We compared different types of phenotypic traits in urban populations of invertebrate and vertebrate species across separate cities. Our main result is that the phenotype of urban animals differs across cities, regardless of the type of phenotypic trait investigated, and this was the case when we considered all taxa together, and when we considered separately birds or invertebrates. We also found that phenotypic differences across cities are more pronounced as the more cities are investigated and the farther away cities are from each other (except for our analyses on reptiles).
Although there have been many recent studies investigating phenotypic changes across cities, it must be noted than in the majority of those studies the focus was in rural-urban comparisons, with the different cities simply providing replicates for those rural-urban comparisons (Evans et al. 2009b; Potvin and Parris 2012; Slabbekoorn and den Boer-Visser 2006; Tyler et al. 2016). Indeed, in some of these studies any potential phenotypic differences across urban populations are not even reported nor discussed (Eggenberger et al. 2019). In a review considering parallel evolution in cities (i.e., whether rural-urban comparisons in different cities show consistent and similar responses driven by urbanization), parallelism was exhibited in only 44% of species across all the cities studied (Santangelo et al. 2020). Even in cases when parallelism across urban-rural comparisons exist, there may be significant differences in phenotypic traits across urban populations, as the changes taking place may be higher in some urban populations than in others. But if episodes of non-parallelism are predominant, in which phenotypic traits increase in some urban populations compared to rural population, but decrease in some others, then substantial differences across urban populations should be expected, and this is confirmed by our results. The emergence and increase of phenotypical differences across urban populations is further exacerbated by the fact that rates of phenotypic change are much higher in urban areas than in natural contexts (Alberti et al. 2017; Hendry et al. 2008).
Phenotypic differences across urban populations may be due to many reasons: adaptation, which has rarely been confirmed to take place in relation to urbanization (Lambert et al. 2021); phenotypic plasticity (Bressler et al. 2020; Thompson et al. 2018); founder effects, i.e., stochastic differentiation following separate colonizations by different subpopulations in different cities (Evans et al. 2009b); decreased gene flow (Johnson and Munshi-South 2017); genetic drift, a nonadaptive, genome-wide process that could lead to random phenotypic differentiation across urban populations (Mueller et al. 2020); and hybridization between native and non-native species, which may potentially increase the distinctiveness of phenotypic traits across cities (Beninde et al. 2018). In the majority of studies in our dataset, the processes involved in any phenotypic differences across urban populations were not investigated, and thus we were not able to determine their relative roles either. We also did not include studies investigating only genetic differences in separate cities, as we could not calculate effect sizes as we did for the phenotypic traits. However, many recent genomic studies have addressed the existence of genetic differentiation across cities. For example, a study on rat populations across four cities, including temperate, subtropical and tropical cities, showed similar genetic diversity across cities but different patterns of gene flow depending on city-specific barriers separating subpopulations within each city (Combs et al. 2018); and a study on bumblebees in nine German cities found in some loci a high degree of genetic differentiation associated to urbanization (Theodorou et al. 2018).
In our models, the most consistent moderator explaining phenotypic differences across cities was the number of cities investigated—as more cities were included in a study, the larger the difference between the smallest and largest mean urban phenotype reported in that study (i.e., a higher standardized mean difference). This was the case for the models containing all taxa, and for models with only birds and only invertebrates, but it was not the case for the models with only reptiles, although this may have been due to the fact that the variation in the number of cities was relatively small in our considered reptile studies (range = 2–5 cities; average = 3.2 cities). However, overall, the more cities for which data from a phenotypic trait were available, the greater the difference was in that phenotypic trait across urban populations. This result supports the idea that separate urban populations of a same species may diverge phenotypically instead of changing in a parallel fashion. Our result also highlights the importance of studying urban populations in many cities, as some biological patterns may only become apparent when doing so. For example, only by studying bird and plant biodiversity across many cities could researchers determine that the density of species was more affected by urban characteristics (e.g., landcover and city age) than by non-anthropogenic factors such as climate and geography (Aronson et al. 2014).
We also found that a greater geographical distance between cities is likely to lead to greater phenotypic differentiation across urban populations. This positive association was the case for the models containing all taxa, and for the models with only birds, but not for the models with only invertebrates (no association) or only reptiles (negative association). Such a difference in the case of invertebrates and reptiles may be due to the fact that geographical distances between studied cities tended to be smaller for invertebrates (range = 22.12–645.79 km; average = 124.13 km) and reptiles (range = 17.4–1661.66 km; average = 162.77 km) than for birds (range = 12.31–9489.13 km; average = 844.68 km). It is also possible that for many invertebrate species distances between cities are magnified compared to birds and reptiles, and that thus there is a smaller distance threshold beyond which any further distance between cities has a superfluous effect. As for reptiles, we found that the difference between phenotypes was greater as distance between cities decreased (for each one-unit increase in distance between cities, the standardized mean difference in phenotypes decreased by 0.0008). However, this result should be taken with caution, as 93% of effect sizes were associated to small distances between cities (average = 78.61 km), whereas the remaining 7% of effect sizes (amounting to only 3 effect sizes) were associated to much larger distances (average = 1284.87 km).
A greater geographical separation between cities does not only minimize the occurrence of genetic flow but it can also maximize abiotic differences between those two cities, e.g., related to latitude and climate conditions. Additionally, small distances between cities will promote a leapfrog process of urban colonization, in which new urban populations are not established by colonizers from adjacent rural populations but by colonizers from urban populations in nearby cities (Evans et al. 2009a; Evans et al. 2010). Cities that are close together in which urban populations were established via a leapfrog process should be more phenotypically similar compared to separate urban populations that were independently established from their respective adjacent rural populations. However, even in species in which the leapfrog process of colonization is at play, separate urban populations will have traversed separate evolutionary paths since their establishments in the different cities (assuming there is no gene flow between them), and phenotypic differences may have still arisen across cities, in this case being greatly determined by the age of those cities and thus the age of the different urban populations.
Differences in the human population densities (a proxy of city size) between the compared cities did not have an effect on the degree of across-city phenotypic differences in the models considering all data, only invertebrates, or only reptiles. However, we found a surprising effect in the case of birds, with the difference in phenotypes between cities being smaller as the difference in population densities increased, although this effect was relatively small (estimate = -0.001). In principle, phenotypic differentiation is likely to be higher in larger cities than in smaller cities. For example, gene flow between rural and urban populations may be more important in smaller cities as the distance between rural and urban populations is reduced (Santangelo et al. 2020). Larger cities will also provide more opportunities for population structuring, with more subpopulations within a city possibly diverging phenotypically from one another (Johnson and Munshi-South 2017). However, it is not clear that city size by itself is a main driver of phenotypic differentiation across cities. For example, the production of hydrogen cyanide in white clover was studied in many cities of different sizes across the same region, and although the trait was consistently less prevalent in urban than in rural sites, the size of the investigated cities did not affect the results (Johnson et al. 2018).
We predicted that morphological traits would be more similar across cities compared to physiological traits, and especially compared to behavioral traits. However, our study does not support this prediction. The overall meta-analyses including moderators did not show significant differences between the types of traits. And the same was the case for the subgroup analyses, with the exception of reptiles. We did find more differentiation in behaviors in reptiles than in morphological traits (there were no physiological traits in the dataset), but behaviors were represented by only two studies on a single species.
Phenotypic differences observed between pairs of cities were similar in cases in which cities were selected by researchers due to some intrinsic difference between those cities (e.g., latitude or city size), and in cases in which the researchers did not mention any a priori differences between the cities. The fact that phenotypic differences between separate urban populations exist even when comparing cities that are not clearly different from one another emphasizes the importance of measuring traits across several cities. When cities are selected so that they differ in some ecological feature (e.g., in relation to latitude, or biome), researchers can concurrently study the effects of urbanization and other ecological factors. This can allow to tackle questions like the effects of urbanization in different ecoregions (e.g., temperate, desert, and tropical cities), or how the combined effects of urbanization and climate change may affect populations differently in separate cities. At the other extreme, if the selected cities are very close together and very similar in many aspects, one minimizes the likelihood of observing major phenotypic differentiation between any two urban populations (Sparkman et al. 2018), which may provide an interesting system to perform experimental approaches that require starting with similar phenotypes.
Our results clearly indicate that separate urban populations of the same species can diverge phenotypically, and that this is the case for any phenotypic trait, no matter if they are morphological, physiological or behavioral traits. In principle, there seem to be two opposing views on whether the responses of animals to urbanization should be consistently similar or dissimilar across cities. First, if several cities under investigation are considered to be similar replicates of the same type of environment, we would predict to find more episodes of convergence than of divergence regarding phenotypic traits, especially when phenotypic differentiation is mostly driven by phenotypic plasticity. Second, if different cities are ecologically distinct (Santangelo et al. 2020), we would expect to find phenotypic differences across them (Ouyang et al. 2018; Thompson et al. 2016), as we did in our meta-analysis. This is likely to be the case the more cities are investigated and the farther apart cities are, which is also mostly supported by our results. As already mentioned, the fact that evolution rates are higher in urban areas than in any other type of environment (Alberti et al. 2017) means that even small differences among cities can lead to measurable phenotypic differentiation across them. Cities can also be highly stochastic, regularly disturbed, and thus variable over time (Sattler et al. 2010). That is, replication may not only be important at the spatial scale (different cities), but also at the temporal scale (populations being studied over time).
In conclusion, most studies on urban ecology have been restricted to one urban center, with researchers tending to conduct studies only in the city in which they live. However, our results support previous pleas from many researchers to conduct urban studies across several urban populations. Those different urban populations would not necessarily act as replicates, as our analysis shows that phenotypic differentiation increases as the more cities are investigated. One approach to implement multi-city studies is by establishing a long-term network of research partners located across many cities (Magle et al. 2019). We also recommend that future studies assess comprehensive sets of traits, as the degree of phenotypic differentiation across cities may vary in different traits (Santangelo et al. 2020). Using a comparative framework would also be important, because different species may have undergone different processes of adaptation to urban environments, given their different ecological requirements. Finally, we recommend that future studies choose cities in different biomes, as urban adaptations may differ substantially in cities sited in different ecological matrices, e.g. cities in desert or tropical regions. Ultimately, a generalized knowledge about how organisms are affected by urbanization will only be possible when comprehensive biological patterns are similarly studied across separate and distinct cities.