Replicated urban mosaics reveal trait- and species-specific shifts in carotenoid and structural plumage colouration of two passerines

Abstract

Urban environments are characterized by markedly altered conditions, which can exert a negative impact on the organisms that inhabit them. Among phenotypic traits most sensitive to urbanization is the colourful plumage of birds. One of the best studied examples might be the urban dullness phenomenon, referring to carotenoid-based colours being subdued in urban areas. In contrast, melanin-based and structural colouration are still understudied in this context. Moreover, research on feather colouration has focused on changes in mean trait expression, while the effect on phenotypic variation was rarely addressed. Here, we examined urbanization-driven differences in carotenoid-based, melanin-based and structural colours of two urban adapters: the great tit (Parus major) and the blue tit (Cyanistes caeruleus). In total, we sampled 309 birds on 8 replicated urban–rural gradients across Poland (Central and Eastern Europe)—an understudied geographical region in the field of evolutionary ecology. In great tits, mean breast carotenoid chroma decreased, while its variance increased, with increasing urbanization. This pattern was not observed in the blue tit. However, blue tits exhibited contrasting patterns of brightness along the urbanization gradient: carotenoid-based breast brightness decreased, whereas structurally-coloured tail brightness increased, suggesting different underlying processes across colouration types (e.g., diet, feather wear and pollution). In contrast, all melanin-based traits (tie area, wing and tail feathers) in the great tit were unaffected. Our results highlight the fact that urbanization influences colour traits in a species and trait-specific manner, cautioning against extrapolating across species.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-44382-y.

Introduction

Urbanization profoundly transforms natural habitats, altering ecological conditions through increased pollution, habitat fragmentation, and changes in food availability, temperature, and light regimes¹. These environmental changes pose physiological and ecological challenges to wildlife, even for species that appear well adapted to urban environments^2–5. Among traits that are influenced by these urban stressors, animal colouration—particularly in birds—has received increasing attention. Plumage colouration plays crucial roles in thermoregulation, camouflage, species recognition, and sexual signalling⁶. It is shaped by a variety of mechanisms, including pigmentation (e.g., carotenoids and melanin), structural colouration, or a combination of both^7–9. All of these are differently sensitive to environmental variation, especially to changes in diet quality, stress levels, and pollutant exposure—factors that are often altered in urban settings^10,11. Plumage colouration variation will also depend on their heritability, with more heritable colouration traits possibly showing weaker responses to urban-driven environmental variation. Urban stressors were already reported to disrupt colour expression, as shown across multiple taxa, and particularly in birds^12,13. A deeper understanding of how urbanization affects plumage colouration, specifically in terms of trait variability and less frequently studied types of colouration, could provide further insights into the ecological and evolutionary responses of animals to urban environments.

Carotenoid-based colouration is by far the most extensively studied class of ornaments and the one most sensitive to anthropogenic factors^11,12. Despite having a genetic component^14–16, carotenoid-based colour traits are usually strongly environmentally mediated¹⁷. Carotenoid pigments cannot be synthesized by birds, thus their deposition in integumentary tissues is strongly related to dietary intake¹⁸. This links environmental quality, specifically the availability of carotenoid-rich food sources, to colour expression, typically measured as carotenoid chroma. In polluted or urbanized areas, the concentration of carotenoids might be lower in both primary carotenoid producers, e.g., trees¹⁹ and leaf-eating caterpillars²⁰, which are important prey for many insectivorous passerines. This may lead to decreased chromaticity of carotenoid-based plumage of urban birds—known as ‘urban dullness’, most comprehensively documented in a recent multianalytical study²¹. Moreover, the expression of carotenoid-based signals may also be mediated by oxidative stress, which is increased in urban environments²². Despite the large number of studies on the urban dullness phenomenon, most focus on the great tit (Parus major) [e.g.,^20,21] and the house finch (Haemorhous mexicanus) [e.g.,^81,82,18,86], with only rare examples investigating other species^24–26. Besides the chromatic component, an important dimension of carotenoid-based colouration is its achromatic component. It is most often quantified as brightness, and is thought to be determined by structure, i.e., density of barbs, variation in their keratin/air matrix, or quality of keratin itself^27,28. These properties may, in turn, depend on the quality of the bird’s diet during moult²⁹. However, although the diet of urban birds differs from that of their rural/forest counterparts³⁰, most studies have not found corresponding changes in the brightness of carotenoid-based feathers^83,21, but see ⁵¹.

In contrast to carotenoid-based colouration, melanin-based colouration is a strongly heritable class of ornaments in birds¹⁷. Urban environments can modify melanin-based traits through selective pressures, a phenomenon known as urban melanism, in which melanic morphs occur more frequently in polluted habitats^12,13. The most famous example of this is industrial melanism in the Peppered moth (Biston betularia) during the Industrial Revolution in England³¹. In birds, evidence largely comes from feral pigeons (Columba livia) where melanic morphs occur more frequently in urban habitats^32,33. One main hypothesis is that melanism confers a selective advantage in polluted areas, as melanized tissues such as feathers can accumulate excess heavy metals, potentially protecting birds from their toxic effects^34–36. Anthropogenic factors such as urban stressors or metal pollution were also proposed to modify the intensity of melanogenesis. Specifically, oxidative stress can upregulate the synthesis of dark eumelanin, while suppressing the production of rusty-brown pheomelanin^34,37. However, despite numerous empirical attempts to demonstrate the impact of anthropogenic factors on the intensity of melanin-based traits, a recent quantitative review found no evidence supporting such an effect¹¹.

Unlike pigment-based ornaments, structural colouration (both iridescent and non-iridescent types) has rarely been studied in the context of urbanization. This type of colouration is characterised by significant but relatively low heritability below 0.2^14,16,38 and was shown to be associated with individual quality, especially body condition and immune function³⁹. However, the mechanism linking environmental conditions and expression of structurally coloured traits is still poorly understood. It was demonstrated that the feathers of feral pigeons exposed to higher levels of lead (both naturally in urban environments and in an experimental setting) had lower iridescent feather brightness⁴⁰. In the case of non-iridescent structural colouration, suburban Florida Scrub-Jays (Aphelocoma coerulescens) were reported to exhibit a greater proportion of total plumage reflectance in the ultraviolet (UV) range than birds from wildland populations, possibly due to greater food availability in suburban habitats⁴¹.

Beyond the effects of urban-related environmental factors on feather development and quality, and/or on the selection of colour traits, air pollution adhering to the feather surface may further alter feather colour as perceived by conspecifics. Such indirect impact of urban-related airborne particles has been demonstrated in experimental studies on structural iridescent colouration of European starling (Sturnus vulgaris)⁴². Specifically, long-term exposure of starling feathers to air-pollution resulted in significant reduction of reflectance, especially in the UV region⁴². Similar results were found in eastern bluebirds (Siala sialis), whose feathers, when exposed to the urban environment, had decreased values of UV and blue chroma⁴³. Despite experimental evidence that airborne pollutants can reduce the reflectance of structurally coloured feathers, particularly in the UV range, studies examining how urbanization affects structural plumage ornaments along natural urban gradients remain scarce, especially in highly polluted Central and Eastern European cities⁴⁴.

Finally, most studies that examined the impact of urbanization on plumage colours aimed to detect differences in phenotypic mean, and often only by comparing urban versus rural/forest populations⁴⁵. However, it is now established that urbanization can also influence phenotypic variation, potentially leading to important eco-evolutionary consequences in terms of selection and response to selection⁴⁶. In urban populations, phenotypic variation might be influenced by several mechanisms, including relaxed selection and higher environmental heterogeneity within urban environments. Accordingly, a recent analysis of 13 European populations of great tits and blue tits revealed a decrease in mean morphological traits and an increase in variance in urban populations⁴⁶. To our knowledge, no studies have explored this issue in relation to avian colour traits.

In this study, we examined urbanization-driven differences in carotenoid-based, melanin-based, and structural colour expression of two urban adapters, the great tit (Parus major) and the blue tit (Cyanistes caeruleus). To account for the spatial heterogeneity of urban landscapes⁴⁵, we sampled birds across six habitat types along urban—forest gradients, replicated across eight Polish cities. Furthermore, to ensure the comparability of urbanization levels with different studies¹³, we characterized urbanization intensity using the percentage of impervious surface cover (ISA) and tree cover.

Specifically, we addressed two research questions:

• (I)How does urbanization affect plumage ornaments with different colour-producing mechanisms?
• (II)Does urbanization modify the phenotypic variation of colour traits?

The inclusion of two study species, collected concurrently at the same sampling sites, across 8 cities in a narrow temporal window of data collection, allowed us to examine a wider palette of colour traits, and to compare their sensitivity to urbanization using highly standardized field data. Based on earlier studies²¹, we predicted that both analysed species would exhibit the urban dullness phenomenon, i.e. lower breast carotenoid chroma in more urbanized habitats, with no corresponding effect on feather brightness. Moreover, considering the high heterogeneity of urban environments⁴⁴ and the established link between carotenoid-based colouration and environmental pigment availability, we also predicted breast colouration to be more variable in more urbanized sites. Regarding melanin-based traits, we expected to observe lower brightness resulting from increased production and deposition of eumelanin driven by elevated levels of oxidative stress in more urbanized habitats²². Despite the scarcity of research on structural colouration in the context of urbanization, we presumed to find decreased UV chroma closer to city centres (Fig. 1).

Fig. 1.

(A) Map of cities where birds from a mosaic of habitats were sampled. In each city, birds were sampled in 4 to 13 sampling sites. (B) Feather sampling location and type of colour trait analysed. Black, yellow and blue circles indicate respectively: melanin-based colouration, carotenoid-based colouration and structural colouration.

Results

Mean expression of colour traits in relation to habitat type

In great tits, carotenoid-based breast plumage differed significantly between habitat types (Fig. 2A, Table S2). Specifically, birds in forest habitats had a higher breast carotenoid chroma than those in city centres (Estimate ± SE; 0.69 ± 0.23, t = 3.00, p = 0.003), residential areas (Estimate ± SE; 0.49 ± 0.22, t = 2.23, p = 0.027) and urban parks (Estimate ± SE; 0.51 ± 0.20, t = 2.48, p = 0.014). While great tit carotenoid-based breast brightness was significantly higher in males than in females, it did not significantly differ between habitats (Fig. 2B, Table S2). Similarly, we observed no differences in melanic wing and tail brightness (which shows an inverse relationship with melanin concentration) between habitat types (Fig. 3A, Table S2). The melanic breast tie area, analysed exclusively in males, was greater in older individuals and positively associated with condition but did not differ between habitat types (Table S2).

Fig. 2.

Boxplots for great tit and blue tit breast feather carotenoid chroma (A,C) and brightness (B,D) in relation to habitat type. Horizontal bars indicate data median, bigger dots indicate mean, box edges denote 25%–75% quartiles, whiskers indicate 1.5 IQR. Plotted on raw data. The red asterisk in the top right corner indicates significant differences in trait means (S2 between habitat types (Table S2), while red “less-than” symbol indicate significant differences in breast feather carotenoid chroma phenotypic variance (Fig. 2A, see also results section for test statistic).

Fig. 3.

Boxplots of great tit (A) and blue tit (B) tail feather brightness in relation to habitat type. Horizontal bars indicate data median, bigger dots indicate mean, box edges denote 25%–75% quartiles, whiskers indicate 1.5 IQR. Plotted on raw data. The red asterisk in the top right corner indicates significant differences between habitat types in the models (Table S2) .

In blue tits, breast carotenoid chroma did not vary between different habitat types (Fig. 2C, Table S2). However, carotenoid-based breast brightness in birds from forest habitats and river corridors was higher than in birds from city centres (Estimate ± SE; 0.77 ± 0.34, t = 2.27, p = 0.025 and 0.82 ± 0.37, t = 2.22, p = 0.029, respectively; Fig. 2D) and in males (Table S2). There were also differences in structurally coloured tail feather brightness between habitat types (Table S2, Fig. 3B), with forest birds expressing lower tail brightness than those from city centres and urban parks (Estimate ± SE; − 0.97 ± 0.32, t = − 3.07, p = 0.003; − 0.62 ± 0.27, t = − 2.22, p = 0.022, respectively) and between those from river corridors and city centres (Estimate ± SE; − 0.71 ± 0.34, t = − 2.07, p = 0.041). Tail brightness was also affected by the interaction between sex and age resulting from higher tail brightness in young females but no sex difference in older individuals (Table S2, Figure S6). Sex- and age- specific interactions in structurally coloured wing feather brightness and UV chroma were also observed (Table S2).

Phenotypic variation of colour traits in relation to habitat type

Interestingly, the coefficient of variation (CVs) of carotenoid chroma in great tits differed across habitat types (D’AD statistic = 36.73, k = 5, p < 0.001), with higher variation in more urbanised habitats (Fig. 2A). However, no other great tit or blue tit colour traits varied in terms of variance between habitat types (Table S3).

Mean expression of colour traits in relation to tree cover and ISA

In great tits, there was a positive association between the amount of tree cover at the sampling site and breast feather carotenoid chroma (Table S4, Fig. 4A). A similar relationship, but in the opposite direction and with a smaller effect size, was observed for the percentage of ISA (Estimate ± SE; 0.01 ± 0.003, t = − 2.58, p = 0.011; Table S5). None of the other great tit colour metrics were related to tree cover and ISA (Table S4, Table S5).

Fig. 4.

Scatterplots of raw data showing association between great tit (A) and blue tit (B) breast carotenoid chroma and tree cover (%).

Unlike great tits, there was no association between tree cover or percentage ISA with blue tit breast feather carotenoid chroma (Fig. 4B, Table S4, Table S5). However, there was a tendency towards a negative association between ISA and carotenoid-based breast feather brightness (Table S5; p = 0.07). Structurally coloured tail feather brightness was explained by the interaction between tree cover and sex (Estimate ± SE; − 0.01 ± 0.01, t = − 2.03, p = 0.045), with males expressing lower values (possibly caused by higher melanin deposition) in habitats with greater tree coverage (Table S4, Figure S4). Furthermore, the association between tail UV chroma and tree cover was age-specific (Estimate ± SE; 0.01 ± 0.005, t = 2.15, p = 0.034), with greater differences between age classes in more forested habitats (Table S4, Figure S5A). Similarly, there was an interaction between ISA and age in the model examining tail UV chroma, with increasing ISA leading to waning differences between older and younger individuals (Table S5, Figure S5B).

Discussion

Our results, based on eight replicated urban gradients in Central and Eastern Europe, strongly support previous findings on the prevalence of the ‘urban dullness’ phenomenon in the great tit –that is, a decrease in carotenoid-based breast feather chroma²¹. Moreover, beyond a shift in trait mean, we also demonstrate that urbanization is associated with increased phenotypic variance in great tit breast carotenoid chroma. In contrast to predictions, the analogous effect was not observed in the blue tit. Instead, carotenoid-based breast feather brightness consistently decreased along the rural–urban gradient, while UV-blue structural tail feathers increased in brightness in more urbanized habitats. Finally, and in contrast to earlier work⁴⁷, we did not find any evidence for an effect of urbanization on melanin-based colour traits of the great tit, including the breast tie area, wing and tail feather colouration (Fig. 5).

Fig. 5.

A graphic summary of main study outcomes. ISA (Impervious Surface Area) is measured as a percentage in a 100 m radius around the sampling site. Similarly, tree cover is measured in % in the same radius as ISA around the sampling site. Habitat types here are ordered from lowest to highest percentage of tree cover on average (see also Table 1). Arrow angles represent direction of association.

Carotenoid-based colouration

In line with our predictions, urbanization was negatively associated with carotenoid chroma of breast plumage in the great tit (Fig. 2A). In contrast, feather brightness—commonly interpreted as a measure of feather structural quality^27,28—was not affected, consistent with most previous studies^21,79,80,83. Carotenoid chroma depends on the amount of pigment deposited in the feather; thus, one of the key factors shaping its expression is the environmental availability of dietary carotenoids. In great tits and blue tits, the main carotenoids are lutein and zeaxanthin, which are obtained from invertebrates (e.g., Lepidoptera caterpillars) that, in turn, derive carotenoids from the leaves of trees such as oak (Quercus robur), birch (Betula spp.), and alder (Alnus spp.) on which they feed^20,48. Thus, the availability of carotenoid-rich food in urbanized environments is limited by the extent of green areas such as parks and urban forests. In line with this hypothesis, we found a positive relationship between tree cover and carotenoid chroma in great tits (Fig. 4A), consistent with observations from another European city where urban great tits consumed fewer arthropods (including Lepidoptera) during the breeding season compared to their rural counterparts³⁰. Although this pattern was not maintained in the long term, deficiencies experienced during reproduction could have prolonged effects during the resource-demanding moult period. The negative effects of limited availability of green spaces in cities may be further aggravated by heavy metal pollution, which can reduce both the carotenoid content and abundance of caterpillars^19,20,48, as well as interfere with pigment deposition²⁴. Supporting this, metal pollution was positively associated with the degree of urbanization at the same study sites as the present research⁴⁴, highlighting metal pollution as a strong candidate for explaining urban-driven variation in plumage colouration.

In addition to reduced mean carotenoid chroma in great tits, our study revealed increased phenotypic variance in this trait in more urbanized habitats, a novel finding in urban colouration research. The most plausible explanation for our findings is that high environmental heterogeneity in urban habitats—characterized by fragmented green areas of varying size and composition, interspersed with extensive impervious surface areas (ISA) limiting movements—creates spatial variation in the availability of carotenoid-rich food. This variation in diet among individuals likely drives the observed increase in phenotypic variance of carotenoid chroma. Local hotspots of the ‘urban heat island’ effect may further exacerbate spatial variation in the availability of caterpillars, amplifying differences in plumage colouration⁴⁹. One important implication of increased phenotypic variation in urban populations is that it may alter the pace and direction of selection on these traits. Supporting this idea, a recent long-term study provided the first evidence for directional selection on carotenoid-based colouration in urban great tit males, showing that paler birds had higher reproductive success⁵⁰. Carotenoid-based colouration in male blue tits also has a significant heritable component (h² = 0.13–0.25 in Corsica and Rouvière populations, respectively¹⁶) and could therefore potentially respond to urban selection. However, in our study, we found no differences in carotenoid chroma variation between urban and forest blue tits, suggesting that limited phenotypic variation may constrain the strength or opportunity for directional selection on this trait in this species.

Despite the fact that yellow colouration in both species is based on the same dietary pigments (specifically, lutein and zeaxanthin, deposited in similar ratios⁴⁸), we found no evidence for an urban dullness effect in the blue tit. However, blue tit colouration is characterized by significantly lower carotenoid chroma, compared to great tits (Figure S2), which suggests that they require less pigment. Therefore, it is possible that the environmental availability of carotenoid-rich dietary sources in more urbanized habitats is not as limiting for colour trait expression in blue tits as it is in great tits²⁰. Another, non-exclusive explanation, might be dietary differences between the two species. Indeed, it was demonstrated that urban great tits shift to a more opportunistic and granivorous diet (including anthropogenic food sources like sunflower seeds and peanuts), while urban blue tits remain largely insectivorous from spring to autumn, thus potentially maintaining their carotenoid intake³⁰. Differences in their foraging strategies may also explain differences in how the achromatic component of carotenoid-based plumage colouration responds to urbanization: in our study, blue tits from city centres displayed lower carotenoid-based breast feather brightness than birds from forests and river corridors (Fig. 2D), whereas this effect was absent in great tits. Similarly, most previous studies have found no effect of urbanisation on carotenoid-based breast feather brightness in great tits, except for one, that also reported a tendency towards lower breast brightness in urban birds⁵¹. Given that brightness is thought to reflect feather structural properties (i.e., microstructure at the barb and barbule levels) and keratin quality^27,28, the observed effect may be driven by differences in the nutritional condition of birds in urban habitats³⁰. In line with this, food composition, specifically its protein content, was shown to affect the size and brightness of white feather ornaments in dark-eyed juncos (Junco hyemalis)⁵². In addition, lower brightness may also result from altered moult timing and duration in urban environments⁵³, as well as increased exposure to oxidative stress and chemical pollution⁵⁴.

Structural colouration

Our study represents one of the first attempts to examine the impact of urbanization on structural UV-blue colouration present in blue tit wing and tail feathers (as well as in the crown, which was not analysed in this study). Two previous experimental studies demonstrated that long-term exposure of structurally coloured feathers to urban air pollution decreases their short-wavelength reflectance due to the absorption properties of airborne particles^42,43. The design of these experiments was intended to eliminate the impact of other factors, including active feather preening. In our study, the most pronounced effect was an increase in brightness of blue tit tail feathers in more urbanized sites (Fig. 3B). Given the direction of this effect, it is unlikely to be caused by the deposition of pollutants on the feather surface, suggesting that birds are capable of efficiently removing urban dust through preening. Moreover, observed differences in brightness may additionally arise from the impact of urban-related factors on feather wear. Specifically, reduced foraging effort in urban birds compared to their rural counterparts may lower mechanical abrasion on feathers, especially tail feathers, allowing structural brightness to be better preserved. Alternatively, feather brightness may vary in response to nutrition during moult, which in turn determines the quality of the emerging feathers. Specifically, the increase in blue tit tail structural colouration brightness in urban birds may be partly explained by the higher availability of anthropogenic food sources in urban habitats. For instance, a previous study using experimental supplementation with mealworms showed that male nestlings of eastern bluebirds (Siala sialis) raised by supplemented parents developed blue primaries with higher brightness⁵⁵. However, Florida Scrub-Jay (Aphelocoma coerulescens) from suburban sites with high availability of anthropogenic foods developed feathers with higher UV chroma, but not brightness, than their wildland counterparts⁴¹. Curiously, in our study, higher brightness in more urbanized habitats was observed in tail feathers but not in secondaries. This pattern likely reflects differences in feather structure, function, and investment⁵⁶. Secondaries are structurally adjusted to the requirements of flight in dense environments and thus more prone to flight-related wear, resulting in consistent brightness across habitats. Tail feathers, by contrast, are subject to lower routine mechanical stress, making their brightness more responsive to variation in moult conditions, diet quality, or urban environmental factors. Additionally, tail feathers were proposed to serve a signalling function⁵⁷, which may allow their appearance to reflect environmental conditions more strongly than wings, which are primarily functional. Future studies could investigate this further by experimentally manipulating diet or quantifying feather wear across urban and rural habitats to test whether differences in tail feather brightness are driven by resource availability or habitat-specific stressors. We also report on an age-specific association between tail UV chroma and tree cover. The disparities between age classes were more pronounced in habitats with higher tree cover, with older individuals expressing higher UV chroma, while in more urbanised habitats this difference was reduced by higher UV chroma of juveniles (Figure S5). A similar pattern was observed for ISA, indicating that ornament elaboration of older individuals becomes less pronounced in urbanized habitats, as juveniles display relatively higher colouration. This may result from easier access to anthropogenic food sources in more urbanized habitats, which eliminates the advantage that older, more experienced individuals, have in competing for food in natural habitats. This could potentially lead to a mismatch between signalled and actual bird quality, possibly resulting in maladaptive mate choices⁴¹.

Pinpointing the proximal causes of variation in blue feathers is inherently complex, as their colouration depends on multiple components. At the microstructural level, brightness is influenced by the melanin/keratin ratio, barb cortex thickness and spatial frequency of the spongy layer^58,59, while at the macrostructure level, it may be linked to the number of barbs or the length of the blue section⁶⁰. Urban-based longitudinal studies that simultaneously examine feather micro- and macrostructure are necessary to confirm the observed effects and clarify their underlying causes. A major limitation of our study is the choice of target feathers: in blue tits, crown feathers—characterized by exceptionally high UV reflectance—represent the optimal trait for assessing urban impacts, and have been extensively studied in sexual selection, quantitative genetics and microstructure research [e.g.^16,58]. However, we were unable to perform robust statistical analysis on crown feather due to limitations in sample size. We therefore recommend that future studies on urban bird colouration prioritize crown feathers to complement the readily available large body of evidence on crown feather colour variation with an urban context.

Melanin-based colouration

In contrast to observed effects of urbanization on carotenoid-based colouration, we found no support for an association between colouration of melanin-based traits and the degree of urbanization. In the great tit, breast tie area is considered to signal social dominance⁶¹, individual quality⁶², and personality traits such as aggressiveness, boldness, and fast exploration⁶⁶. Therefore, differences in tie area associated with the degree of habitat urbanization could suggest directional selection on personality traits correlated with this trait, potentially leading to local adaptation. In line with this expectation, breast tie width was shown to be lower in the city of Barcelona compared to the birds of the forest population⁴⁷. In contrast, there was no effect of urbanization on breast tie measured in 13 cities in the Czech Republic⁶³. Similarly, Grunst et al. 2020 found no effect of metal pollution and proximity to roads on great tit breast tie area²³ (but see⁶⁴ for contrasting results). Other studies have suggested that oxidative stress could influence melanogenesis underlying the tie area²². Although urbanization has been linked to increased oxidative stress⁵⁴ our study found no evidence of an association between urbanization and melanogenesis. Based on gathered evidence presented here and in⁶³ great tit melanin-based traits are unlikely to be affected by urban-related factors.

Conclusions

Our study demonstrates that urbanization influences feather colouration in a species- and trait-specific manner. In great tits, reduced carotenoid chroma likely reflects plastic responses to limited dietary carotenoid availability and physiological stress, consistent with the classic ‘urban dullness’ phenomenon. Importantly, we report for the first time an increase in phenotypic variance in a colour trait in the urban environment, which is in line with increased trait variance in the city reported for morphological traits⁴⁶. In blue tits, urban-related changes in brightness, but not in carotenoid chroma as in the great tit, may result from differences in feather wear, pollutant deposition, or feather quality, highlighting that distinct mechanisms can underlie colour trait variation across traits and species. These findings underscore that even closely related species with overlapping ecological niches can differ in their sensitivity to anthropogenic stressors, and caution is warranted when extrapolating urbanization effects on colour traits across taxa. Given the widespread role of carotenoid-based ornaments in sexual selection, further studies on feather colour traits in a broader range of species are needed to fully understand the ecological and evolutionary consequences of urban environments on colour trait variation.

Methods

Study species

Great tits and blue tits are closely related passerine species, abundant in forests and urban habitats. Although they occupy a similar ecological niche, there are some differences in their foraging strategy, reproductive ecology, or sensitivity to urban conditions^2,30,65. Both species express conspicuous plumage patterns with carotenoid-based colouration on the breast and belly. The great tit exhibits melanin-based colouration on the wing flight feathers and coverts, tail, cap and breast tie, with additional structural reflectance on the cap and breast. The melanin-based colouration of the great tit’s breast is referred to as the breast tie, a stripe extending from the throat to the vent along the breast midline; this feature is generally wider in males than in females and is known to be a signal of social dominance^61,66. In contrast, the blue tit displays non-iridescent structural UV-blue colouration on the crown, wing flight feathers and coverts and tail. Blue tits only have a narrow breast stripe, which is not known to play any signalling function, and was not analysed in this study.

Study sites

Great tits and blue tits were caught in 2018, in 44 study sites across urban-forest gradients in 8 Polish cities: Białystok, Katowice, Łódź, Toruń, Lublin, Poznań, Warszawa and Wrocław (Fig. 1A). For each city, sampling sites were chosen to represent each of the following urban habitats: city centres, residential areas, urban parks and suburban forests. Sampling locations for each type of habitat were triplicated in Warsaw—the capital city of Poland and largest city in the dataset. Additionally, in 4 cities, we established four sampling sites that were categorized as river corridors, with sampling sites in Warszawa and Toruń (Vistula river), Poznań (Warta river) and Wrocław (Odra river).

We used two metrics characterizing the magnitude of urbanization for each sampling site: the percentage of impervious surfaces (ISA—impervious surface area) and tree cover within a radius of 100 m from the sampling site⁴⁵. Both metrics were quantified from satellite images from the Copernicus Land Monitoring Services (Imperviousness Density 2015 and Tree Cover Density 2015, with the spatial resolution of 20 m per pixel) using the function Zonal Statistics in QGIS⁴⁵. The averaged urbanization metrics for each habitat type are presented in Table 1.

Table 1.

Summary statistics for the number of (i) study sites across 8 cities, (ii) sampled individuals and (iii) urbanization metrics for each habitat type (mean ± SE). The order of habitat types follows decreasing tree cover.

Habitat type	Sites	Blue tits	Great tits	ISA (%)	Tree cover (%)
Forest	10	17	33	0.09 (± 0.03)	72.77 (± 1.89)
River corridor	4	13	13	6.82 (± 2.0)	53.37 (± 4.29)
Urban park	10	38	72	2.37 (± 0.44)	50.98 (± 2.22)
Residential area	10	25	46	27.98 (± 1.14)	16.88 (± 1.87)
City centre	10	15	37	52.09 (± 1.57)	15.45 (± 1.76)

Bird sampling

The sampling took place between March 6th and April 11th, 2018—a very narrow temporal window to maximise field data standardisation. This sampling period is optimal as it does not coincide with the moulting period of both species (spanning from mid-May to the beginning of October⁶⁷) and the breeding season—a period characterised by increased feather abrasion⁶⁸. Birds were caught using mist-nets and blue tit and great tit call playbacks through loudspeakers. Each bird was ringed with individually numbered rings (Polish Ringing Centre, Ornithological Station, MiIZ PAS, Gdańsk, Poland). Birds were sexed according to the presence of the brood patch and plumage features (the latter in great tits only) and aged (1^st year breeders or older) based on the moult limit between primary and greater coverts⁶⁷. Mass was measured with a digital scale to the nearest 0.1 g. The length of the right tarsus was measured three times with a calliper, and the mean value was used for further analysis. Residuals from a regression of body mass on tarsus length were calculated across the entire dataset (separately for great tit and blue tit) and used as an index of current body condition (hereafter: condition)⁶⁹. Yellow breast feathers (approximately eight feathers), the second left tail feather, and the innermost secondary feather of the left wing were collected. Finally, great tit males were photographed with a digital camera (Nikon D3200 with AF-S Nikkor 18–55 mm lens) for tie area measurement⁷⁰.

In total, we collected feather samples from 309 birds, 201 great tits (200 and 198 of wing and tail feather samples, respectively) and 108 blue tits (Table 1). Birds were caught at the early stage of the breeding season, which could have contributed to the significant male bias in sample size (females constituted 36,1% and 36,14% of the caught individuals, respectively, in great tits and blue tits).

Ethics declaration

Bird sampling was conducted based on the approval of the Local Ethical Committee nr I for Animal Experimentation in Warsaw (permission no. 220/2016). All procedures were performed in compliance with ARRIVE guidelines (PLoS Bio 8(6), e1000412, 2010) and in accordance with the applicable rules and regulations.

Feather colouration analysis

Feather samples (Fig. 1B) were preserved in parchment envelopes and protected from light. Shortly before measurements, breast feathers were stuck on black paper in tiled stacks using double-adhesive transparent tape (approximately 8 feathers in a pile). Such samples were labelled with the bird’s ring number only, so that the measurements were blind to bird origin or sex. Wing and tail feathers were measured against black background paper, with measurements carried out on a fragment of the inner vane, at a height of 5—15 mm from the top of the feather. Reflectance was measured with the spectrophotometer Maya 2000 Pro with a PX-2 pulsed xenon light source (Ocean Optics, USA) and a fiber-optic cable 6 × 400 µm with a custom-made 2-mm collar. Measurements were repeated five times for tail and wing feathers, and ten times for breast feathers, with a white standard reference scan (Labsphere) taken every 10 min.

The spectra were processed, smoothed, and averaged within each sample in the package pavo2.0 (v. 2.9.0⁷¹ ). For breast feathers of both species, we calculated mean brightness and carotenoid chroma ([R₇₀₀ − R₄₅₀]/R₇₀₀). For structurally coloured, wing and tail feathers of the blue tit, we calculated mean brightness and UV chroma (R_300−400/R_300−700). Only mean brightness was calculated from melanic great tit wing and tail feathers (Fig. 1B). Within-sample repeatability of measurements was calculated in the package R-rptR⁷³ (v. 0.9.22 ) and is presented in Table S1, which varied between 0.21 and 0.76 depending on the colour metric. Reasons for low repeatability values of wing and tail feather brightness stem from the fact that there is (i) uneven brightness in those feathers caused by the presence of the growth bars and (ii) that the probes were moved between measurements. Breast carotenoid chroma was positively correlated with mean brightness in both species (r = 0.22, p = 0.001 and r = 0.24, p = 0.01, respectively in great tit and blue tit; Figure S1). In the blue tit, there was a non-significant tendency for negative correlation between tail brightness and UV chroma (r = − 0.17, p = 0.08), while colour metrics describing wing feathers were not significantly correlated (r = 0.09, p = 0.34).

The extent of black breast tie area was analysed with Gimp 2 version 2.8.14 (The GIMP Development Team, 2024) using photographs of 139 male great tits. Photographs were standardized using a black and a white reference on the picture. The tie area was measured using a slightly modified protocol from⁷⁰ . It was calculated as the black surface comprising a 4-cm-long rectangle starting at the lowest point of yellow on the chest and as broad as the width of the belly.

Statistical analysis

All statistical analysis and graphs were generated in the R environment v. 4.4.1⁷² .

We examined species-specific differences in carotenoid-based breast plumage colouration (i.e., mean brightness and carotenoid chroma) using linear models with colour metrics as a response and species, sex, and age (first-year breeders or older) as categorical predictors. Both carotenoid chroma (F_1;301 = 50.06, p < 0.001) and brightness (F_1;301 = 210.31, p < 0.001) was significantly higher in the great tit than in the blue tit (Figure S2). Therefore, we decided to analyse the impact of urbanization on plumage colour traits separately for great tits and blue tits.

To analyse great tit and blue tit colour traits expression in relation to different urban habitat types, we tested a set of linear mixed effects models, fitted using the lmer function in the lmerTest package (v. 3.1–384). The models included Z-score scaled colour metrics as response variables and habitat type (forest, river corridor, urban park, residential area and city centre), sex, age (birds in their second year or older), and condition as explanatory variables. The city (location) was introduced as a random intercept to account for the variability resulting from local urban characteristics. To account for potential differences in the response to urbanization between sexes and age classes, we initially included interactions between habitat type and sex, habitat type and age, and the three-way interaction between habitat type, sex, and age in each model. Non-significant interactions were sequentially removed.

The models’ assumptions were tested using the package DHARMa (v. 0.4.7⁷⁴ ). We observed significant quantile deviations in several models; appropriate transformations were consequently applied (great tit breast carotenoid chroma was squared, great tit tie area and blue tit breast carotenoid chroma were log-transformed). Conditional and marginal R² were calculated using the package performance (v. 0.12.4⁷⁵). In models with significant differences between habitat types, post-hoc least square mean pairwise comparisons were performed using lsmeans package (v. 2.30-0⁷⁶ ). Moreover, to examine differences in phenotypic variation of colour traits between different habitat types, we performed the asymptotic test for equality of coefficients of variation using the package cvequality ⁷⁸.

A similar set of models was constructed to examine the relationship between colour traits and quantitative proxies of urbanization (i.e., tree cover and ISA). Given that tree cover and ISA were highly, negatively correlated (r = -0.70, df = 332, p < 0.001), their impact was analysed in separate models. The models were analogous as described above but included tree cover / ISA, instead of habitat type, in interaction with sex, and age. We detected quantile deviations in several models; thus, appropriate transformations were applied in the response variables (see Table 1 and S3). Given the similarity of model outcomes stemming from high negative correlation between the extent of tree cover and amount of impervious surface (ISA) across the sampling sites (Figure S3), the results of models examining the impact of ISA are presented in SI (Table S4).

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Conceptualization, M. S., A.D.S., M.Ch., K.J.; Data curation, K.J., M.S.; Formal analysis, K.J.; Funding acquisition, M.S.; Investigation, K.J., J.S., Ł.W., M.Co., M.Ch.; Methodology, K.J., M.Co., M.S.; Project administration, M.S.; Resources, M.S.; Software, K.J.; Supervision, M.S.; Validation, M.S.; Visualization, K.J.; Writing—original draft, K.J.; Writing—review & editing, M.Ch., M.Co., A.D.S., Ł.W., J.S., M.S.

Funding

The study was funded with a Sonata Bis Grant (2014/14/E/NZ8/00386) from the Polish National Science Centre awarded to Marta Szulkin.

Data availability

Full set of raw data is available on the Figshare platform: 10.6084/m9.figshare.27925305.

Competing interests

The author reports no conflicts of interest.

Declaration of AI use

We have not used AI-assisted technologies in creating this article.

Preprint statement

This paper is available publicly as a preprint via a bioRxiv preprint server⁸⁵ .