C: exploration, D: tarsus length, and E: body mass).</caption><thead><tr><th rowspan="3"></th><th colspan="8">(1) Wild</th></tr><tr><th colspan="2">(A) Aggression<br>N = 1,308 obs, 772 ind</th><th colspan="2">(B) Breath rate<br>N = 702 obs, 531 ind</th><th colspan="2">(C) Exploration<br>N = 581 obs, 472 ind</th><th colspan="2">(D) Tarsus length<br>N = 1,437 obs, 837 ind</th><th colspan="2">(E) Body mass<br>N = 1,375 obs, 810 ind</th></tr><tr><th>Est</th><th>CI</th><th>Est</th><th>CI</th><th>Est</th><th>CI</th><th>Est</th><th>CI</th><th>Est</th><th>CI</th></tr></thead><tbody><tr><td>Fixed effects</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Intercept</td><td>2.29</td><td>1.68-2.84</td><td>12.74</td><td>10.53-15.19</td><td>3.32</td><td>0.78-5.97</td><td>19.42</td><td>19.25-19.58</td><td>15.99</td><td>15.35-16.65</td></tr><tr><td>Habitat (urban)</td><td>0.03</td><td>-0.3-0.46</td><td>-0.70</td><td>-1.71-0.35</td><td>1.60</td><td>0.84-2.2</td><td>-0.29</td><td>-0.48-0.11</td><td>-0.41</td><td>-1.14-0.18</td></tr><tr><td>Sex (male)</td><td>0.13</td><td>-0.04-0.3</td><td>0.06</td><td>-0.3-0.4</td><td>-0.06</td><td>-0.47-0.32</td><td>0.54</td><td>0.48-0.62</td><td>0.60</td><td>0.5-0.7</td></tr><tr><td>Age (yearling)</td><td>-0.09</td><td>-0.2-0.02</td><td>-0.008</td><td>-0.32-0.29</td><td>0.13</td><td>-0.19-0.48</td><td></td><td></td><td>-0.31</td><td>-0.39-0.24</td></tr><tr><td>Time of day</td><td>-0.04</td><td>-0.06-0.01</td><td></td><td></td><td>-0.01</td><td>-0.1-0.07</td><td></td><td></td><td>0.04</td><td>0.02-0.06</td></tr><tr><td>Date of measure</td><td>-0.001</td><td>-0.003-0.001</td><td>-0.003</td><td>-0.02-0.01</td><td>-0.01</td><td>-0.03-0.01</td><td></td><td></td><td>0.003</td><td>0.001-0.005</td></tr><tr><td>Protocol (old)</td><td></td><td></td><td>0.16</td><td>-0.39-0.71</td><td>0.16</td><td>-0.35-0.63</td><td></td><td></td><td></td><td></td></tr><tr><td>Temperature</td><td></td><td></td><td>0.09</td><td>0.06-0.13</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Habitat * Sex</td><td>0.28</td><td>0.03-0.53</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Random effects</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Individual ID (V<sub>IND</sub>)</td><td>0.43</td><td>0.34-0.52</td><td>2.61</td><td>2.06-3.19</td><td>2.73</td><td>1.99-3.5</td><td>0.28</td><td>0.25-0.31</td><td>0.39</td><td>0.33-0.45</td></tr><tr><td>Site ID (V<sub>SITE</sub>)</td><td>0.02</td><td>&lt;0.001-0.08</td><td>0.25</td><td>&lt;0.001-0.74</td><td>0.08</td><td>&lt;0.001-0.3</td><td>0.008</td><td>&lt;0.001-0.02</td><td>0.09</td><td>0.001-0.3</td></tr><tr><td>Year ID (V<sub>YEAR</sub>)</td><td>0.03</td><td>&lt;0.001-0.07</td><td>0.05</td><td>&lt;0.001-0.19</td><td>0.06</td><td>&lt;0.001-0.22</td><td>0.002</td><td>&lt;0.001-0.004</td><td>0.06</td><td>0.01-0.13</td></tr><tr><td>Observer ID (V<sub> OBS</sub>)</td><td>0.04</td><td>0.01-0.08</td><td>0.52</td><td>0.1-1.26</td><td></td><td></td><td>0.005</td><td>0.001-0.01</td><td></td><td></td></tr><tr><td>Residual variance (V<sub>R</sub>)</td><td>0.55</td><td>0.48-0.62</td><td>1.75</td><td>1.4-2.1</td><td>1.61</td><td>1.08-2.14</td><td>0.02</td><td>0.02-0.02</td><td>0.27</td><td>0.24-0.31</td></tr><tr><td>V<sub>IND</sub></td><td>0.48</td><td>0.38-0.70</td><td>2.89</td><td>2.28-4.36</td><td>3.44</td><td>2.57-4.61</td><td>0.38</td><td>0.34-0.44</td><td>0.57</td><td>0.47-1.15</td></tr><tr><td>R<sub>WILD</sub></td><td>0.45</td><td>0.37-0.56</td><td>0.62</td><td>0.52-0.73</td><td>0.13</td><td>0.06-0.20</td><td>0.94</td><td>0.93-0.95</td><td>0.62</td><td>0.53-0.78</td></tr></tbody></table>
<table><caption>**Table 3. Continued**</caption><caption>(2) Common Garden</caption><thead><tr><th rowspan="2">Fixed effects</th><th colspan="2">(A) Aggression<br>N = 285 obs, 73 ind</th><th colspan="2">(B) Breath rate<br>N = 283 obs, 73 ind</th><th colspan="2">(C) Exploration<br>N = 203 obs, 70 ind</th><th colspan="2">(D) Tarsus length<br>N = 211 obs, 72 ind</th><th colspan="2">(E) Body mass<br>N = 210 obs, 71 ind</th></tr><tr><th>Est</th><th>CI</th><th>Est</th><th>CI</th><th>Est</th><th>CI</th><th>Est</th><th>CI</th><th>Est</th><th>CI</th></tr></thead><tbody><tr><td>Intercept</td><td>1.40</td><td>0.23-2.59</td><td>9.43</td><td>7.21-11.62</td><td>4.02</td><td>1.91-6.07</td><td>19.43</td><td>19.1-19.76</td><td>15.84</td><td>14.71-16.98</td></tr><tr><td>Habitat (urban)</td><td>0.12</td><td>-0.2-0.45</td><td>-1.16</td><td>-2.32 to -0.02</td><td>-0.05</td><td>-1.06-0.81</td><td>-0.22</td><td>-0.64-0.17</td><td>-0.50</td><td>-0.96-0.03</td></tr><tr><td>Sex (male)</td><td>-0.23</td><td>-0.5-0.05</td><td>0.81</td><td>0.03-1.6</td><td>-0.36</td><td>-0.84-0.14</td><td>0.46</td><td>0.24-0.67</td><td>1.03</td><td>0.75-1.33</td></tr><tr><td>Time of day</td><td>0.04</td><td>-0.07-0.16</td><td>0.47</td><td>0.27-0.67</td><td>-0.05</td><td>-0.24-0.12</td><td></td><td></td><td>-0.10</td><td>-0.2-0.02</td></tr><tr><td>Measurement (2)</td><td>-0.17</td><td>-0.44-0.12</td><td>0.94</td><td>0.5-1.37</td><td>-0.18</td><td>-0.67-0.24</td><td></td><td></td><td>0.12</td><td>-0.04-0.28</td></tr><tr><td>Measurement (3)</td><td>-0.25</td><td>-0.52-0.01</td><td>0.50</td><td>0.04-0.9</td><td>-0.13</td><td>-0.58-0.3</td><td></td><td></td><td>0.50</td><td>0.37-0.67</td></tr><tr><td>Measurement (4)</td><td>-0.38</td><td>-0.62-0.12</td><td>-0.38</td><td>-0.8-0.02</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Observer (2)</td><td></td><td></td><td></td><td></td><td></td><td></td><td>0.07</td><td>0.04-0.11</td><td></td><td></td></tr><tr><td>Random effects</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Individual ID (V<sub>ID</sub>)</td><td>0.19</td><td>0.08-0.32</td><td>2.15</td><td>1.1-3.18</td><td>0.47</td><td>&lt;0.001-0.98</td><td>0.18</td><td>0.11-0.27</td><td>0.26</td><td>0.13-0.41</td></tr><tr><td>Origin nest ID (V<sub>NO</sub>)</td><td>0.02</td><td>&lt;0.001-0.06</td><td>0.58</td><td>&lt;0.001-1.66</td><td>0.20</td><td>&lt;0.001-0.71</td><td>0.10</td><td>&lt;0.001-0.22</td><td>0.12</td><td>&lt;0.001-0.3</td></tr><tr><td>Foster nest ID (V<sub>NF</sub>)</td><td>0.01</td><td>&lt;0.001-0.05</td><td>0.15</td><td>&lt;0.001-0.68</td><td>0.28</td><td>&lt;0.001-1.1</td><td>0.03</td><td>&lt;0.001-0.1</td><td>0.04</td><td>&lt;0.001-0.13</td></tr><tr><td>Aviary ID (V<sub>AV</sub>)</td><td>0.01</td><td>&lt;0.001-0.05</td><td>0.14</td><td>&lt;0.001-0.62</td><td>0.05</td><td>&lt;0.001-0.21</td><td></td><td></td><td></td><td></td></tr><tr><td>Residual variance (V<sub>R</sub>)</td><td>0.56</td><td>0.46-0.67</td><td>1.58</td><td>1.29-1.87</td><td>1.66</td><td>1.22-2.17</td><td>0.01</td><td>0.01-0.02</td><td>0.18</td><td>0.14-0.23</td></tr><tr><td>V<sub>I</sub></td><td>0.25</td><td>0.14-0.45</td><td>3.74</td><td>2.54-5.83</td><td>1.01</td><td>0.39-2.49</td><td>0.38</td><td>0.26-0.60</td><td>0.74</td><td>0.53-1.11</td></tr><tr><td>R<sub>CG</sub></td><td>0.30</td><td>0.17-0.44</td><td>0.68</td><td>0.54-0.78</td><td>0.10</td><td>0.03-0.17</td><td>0.97</td><td>0.95-0.98</td><td>0.80</td><td>0.73-0.87</td></tr></tbody></table>

Note. Exploration estimates are from a Poisson generalized mixed-effect model, while all other traits were fit with Gaussian mixed-effect models. Common garden models estimated Individual ID ($V_{ID}$), origin nest ID ($V_{NO}$), foster nest ID ($V_{NF}$), aviary ID ($V_{AV}$), and residual variance ($V_{R}$). The number of observations (obs) and individuals (ind) for each trait and context are shown. Shown in bold are fixed effects whose Cls exclude zero or random effects whose lower CI is ≥ 0.001. Computed among-individual variance ($V_{IND} = V_{ID} + V_{SITE} + V_{SEX} + V_{HAB} + V_{I} = V_{ID} + V_{NO} + V_{NF} + V_{AV} + V_{SEX} + V_{HAB}$) Equations 2 and 5) and repeatability (R) are shown for both contexts for comparison.
<figure><img src="image_3.png" /><figcaption>**Figure 2.** Habitat type model estimate and 95% credible intervals (CI) on phenotypic traits of wild and common garden birds across (A) aggression in hand, (B) breath rate index, (C) exploration score, (D) tarsus length, and (E) body mass. Habitat differences varied clearly by sex only in one case (A: aggression in wild birds), and these sex differences are shown (wild males: squares, wild females: triangles); aggression did not differ clearly by sex in the common garden. Phenotypes were measured in the wild annually during the breeding season between 2011 and 2022, whereas we measured phenotypes in the common garden between June 6, 2022 and January 31, 2023. See also Supplementary Figure S1 for plots that show raw data.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Figure 3.** Proportion of impervious surface area (ISA) model estimate and 95% credible intervals (CI) on phenotypic traits of wild and common garden birds across (A) aggression in hand, (B) breath rate index, (C) exploration score, (D) tarsus length, and (E) body mass. ISA effects varied clearly by sex only in one case (A: aggression in wild birds), and these sex differences are shown (wild males: orange, wild females: red); aggression over the ISA gradient in the common garden did not differ clearly by sex. Phenotypes were measured in the wild annually during the breeding season between 2011 and 2022, whereas we measured phenotypes in the common garden between June 6, 2022 and January 31, 2023. See also Supplementary Figure S2 for plots that show raw data.</figcaption></figure>
(Table 3.2E; Figure 2E). Results in the CG across the urban gradient were consistent with this conclusion where the weight of CG birds decreased with increasing urbanization of the origin habitat (Supplementary Table S4; Figure 3E). Individual ID explained 28% variation in CG body mass, followed by origin nest which explained 12% variation, and foster nest effects which explained 4% (Table 3.2E). On average CG birds were significantly lighter than wild birds, reflecting an experimental effect on body mass (Welch's *t*-test: *t* = -22.30, *df* = 272, *p* < 0.001, Figure 2E; Figure 3E).

## Additive genetic variance and heritability of CG traits

The complementary quantitative genetic models (Supplementary Table S3) revealed that breath rate index, tarsus length, and body mass measured in CG birds were moderately heritable (breath rate: $V_A = 1.53, h^2 = 0.23$ [0.001–0.57]; tarsus length: $V_A = 0.16, h^2 = 0.33$ [0.003–0.72]; body mass: $V_A = 0.28, h^2 = 0.28$ [0.01–0.49]), while aggression in hand and exploration were lowly heritable (aggression: $V_A = 0.07, h^2 = 0.05$ [<0.001–0.28]; exploration: $V_A = 0.43, h^2 = 0.02$ [<0.001–0.06]). Although there was uncertainty around variance estimates in these models (i.e., CIs close to zero), estimates of $V_{NO}$ were low across all traits (Supplementary Table S3) providing additional insight into the minimal contributions from early maternal effects in the experiment.

## Discussion

We found evidence that both genetic and plastic changes have contributed to phenotypic shifts in wild urban tits, but the relative contributions of these drivers depend on the trait (aim 1). Specifically, our results provide evidence that genetic differences between populations have strongly driven the divergence observed in breath rate and body mass, while plasticity to urban conditions predominately contributes to divergences in aggression and exploration. Further, we found that individual differences tended to explain the most trait variation in the experiment, whereas nest of origin and foster nest variation had minimal contributions (aim 2).

We found support that genetic change or very early material investment in eggs has driven population divergences in breath rate index as phenotypic differences between birds from urban and forest origins were clearly maintained in our experiment (but note difference not statistically supported along gradient). In line with findings in the wild populations (Caizergues et al., 2022), birds from urban origins had faster breath rates than birds from forest origins. As breath rate index correlates with heart rate under constraint (Dubuc-Messier et al., 2016) and has previously been associated with physiological stress responses in this species (Carere & van Oers, 2004; Krams et al., 2014), our results could indicate that genetic change in urban populations has contributed to a more proactive coping style in urban environments (Koolhaas et al., 2010, 2011). Our results differ from those in urban European blackbirds (Turdus merula; Partecke et al., 2006) and juncos (Junco hyemalis; Atwell et al., 2012), where lower stress responses of urban birds were maintained when individuals were reared under common conditions. Indeed, there is no general consensus on how urbanization impacts stress responses in birds (reviewed in Bonier, 2012) and so our results make a useful contribution towards understanding how physiology might impact adaptation to urban contexts.

We also found support that being smaller may have a genetic basis in cities. Specifically, birds from urban origins were lighter than birds from forest origins despite being fed the same diet *ad libitum*. This habitat difference in body mass was statistically clear and higher in the CG than the wild supporting a genetic basis for shifts to smaller urban body mass, rather than plasticity, which could possibly reduce this wild phenotypic difference. Lighter urban body mass in the experiment could also be explained by early maternal investment in the egg, especially since the urban eggs collected for the experiment were on average lighter than forest eggs. Since egg size is highly heritable (e.g., egg volume $h^2 = 0.6–0.8$, Van Noordwijk et al., 1981; Hörak et al., 1995; see also Christians, 2002) and female body size can positively correlate with their egg size in this species (Hörak et al., 1995), it is possible that genetic differences between females in material egg investment could shape body mass variation in the wild. A very recent CG study found evidence that shorter urban tarsus length is driven by genetic differences between Dutch populations of urban and forest great tits (Tomotani et al., 2025), suggesting smaller urban size has a genetic basis in other European populations.

In this study, birds from urban origins also tended to have smaller tarsi than birds from forest origins in line with the phenotypic shift from the wild (Caizergues et al., 2018), but this was not supported statistically. We could lack statistical power to make firm conclusions on whether this difference in tarsus length was maintained in the CG, especially since the wild phenotypic difference in tarsus length is small and would require large samples to detect. The habitat difference for tarsus length was weaker in the CG than the wild (i.e., difference of 0.22 mm in CG vs 0.29 mm in wild), which may indicate that a combination of plastic and genetic effects explain the tarsus length difference. Tarsus length and body mass are heritable traits and tend to strongly correlate with each other in this species (Gebhardt-Henrich & Van Noordwijk, 1991; Gosler & Harper, 2000; Hörak, 1994; Young & Postma, 2023), suggesting that parallel genetic change for tarsus alongside body mass is possible (but see Tomotani et al., 2025). However, tarsus development is also strongly influenced by environmental conditions in early life (Dhondt, 1982; Merilä & Fry, 1998; Seress et al., 2020; Talloen et al., 2010), and so we hypothesize that genetic and plastic effects both contribute to smaller tarsus lengths in these urban birds. Further quantitative genetic approaches using long-term datasets on wild populations and observed or genetically reconstructed pedigrees will provide a useful complementary exploration on the underlying drivers behind shifts to smaller urban tarsus lengths in tits.

Decreases in traits associated with body size are documented across diverse taxa in cities (Hahs et al., 2023; Merckx et al., 2018), and this phenotypic shift is hypothesized to facilitate heat dissipation and be an adaptive response to rising global temperatures that are pronounced in urban areas via the heat island effect (Sumasgutner et al., 2023; Youngflesh et al., 2022). For example, *Daphnia* from urban origins had smaller body sizes and higher heat tolerance in a CG experiment than those from nonurban origins, and there was evidence that smaller urban body sizes could indirectly increase heat tolerance in this species (Brans et al., 2017a). Further, city great tits tend to be lighter than forest tits across Europe (Thompson et al., 2022) and, in Veszprém, reproduction of city tits is less affected by extreme temperature than their
forest counterparts (Pipoly et al., 2022). These results suggest that city great tits could be adapted to warming conditions and our results could imply that decreases in urban body size are an adaptive response to heat island effects (but see Playà-Montmany et al., 2021). Smaller body size does not appear to afford urban great tits in Montpellier reproductive benefits (Caizergues et al., 2018); hence further work will be needed to evaluate whether smaller body sizes or other correlated traits are associated with higher survival in urban habitats.

Genetic change between populations that contribute to phenotypic differences can also arise via neutral evolutionary processes like genetic drift or founder effects (Leinonen et al., 2013), and differentiating these processes from local adaptation is informative to evaluate whether populations are adapting in pace with environmental change (de Villemereuil et al., 2020). We found evidence that breath rate and body mass differences are likely driven by genetic change or very early maternal investment, but we are unable to completely dismiss the role of neutral evolutionary processes toward genetic differences between populations. Using a complementary quantitative genetics approach, we estimated higher genetic differences underlying these traits (computed $Q_{ST}$ values in Supplementary Table S3: 0.06 and 0.08) than would be expected by neutral genetic variation between these populations ($F_{ST}$ values between 0.006 and 0.008; Perrier et al., 2018). However, the uncertainty around these $Q_{ST}$ estimates (CIs crossed 0.006) prevent us from completely excluding neutral evolutionary processes here. In future, rearing individuals from multiple city and forest comparisons in a CG experiment would further strengthen our evidence against processes of neutral evolution and possibly demonstrate parallel evolutionary trajectories across multiple city populations.

We did not find evidence that genetic change has considerably contributed to urban behavioral shifts as birds from urban and forest origins did not clearly differ in their aggressive or exploratory behaviors in the experiment. It is commonly assumed that urban populations are evolving and adapting to novel urban conditions (Lambert et al., 2021), and behavioral adaptations may be particularly important in this process (Miranda et al., 2013). Alternatively, it has been argued that phenotypic adjustments through plasticity are probably more frequent (Hendry et al., 2008), especially for behavioral traits (Caspi et al., 2022; Sol et al., 2013). Our results provide support for the latter argument and contrast findings in urban blackbirds and juncos where behavioral differences were assumed to be a result of local adaptation (Atwell et al., 2012; Miranda et al., 2013). We therefore conclude that more aggressive and exploratory behaviors of wild urban great tits are most strongly driven by plastic adjustments to life in cities. Habitat matching behaviors (Camacho et al., 2020; Edelaar et al., 2008) could contribute to these urban differences in the wild, whereby more aggressive and exploratory individuals disperse and settle in more urbanized habitats, if these behaviors provide them an advantage in urban habitats. However, these behaviors were not found to covary at the individual level in an urban behavioral syndrome and do not seem to afford reproductive or survival benefits in the urban population (Caizergues et al., 2022). Dispersal dynamics and habitat matching behaviors in an urban context are still poorly understood but, as these behaviors do not covary or improve fitness, we so far have limited evidence that adaptive habitat matching contributes to these urban phenotypic shifts.

Our second aim was to investigate how different sources of environmental and genetic variation contributed to repeatable individual differences across traits in our experimental context. The estimated among-individual variance and repeatability of traits in the CG were remarkably similar to those estimated in the wild (i.e., similar estimates and overlapping CIs). Trait variation in the experiment tended to be shaped predominately by differences between individuals (i.e., Individual ID). Individual ID in the experiment could comprise both individual-specific genetic and environmentally induced individual differences, and our complementary quantitative genetic analysis suggested that individual genetic variation explains between 23% and 33% of the variation across breath rate, tarsus length and body mass, but only 2%–5% for aggression and exploration behaviors (i.e., estimates of $V_A$ in Supplementary Table S3, but note wide CIs). Variation attributed to foster nest and aviary, likely related to brood and social environmental conditions, respectively, remained low across traits (3%–14%). Specifically, early environmental conditions can affect tarsus development and growth (e.g., Seress et al., 2020), but we found limited support that foster parents and nests contributed to individual differences in tarsus. Overall, estimated individual differences were similar between CG and wild contexts, especially in those traits where we find evidence for underlying genetic differences between populations.

Finally, a few caveats should be considered when interpreting our results. First, we are unable to fully discount the contribution of very early maternal effects towards the maintained breath rate and body mass differences in the CG. By collecting unincubated eggs we limited maternal contributions to egg investment, which could influence morphological traits like body mass (Hōrak et al., 1995). Although we found limited (but unclear) support for maternal effects in our experiment (i.e., negligible $V_{NO}$; Supplementary Table S3), our results should be interpreted with this in mind. Second, birds in our experiment were assayed at a relatively young age (between 38 and 261 days old) compared to when they are usually assayed in the wild (73% observations at 1 year old), which may affect how our CG estimates compare to our wild populations. However, measuring phenotypic traits earlier in our experiment seemed to have limited impact on results as most CG phenotypes were similar to wild phenotypes. Body mass in the CG was the only trait that seemed to differ from the wild. Wild juvenile birds (1 year of age) tend to be on average 0.3 g lighter than wild adults in this population, so this may indicate that age could at least partially contribute to the observed difference in mass between contexts. Third, we monitor forest great tits in one larger study site and, although this nonurban area contains different forest types, we lack replication to draw broad inferences on the phenotypes of forest great tits more generally. Fourth, although the effects of urbanization on wild trait divergences were in the same direction and of similar magnitude to what has been previously reported in these populations, there were three cases in this study where statistical support varied (statistically unclear effect of habitat on breath rate and body mass, and ISA on exploration). In all these cases >93% of the posterior was negative (positive for exploration) indicating weak tendencies, which were likely driven by methodological differences between this and previous studies (i.e., different subset of data, additional years of data, Bayesian approach). Finally, rearing individuals under the same restricted and benign
conditions (e.g., *ad libitum* food supply) may have prevented us from detecting phenotypic differences if they are impacted by genetic and environmental interactions (G × E; Conover *et al.*, 2009). Although difficult to conduct, multitreatment CGs where food or temperature are manipulated could be especially valuable for teasing apart genetic and environmental interactions acting on phenotypic shifts in urban great tits (*Tomotani et al.*, 2025).

In conclusion, our survey of the literature for urban CG experiments indicates that both plastic and genetic divergences between urban and nonurban populations are common. In our study, we find evidence that urban phenotypic divergences in stress physiology and morphology are mainly driven by genetic change or very early maternal investment in eggs. CGs are not able to affirm local adaptation, unless realistic multitreatment or reciprocal transplant approaches are used (e.g., Gorton et al., 2018; Tüzün & Stoks, 2021), and evaluating reproductive and survival benefits of our CG birds in aviaries would not be appropriate. Thus, investigating whether these genetic differences between populations are adaptive remains an avenue for future research. We did not find evidence that genetic change strongly drives urban behavioral shifts, which provides contrary evidence to urban CG studies in other bird species (Atwell et al., 2012; Miranda et al., 2013). Further work will be needed to uncover whether plasticity predominantly drives other urban behaviors in great tits (e.g., neophilia or boldness) and determine the mechanisms underlying discrepancies with other studies. Our results highlight that phenotypic shifts in urban populations can be impacted by both genetic and plastic changes and make a valuable contribution in filling a fundamental gap concerning the urban evolution of a model species. Examining how evolutionary processes in urban contexts impact phenotypic and genetic variation will have important applications for conserving urban wildlife populations and their ecological roles in communities (Des Roches et al., 2021; Lambert & Donihue, 2020), but will also improve our fundamental understanding of ecology and evolution in wild systems more broadly, especially in light of global environmental change.

## Supplementary material

Supplementary material is available online at *Evolution*.

## Data availability

The data and code to reproduce the results of this article are available in the Dryad Digital Repository at https://doi.org/10.5061/dryad.5hqbzkhf5.

## Author contributions

M.J.T., D.R., S.P.C., and A.C. conceived the study. All authors developed the study methodology and collected data. M.J.T. compiled the data, performed the analyses, and wrote the first draft of the manuscript. M.J.T., D.R., S.P.C., and A.C. interpreted the results. All authors contributed substantially to reviewing and editing drafts of the manuscript.

## Funding

This work was supported by a Canadian Graduate Scholarship from the Natural Sciences and Engineering

Research Council of Canada, a Fonds de Recherche du Québec Nature et Technologie PhD scholarship, and a mobility grant from le Centre Méditerranéen de l'Environnement et de la Biodiversité (to M.J.T.); the Agence Nationale de la Recherche (URBANTIT grant ANR-19-CE34-0008-05), the OSU-OREME, the long-term Studies in Ecology and Evolution (SEE-Life) program of the Centre National de la Recherche Scientifique (to A.C.); and a Fonds de Recherche du Québec Nature et Technologie grant (to D.R.).

*Conflict of interest:* The authors declare no conflict of interest.

## Acknowledgments

We would like to thank everyone who participated in the common garden and is not an author: Laura Gervais, Barbara Class, Dhanya Bharath, Christophe de Franceschi, Sam Perret from the CEFE and Vivian Espinasse, Thibault Pujol, Flavien Daunis, Cathie Toussier, Jérôme Brière, Laetitia Boscardin, Sébastien Pouvreau, Lucas Boussioux, Charlotte Gay, Marion Darde from the Montpellier Zoo. Thanks to Pierre de Villemereuil for useful discussions and coding help. Comments and suggestions on a previous version of the manuscript from Timothée Bonnet and an anonymous reviewer improved the content and clarity of the paper.

## References

Alberti, M., Marzluff, J., & Hunt, V. M. (2017). Urban driven phenotypic changes: Empirical observations and theoretical implications for eco-evolutionary feedback. *Philosophical Transactions of the Royal Society of London, Series B: Biological Sciences*, 372(1712), 20160029. https://doi.org/10.1098/rstb.2016.0029

Altermatt, F., & Ebert, D. (2016). Reduced flight-to-light behaviour of moth populations exposed to long-term urban light pollution. *Biology Letters*, 12(4), 20160111. https://doi.org/10.1098/rslb.2016.0111

Antonio-Nkondjio, C., Fossog, B. T., Ndo, C., Djantio, B. M., Togouet, S. Z., Awono-Ambene, P., Costantini, C., Wondji, C. S., & Ranson, H. (2011). *Anopheles gambiae* distribution and insecticide resistance in the cities of Douala and Yaoundé (Cameroon): Influence of urban agriculture and pollution. *Malaria Journal*, 10(1), 1–13.

Atwell, J. W., Cardoso, G. C., Whittaker, D. J., Campbell-Nelson, S., Robertson, K. W., & Ketterson, E. D. (2012). Boldness behavior and stress physiology in a novel urban environment suggest rapid correlated evolutionary adaptation. *Behavioral Ecology : official journal of the International Society for Behavioral Ecology*, 23(5), 960–969. https://doi.org/10.1093/beheco/ars059

Atwell, J. W., Cardoso, G. C., Whittaker, D. J., Price, T. D., & Ketterson, E. D. (2014). Hormonal, behavioral, and life-history traits exhibit correlated shifts in relation to population establishment in a novel environment. *The American Naturalist*, 184(6), E147–E160. https://doi.org/10.1086/678398

Baxter-Gilbert, J., Riley, J. L., & Whiting, M. J. (2019). Bold new world: Urbanization promotes an innate behavioral trait in a lizard. *Behavioral Ecology and Sociobiology*, 73(8), 1–10.

Biard, C., Brischoux, F., Meillère, A., Michaud, B., Nivière, M., Ruault, S., Vaugoyeau, M., & Angelier, F. (2017). Growing in cities: An urban penalty for wild birds? A study of phenotypic differences between urban and rural great tit chicks (*Parus major*). *Frontiers in Ecology and Evolution, 5*, 1–14. https://doi.org/10.3389/fevo.2017.00079

Bókony, V., Balogh, E., Ujszegi, J., Ujhegyi, N., Szederkényi, M., & Hettyey, A. (2024). Tadpoles develop elevated heat tolerance in urban heat islands regardless of sex. *Evolutionary Biology*, 51(1), 209–216. https://doi.org/10.1007/s11692-024-09626-7
Bókony, V., Ujhegyi, N., Hamow, K. A., Bosch, J., Thumsová, B., Vörös, J., Aspbury, A. S., & Gabor, C. R. (2021). Stressed tadpoles mount more efficient glucocorticoid negative feedback in anthropogenic habitats due to phenotypic plasticity. *The Science of the Total Environment*, 753, 141896. https://doi.org/10.1016/j.scitotenv.2020.141896

Bókony, V., Üveges, B., Verebélyi, V., Ujhegyi, N., & Móricz, A. M. (2019). Toads phenotypically adjust their chemical defences to anthropogenic habitat change. *Scientific Reports*, 9(1), 1–8.

Bonier, F. (2012). Hormones in the city: Endocrine ecology of urban birds. *Hormones and Behavior*, 61(5), 763–772. https://doi.org/10.1016/j.yhbeh.2012.03.016

Brans, K. I., Almeida, R. A., & Fajgenblat, M. (2021). Genetic differentiation in pesticide resistance between urban and rural populations of a nontarget freshwater keystone interactor, *Daphnia magna*. *Evolutionary Applications*, 14(10), 2541–2552. https://doi.org/10.1111/eva.13293

Brans, K. I., & De Meester, L. (2018). City life on fast lanes: Urbanization induces an evolutionary shift towards a faster lifestyle in the water flea *Daphnia*. *Functional Ecology*, 32(9), 2225–2240. https://doi.org/10.1111/1365-2435.13184

Brans, K. I., Jansen, M., Vanoverbeke, J., Tüzün, N., Stoks, R., & De Meester, L. (2017a). The heat is on: Genetic adaptation to urbanization mediated by thermal tolerance and body size. *Global Change Biology*, 23(12), 5218–5227. https://doi.org/10.1111/gcb.13784

Brans, K. I., Govaert, L., Engelen, J. M. T., Gianuca, A. T., Souffreau, C., & De Meester, L. (2017b). Eco-evolutionary dynamics in urbanized landscapes: Evolution, species sorting and the change in zooplankton body size along urbanization gradients. *Philosophical Transactions of the Royal Society of London, Series B: Biological Sciences*, 372(1712), 20160030. https://doi.org/10.1098/rstb.2016.0030

Brans, K. I., Stoks, R., & De Meester, L. (2018). Urbanization drives genetic differentiation in physiology and structures the evolution of pace-of-life syndromes in the water flea *Daphnia magna*. *Proceedings of the Royal Society B: Biological Sciences*, 285(1883), 20180169. https://doi.org/10.1098/rspb.2018.0169

Brans, K. I., Tüzün, N., Sentis, A., De Meester, L., & Stoks, R. (2022). Cryptic eco-evolutionary feedback in the city: Urban evolution of prey dampens the effect of urban evolution of the predator. *Journal of Animal Ecology*, 91(3), 514–526. https://doi.org/10.1111/1365-2656.13601

Breitbart, S. T., Agrawal, A. A., Wagner, H. H., & Johnson, M. T. (2023). Urbanization and a green corridor do not impact genetic divergence in common milkweed (*Asclepias syriaca* L.). *Scientific Reports*, 13(1), 20437.

Caizergues, A. E., Charmantier, A., Lambrechts, M. M., Perret, S., Demeyrier, V., Lucas, A., & Grégoire, A. (2021). An avian urban morphotype: How the city environment shapes great tit morphology at different life stages. *Urban Ecosystems*, 24(5), 929–941. https://doi.org/10.1007/s11252-020-01077-0

Caizergues, A. E., Grégoire, A., & Charmantier, A. (2018). Urban versus forest ecotypes are not explained by divergent reproductive selection. *Proceedings Biological Sciences*, 285(1882), 20180261. https://doi.org/10.1098/rspb.2018.0261

Caizergues, A. E., Grégoire, A., Choquet, R., Perret, S., & Charmantier, A. (2022). Are behaviour and stress-related phenotypes in urban birds adaptive? *The Journal of Animal Ecology*, 91(8), 1627–1641. https://doi.org/10.1111/1365-2656.13740

Caizergues, A. E., Robira, B., Perrier, C., Jeanneau, M., Berthomieu, A., Perret, S., Gandon, S., & Charmantier, A. (2024). Cities as parasitic amplifiers? Malaria prevalence and diversity in great tits along an urbanization gradient. *Peer Community Journal*, 4, article no. e38. https://doi.org/10.24072/pcjournal.405

Camacho, C., Sanabria-Fernández, A., Baños-Villalba, A., & Edelaar, P. (2020). Experimental evidence that matching habitat choice drives local adaptation in a wild population. *Proceedings Biological Sciences*, 287(1927), 20200721. https://doi.org/10.1098/rspb.2020.0721

Campbell-Staton, S. C., Velotta, J. P., & Winchell, K. M. (2021). Selection on adaptive and maladaptive gene expression plasticity during thermal adaptation to urban heat islands. *Nature Communications*, 12(1), 6195. https://doi.org/10.1038/s41467-021-26334-4

Capilla-Lasheras, P., Thompson, M. J., Sánchez-Tójar, A., Haddou, Y., Branston, C. J., Réale, D., Charmantier, A., & Dominoni, D. M. (2022). A global meta-analysis reveals higher variation in breeding phenology in urban birds than in their non-urban neighbours. *Ecology Letters*, 25(11), 2552–2570. https://doi.org/10.1111/ele.14099

Carere, C., & van Oers, K. (2004). Shy and bold great tits (*Parus major*): Body temperature and breath rate in response to handling stress. *Physiology and Behavior*, 82(5), 905–912. https://doi.org/10.1016/j.physbeh.2004.07.009

Caspi, T., Johnson, J. R., Lambert, M. R., Schell, C. J., & Sih, A. (2022). Behavioral plasticity can facilitate evolution in urban environments. *Trends in Ecology & Evolution*, 37(12), 1092–1103. https://doi.org/10.1016/j.tree.2022.08.002

Charmantier, A., Demeyrier, V., Lambrechts, M., Perret, S., & Grégoire, A. (2017). Urbanization is associated with divergence in pace-of-life in great tits. *Frontiers in Ecology and Evolution*, 5, 1–13. https://doi.org/10.3389/fevo.2017.00053

Charmantier, A., Garant, D., & Kruuk, L. E. (2014). *Quantitative genetics in the wild*. OUP Oxford.

Cheptou, P.-O., Carrue, O., Rouifed, S., & Cantarel, A. (2008). Rapid evolution of seed dispersal in an urban environment in the weed *Crepis sancta*. *Proceedings of the National Academy of Sciences of the United States of America*, 105(10), 3796–3799. https://doi.org/10.1073/pnas.0708446105

Chick, L. D., Strickler, S. A., Perez, A., Martin, R. A., & Diamond, S. E. (2019). Urban heat islands advance the timing of reproduction in a social insect. *Journal of Thermal Biology*, 80, 119–125. https://doi.org/10.1016/j.jtherbio.2019.01.004

Chick, L. D., Waters, J. S., & Diamond, S. E. (2021). Pedal to the metal: Cities power evolutionary divergence by accelerating metabolic rate and locomotor performance. *Evolutionary Applications*, 14(1), 36–52. https://doi.org/10.1111/eva.13083

Christians, J. K. (2002). Avian egg size: Variation within species and inflexibility within individuals. *Biological Reviews of the Cambridge Philosophical Society*, 77(1), 1–26. https://doi.org/10.1017/s1464793101005784

Conover, D. O., Duffy, T. A., & Hice, L. A. (2009). The covariance between genetic and environmental influences across ecological gradients. *The Year in Evolutionary Biology* 2009, 1168, 100.

Corsini, M., Schöll, E. M., Di Lecce, I., Chatelain, M., Dubiec, A., & Szulkin, M. (2021). Growing in the city: Urban evolutionary ecology of avian growth rates. *Evolutionary Applications*, 14(1), 69–84. https://doi.org/10.1111/eva.13081

Czaczkes, T. J., Bastidas-Urrutia, A. M., Ghislandi, P., & Tuni, C. (2018). Reduced light avoidance in spiders from populations in light-polluted urban environments. *The Science of Nature*, 105(11-12), 1–5.

de Villemereuil, P. (2018). Quantitative genetic methods depending on the nature of the phenotypic trait. *Annals of the New York Academy of Sciences*, 1422(1), 29–47. https://doi.org/10.1111/nyas.13571

de Villemereuil, P., Gaggiotti, O. E., & Goudet, J. (2020). Common garden experiments to study local adaptation need to account for population structure. *Journal of Ecology*, 110(5), 1005–1009. https://doi.org/10.1111/1365-2745.13528

de Villemereuil, P., Mouterde, M., Gaggiotti, O. E., & Till-Bottraud, I. (2018a). Patterns of phenotypic plasticity and local adaptation in the wide elevation range of the alpine plant *Arabis alpina*. *Jacquemyn H*, editor. *Journal of Ecology*, 106(5), 1952–1971. https://doi.org/10.1111/1365-2745.12955

de Villemereuil, P., Morrissey, M. B., Nakagawa, S., & Schielzeth, H. (2018b). Fixed-effect variance and the estimation of repeatabilities and heritabilities: Issues and solutions. *Journal of Evolutionary Biology*, 31(4), 621–632. https://doi.org/10.1111/jeb.13232

de Villemereuil, P., Schielzeth, H., Nakagawa, S., & Morrissey, M. (2016). General methods for evolutionary quantitative genetic
inference from generalized mixed models. *Genetics*, 204(3), 1281-1294. https://doi.org/10.1534/genetics.115.186536

Demeyrier, V., Lambrechts, M. M., Perret, P., & Grégoire, A. (2016). Experimental demonstration of an ecological trap for a wild bird in a human-transformed environment. *Animal Behaviour*, 118, 181–190. https://doi.org/10.1016/j.anbehav.2016.06.007

Des Roches, S., Pendleton, L. H., Shapiro, B., & Palkovacs, E. P. (2021). Conserving intraspecific variation for nature's contributions to people. *Nature Ecology and Evolution*, 5(5), 574–582. https://doi.org/10.1038/s41559-021-01403-5

Dhondt, A. A. (1982). Heritability of blue tit tarsus length from normal and cross-fostered broods. *Evolution*, 36(2), 418–419. https://doi.org/10.1111/j.1558-5646.1982.tb05061.x

Diamond, S. E., Chick, L., Perez, A. B. E., Strickler, S. A., & Martin, R. A. (2017). Rapid evolution of ant thermal tolerance across an urban-rural temperature cline. *Biological Journal of the Linnean Society*, 121(2), 248–257. https://doi.org/10.1093/biolinnean/blw047

Diamond, S. E., & Martin, R. A. (2021). Evolution in cities. *Annual Review of Ecology, Evolution, and Systematics*, 52(1), 519–540. https://doi.org/10.1146/annurev-ecolsys-012021-021402

Diamond, S. E., Martin, R. A., Bellino, G., Crown, K. N., & Prileson, E. G. (2022). Urban evolution of thermal physiology in a range-expanding, mycophagous fruit fly, *Drosophila tripunctata*. *Biological Journal of the Linnean Society*, 137(3), 409–420. https://doi.org/10.1093/biolinnean/blac094

Diamond, S. E., Chick, L. A., Perez, A., Strickler, S. A., & Zhao, C. (2018a). Evolution of plasticity in the city: Urban acorn ants can better tolerate more rapid increases in environmental temperature. *Conservation Physiology*, 6(1), 1–12. https://doi.org/10.1093/conphys/coy030

Diamond, S. E., Chick, L. D., Perez, A., Strickler, S. A., & Martin, R. A. (2018b). Evolution of thermal tolerance and its fitness consequences: Parallel and non-parallel responses to urban heat islands across three cities. *Proceedings of the Royal Society B: Biological Sciences*, 285(1882), 20180036.

Dubuc-Messier, G., Caro, S. P., Perrier, C., van Oers, K., Réale, D., & Charmantier, A. (2018). Gene flow does not prevent personality and morphological differentiation between two blue tit populations. *Journal of Evolutionary Biology*, 31(8), 1127–1137. https://doi.org/10.1111/jeb.13291

Dubuc-Messier, G., Réale, D., Perret, P., & Charmantier, A. (2016). Environmental heterogeneity and population differences in blue tits personality traits. *Behavioral Ecology*, 28, arw148. https://doi.org/10.1093/beheco/arw148

Edelaar, P., Siepielski, A. M., & Clobert, J. (2008). Matching habitat choice causes directed gene flow: A neglected dimension in evolution and ecology. *Evolution*, 62(10), 2462–2472. https://doi.org/10.1111/j.1558-5646.2008.00459.x

Fudickar, A. M., Greives, T. J., Abolins-Abols, M., Atwell, J. W., Meddle, S. L., Friis, G., Stricker, C. A., & Ketterson, E. D. (2017). Mechanisms associated with an advance in the timing of seasonal reproduction in an urban songbird. *Frontiers in Ecology and Evolution*, 5, 85.

Fukano, Y., Guo, W., Uchida, K., & Tachiki, Y. (2020). Contemporary adaptive divergence of plant competitive traits in urban and rural populations and its implication for weed management. *Journal of Ecology*, 108(6), 2521–2530. https://doi.org/10.1111/1365-2745.13472

Gebhardt-Henrich, S. G., & Van Noordwijk, A. J. (1991). Nestling growth in the great tit *I.* Heritability estimates under different environmental conditions. *Journal of Evolutionary Biology*, 4(3), 341–362.

Géron, C., Lembrechts, J. J., Hamdi, R., Berckmans, J., Nijs, I., & Monty, A. (2022). Phenotypic variation along urban-to-rural gradients: An attempt to disentangle the mechanisms at play using the alien species *Matricaria discoidea* (Asteraceae). *Plant Ecology*, 223(10-12), 1219–1231. https://doi.org/10.1007/s11258-022-01269-y

Ghalambor, C. K., McKay, J. K., Carroll, S. P., & Reznick, D. N. (2007). Adaptive versus non-adaptive phenotypic plasticity and the potential for contemporary adaptation in new environments. *Functional Ecology*, 21(3), 394–407. https://doi.org/10.1111/j.1365-2435.2007.01283.x

Gorton, A. J., Moeller, D. A., & Tiffin, P. (2018). Little plant, big city: A test of adaptation to urban environments in common ragweed (*Ambrosia artemisiifolia*). *Proceedings Biological Sciences*, 285(1881), 20180968. https://doi.org/10.1098/rspb.2018.0968

Gosler, A. G., & Harper, D. G. C. (2000). Assessing the heritability of body condition in birds: A challenge exemplified by the great tit *Parus major* L. (Aves). *Biological Journal of the Linnean Society*, 71(1), 103–117. https://doi.org/10.1111/j.1095-8312.2000.tb01245.x

Hadfield, J. D. (2010). MCMC methods for multi-response generalized linear mixed models: The {MCMCglmm} [R] package. *Journal of Statistical Software*, 33(2), 1–22.

Hahs, A. K., Fournier, B., Aronson, M. F., Nilon, C. H., Herrera-Montes, A., Salisbury, A. B., Threlfall, C. G., Rega-Brodsky, C. C., Lepczyk, C. A., & La Sorte, F. A. (2023). Urbanisation generates multiple trait syndromes for terrestrial animal taxa worldwide. *Nature Communications*, 14(1), 4751.

Harten, L., Gonceer, N., Handel, M., Dash, O., Fokidis, H. B., & Yovel, Y. (2021). Urban bat pups take after their mothers and are bolder and faster learners than rural pups. *BMC Biology*, 19(1), 190. https://doi.org/10.1186/s12915-021-01131-z

Hendry, A. P. (2017). *Eco-evolutionary dynamics*. Princeton University Press. [accessed 2024 Apr 12]. https://www.degruyter.com/document/doi/10.1515/9781400883080/html

Hendry, A. P., Farrugia, T. J., & Kinnison, M. T. (2008). Human influences on rates of phenotypic change in wild animal populations. *Molecular Ecology*, 17(1), 20–29. https://doi.org/10.1111/j.1365-294X.2007.03428.x

Hörak, P. (1994). Effect of nestling history on adult size and reproduction in the Great Tit. *Ornis Fennica*, 71(2), 47–54.

Hörak, P., Mänd, R., Ots, I., & Leivits, A. (1995). Egg size in the Great Tit *Parus major*: individual, habitat and geographic differences. *Ornis Fennica*, 72(3), 97–114.

Hwang, C. C., & Turner, B. D. (2009). Small-scaled geographical variation in life-history traits of the blowfly *Calliphora vicina* between rural and urban populations. *Entomologia Experimentalis et Applicata*, 132(3), 218–224. https://doi.org/10.1111/j.1570-7458.2009.00891.x

Ichikawa, I., & Kuriwada, T. (2023). The combined effects of artificial light at night and anthropogenic noise on life history traits in ground crickets. *Ecological Research*, 38(3), 446–454. https://doi.org/10.1111/1440-1703.12380

Irwin, R. E., Warren, P. S., Carper, A. L., & Adler, L. S. (2014). Plant–animal interactions in suburban environments: Implications for floral evolution. *Oecologia*, 174(3), 803–815. https://doi.org/10.1007/s00442-013-2797-2

Jacquier, L., Molet, M., Bocquet, C., & Doums, C. (2021a). Hibernation conditions contribute to the differential resistance to cadmium between urban and forest ant colonies. *Animals*, 11(4), 1050. https://doi.org/10.3390/ani11041050

Jacquier, L., Doums, C., Four-Chaboussant, A., Peronnet, R., Tirard, C., & Molet, M. (2021b). Urban colonies are more resistant to a trace metal than their forest counterparts in the ant *Temnothorax nylan-deri*. *Urban Ecosystems*, 24(3), 561–570. https://doi.org/10.1007/s11252-020-01060-9

Kaiser, A., Merckx, T., & Van Dyck, H. (2018). Urbanisation and sex affect the consistency of butterfly personality across metamorphosis. *Behavioral Ecology and Sociobiology*, 72(12), 1–11.

Kaiser, A., Merckx, T., & Van Dyck, H. (2020). An experimental test of changed personality in butterflies from anthropogenic landscapes. *Behavioral Ecology and Sociobiology*, 74(7), 1–8.

Kawecki, T. J., & Ebert, D. (2004). Conceptual issues in local adaptation. *Ecology Letters*, 7(12), 1225–1241. https://doi.org/10.1111/j.1461-0248.2004.00684.x
Kern, E., & Langerhans, R. B. (2019). Urbanization alters swimming performance of a stream fish. *Frontiers in Ecology and Evolution*, **6**, 229.

Kern, E. M. A., & Langerhans, R. B. (2018). Urbanization drives contemporary evolution in stream fish. *Global Change Biology*, **24**(8), 3791–3803. https://doi.org/10.1111/gcb.14115

Kinnison, M. T., & Hendry, A. P. (2001). The pace of modern life II: From rates of contemporary microevolution to pattern and process. *Genetica*, **112-113**, 145–164. https://doi.org/10.1007/978-94010-0585-2_10

Koolhaas, J. M., Bartolomucci, A., Buwalda, B., de Boer, S. F., Flügge, G., Korte, S. M., Meerlo, P., Murison, R., Olivier, B., Palanza, P., Richter-Levin, G., Sgoifo, A., Steimer, T., Stiedl, O., van Dijk, G., Wöhr, M., & Fuchs, E. (2011). Stress revisited: A critical evaluation of the stress concept. *Neuroscience & Biobehavioral Reviews*, **35**(5), 1291–1301. https://doi.org/10.1016/j.neubiorev.2011.02.003

Koolhaas, J. M., De Boer, S. F., Coppens, C. M., & Buwalda, B. (2010). Neuroendocrinology of coping styles: Towards understanding the biology of individual variation. *Frontiers in Neuroendocrinology*, **31**(3), 307–321. https://doi.org/10.1016/j.yfrne.2010.04.001

Kostanecki, A., Gorton, A. J., & Moeller, D. A. (2021). An urban–rural spotlight: Evolution at small spatial scales among urban and rural populations of common ragweed. *Journal of Urban Ecology*, **7**(1), juab004.

Krams, I. A., Vrublevska, J., Sepp, T., Abolins-Abols, M., Rantala, M. J., Mierauskas, P., & Krama, T. (2014). Sex-specific associations between nest defence, exploration and breathing rate in breeding pied flycatchers. *Ethology*, **120**(5), 492–501. https://doi.org/10.1111/eth.12222

Kuriwada, T. (2023). Differences in male calling song and female mate location behaviour between urban and rural crickets. *Biological Journal of the Linnean Society*, **139**(3), 275–285. https://doi.org/10.1093/biolinnean/blad027

Lambert, M. R., Brans, K. I., Roches, S. D., Donihue, C. M., & Diamond, S. E. (2021). Adaptive evolution in cities: Progress and misconceptions. *Trends in Ecology & Evolution*, **36**(3), 239–257.

Lambert, M. R., & Donihue, C. M. (2020). Urban biodiversity management using evolutionary tools. *Nature Ecology and Evolution*, **4**(7), 903–910. https://doi.org/10.1038/s41559-020-1193-7

Lambrecht, S. C., Mahieu, S., & Cheptou, P. -O. (2016). Natural selection on plant physiological traits in an urban environment. *Acta Oecologica*, **77**, 67–74. https://doi.org/10.1016/j.actao.2016.09.002

Lampe, U., Reinhold, K., & Schmoll, T. (2014). How grasshoppers respond to road noise: Developmental plasticity and population differentiation in acoustic signalling. *Functional Ecology*, **28**(3), 660–668. https://doi.org/10.1111/1365-2435.12215

Leinonen, T., Mccairns, R. J. S., O'Hara, R. B., & Merilä, J. (2013). QST–FST comparisons: evolutionary and ecological insights from genomic heterogeneity. *Nature Reviews Genetics*, **14**(3), 179–190. https://doi.org/10.1038/nrg3395

Martin, R. A., Chick, L. D., Yilmaz, A. R., & Diamond, S. E. (2019). Evolution, not transgenerational plasticity, explains the adaptive divergence of acorn ant thermal tolerance across an urban–rural temperature cline. *Evolutionary Applications*, **12**(8), 1678–1687. https://doi.org/10.1111/eva.12826

McLean, M. A., Angilletta, M. J. Jr, & Williams, K. S. (2005). If you can't stand the heat, stay out of the city: Thermal reaction norms of chitinolytic fungi in an urban heat island. *Journal of Thermal Biology*, **30**(5), 384–391. https://doi.org/10.1016/j.jther-bio.2005.03.002

Merckx, T., Nielsen, M. E., Heliölä, J., Kuussaari, M., Pettersson, L. B., Pöyry, J., Tiainen, J., Gotthard, K., & Kivelä, S. M. (2021). Urbanization extends flight phenology and leads to local adaptation of seasonal plasticity in Lepidoptera. *Proceedings of the National Academy of Sciences*, **118**(40), e2106006118. https://doi.org/10.1073/pnas.2106006118

Merckx, T., Nielsen, M. E., Kankaanpää, T., Kadlec, T., Yazdanian, M., & Kivelä, S. M. (2023). Dim light pollution prevents diapause

induction in urban and rural moths. *Journal of Applied Ecology*, **60**(6), 1022–1031. https://doi.org/10.1111/1365-2664.14373

Merckx, T., Nielsen, M. E., Kankaanpää, T., Kadlec, T., Yazdanian, M., & Kivelä, S. M. (2024). Continent-wide parallel urban evolution of increased heat tolerance in a common moth. *Evolutionary Applications*, **17**(1), e13636. https://doi.org/10.1111/eva.13636

Merckx, T., Souffreau, C., Kaiser, A., Baardsen, L. F., Backeljau, T., Bonte, D., Brans, K. I., Cours, M., Dahirel, M., Debortoli, N., De Wolf, K., Engelen, J. M. T., Fontaneto, D., Gianuca, A. T., Govaert, L., Hendrickx, F., Higuti, J., Lens, L., Martens, K., ... Van Dyck, H. (2018). Body-size shifts in aquatic and terrestrial urban communities. *Nature*, **558**(7708), 113–116. https://doi.org/10.1038/s41586-018-0140-0

Merilä, J., & Fry, J. D. (1998). Genetic variation and causes of genotype-environment interaction in the body size of blue tit (*Parus caeruleus*). *Genetics*, **148**(3), 1233–1244. https://doi.org/10.1093/genetics/148.3.1233

Mertens, J. A. L. (1977). Thermal conditions for successful breeding in great tits (*Parus major* L.) I. Relation of growth and development of temperature regulation in nestling great tits. *Oecologia*, **28**(1), 1–29. https://doi.org/10.1007/BF00346834

Miranda, A. C., Schielzeth, H., Sonntag, T., & Partecke, J. (2013). Urbanization and its effects on personality traits: A result of micro-evolution or phenotypic plasticity? *Global Change Biology*, **19**(9), 2634–2644. https://doi.org/10.1111/gcb.12258

Mitchell-Olds, T., Willis, J. H., & Goldstein, D. B. (2007). Which evolutionary processes influence natural genetic variation for phenotypic traits? *Nature Reviews. Genetics*, **8**(11), 845–856. https://doi.org/10.1038/nrg2207

Mohajerani, A., Bakaric, J., & Jeffrey-Bailey, T. (2017). The urban heat island effect, its causes, and mitigation, with reference to the thermal properties of asphalt concrete. *Journal of Environmental Management*, **197**, 522–538. https://doi.org/10.1016/j.jenvman.2017.03.095

Mühlenhaupt, M., Baxter-Gilbert, J., Makhubo, B. G., Riley, J. L., & Measey, J. (2022). No evidence for innate differences in tadpole behavior between natural, urbanized, and invasive populations. *Behavioral Ecology and Sociobiology*, **76**(1), 11. https://doi.org/10.1007/s00265-021-03121-1

Nicolaus, M., & Edelaar, P. (2018). Comparing the consequences of natural selection, adaptive phenotypic plasticity, and matching habitat choice for phenotype–environment matching, population genetic structure, and reproductive isolation in meta-populations. *Ecology and Evolution*, **8**(8), 3815–3827. https://doi.org/10.1002/ece3.3816

Palomar, G., Wos, G., Stoks, R., & Sniegula, S. (2023). Latitude-specific urbanization effects on life history traits in the damselfly *Ischnura elegans*. *Evolutionary Applications*, **16**(8), 1503–1515. https://doi.org/10.1111/eva.13583

Partecke, J., & Gwinner, E. (2007). Increased sedentariness in European blackbirds following urbanization: A consequence of local adaptation? *Ecology*, **88**(4), 882–890. https://doi.org/10.1890/06-1105

Partecke, J., Schwabl, I., & Gwinner, E. (2006). Stress and the city: Urbanization and its effects on the stress physiology in European blackbirds. *Ecology*, **87**(8), 1945–1952. https://doi.org/10.1890/0012-9658(2006)87[1945:satcua]2.0.co;2

Perrier, C., Lozano del Campo, A., Szulkin, M., Demeyrier, V., Gregoire, A., & Charmantier, A. (2018). Great tits and the city: Distribution of genomic diversity and gene–environment associations along an urbanization gradient. *Evolutionary Applications*, **11**(5), 593–613. https://doi.org/10.1111/eva.12580

Pipoly, I., Preiszner, B., Sándor, K., Sinkovics, C., Seress, G., Vincze, E., Bókony, V., & Liker, A. (2022). Extreme hot weather has stronger impacts on Avian reproduction in forests than in cities. *Frontiers in Ecology and Evolution*, **10**, 825410.

Pisman, M., Bonte, D., & De La Peña, E. (2020). Urbanization alters plastic responses in the common dandelion *Taraxacum officinale*. *Ecology and Evolution*, **10**(9), 4082–4090. https://doi.org/10.1002/ece3.6176
Playà-Montmany, N., González-Medina, E., Cabello-Vergel, J., Parejo, M., Abad-Gómez, J. M., Sánchez-Guzmán, J. M., Villegas, A., & Masero, J. A. (2021). The thermoregulatory role of relative bill and leg surface areas in a Mediterranean population of Great tit (Parus major). *Ecology and Evolution*, *11*(22), 15936–15946. https://doi.org/10.1002/ece3.8263

Qu, J., Bonte, D., & Vandegehuchte, M. L. (2022). Phenotypic and genotypic divergence of plant-herbivore interactions along an urbanization gradient. *Evolutionary Applications*, *15*(5), 865–877. https://doi.org/10.1111/eva.13376

R Core Team. (2024). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria.

Rasner, C. A., Yeh, P., Eggert, L. S., Hunt, K. E., Woodruff, D. S., & Price, T. D. (2004). Genetic and morphological evolution following a founder event in the dark-eyed junco, *Junco hyemalis thurberi*. *Molecular Ecology*, *13*(3), 671–681. https://doi.org/10.1046/j.1365-294x.2004.02104.x

Reichard, D. G., Atwell, J. W., Pandit, M. M., Cardoso, G. C., Price, T. D., & Ketterson, E. D. (2020). Urban birdsongs: Higher minimum song frequency of an urban colonist persists in a common garden experiment. *Animal Behaviour*, *170*, 33–41. https://doi.org/10.1016/j.anbehav.2020.10.007

Rivkin, L. R., Santangelo, J. S., Alberti, M., Aronson, M. F. J., de Keyzer, C. W., Diamond, S. E., Fortin, M. J., Frazee, L. J., Gorton, A. J., Hendry, A. P., Liu, Y., Losos, J. B., MacIvor, J. S., Martin, R. A., McDonnell, M. J., Miles, L. S., Munshi-South, J., Ness, R. W., Newman, A. E. M., ... Johnson, M. T. J. (2019). A roadmap for urban evolutionary ecology. *Evolutionary Applications*, *12*(3), 384–398. https://doi.org/10.1111/eva.12734

Salmón, P., Jacobs, A., Ahrén, D., Biard, C., Dingemanse, N. J., Dominoni, D. M., Helm, B., Lundberg, M., Senar, J. C., Sprau, P., Visser, M. E., & Isaksson, C. (2021). Continent-wide genomic signatures of adaptation to urbanisation in a songbird across Europe. *Nature Communications*, *12*(1), 2983. https://doi.org/10.1038/s41467-021-23027-w

Samocha, Y., & Scharf, I. (2020). Comparison of wormlion behavior under man-made and natural shelters: Urban wormlions more strongly prefer shaded, fine-sand microhabitats, construct larger pits and respond faster to prey. *Current Zoology*, *66*(1), 91–98. https://doi.org/10.1093/cz/zoz006

Sanderson, S., Bolnick, D. I., Kinnison, M. T., O'Dea, R. E., Gorné, L. D., Hendry, A. P., & Gotanda, K. M. (2023). Contemporary changes in phenotypic variation, and the potential consequences for eco-evolutionary dynamics. *Ecology Letters*, *26*(Suppl 1), S127–S139. https://doi.org/10.1111/ele.14186

Santangelo, J. S., Miles, L. S., Breitbart, S. T., Murray-Stoker, D., Rivkin, L. R., Johnson, M. T. J., & Ness, R. W. (2020). Urban environments as a framework to study parallel evolution. In M. Szulkin, J. Munshi-South, & A. Charmantier (Eds.), *Urban evolutionary biology* (pp. 36–53). Oxford University Press.

Sato, A., & Takahashi, Y. (2022). Responses in thermal tolerance and daily activity rhythm to urban stress in *Drosophila suzukii*. *Ecology and Evolution*, *12*(12), e9616. https://doi.org/10.1002/ece3.9616

Schmitz, G., Linstädter, A., Frank, A. S. K., Dittberner, H., Thome, J., Schrader, A., Linne von Berg, K., Fulgione, A., Coupland, G., & De Meaux, J. (2024). Environmental filtering of life-history trait diversity in urban populations of *Arabidopsis thaliana*. *Journal of Ecology*, *112*(1), 14–27. https://doi.org/10.1111/1365-2745.14211

Seress, G., Sándor, K., Evans, K. L., & Liker, A. (2020). Food availability limits avian reproduction in the city: An experimental study on great tits *Parus major*. *The Journal of Animal Ecology*, *89*(7), 1570–1580. https://doi.org/10.1111/1365-2656.13211

Snell-Rood, E. C. (2013). An overview of the evolutionary causes and consequences of behavioural plasticity. *Animal Behaviour*, *85*(5), 1004–1011. https://doi.org/10.1016/j.anbehav.2012.12.031

Sol, D., Lapiedra, O., & González-Lagos, C. (2013). Behavioural adjustments for a life in the city. *Animal Behaviour*, *85*(5), 1101–1112. https://doi.org/10.1016/j.anbehav.2013.01.023

Stirling, D. G., Réale, D., & Roff, D. A. (2002). Selection, structure and the heritability of behaviour. *Journal of Evolutionary Biology*, *15*(2), 277–289. https://doi.org/10.1046/j.1420-9101.2002.00389.x

Sumasgutner, P., Cunningham, S. J., Hegemann, A., Amar, A., Watson, H., Nilsson, J. F., Andersson, M. N., & Isaksson, C. (2023). Interactive effects of rising temperatures and urbanisation on birds across different climate zones: A mechanistic perspective. *Global Change Biology*, *29*(9), 2399–2420. https://doi.org/10.1111/gcb.16645

Svensson, L. (1992). *Identification guide to European passerines*. The author.

Szulkin, M., Munshi-South, J., & Charmantier, A. (2020). *Urban evolutionary biology*. Oxford University Press.

Taichi, N., & Ushimaru, A. (2024). Trait variation along an urban-rural gradient in Asian dayflower: The contribution of phenotypic plasticity and genetic divergence. *Plant Biology*, *26*(1), 74–81. https://doi.org/10.1111/plb.13595

Talloen, W., Lens, L. U. C., Van Dongen, S., Adriaensen, F., & Matthysen, E. (2010). Mild stress during development affects the phenotype of great tit *Parus major* nestlings: A challenge experiment. *Biological Journal of the Linnean Society*, *100*(1), 103–110. https://doi.org/10.1111/j.1095-8312.2010.01422.x

Tene Fossog, B., Antonio-Nkondjio, C., Kengne, P., Njiokou, F., Besan sky, N. J., & Costantini, C. (2013a). Physiological correlates of ecological divergence along an urbanization gradient: differential tolerance to ammonia among molecular forms of the malaria mos quito *Anopheles gambiae*. *BMC Ecology*, *13*(1), 1–12. https://doi.org/10.1186/1472-6785-13-1

Tene Fossog, B., Poupardin, R., Costantini, C., Awono-Ambene, P., Wondji, C. S., Ranson, H., & Antonio-Nkondjio, C. (2013b). Resistance to DDT in an urban setting: Common mechanisms impli cated in both M and S forms of *Anopheles gambiae* in the city of Yaoundé Cameroon. *PLoS One*, *8*(4), e61408.

Thompson, K. A., Renaudin, M., & Johnson, M. T. J. (2016). Urbanization drives the evolution of parallel clines in plant populations. *Proceedings of the Royal Society B: Biological Sciences*, *283*(1845), 20162180. https://doi.org/10.1098/rspb.2016.2180

Thompson, M. J., Capilla-Lasheras, P., Dominoni, D. M., Réale, D., & Charmantier, A. (2022). Phenotypic variation in urban environments: Mechanisms and implications. *Trends in Ecology & Evolution*, *37*(2), 171–182. https://doi.org/10.1016/j.tree.2021.09.009

Tomotani, B. M., Couweleers, M., ten Brinke, B., Walboom, A., van Oers, K., & Visser, M. (2025). Size in the city: Morphological differences between city and forest great tits have a genetic basis. *Evolution Letters*, *1*–26. https://doi.org/10.1093/evlett/qraf006

Tüzün, N., Debecker, S., de Beeck, L. O., & Stoks, R. (2015). Urbanisation shapes behavioural responses to a pesticide. *Aquatic Toxicology*, *163*, 81–88.

Tüzün, N., Op de Beeck, L., Brans, K. I., Janssens, L., & Stoks, R. (2017a). Microgeographic differentiation in thermal performance curves between rural and urban populations of an aquatic insect. *Evolutionary Applications*, *10*(10), 1067–1075. https://doi.org/10.1111/eva.12512

Tüzün, N., Mueller, S., Koch, K., & Stoks, R. (2017b). Pesticide-induced changes in personality depend on the urbanization level. *Animal Behaviour*, *134*, 45–55.

Tüzün, N., & Stoks, R. (2021). Lower bioenergetic costs but similar immune responsiveness under a heat wave in urban compared to rural damselflies. *Evolutionary Applications*, *14*(1), 24–35. https://doi.org/10.1111/eva.13041

Van de Schoot, E., Merckx, T., Ebert, D., Wesselingh, R. A., Altermatt, F., & Van Dyck, H. (2024). Evolutionary change in flight-to-light response in urban moths comes with changes in wing morphology. *Biology Letters*, *20*(3), 20230486. https://doi.org/10.1098/rslb.2023.0486

Van Noordwijk, A. J., Keizer, L. C. P., Van Balen, J. H., & Scharloo, W. (1981). Genetic variation in egg dimensions in natural
populations of the Great Tit. *Genetica*, **55**(3), 221-232. https://doi.org/10.1007/bf00127206

Vaugoyeau, M., Adriaensen, F., Artemyev, A., Bańbura, J., Barba, E., Biard, C., Blondel, J., Bouslama, Z., Bouvier, J. -C., Camprodon, J., Cecere, F., Charmantier, A., Charter, M., Cichoń, M., Cusimano, C., Czeszczewik, D., Demeyrier, V., Doligez, B., Doutrelant, C., ... Møller, A. P. (2016). Interspecific variation in the relationship between clutch size, laying date and intensity of urbanization in four species of hole-nesting birds. *Ecology and Evolution*, **6**(16), 5907-5920. https://doi.org/10.1002/ece3.2335

Westby, K. M., & Medley, K. A. (2020). Cold nights, city lights: Artificial light at night reduces photoperiodically induced diapause in urban and rural populations of *aedes albopictus* (Diptera: *Culicidae*). *Journal of Medical Entomology*, **57**(6), 1694-1699. https://doi.org/10.1093/jme/tjaa139

Weston, L. M., Mattingly, K. Z., Day, C. T. C., & Hovick, S. M. (2021). Potential local adaptation in populations of invasive reed canary grass (*Phalaris arundinacea*) across an urbanization gradient. *Ecology and Evolution*, **11**(16), 11457-11476. https://doi.org/10.1002/ece3.7938

Whitehead, A., Pilcher, W., Champlin, D., & Nacci, D. (2012). Common mechanism underlies repeated evolution of extreme pollution tolerance. *Proceedings Biological Sciences*, **279**(1728), 427-433. https://doi.org/10.1098/rspb.2011.0847

Wilson, A. J. (2018). How should we interpret estimates of individual repeatability? *Evolution Letters*, **2**(1), 4-8. https://doi.org/10.1002/evl3.40

Winchell, K. M., Reynolds, R. G., Prado-Irwin, S. R., Puente-Rolón, A. R., & Revell, L. J. (2016). Phenotypic shifts in urban areas in the tropical lizard *Anolis cristatellus*. *Evolution*, **70**(5), 1009-1022.

y Gomez, G. S. M., & Van Dyck, H. (2012). Ecotypic differentiation between urban and rural populations of the grasshopper *Chorthippus brunneus* relative to climate and habitat fragmentation. *Oecologia*, **169**(1), 125-133.

Yakub, M., & Tiffin, P. (2017). Living in the city: Urban environments shape the evolution of a native annual plant. *Global Change Biology*, **23**(5), 2082-2089. https://doi.org/10.1111/gcb.13528

Yeh, P. J. (2004). Rapid evolution of a sexually selected trait following population establishment in a novel habitat. *Evolution*, **58**(1), 166-174. https://doi.org/10.1111/j.0014-3820.2004.tb01583.x

Yeh, P. J., & Price, T. D. (2004). Adaptive phenotypic plasticity and the successful colonization of a novel environment. *The American Naturalist*, **164**(4), 531-542. https://doi.org/10.1086/423825

Yilmaz, A. R., Diamond, S. E., & Martin, R. A. (2021). Evidence for the evolution of thermal tolerance, but not desiccation tolerance, in response to hotter, drier city conditions in a cosmopolitan, terrestrial isopod. *Evolutionary Applications*, **14**(1), 12-23. https://doi.org/10.1111/eva.13052

Young, E. A., & Postma, E. (2023). Low interspecific variation and no phylogenetic signal in additive genetic variance in wild bird and mammal populations. *Ecology and Evolution*, **13**(11), e10693. https://doi.org/10.1002/ece3.10693

Youngflesh, C., Saracco, J. F., Siegel, R. B., & Tingley, M. W. (2022). Abiotic conditions shape spatial and temporal morphological variation in North American birds. *Nature Ecology & Evolution*, **6**(12), 1860-1870. https://doi.org/10.1038/s41559-022-01893-x

Zhang, H., He, Y., Yang, J., Mao, H., & Jiang, X. (2022). Contemporary adaptive evolution in fragmenting river landscapes: Evidence from the native waterflea *Ceriodaphnia cornuta*. *Journal of Plankton Research*, **44**(1), 88-98. https://doi.org/10.1093/plankt/fbab077