Monitoring of broods started before nest construction and ended after the last nestling had fledged. Weekly nest-box field visits to each brood resulted in the observation of the exact date when the first egg was laid (lay date, March 1st = 1), the number of eggs laid (clutch size), the number of hatchlings, and the number of fledglings (nests were visited at 15 days, and then 21 days after hatching to check fledging). In order to compare with previous studies (e.g., Vaugoyeau et al., 2016), only data collected on first broods were considered in the analyses. Parents were captured and measured when their nestlings were 9–15 days old and uniquely identified with metal rings provided by the CRBPO (Centre de Recherches sur la Biologie des Populations d'Oiseaux, Museum National d'Histoire Naturelle, Paris). Nestlings were also uniquely ringed and measured at 14–15 days old.

### Urbanization Measures

As previously described in Demeyrier et al. (2016), four features of the urban matrix were measured within a 50 m radius around each nest-box: vegetation cover, car traffic, pedestrian count, and intensity of artificial light. Demeyrier et al. (2016) provide all the details on how these measures were performed, but also how they were analyzed in a principal component analysis (PCA, see Figure 4 in Demeyrier et al., 2016). From this PCA, we extracted the two major axes: PC1 reflecting habitat naturalness and hereafter abbreviated "naturalness" (positively related to vegetation cover and negatively correlated with car traffic and artificial lights, explaining 56.6% of the variance), and PC2 reflecting the pedestrian frequency, hereafter abbreviated "pedestrian" (explaining 16.7% of the variance in the urban environmental features).

#### Behavioral Assays

The behavioral trials were performed in both La Rouvière forest and Montpellier in 2014, 2015, and 2016. Great tit parents were caught in their breeding nest-boxes while feeding their 9–15 days old nestlings. A captured bird was kept for around 20 min in total, during which time it was submitted to the following sequence of events: capture in the nest-box, handling aggression scoring, resting phase in cloth bag for 5 min, measure of breath rate during handling, resting phase of 2 min in small acclimatization compartment adjacent but separated from the main open-field cage (**Figure 1**), exploratory scoring in open-field trial during 4 min, ringing (if not already ringed), morphological measurements (body mass, tarsus, tail and wing lengths), and blood sampling.

First, immediately after its capture, a bird was tested for handling aggression by the capturer. The handler held the bird high up in the legs with one hand and nagged the bird with one finger from his/her other hand following a standardized procedure where the finger approaches the bird but does not touch it. After ca. 15 s of test and observation, the handler attributed a handling aggression score (HA, following the standardized criteria list in Table S4 of Dubuc-Messier et al., 2016, see also Brommer and Kluen, 2012; Class et al., 2014), ranging from 0 (completely unreactive bird) to 3 (bird aiming for the handler's finger all the time, spreading its wings and tail), with 0.5 increments. Following this first behavioral assay, the bird was placed in a bag for 5 min and brought to the open-field apparatus which was less than 5 min walking distance from the nest-box. Second, right after it was removed from the cloth bag, the bird was held still on its back by the handler who counted twice in a row the number of breaths during 30 s (breaths were visually expressed via fast movements of the chest). Handling breath rate (HBR) was the number of breaths per minute, averaged over the two measures (Carere and van Oers, 2004; Fucikova et al., 2009; Brommer and Kluen, 2012; Kluen et al., 2014). HBR has been shown in other tit populations to be

positively related to stress response (Carere and van Oers, 2004). Third, a "novel environment test" was performed in a unique experimental open-field cage built following the exact set up and dimensions described in Stuber et al. (2013) (**Figure 1**). This kind of set-up has been classically used to measure exploration behavior in great tits (e.g., Stuber et al., 2013) and blue tits (e.g., Mutzel et al., 2013; Dubuc-Messier et al., 2016), and provides exploratory scores that are repeatable and well correlated to those performed in the laboratory (e.g., in Dutch and German great tits, Dingemanse et al., 2002; Stuber et al., 2013). Right after the HBR measure, the bird was placed in a double door small compartment on the side of the open-field cage for acclimatization during 2 min with no visual cue other than the small cage of dimensions 11 × 12 × 11 cm. Following this resting time, the bird was released through a sliding door into the exploration cage of 61 × 39 × 40 cm dimensions, where it was filmed during 4 min by a camcorder (CH4 Sony Handycam) fixed 110 cm away from the wired side of the open-field cage. The bird was free to move around the cage and/or to settle on three perches. The light was standardized since the open-field cage was placed in a wider cage of dimension 61 × 150 × 40 cm, lighted by two led lights. Following previous studies on great tits, the total number of flights and hops during the open-field trial was used as a proxy of exploratory behavior (e.g. Dingemanse et al., 2002). This number was square root transformed (to minimize deviation from normality and following Nicolaus et al., 2016) and hereafter called exploration score. VD analyzed all videos using the software JWatcher (Blumstein et al., 2006) and scored this behavior.

#### Data Analysis

All statistical analyses were run with the software R (version 3.3.2, R Core Team, 2016). We used several datasets to restrict the limitations of missing data in the various variables. In a first step of the analyses, we compared breeding, personality and physiological traits between the forest and the urban habitats using two-sided t-tests on the entire dataset available (considering the data from both known and unknown parents), hence not restricting to observations where the identity of one parent was known. For this and all other analyses, we removed estimations of number of hatchlings and number of fledglings for all broods that had been (experimentally) manipulated after the egg-laying stage, which explains smaller sample sizes for these two variables compared to lay date and clutch size.

The first step in the statistical analysis compared population means using a dataset where many female identities were unknown (since parents were not captured during the first 2 years of monitoring) providing an idea of results that can be obtained without comprehensive monitoring and individual ringing.

In a second step, we more formally tested for differences between forest and urban birds with univariate linear mixed models (LMM, package lme4, Bates et al., 2015) which included habitat type (forest versus city) as a fixed effect and female identity as a random effect. All LMMs initially included as fixed effects habitat (forest versus urban), year, a habitat × year interaction and other trait-specific fixed effects (see below). The fixed effect model was selected in a backward stepwise procedure starting with a model including all variables. The significance of each fixed effect was tested using likelihood-ratio tests (Bates et al., 2015). While all behavioral and physiological traits showed no significant correlations (**Tables 1C,D**), the three reproductive performance traits were highly correlated (**Tables 1A,B** and Table S1). Hence clutch size was included as a fixed effect in brood size models (hereafter called hatching success), and brood size was included as a fixed effect in number of fledglings models TABLE 1 | Phenotypic correlations (above the diagonal) among (A,B) breeding traits (in forest and city) and (C,D) behavior and physiological traits (in forest and city), and their associated P-values (below the diagonal).

Lay date is calendar date of first egg laid; clutch size is the number of eggs laid; nb hatchlings is the number of nestlings hatched; nb fledglings is the number of nestlings that survived until fledging; HA is handling aggression; exploration is the exploration score from open-field trials and HBR is handling breath rate. Pearson's correlation coefficients are provided above the diagonal and associated P-values below the diagonal. In bold are highlighted significant coefficients (P < 0.05). For values of the t statistics and degrees of freedom, see Table S1.

(hereafter called fledgling success). For handling aggression and breath rate, the initial full model included fixed effects for sex (and sex × habitat interaction), age, individual life-long capture rank (from 1 to 3, for first to third capture of the same individual) and ambient temperature, and observer/manipulator identity as a random effect. For exploration score, we included sex (and sex × habitat interaction), age, test rank (from 1 to 3), and temperature as fixed effects. As our factor of interest, habitat was kept in all models, even if not significant, to provide the effect size and statistics associated with its fit in the model. Since these LMMs included a female random effect, the dataset used in this second step included breeding parameters that had been recorded for broods where the female was captured only, hence the number of observations was reduced compared to the first dataset.

Based on the best models retained in this step 2, we estimated an adjusted repeatability with the package rptR (Nakagawa and Schielzeth, 2010) for all focal traits. We estimated overall repeatabilities across the whole dataset but also sex-specific and habitat-specific repeatabilities.

In a third and final step, we used only data collected in the city of Montpellier to contrast the measured traits against the two urbanization gradients revealed by the PCA of environmental features in the city: habitat naturalness, abbreviated "naturalness" (PC1) and pedestrian frequency, abbreviated "pedestrian" (PC2). Model selection was similar to that in step 2 (with similar fixed and random effects structure), apart from the use of naturalness and pedestrian instead of the habitat variable which previously was used to compare forest and city. As previously, naturalness was kept in all models since it was our main variable of interest.

Sample sizes are indicated in **Table 2** (for step 1), **Table 3** (for step 2), and **Table 4** (for step 3). While models for phenology and reproductive success were female-specific, models on behavior and physiology pooled data on both sexes, with balanced sampling across sexes since 47–48% of observations were on males (detailed sex-specific sample sizes are in Table S3).

#### RESULTS

Comparison of mean traits between forest and city using the largest dataset (2011–2016) without controlling for the identity of individuals showed strong differences in mean lay dates, clutch sizes, number of hatchlings, and exploration score, but no significant differences in number of fledglings, handling aggression, and breath rate during constraint (**Table 2**, Figure S2).

An interesting result from this investigation on all monitoring data collected (after removing experimental broods) was a lower percentage of complete brood failure (i.e., no fledgling) in the city: 31% of complete brood failure in the forest versus 21% in the city (t = −2.167, df = 253.82, P = 0.03). Unfortunately, this data could not be analyzed using Generalized Linear Mixed Models with individual identity as random effect since most failed broods did not allow parent capture and identification. In the latter dataset used in the LMM for breeding traits, brood failure was 32% in the forest vs. 29% in the city. Unfortunately, the large majority of these failures were of unknown cause, but we can hypothesize predation on a parent, catastrophic climatic event or abandonment by parents because of high levels of stress and parental care. Future research should investigate whether some of these drivers of brood failure differ between the forest and city environments.

Hence at first glance when comparing mean traits without controlling for fixed effects that may be important or the nonindependence of data collected on the same individuals, city birds laid earlier, and smaller first clutches, with decreased hatching success yet decreased occurrence of complete brood failure, and they did not fledge a lower number of offspring (see statistical details in **Table 1**). They also presented significantly higher exploration activity as well as slightly higher handling aggression and breath rate, although not significantly so.

When accounting in step 2 for the non-independence of data collected on the same individuals, restricting to records from known individuals (2013–2016, sample sizes in **Table 3**), and controlling for fixed effects, LMMs confirmed strong differences between forest and city great tits in their lay date, clutch size, hatching success (number of hatchlings when controlling for clutch size) (**Table 3** and **Figure 2**), and interestingly still no significant difference between forest and city birds in their fledging success (number of fledglings controlling for number of hatchlings, **Table 3** and **Figure 2**). Note that this non-significant result remained when modeling number of fledglings without controlling for the strong positive correlation with number of hatchlings, that is when comparing absolute fledgling number (Table S2). As in step 1, step 2 confirmed that city birds laid earlier and smaller first clutches, with decreased hatching success (**Table 3**). Urban great tits also displayed faster exploration scores and faster breath rates, but did not differ from their forest conspecifics in handling aggression (see statistical details in **Table 3** and **Figure 2**).

Both estimations of reproductive success were not repeatable among females (adjusted repeatabilities of 0.31 (SE = 0.14) for hatching success and 0.16 (SE = 0.14) for fledgling success, P = 1, Nakagawa and Schielzeth, 2010). All other traits were significantly repeatable with adjusted repeatabilities of 0.43 (SE = 0.10, P = 2.3e-5) for lay date, 0.55 (SE = 0.08, P = 4.8e-3) for clutch size, 0.34 (SE = 0.09, P = 2.6e-4) for handling aggression, 0.55 (SE = 0.07, P = 5.2e-7) for exploration score and 0.35 (SE = 0.08, P = 8.6e-6) for breath rate during constraint. While sex- and habitat-specific repeatabilities were based on small sample sizes, they suggested that behavioral and physiological traits might be more repeatable in females than in males and more repeatable in forest than urban habitats (see Table S3). In contrast, laying date

TABLE 2 | Comparison of mean traits between forest and city great tits based on the full monitoring dataset (hence including broods with unknown parents).

Boxplots for these comparisons are shown in Figure S2. In bold, traits showing significant differences between forest and city (P < 0.05).

TABLE 3 | Contrasting phenology, reproductive success, behavior, and physiology between forest and city birds (Habitat) using linear mixed models with female/individual identity and manipulator identity (for handling aggression and breath rate) as random effects.

Intercept 1.97 0.15 13.08 6e-13 3.66 0.42 8.62 4e-16 133.04 4.35 30.55 <2e-16 Habitat - urban 0.07 0.12 0.60 0.55 2.78 0.49 5.65 3e-8 4.02 1.95 2.06 0.04 Sex-Female −0.43 0.10 −4.17 4e-05 ns ns ns ns ns ns ns ns

Gray areas highlight the focal effect of habitat type (forest vs. city), with significant results in bold (P < 0.05). ns: non-significant (P > 0.05). Sample sizes are provided for the number of females (Nb ♀) or individuals (Nb ind.), the number of observations (Nb obs.) and the number of manipulators (Nb manipul.).

and clutch size were repeatable in urban birds but not in forest birds (where again, sample sizes were small, Table S3).

The step 3 analysis along the gradients of naturalness and pedestrian pressure showed no influence of pedestrian frequency on any of the focal traits (**Table 4**). Three out of the seven traits studied showed variation across the naturalness gradient: in more urbanized areas great tit females laid earlier and smaller broods and birds had generally (see below) faster breath rates during handling (i.e., similar trends as found when contrasting urban and forest birds). In all three cases however, the strength (and even direction in the case of breath rate) of the urbanization effect showed significant variation across years (**Table 4**, **Figure 3**): for example, later laying in more natural habitats was true for 3 years out of four (**Figure 3A**) and breath rate showed inconsistent changes along the urban gradient across the 3 years of study (**Figure 3C**).

#### DISCUSSION

Based on 6 years of great tit monitoring in an oak forest and the nearby city of Montpellier in southern France, our study reveals strong phenotypic divergence for a suite of breeding, behavioral and physiological traits, both when comparing forest and city birds, but also for some of the traits, within the urban gradient. We discuss below how these results fit with the pace-oflife syndrome hypothesis, and what processes could be involved in these divergences.

#### Phenotypic Divergence between Forest and City and along the Urbanization Gradient

We confirm here a commonly found breeding divergence, which is that city birds breed earlier and produce smaller clutches, and even more so in more urbanized areas of the city (**Tables 3**, **4**, **Figures 2A,B** and **3A,B**). Such urban effects on the timing of breeding and clutch size have been demonstrated before in great tits (e.g., Dhondt et al., 1984; Wawrzyniak et al., 2015) and many other avian species (see review in Marzluff, 2017). Note however that in a recent comparative analysis of forest and urban passerines at a macro-geographic scale (including 138 great tit populations), Vaugoyeau et al. (2016) found that average

Gray areas highlight the focal effect of the naturalness or pedestrian gradients, with significant results in bold (P < 0.05). ns, non-significant (P > 0.05). Sample sizes are provided for the number of females (Nb ♀) or individuals (Nb ind.), the number of observations (Nb obs.) and the number of manipulators (Nb manipul.).

laying date was not correlated with the degree of urbanization, while average clutch size decreased with urbanization in collared (Ficedula albicollis) and pied flycatchers (Ficedula hypoleuca) but not in great tits. Later laying and smaller clutches in cities have been attributed to the urban heat island effect, as well as the artificial lights and the lack of adequate resources (i.e., arthropods) during the breeding season (Chamberlain et al., 2009; Kempenaers et al., 2010).

Hatching success was lower in urban broods, yet this did not lead to lower fledging success (**Table 3**, **Figure 2D**) nor to lower fledgling numbers (Table S2). Within the city, fledging success varied significantly across years but again was not significantly lower in more urbanized areas. It is possible that the slightly lower fledgling productivity in urban broods (**Table 2**, Table S2) was not significant in our 6 years study because of insufficient power. However, it remains that while the other breeding traits show strong significant differences between forest and city, it did not translate in a similarly strong divergence for fledgling success. This result contrasts with previous findings in great tits (Bailly et al., 2016) and with the common assessment that the urban environment induces strong constraints on avian reproduction (Gil and Brumm, 2014). It raises the very interesting question of whether urban birds might display adaptive breeding strategies that differ from birds in the forest, yet do not lead to lower reproductive success (see further discussion on this possibility, below). Reduced clutch size

FIGURE 2 | Predicted contrasts in breeding [A, lay date; B, clutch size; C, number of hatchlings (from a model controlling for clutch size), D, number of fledglings (controlling for number of hatchlings)], behavior (E, handling aggression; F, exploration score) and physiological traits (G, breath rate) between great tits from the forest of La Rouvière and great tits from the city of Montpellier. The graphs present the boxplot of predicted values from models detailed in Table 3. For lay date, 1 = 1st March. Breath rate is in number of breaths/min.

would hence be the result of an adaptive process rather than the expression of energetic constraints. Overall in terms of POLS, the breeding data suggested that city birds displayed one element pointing to slower life histories (smaller clutches) yet did not differ consistently from forest birds in their reproductive output.

Our results on personality traits and breath rate were more consistent with the prediction of faster behavioral syndromes in urban compared to forest habitats. Handling aggression was not significantly influenced by the urban habitat, yet city birds were faster explorers with generally faster breath rate under constraint (**Table 3**). Breath rate also varied across the urban gradient, yet with important annual variation (**Table 4**, **Figure 3C**). Previous rural vs. urban avian comparisons for these traits found: lower short-term stress response in urban European blackbirds (Partecke et al., 2006), reduced physiological stress and faster exploratory behavior in urban dark-eyed juncos Junco hyemalis (Atwell et al., 2012; Abolins-Abols et al., 2016), higher territorial aggression in urban song sparrows Melospiza melodia (Evans et al., 2010), urban Australian magpies Gymnorhina tibicen (Cilento and Jones, 1999), urban Eurasian coot Fulica atra (Minias, 2015), and urban noisy miners Manorina melanocephala (Lowry et al., 2011), and finally higher breath rates in urban great tits during handling (Torne-Noguera et al., 2014, but see Senar et al. from this issue).

Mean breath rate during handling for Montpellier great tits was similar to that found in the study by Torne-Noguera et al. (2014, i.e., around 130 breaths/min) yet it was only around 2% higher than breath rate in forest birds, compared to a 14% difference in Barcelona (Torne-Noguera et al., 2014). Our singular analysis along the urban gradient revealed strong annual variation in the breath rate response to urbanization level (**Figure 3C**), which could partly explain the discrepancies between previous studies (Partecke et al., 2006; Torne-Noguera et al., 2014; Senar et al. this special issue). While heart rate and breath rate are highly related (see e.g., Dubuc-Messier et al., 2016), their interpretation is complicated by the fact that they reflect both sympathetic and parasympathetic reactivity (e.g., Koolhaas et al., 1999), and that responses to long term or acute stresses differ (Abolins-Abols et al., 2016). In rodents more aggressive and active individuals have a strong sympathetic response yet a low parasympathetic reactivity to acute stress, which translates into increased heart rate response (Koolhaas et al., 1997). Similarly, domestic hens with active coping styles (high-feather peckers) have faster heart rates during manual restraint (Korte et al., 1999). Hence overall, in the context of POLS, higher breath rate and faster exploration scores in urban great tits are suggestive of a faster pace-of-life.

### Plastic vs. Genetic Origin and Adaptive Nature of Divergence

While we have demonstrated in this study that forest and city great tits display strong phenotypic differences for breeding parameters, behavioral traits and physiological reaction to stress, two fundamental questions remain unanswered. The first question is whether these physiological differences emanate from underlying genetic differences, rather than plasticity. This would suggest that urban birds form a genetically distinct ecotype compared to forest birds, possibly because of a process of local adaptation. The second, related question, is whether the phenotypic differences we have described here result from an adaptive process since both plasticity and genetic processes (such as drift) can result in maladaptive responses.

As regards the first issue of a plastic vs. genetic origin in the phenotypic differences, a genetic difference can occur only if the focal traits are heritable, and in the case of restricted gene flow (Lenormand, 2002). An interesting point to make here is that in our study populations, hatching and fledging success were not repeatable, and there is very little evidence for heritability of reproductive success in the literature (review in Postma, 2014). All other focal traits studied here were repeatable and they were previously shown heritable, in great tits or in closely related avian species (for lay date and clutch size see review in Postma, 2014, for aggression and exploration behavior see review in Dingemanse and Dochtermann, 2014, for breath rate see Class et al., 2014). This overview on heritability suggests that a rapid evolution of the focal traits is possible except for hatching and fledging success. Recent results in population genomics also provide useful knowledge on the neutral and adaptive genomic divergence of Montpellier great tits at both scales studied here (forest vs. city and urban gradient). Using a RAD sequencing analysis of nearly 50,000 SNPs in 140 great tits, Perrier and colleagues (Perrier et al. in review) show that the neutral genetic structure is weakly impacted by urbanization, with an average FST between La Rouvière forest and Montpellier city of 0.009 and an FST of 0.012 between the most and least urbanized sites within the city of Montpellier. While all FST were significant, their low value suggests that gene flow is common along the urban gradient and between forest and city. However, it could well be that non-random gene flow, e.g., via individuals matching their habitat choice to their phenotype (Edelaar et al., 2008; Edelaar and Bolnick, 2012) reinforces local adaptation. In fact it has been suggested that urbanization should favor individuals with high behavioral plasticity (Lowry et al., 2013). Our preliminary results on habitatspecific repeatability (Table S3) show lower repeatability in the behavior and physiology of urban compared to forest birds which could be indicative of higher plasticity, although this is not true for breeding traits. An interesting avenue would hence be to focus on plasticity as a trait per se, evaluating how/whether it is heritable and selected for in the urban landscape and across the range of traits studied here (e.g., following the approach of Nussey et al., 2005 for plasticity of timing of breeding). We also envisage in the future a common garden study to experimentally test whether birds from more or less urbanized areas yet raised in aviaries display genetically based differences in the traits studied here.

Addressing the second question to find out whether the phenotypic variation described here is adaptive or not will require more long term data (including survival and reproductive data) to compare the direction and force of selection acting on the focal traits across the habitats (Isaksson, 2015). It is commonly assumed that the novelty of the urban environmental features induces new and strong selection pressures providing the potential for rapid trait change (Markó et al., 2013; Alberti, 2015; Hendry et al., 2017). However, direct empirical measures of urban natural selection remain scarce (but see e.g., the emblematic peppered moth case, Cook and Saccheri, 2013). New genomic resources offer a powerful way to bypass the data consuming classical selection analyses by testing for genomewide signatures of selection associated with urbanization, even in non-model species (Harris et al., 2015). While at first hand, breeding parameters show decreased clutch size and hatching success in city great tits compared to their forest conspecifics (**Table 3**), a very important result from our study is that these city birds do not have lower fledgling rate or success (**Table 3** and Table S2). This stability of reproductive performance in the nest despite strong differences in breeding traits, suggests that city birds might have evolved an adapted breeding strategy in response to urbanization with smaller clutches and broods to adjust to the lack of food for nestlings in the urban habitat (Chamberlain et al., 2009). A comparison of morphological traits in our focal populations also points to possibly adaptive changes since urban great tits are smaller and lighter than forest birds yet they do not differ in body condition (Demeyrier et al. in prep.).

## Perspectives on a Multivariate Long-Term Process

Previous studies have shown enhanced annual survival in urban adult birds, in particular because of higher resource availability over winter (Rebolo-Ifran et al., 2015; Marzluff et al., 2016). In the context of the pace-of-life theory, such decreased probability of mortality would be indicative of a slower pace-of-life (Réale et al., 2010). However, new predation pressures in the city are also thought to affect avian survival, especially nestlings', and overall rural/urban survival comparisons are still scarce and show no consistent pattern (Chamberlain et al., 2009). With only 3 years of breeding captures, our study lacked power to conduct capturemark-recapture analyses, yet it is most definitely our objective to pursue the long term research and compare survival rates across the urbanization gradient in the future. In the same vein, a longer monitoring will enable a comparison of recruitment success across urbanization, providing a complementary perspective to our results on fledgling success. Survival data will also be crucial to understand selection pressures acting on urban great tits, and to compare them with selection in more natural habitats.

While the present analysis focused on first broods following classical approaches in the literature, considering second broods in the study of forest and urban reproductive strategies could reveal further divergences. Between 2011 and 2016, 11.6% of Montpellier urban females laid second broods vs. 4.5% in La Rouvière forest (along with 20.6 and 23.1% of replacement broods). In our study sites in the city of Montpellier, Pinus spp. and the evergreen oak Quercus ilex are dominant tree species. In these breeding habitats, later breeding could be favored because the food required to rear nestlings peaks later in the season (Blondel et al., 2006). Also, in such habitats, second clutches can be typically larger than first broods, while the reverse is true in deciduous habitats such as found in La Rouvière forest (Lambrechts et al., 2008). Hence a complementary analysis involving both first and second broods might confirm that city birds have adopted a different yet adaptive breeding strategy.

Finally, the colonization of an urban landscape by an avian species is likely to be a long process, possibly leading to a succession of adaptive phenotypes. In line with the general finding that individuals that are superior colonizers are inferior competitors (Yu and Wilson, 2001), it has been suggested that while a proactive fast lifestyle would be favored during the colonization stage of urban habitats (Sol et al., 2013), the higher sensitivity of reactive slow individuals to the environmental conditions might favor them on the long run when facing ecological novelty (Robertson et al., 2013). In the case of such a sequence of colonization, findings regarding the pace-of-life of birds adapted to the urban habitat will depend a. on how advanced the colonization process is, and b. on how much the urban population is isolated from surrounding rural populations. Although Montpellier is a millenary city where great tits have a long history of presence, it is still undergoing a rapid urbanization process, which could explain the high prevalence of bold birds in the most urbanized areas. Also, recent genetic analyses based on genome-wide RAD-sequences have revealed that Montpellier and La Rouvière great tits show significant but low genetic differentiation (Perrier et al. in review). Uncovering the direction of gene flow between city and forest, and testing for a sourcesink dynamic would reveal whether the city population could be regularly supplied with new, possibly bold, individuals.

Overall, our study shows strong phenotypic divergence between forest and urban great tits, but also across the urbanization gradient. Behavioral and physiological traits suggested that urban birds were on a faster end of the pace-of-life continuum than their forest conspecifics, yet breeding data did not support this conclusion. Further investigations on the plastic vs. genetic origin of the differences revealed here, along with the selection forces on the focal trait, will improve our understanding of how urban life shapes great tit pace-of-life in the future.

## ETHICS STATEMENT

Capture and ringing was performed under individual ringing permits delivered by the CRBPO and all the protocols involved, including the behavioral assays described below, were approved by the Animal Care and Use Committee Languedoc Roussillon (CEEA-LR-12066) as well as by Regional Institutions (bylaw issued by the Prefecture of Montpellier number 2012167-0003).

## AUTHOR CONTRIBUTIONS

Conceived study: AC, VD, ML, AG. Collected data: AC, VD, ML, SP, AG. Analyzed videos: VD. Statistical analyses: AC. Wrote the manuscript: AC. Improved the manuscript: VD, ML, AG. Contributed funding and materials: AC, AG.

## FUNDING

This research was supported by a recurrent annual funding from the OSU-OREME and by the European Research Council (Starting grant ERC-2013-StG-337365-SHE to AC).

## ACKNOWLEDGMENTS

We thank the many participants to the field monitoring, the City of Montpellier (Direction Paysage et Biodiversité) and the Zoo de Lunaret for logistic help. We thank Aude Caizergues, Arnaud Béchet, Gabrielle Dubuc Messier, and Denis Réale for useful discussions.

## SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fevo. 2017.00053/full#supplementary-material

## REFERENCES

urban disturbance? Ecol. Indic. 15, 30–35. doi: 10.1016/j.ecolind.2011. 09.018

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The handling Editor declared a past collaboration with one of the authors AG and states that the process nevertheless met the standards of a fair and objective review.

Copyright © 2017 Charmantier, Demeyrier, Lambrechts, Perret and Grégoire. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Humans and Tits in the City: Quantifying the Effects of Human Presence on Great Tit and Blue Tit Reproductive Trait Variation

Michela Corsini <sup>1</sup> \*, Anna Dubiec<sup>2</sup> , Pascal Marrot <sup>3</sup> and Marta Szulkin<sup>1</sup>

<sup>1</sup> Wild Urban Evolution and Ecology Lab, Centre of New Technologies, University of Warsaw, Warsaw, Poland, <sup>2</sup> Museum and Institute of Zoology, Polish Academy of Sciences, Warsaw, Poland, <sup>3</sup> Laboratoire Évolution et Diversité Biologique UMR 5174, Université Toulouse III Paul Sabatier, Toulouse, France

Environmental conditions are key drivers of life-history evolution, and the urban environment is an extreme form of land-use readily inhabited by avian wildlife, whose life-history variation in such altered environment is still poorly understood. Recently, the study of environmental variables associated with urban living—which include shifts in temperature, light, noise or food availability—has attracted increased attention. Another environmental axis that sets the urban space at odds relative to natural habitats is high human abundance, yet very little is known about its effect on avian fitness. We developed a protocol to quantify human presence by performing repeated counts of humans on the ground within a 15 m radius of nestboxes monitored in two centrally-located study areas of a European capital city. In parallel, a GIS-based approach was used to infer nestbox distance to the nearest path and road. Multiple counts of human presence around each nestbox yielded moderate to high repeatabilities (0.6 ≤ r ≤ 0.8) while requiring considerable resources time- and people- wise. In contrast, GIS-based estimates of nestbox distance to paths and roads were time efficient and generated highly repeatable results. The effects of (i) human presence around each nestbox, (ii) nestbox distance to the nearest path and (iii) nestbox distance to the nearest road were tested on reproductive traits of blue tits Cyanistes caeruleus and great tits Parus major breeding in two urban sites. Human presence did not influence blue tit or great tit life-history traits and reproductive success, suggesting reproductive habituation to humans in an urban landscape. In contrast, nestbox distance to roads shortened incubation time in great tits while nestbox distance to paths increased incubation time in blue tits. Moreover, blue tit offspring 2 weeks after hatching were lighter closer to roads. Our study confirms the reliability of a field protocol capturing human presence around multiple fixed locations that can be easily implemented in either urban or rural landscapes. At the same time, it appears that when applied to two urban sites where habituation to humans might have occurred, it is infrastructural networks rather than human presence per se that played a greater role in tit reproductive trait variation.

Keywords: human disturbance, habituation, Parus major, Cyanistes caeruleus, life-history traits, paths, roads, incubation

#### Edited by:

Amanda D. Rodewald, Cornell University, United States

#### Reviewed by:

Carolina Remacha, Complutense University of Madrid, Spain Davide M. Dominoni, Netherlands Institute of Ecology (NIOO-KNAW), Netherlands

\*Correspondence:

Michela Corsini<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Urban Ecology, a section of the journal Frontiers in Ecology and Evolution

> Received: 10 February 2017 Accepted: 11 July 2017 Published: 09 August 2017

#### Citation:

Corsini M, Dubiec A, Marrot P and Szulkin M (2017) Humans and Tits in the City: Quantifying the Effects of Human Presence on Great Tit and Blue Tit Reproductive Trait Variation. Front. Ecol. Evol. 5:82. doi: 10.3389/fevo.2017.00082

## INTRODUCTION

Humans are a keystone species in an urban habitat, modifying and shaping original ecosystem structures with often drastic consequences for their functionalities (Rees, 1997). Urban space is a habitat that attracts humans: while 54% of the world human population already resides in urban settlements, it is likely to increase to 66% by 2050 (United Nations, 2014). Consequently, 0.5% of the planet's land area has become urban space (Schneider et al., 2009), a value that is expected to grow several-fold in the next decades (Seto et al., 2011).

Urban growth is one of the major causes of natural habitat loss and fragmentation at a global scale (Grimm et al., 2008) as it modifies extensively the original landscapes (Shochat et al., 2006), leading to an overall rearrangement of habitats and animal communities (McDonnell and Pickett, 1990; Chamberlain et al., 2009). Urban areas are characterised by impervious surfaces including buildings and infrastructural networks, but also modified greenspaces; they encapsulate a multitude of ecological niches at a local scale, set in a multidimensional space of modified biotic and abiotic elements and processes (Sprau et al., 2016). Unavoidable interactions between urban wildlife and humans are thus increasing in prevalence due to growing urban sprawl taking over rural areas (Ditchkoff et al., 2006; Grimm et al., 2008). Birds are a convenient model to study the effects of urbanization on wildlife, due to their conspicuousness and ubiquity in both cities and rural sites. Moreover, much knowledge on bird biology is readily available from long-term studies carried out in the wild, thus offering a valuable background to infer change occurring in an urban environment (Savill et al., 2011).

A large number of previous studies have reported changes in bird ecology triggered by the urban environment (reviewed in Chace and Walsh, 2006; Gil and Brumm, 2013); these pointed out how this distinctive habitat may be defined by a multitude of environmental factors which rarely act in isolation. Overall, even though cities and towns are generally characterised by altered climatic profiles (increased minimum temperatures, Marzluff, 2001), predator communities (Churcher and Lawton, 1987) and food resources (i.e., supplementary feeding by humans; Fuller et al., 2008; Robb et al., 2008), the latter is emerging as a paramount in terms of its influence on habitat quality (Solonen, 2001; Robb et al., 2008; Chamberlain et al., 2009).

Urban-driven abiotic factors are also known to impact avian ecology and breeding biology, such as noise, light and chemical pollution (Slabbekoorn and Ripmeester, 2008; Dominoni et al., 2013). Thus, bird species were shown to decline in terms of richness, density and abundance due to noise pollution (Stone, 2000; Bayne et al., 2008; Mockford and Marshall, 2009). At the population level, noise pollution was found to induce an increased pitch in birds or a shift in their singing activity to the night time, avoiding noisy periods (Slabbekoorn and Peet, 2003; Kirschel et al., 2009; Nemeth and Brumm, 2009). Artificial night lighting—light pollution—may attract many birds during their nocturnal migration: harsh consequences for such important detours are an increased number of predation events and/or collisions with artificial structures in cities (van de Laar, 2007, reviewed in Erritzoe et al., 2003), as well as a reduction in energy storage or delayed arrival at wintering or breeding areas (Seress and Liker, 2015). Light pollution can also affect bird behaviour by advancing avian singing time at dawn and dusk (Da Silva et al., 2014). Finally, the increased exposure to environmental toxic chemicals—chemical pollution—can affect birds' physiology and phenotype directly but also indirectly because of habitat alterations or parasites spread (Morrison, 1986; Eeva et al., 1994; Fry, 1995; Isaksson, 2015).

While the effect of a large range of human triggered, biotic and abiotic variables specific to urban landscapes have been tested on avian reproductive traits, knowledge on the "direct" effect of human presence on avian reproduction is much more limited. Humans (often deterministically defined as "human disturbance") are known to trigger various responses in terms of changes in animal distribution, demography and population size (as reviewed in Gill, 2007). Earlier studies often tested the effect of human disturbance on bird species by inducing artificially different levels of noise, but without explicitly linking noise treatment levels to real life distribution of visitors on the ground (Lord et al., 2001; Verhulst et al., 2001; Baines and Richardson, 2007). Moreover, the direct effect of human disturbance at the individual level has largely focused on behavioural responses, where flush distance (Fernández-Juricic and Tellería, 2000; Miller et al., 2001; Tarjuelo et al., 2015), feeding patterns (Fernández-Juricic and Tellería, 2000) or physiological responses (e.g., increased corticosterone levels; Fowler, 1999; Walker et al., 2006; Ellenberg et al., 2007; Almasi et al., 2015) were measured. However, knowledge on the direct effect of human presence on individual fitness in an urban habitat is limited, as variation in life-history traits and reproductive success caused by human presence has been largely investigated in non-urban habitats and largely in noncavity nesting bird species (**Table 1**). While we may expect that birds breeding in urbanised habitats are continuously exposed to humans, thus habituated to their often not-threatening activities, no evidence in terms of life-history trait variations for such specific stressor was reported in cities. Even though many studies underlined how avian populations breeding in habitat characterised by frequent human disturbance, might often reduce their flee distance as a result of habituation (Metcalf et al., 2000; Rodriguez-Prieto et al., 2009; Clucas and Marzluff, 2012), it is also known that the presence of humans near the nest might ordinarily trigger strong behavioural responses in parental behaviour while feeding their young—for example, by alarm calling and avoiding entering nests for a certain time (Müller et al., 2006).

Human presence at the site and at the nest level is not often easy to monitor due to its inherent property of being variable in space and time. It can depend on season and weather conditions (sunny vs. rainy days; Gla¸dalski et al., 2016), days of the week (weekends vs. working days; Remacha et al., 2016) or hours of the day (morning peak hour vs. afternoon). It is thus important that such variability is taken into account when quantifying human presence and its possible disturbance to wildlife. Moreover, and in order to detect the potential biological effect of human presence on avian reproduction, protocols for capturing human disturbance need to be repeatable—an information that is only

bDisturbancequantifiedasobservationaldataorexperimentally

 induced. cSpecifieswhetherarepeatabilitytestforhumanpresencedetectionwasperformed.

 dReports whether human presence affected the inferred life-history traits positively (+), negatively (−) or had no effect (ns). The symbols' order refers to life-history traits listed in

 "a."

TABLE

1

rarely reported in studies of human disturbance on wildlife (**Table 1**).

This study quantifies the biological effect of humans on great tit (Parus major) and blue tit (Cyanistes caeruleus) life-history traits and reproductive success, characterised at the nestbox level and estimated in two contrasted yet centrally located urban study sites. Great tits and blue tits are two well-studied songbird species that breed in nestboxes in a large range of environments, from undisturbed rural areas to the chaos of urban settlements. The two study sites presented in this study—an urban park and a cemetery—offer the opportunity to compare two different urban habitat structures whose distributions of visitors vary gradually from a regularly visited site during specific opening hours (cemetery) to frequently visited without specific rules (urban park). Here, the effects of human presence—quantified with an easily implemented and repeatable protocol capturing human presence on the ground and at the nestbox level—is inferred and contrasted with the effects of nestbox distance to the nearest path and road, which are infrastructural networks acting as key sources of human presence in urban areas.

### METHODS

#### Study Sites

This study was conducted in 2016 as part of a new long-term research project whose main aim is to study the effect of cities on the genotype and phenotype of two wild passerine species—great tits P. major and blue tits C. caeruleus. 196 Schwegler woodcrete nestboxes (type 1b with an entrance hole of 32 mm and suitable for both great tits and blue tits) were erected in two study sites— 91 in a cemetery and 105 in an urban park (Pole Mokotowskie), located in the city centre of Warsaw, Poland. Warsaw (52◦ 14′N, 21◦ 1 ′E) is the largest city of Poland, and ranks as the 9th most populous capital city in the European Union with a population of over 1.7 million people (Eurostat, 2017). While both study sites are centrally located, the two areas are defined by different habitat structures, whose spatial location and actual surface use leads wildlife to face contrasted environmental pressures, especially in terms of human presence. Nestboxes were laid out in a grid, with an inter-nestbox distance of c. 50 m, an average south-east orientation (N = 196, mean = 106.7 degrees, SE = 5.6), and fixed at a height ranging from 2.5 to 3.0 m above ground level.

The urban park Pole Mokotowskie (52◦ 12′N, 20◦ 59′E) covers c. 65 hectares. Despite its current design, the park lays on a surface initially used as an airport until 1945. Pole Mokotowskie is now a typical managed green space framed by four large roads, where grass, flower beds and trees alternate to generate covered and open areas. Due to both size and central location, this urban public park plays an important role for city dwellers both as recreational area and a bike-friendly commuting space.

The Jewish Cemetery (52◦ 14′N, 20◦ 58′E), with its area of 34 hectares, is one of the largest Jewish necropolis in the world and still serves as burial place today. Established in 1806, the cemetery was largely neglected and abandoned during the German invasion. Due to a strongly reduced number of visitors after the II World War, only a small portion of the site was regularly visited, leading to a general overgrowth of trees and shrubs in the rest of the area. Because of selective forces mainly driven by important historical events, the Jewish cemetery is now formed by a naturally regenerating habitat. It is distinguished by a particular landscape of moss-covered tombstones in a large wild urban forest mainly composed by a mixture of both native and exotic tree species; oaks (Quercus spp.), silver birch (Betula pendula), Norway maples (Acer platanoides), elms (Ulmus spp.) and black locust (Robinia spp.) are the most common tree species in the site.

Human abundance and space use is highly contrasted in the two study sites: human presence in the cemetery is heterogeneous (many areas have few or hardly no visitors), and it is limited in terms of opening hours to daytime and outside of religious holidays. Cemetery visitors often arrive in groups and remain on marked paths. In contrast, the urban park is an open site where one can roam freely, and is visited by numerous commuting urban dwellers (pedestrians or cyclists) or those interested in recreational activities.

#### Life-History Data

Starting from mid-March, each nestbox was visited at weekly intervals to record the date of the first egg laid. The following breeding variables were compiled: nest occupancy (a nestbox was considered occupied if at least one egg was laid by a blue tit or great tit; empty nestboxes and those occupied by other species were considered as not occupied in our analyses), egg laying date (lay date recorded as day 1 = 1st of April), standardised lay date (standardised for each site and species), clutch size, incubation duration (defined as the number of days between the last egg laid to the first egg hatched), hatching success (number of hatchlings/clutch size), fledging success (number of fledglings/number of hatchlings) and chick mass 15 days after hatching (hatching day = day 1). Birds were ringed at day 15 using standard-numbered metal rings supplied by the Polish Bird Ringing Centre (Museum and Institute of Zoology, Polish Academy of Sciences). Nestboxes were checked c. 25 days after hatching to establish fledging success. Key summary statistics of reproductive success for each study site are presented in Supplementary Material Table 1.

#### Ethics Statement

Research was carried out under permit from the Regional Directorate for Environmental Protection in Warsaw, Poland.

## Quantifying Human Presence

The possible disturbance to great tit and blue tit reproductive biology caused by human presence was quantified in both sites and for each nestbox by measuring: (i) the number of humans and dogs present around each nestbox in a 15 m radius, (ii) nestbox distance from the nearest path and (iii) nestbox distance from the nearest road.

#### Human Presence Protocol

The ground-based data collection for human presence was performed from March until July 2016 on specific days throughout the season and aimed to capture human and dog presence at each nestbox. Each site was split in two paths grouping a similar number of nestboxes to minimize the time between the start and the end of each count. During each trial, two fieldworkers per site were counting for 30 s all humans (i.e., bikers and pedestrians) and dogs within a 15 m radius of each nestbox in the two study sites, following two assigned paths (i.e., track 1 and track 2, see maps in Supplementary Material Figure 1). In order to guarantee that all counts at each nestbox were performed at similar timeframes during the respective counts, the tracks' directions followed by the two fieldworkers during the human presence protocol were switched at each trial. The time needed to perform counts of human presence around each nestbox in the field did not vary significantly between sites: fieldworkers spent on average 83.2 min in the cemetery (SE = 1.75, N = 36) and 80.15 min in the urban park (SE = 1.45, N = 96) on their assigned path. To capture variation in human presence that can be influenced by human activities, and which might depend on time of day and working vs. weekend day, counts in the urban park were replicated across 2 working days (Mondays and Fridays) and 2 days during the weekend (Saturdays and Sundays). In each of these days, counts of human presence for each nestbox were performed 4 times throughout the day (at 8:00 a.m., 12:00 p.m., 3:00 p.m., and 6:00 p.m. for the urban park). Due to restricted opening hours, the cemetery counts were performed twice daily, and were restricted to Mondays, Fridays, and Sundays (**Table 3**). Dogs represented only 6% of the total number of humans and dogs (N = 7,586) recorded in a 15-m radius to each nestbox, we therefore use for simplicity the term "human presence" to refer to any human (pedestrian or biker) or dog presence around any given nestbox.

#### Distances from Paths and Roads

We recorded the spatial location of nestboxes using a GPS Garmin Map 64s and all the nestboxes coordinates were downloaded using the open source Software DNRGPS Minnesota. All nestbox distances to the closest path and road were calculated using the free and Open Source Geographic Information System Quantum GIS 2.8.2 "Wien," (version released on the 9th of May 2015), with a default projection of WGS84-Geographic Coordinate System. Built on the Plugin OpenLayers, the OpenStreetMap has been used as base map to measure all distances with the Distance Matrix tool of the software. Specifically, the distances were measured in meters from the nestbox point to the middle of both the closest path and road. Since the measurements recorded with the Distance Matrix tool may change in precision due to the zoom function manually selected by the observer at each record, the repeatability of the process was tested comparing the measurements taken in two different days by the same person (M.C.).

## Statistical Analyses

#### Repeatability

The repeatability of human presence around each nestbox measured on the ground as well as the repeatability of distance measurement of every nestbox to the closest path and road computed in qGIS was calculated using the "rptR-package" in R following Stoffel et al. (2017). This package allowed for repeatability estimation using a generalised linear model. Specifically, the repeatability test to infer human presence on the ground was performed for four different temporal combinations (early vs. late in the day, between working days, weekend vs. working days, early vs. late in the season) within which the total number of visitors around each nestbox was inferred and the nestbox ID was fitted as random effect in the model. The same method was extended to verify the reliability of the GIS-based approach, where the same observer (M.C.) measured nestbox distances to roads and paths in two separate days. Finally, correlation tests were performed to quantify the strength of association between human presence and distance from the closest path and from the closest road within sites.

#### Statistical Analyses

Statistical analyses were implemented using the computing environment R (version 3.3.1, released on the 21st of June 2016). To test the effects of human presence and the effects of distance to paths and roads on blue tit and great tit lifehistory traits and reproductive success, we used the glm function of Generalised Linear Models (GLM) in R. All interactions between human presence index, distance from paths and roads and site were tested; however, these were found non-significant and consequently removed from the final models. The following traits were inferred, tested for blue tits and great tits in separate analyses: egg laying date, clutch size and incubation time were analysed with a general linear model with normal error distribution. Occupancy rate, hatching success (i.e., the number of hatchlings on clutch size) and fledging success (i.e., the number of fledglings on the number of hatchlings in nests with at least one hatched offspring) were tested with a quasi-binomial distribution to control for over-dispersion. Human presence and nestbox distance to paths and roads were fitted as a continuous variable in all models while the site was fitted as a fixed effect with two levels corresponding to the two study areas in blue tits: the cemetary was excluded as predictor in great tits due to the low sample size recorded in the site (N = 2). Egg laying date (standardised for site and species) was fitted in all models with the exception of two models where occupancy rate and lay date were fitted as response variables. Finally, we used a linear mixed model (lme function in R) to test the effects of human presence and the effects of nestbox distance to paths and roads on individual nestling mass at day 15 (response variable), where nestbox ID was fitted as random effect to control for non-independence of nestlings.

#### RESULTS

#### Repeatability of Human Presence on the Ground and With a GIS-Based Approach

Human presence recorded in a 15 meters radius around each nestbox during a 30 second long scan varied from 0 to 33 detected humans, and was on average 7.6 times higher in the urban park than in the cemetery (**Figures 1A,B**, **Table 2**). Interpolated maps illustrating average human presence for each nestbox (**Figure 1**) show that the flux of people varied in terms of intensity and scatter between and within sites: while people were heterogeneously distributed and most abundant in the southern areas of the urban park (**Figure 1C**), visitors in the cemetery were usually detected while walking on the main path near the entrance gate (**Figure 1D**). Due to renovation works in the cemetery, workers were systematically detected in the North-Eastern part of the site during the trials.

The repeatability of human presence, measured on the ground for each site separately, was high in the urban park, reaching values between 0.808 and 0.840 and overlapping confidence intervals across the four temporal combinations tested (**Table 3**). The repeatability of human presence in the cemetery was lower, with values ranging between 0.205 and 0.563, which is likely to be the consequence of both reduced human presence and fewer counts (**Table 3**).

Distances from paths and roads at each nestbox, estimated using a computer-based GIS approach, showed near-perfect measurement repeatability, which ranged between 0.991 and 1 (**Table 3**). Additional tests were carried out to determine the strength and direction of any possible linear relationship between human presence (human abundance detected at each nestbox) and distances from paths and roads (obtained via GIS-based approaches). Correlations were tested at a within site levels and were all found non-significant but for the exception of the urban park where there was a weak yet significant negative correlation between the number of people around any given nestbox and its distance to the nearest path (**Table 4**).

#### Effects of Human Presence, Distance to Paths and Roads on Life-History Traits and Reproductive Success

Human presence estimated in a 15 m radius around each nestbox did not influence any of the investigated blue tit or great tit lifehistory and reproductive traits (**Table 5**). In contrast, in blue tits incubation duration was significantly longer closer to paths while chick mass 15 days after hatching was significantly lower closer to roads. In great tits, incubation duration was significantly shorter closer to roads (**Table 5**). No effect of human presence, distance

"N counts" refers to the number of times human presence was assessed around each nestbox on any given site.

to paths and roads was detected on hatching and fledging success (Supplementary Material Table 2).

#### DISCUSSION

This study is one of the very few testing the biological effect of human presence on great tit and blue tit life-history and reproductive success in an urban environment (see also **Table 1**). To the extent of our knowledge, this is the first study that takes into consideration both the intensity of human presence at the nestbox level and its repeatability. The nestbox-based protocol of human presence quantification, as well as the heterogeneous human distribution in space reported in this study that occurred both at a within-site and between-site levels (**Figure 1**), allows for the testing of human disturbance at the finest biological level—the breeding event itself.

Available reports testing the effects of human presence were usually carried out in natural and semi-natural areas, and the methods applied in quantifying such dynamics varied among studies. Human presence was often induced experimentally (Lord et al., 2001; Verhulst et al., 2001; Baines and Richardson, 2007), and the relationship to a natural distribution of these variables is not always explicit. In addition, while most of the previous studies were conducted in colonial waterbirds far from an urban context (see review by Carney and Sydeman, 1999) only a few studies testing the effect of human presence were carried out in urban settings (Fernández-Juricic, 2000; Fernández-Juricic and Tellería, 2000).

The protocol for capturing human presence on the ground required considerable effort in terms of time and number of observers relative to a computer-based GIS approach. Repeatability tests were performed for several temporal combinations that could detect whether human presence is consistent (or not) between times of the day (mornings vs. afternoons), between working days (Mondays vs. Fridays), between working days and weekends, and finally testing whether human presence is stable at the nestbox level throughout the season. The 4 types of combinations tested generated highly equivalent repeatabilities (0.81 < r < 0.84) with overlapping confidence intervals, overall revealing that human presence in the urban park is temporally stable. In the cemetery site in contrast, we detected lower intra-day and intra-week repeatabilities (**Table 3**), yet a satisfactory repeatability of r = 0.56 when human presence was compared early vs. late in the breeding season. The lower repeatability of human presence recorded in the cemetery is likely to be the result of fewer counts combined with a low and irregular distribution of humans within the study site (**Figure 1**). Thus, our study reveals that the repeatability of human presence was considerably larger in the site with higher human presence (the urban park) and with a greater heterogeneity in human presence between nestboxes. In the urban park where human presence was high, the high repeatability of ground-based counts (r > 0.8) confirmed the reliability of our protocol to accurately capture human presence, thereby revealing a stable yet heterogeneous human distribution in both urban sites.

FIGURE 1 | (A,B) Histograms of average human presence around each nestbox in the urban park (A) and in the cemetery (B). Average human presence was calculated as the total number of humans and dogs in a 15 m radius to each nestbox standardised by the number of counts performed within each site (i.e., N = 48 in A, N = 18 in B). (C,D) Interpolated maps of average human presence around each nestbox (black dots) are indicated for the urban park (C) and the cemetery (D), respectively.

a Intra-day variation: all counts of human presence performed in the morning were tested against counts of human presence performed in the afternoon.

<sup>b</sup>Seasonal variation: the first half of the dataset was tested against the second half of the dataset.

<sup>c</sup>Weekday variation: counts of human presence made on Saturdays and Sundays were compared with counts performed on working days, e.g., Mondays or Fridays.

<sup>d</sup>Working day variation: counts performed on Mondays were compared with counts performed on Fridays. For GIS-based repeatabilities.

<sup>e</sup>Computer-based distance measurements collected on the first day were tested against measurements collected on the second day.

Further work is required to test (i) the extent to which human presence can be captured with high repeatability in a gradient of urbanization with contrasted human presence at the nestbox and site level (indeed, repeatability values for human presence in the cemetery, characterised by considerably lower human presence, were also lower) and (ii) to define a minimum number of counts that would reduce ground effort while securing satisfactory repeatability (r ≥ 0.6) of human presence at the level

TABLE 4 | Pearson's correlations and significance tests investigating collinearity between human presence and distances to infrastructural facilities.

Significance levels: \*p < 0.05, \*\*\*p < 0.001.

of individual breeding events (Corsini et al., in preparation). Human preferences in terms of space use were particularly marked in the urban park, which was also the site with the highest number of visitors (**Figures 1A,C**). Indeed, the pattern of space use within this area was associated with the presence of recreational facilities, mostly located in the southern part of the study site. The GIS-based approach, in contrast, was a considerably less-time consuming method compared to the protocol for detecting human presence on the ground (involving only one observer and 2 days of measurements), and proved to be highly repeatable (r ≥ 0.991). Importantly, the easily-computed GIS-based approach for measuring nestbox-level distances to paths and roads provided an alternative approach to capture human presence on the ground, and was found to have a much greater influence on tit life-history trait variation than human presence per se: while no direct effect of human presence was detected in great and blue tit life-history, incubation time in blue tits and great tits, as well as blue tit offspring mass 2 weeks after hatching were associated with distance to infrastructural facilities such as paths and roads.

Policy regulations of a given area are likely to play an important role in shaping the dynamics of people flow within sites. While in both sites visitors can roam freely, paths remain the major communication channels throughout both study sites. However, human presence correlated with distance to paths only in the urban park, which may be caused by a larger detection power of such associations due to a larger number of visitors overall. Our results are thus in line with the findings of Remacha et al. (2016), who reported that distance to paths is a reliable index of human disturbance. In the case of roads, there is no evidence that this infrastructure is correlated with distribution of human presence detected on the ground, thereby suggesting that the presence of such infrastructural facilities at the site level is not always an important source of human presence, although it can impact reproduction through independent means, as explained below.

In this study, variation in blue tit or great tit life-history traits and reproductive success was not explained by human presence estimated using the ground-based protocol, suggesting that human presence does not affect birds' survival or lifehistory traits in the long-term. In contrast with our findings, Remacha et al. (2016) showed that blue tit nestlings from forested areas used as a recreational site that hatched on days with increased activity of visitors (weekends and other nonworking days) were found to grow slower and fledge with lower body mass; this difference may be due to differences in birds' environmental backgrounds: in a more semi-natural context, where human presence is higher during the breeding season, it is possible that birds are more sensitive to human disturbance. Birds inhabiting rural realities already showed a higher physiological and behavioural stress response than their urban counterparts, often revealed through a higher flightinitiation distance (McGiffin et al., 2013; Abolins-Abols et al., 2016). On the other hand, birds inhabiting urban parks are almost continuously exposed to humans—so despite the fact that each nestbox can be consistently exposed to high or low human presence, birds may become habituated through their daily experience of humans while foraging. Studies of the effect of human presence in other bird species reveal mixed results. Similarly to our findings, in a wild population of black grouse located in a recreational area, the presence of visitors did not affect productivity in terms of clutch size, hatching success and breeding success (Baines and Richardson, 2007). In the woodlark, on the other hand, productivity was related to the level of human disturbance, although in the opposite direction than expected, with birds breeding in more disturbed areas raising more fledglings (Mallord et al., 2007). Human presence was also found to extend the duration of incubation period in the dotterel and the European oystercatcher (Lord et al., 2001; Verhulst et al., 2001), while recreational activities had a negative-marginal effect on incubation behaviour in American oystercatchers (McGowan and Simons, 2006).

These results suggest that behavioural responses are usually context-dependent and related to the trade-offs experienced by individuals: indeed, it is easier to expect habituation in birds that are used to human presence, which is a common scenario in an urban park, while birds in rural environments may react to human presence adversely (Remacha et al., 2016). Furthermore, the impact of human disturbance is likely to change among avian species or species with variable ecological niches: ground and shrub nesting birds, for example, are likely to be more sensitive to human disturbance. The same pattern was reported for ground-foraging species (Rodgers and Smith, 1995; Gill, 2007). In addition, while there is an often-held assumption that human presence triggers changes in behaviour [like flush responses, reduction in incubation duration or changes in foraging behaviour (Lord et al., 2001; Verhulst et al., 2001; Thiel et al., 2007)], which in turn may be translated into reduced fitness, those fitness consequences are in fact rarely tested (Gill et al., 2001). Here, we did not find any evidence that human presence affects the life-history or reproductive success of birds in an urban context, and suggest that structural properties of the environment, such as paths and roads, overrides the weak or non-existent effect of human presence in cities.

In contrast to a lack of effect caused by human presence, nestbox distance to paths and roads influenced incubation duration and chick mass on day 15 after hatching. A larger dataset (in terms of study sites and years of study) is likely to be required to detect finer scale effects of the effect of human TABLE 5 | Effects of human presence, paths and roads on blue tit and great tit reproductive traits.

Nest occupancy was fitted as a quasi-binomial response variable, while egg laying date, clutch size and incubation duration were modelled with a Gaussian error distribution. Chick mass was modelled with a linear mixed model and nest ID as random effect. In blue tits, the cemetery was used as reference level to test differences between sites. Significance levels: \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001.

presence or distance to paths and roads on tit reproductive trait variation. Roads have already been shown to have a strong impact on fitness in birds (e.g., Halfwerk et al., 2011; Dietz et al., 2013; but see review by Kociolek et al., 2011). Their negative effect may arise through noise pollution leading to disruption in communication, for example in the context of mate attraction, predator avoidance or parent-offspring communication (Slabbekoorn and Ripmeester, 2008; Francis, 2015). Thus, communication breakdown caused by noise may at least partly be responsible for lower body mass in nestlings located closer to roads. A more direct mechanism associating proximity to roads with lower offspring body mass is the fact that a greater proportion of impervious surface close to the nestbox automatically results in lower levels of biomass and resulting food availability for our focal species (but see the opposite effect in Florida scrub jays Morgan et al., 2010). Moreover, roads are associated with many biotic and abiotic changes, for example: increased mortality in birds due to direct collisions with vehicles, but also the general alteration of physical and chemical properties in these novel environments (Reijnen et al., 1995; Erritzoe et al., 2003; but see review by Trombulak and Frissell, 2000).

The fact that blue tits significantly increased their incubation time closer to paths (while great tits show an equivalent yet non-significant trend in the same direction) independently of human presence is surprising and suggests that paths act as a disruptor to incubation that may be independent of human presence. Indeed, the correlation between human presence and distance from paths was significant but weak (r = −0.29, p = 0.002, **Table 4**), suggesting that due to the frequency of off-trail events, the presence of visitors in the urban park is not strictly connected with infrastructural networks. However, previous studies reported that habituation to walkers is common in several species, especially when people remain on marked paths: however, this level of habituation may change during the breeding season, whenever off-trail events occur (Nisbet, 2000). In addition, it is possible that the mere presence of humans in proximity of a nest can induce increased alertness in both adults affecting parental care (while feeding the nestlings or, as in this case, during incubation). In contrast, incubation duration was significantly shorter in great tits breeding in proximity to paved roads, which also coincides with c. 0.5◦C increase every 100 m closer to the largest road neighbouring the urban park (daily averages over 4 months starting on the 25th of March; Corsini, unpublished data).

In conclusion, we confirm that the protocol for quantifying human presence developed in this study proved to be highly repeatable in a site with high intra-site variance of human presence, thereby confirming its usefulness to other studies

#### REFERENCES

testing the effects of human presence around fixed point locations in avian and non-avian species alike. At the same time, our results suggest that urban great tits and blue tits are habituated to human presence, a keystone species in the urban environment, and generally perceive humans as harmless stimuli (Lowry et al., 2013). While the process of habituation is known to reduce stress responses in avian species (Walker et al., 2006), fitness consequences due to a long-term exposure to human generated stressors may be complex, especially in an urban context where several environmental variables other than human presence interact. Finally, in the context of urban study sites, it is mostly infrastructural networks rather than human presence per se that played a greater role in tit reproductive trait variation.

#### AUTHOR CONTRIBUTIONS

MS designed the study; MC, AD, and MS collected the data; MC, PM, and MS performed the analyses, MC prepared a first draft of the manuscript; MC, AD, PM, and MS edited and approved the final version of the manuscript.

## FUNDING

This study was financed with a Sonata Bis grant 2014/14/E/NZ8/00386 from the National Science Centre, Poland.

#### ACKNOWLEDGMENTS

We thank Tomasz Mazgajski, Jerzy Banbura and Anne ´ Charmantier for constructive discussions on urban ecology. We also thank Karol Kobiałka, Marta Celej, Justyna Kubacka and Fatima Hayatli for help in the field. We are grateful to ground managers for enabling research on their sites.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fevo. 2017.00082/full#supplementary-material

boreal forest. Conserv. Biol. 22, 1186–1193. doi: 10.1111/j.1523-1739.2008. 00973.x

responses of an endangered steppe bird. Behav. Ecol. 26, 828–838. doi: 10.1093/beheco/arv016

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Corsini, Dubiec, Marrot and Szulkin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Does Seasonal Decline in Breeding Performance Differ for an African Raptor across an Urbanization Gradient?

#### Sanjo Rose † , Petra Sumasgutner † \*, Ann Koeslag and Arjun Amar

FitzPatrick Institute of African Ornithology, DST-NRF Centre of Excellence, University of Cape Town, Cape Town, South Africa

As human populations have increased globally, so too has the transformation of natural landscapes into more urban areas. Within Africa, population growth rates and urbanization rates are amongst the highest in the world, but the impacts of these processes on Africa's wildlife are largely un-explored. In this study, we focus on a recently established population of black sparrowhawks Accipiter melanoleucus in Cape Town, South Africa. Using long-term data (16 years), we investigate the relationship between the timing of breeding and breeding performance along an urban-rural habitat gradient. We found no evidence for a direct effect of urbanization on either timing of breeding or breeding performance. However, we did find some evidence for a differential seasonal decline in breeding performance dependent on the levels of urbanization. Thus, higher productivity was found in more urbanized habitats earlier in the season, but towards the end of the breeding season, birds in less urbanized habitats performed better. Our study represents one of the first to examine avian productivity in relation to urbanization in Africa, and also represents one of the first studies to suggest that seasonal declines in productivity may differ according to levels of urbanization. These results have considerable implications for potential changes in phenology or productivity for the regions' avifauna as African urbanization continues.

#### Edited by:

Diego Gil, Consejo Superior de Investigaciones Científicas, Spain

#### Reviewed by:

Davide M. Dominoni, Netherlands Institute of Ecology (NIOO-KNAW), Netherlands Meritxell Genovart, Mediterranean Institute for Advanced Studies (CSIC), Spain

#### \*Correspondence:

Petra Sumasgutner<EMAIL_ADDRESS>
† These authors have contributed equally to this work.

#### Specialty section:

This article was submitted to Urban Ecology, a section of the journal Frontiers in Ecology and Evolution

> Received: 31 January 2017 Accepted: 28 April 2017 Published: 16 May 2017

#### Citation:

Rose S, Sumasgutner P, Koeslag A and Amar A (2017) Does Seasonal Decline in Breeding Performance Differ for an African Raptor across an Urbanization Gradient?. Front. Ecol. Evol. 5:47. doi: 10.3389/fevo.2017.00047 Keywords: urban ecology, timing of breeding, reproduction, South Africa, Accipiter melanoleucus, raptors

## INTRODUCTION

Urbanization is a rapid and global process in which natural environments are transformed into new urban systems (Berry et al., 1998) which are characterized by an increase of unproductive sealed surfaces in highly disturbed fragmented landscapes (McDonnell and Pickett, 1990). By 2050, 66% of the global population is predicted to live in urban areas (United Nations, 2015). Urbanization significantly alters the physical, chemical, and biotic environment (McDonnell and Pickett, 1990). The resulting decrease of natural habitat forces ecosystems and species to adapt, avoid or tolerate these new conditions (Blair, 1996; Isaksson, 2015).

Key processes that contribute to the impact of urbanization on birds include vegetation changes, habitat fragmentation, introduction of exotic species, changes in food abundance and quality, and novel predator assemblages (Chace and Walsh, 2006). An urban gradient characterizes different levels of urban stressors, such as pollution (light, noise, and air), the abundance of novel predators like cats and corvids (e.g., Stracey, 2011), and of anthropogenic food resources that may be of lower quality ("urban junk food," Pierotti and Annett, 2001; Schoech and Bowman, 2001). All of these factors can potentially affect demographic parameters of city-dwelling species (Isaksson, 2015). In order to anticipate the costs and benefits for birds living in urban areas, it is useful to quantify the relative breeding performance within urbanized areas compared to more natural habitats.

There is a growing body of literature comparing breeding performance of birds living in urban and rural habitats, with some general patterns emerging. The onset of breeding tends to be earlier in urban areas as a potential result of increased food availability (Chamberlain et al., 2009), the urban heat island effect (i.e., higher temperatures in the city center, Shochat et al., 2006; since the beginning of incubation can be temperature dependent, Visser et al., 2009) and light pollution (Spoelstra and Visser, 2014). Clutch sizes tend to be smaller and nestling conditions tend to be poorer in urban vs. natural habitats (reviewed by Chamberlain et al., 2009). However, these studies have mostly been conducted in the Northern Hemisphere with very different climatic conditions. Despite an increased focus on urban avian ecology, as far as we know no studies have yet focused on these questions for any bird species in Africa.

Many bird species show seasonal declines in breeding performance during the course of a breeding season (e.g., Both et al., 2004; Grüebler and Naef-Daenzer, 2010), and this has also been found for some urban nesting species (Chamberlain et al., 2009), including urban raptors (e.g., Sumasgutner et al., 2014a). The explanation for these patterns often focuses on declines in food availability that might also influence sibling competition (Gil et al., 2008), which may in theory vary across levels of urbanization. If so, it might be expected that the patterns of such seasonal declines might vary across an urban gradient. In the only study to explore this issue so far, Wawrzyniak et al. (2015) found seasonal declines in clutch size of blue tits (Parus major) nesting in urban habitats but not for those nesting in forests.

Because raptors belong to the upper trophic level, they can be particularly susceptible to changes in the environment (Newton, 1998). Despite this, some raptors are increasingly associated with urbanized areas (e.g., Falco peregrinus, Cade and Bird, 1990; F. columbarius, Sodhi et al., 1992; F. tinnunculus, Village, 1990; Charter et al., 2007; Accipiter cooperii, Boal and Mannan, 1999, 2000; A. gentilis, Rutz, 2008). This is potentially due to their relatively high mobility that could allow them to escape from some of the limitations of urban areas (Chace and Walsh, 2006), while still benefitting from the advantages, such as lower direct persecution (Rutz, 2008) and novel resources like higher abundance of avian prey or nesting sites (e.g., Palomino and Carrascal, 2007; Cavalli et al., 2014; Rullman and Marzluff, 2014; Sumasgutner et al., 2014b). However, some rodent specialists such as Barn owls Tyto alba or Eurasian kestrels F. tinnunculus show decreased fledgling success with increasing urbanization, probably due to decreased prey availability connected to sensory interference due to noise pollution for owls (Senzaki et al., 2016), or nocturnal rodents being harder to locate for a diurnal raptor in an urban environment (Hindmarch et al., 2014; Sumasgutner et al., 2014a). Dietary requirements may be an important factor in determining the success of an urban raptor, specifically the degree of specialization in their diet choice. Highly specialized raptors may have to spend more energy and time seeking preferred prey, whereas generalists may have more flexibility in coping with fluctuating food availability (see Terraube et al., 2011 for a natural setting).

In this study we focus on the black sparrowhawk Accipiter melanoleucus in South Africa, a raptor that colonized the city of Cape Town in the last two decades after expanding its distribution range from the North-East into the South-West of Southern Africa (Amar et al., 2014). Black sparrowhawks appear to have adapted well to urban areas, with more than 50 breeding pairs (Martin et al., 2014b) within our study area. Together with the colonization of the urban habitat, the species extended its breeding season toward earlier egg-laying, resulting in a breeding season of 9 months (Mar-Nov; Martin et al., 2014b). Potentially contributing to the success of the species in this area, the health of black sparrowhawks' chicks does not appear to be negatively influenced by levels of urbanization (Suri et al., 2017, but see van Velden et al., 2017 for a specific Knemidokoptes mite infection in adults). This might be linked to the high abundance of suitable prey species across all habitat types that may buffer any potential negative health effects of urban stressors (Suri et al., 2017). Building on these results, our study aims to explore how the breeding performance of urban black sparrowhawks varies with urbanization in Cape Town. Using a long-term study (16 years), we investigated the timing of breeding and breeding performance along an urban-rural habitat gradient. Additionally, we specifically explore whether the established relationship between timing of breeding and breeding performance (Martin et al., 2014b) differed depending on levels of urbanization. This question is particularly relevant to our study area, since it is unique within a South African context in having an extended 9 months long breeding season (Martin et al., 2014b). Given the results of several other studies exploring timing of breeding, we predicted the onset of breeding to be earlier in more urbanized areas. The expected relationship between urbanization and breeding performance is harder to predict, however, given that Suri et al. (2017) tended to find higher abundance of doves and pigeons in more urbanized habitats, we might expect higher productivity in more urban areas. Likewise the precise relationship between timing of breeding and breeding performance in relation to urbanization was difficult to predict, but if urbanized areas do have higher or more regular prey availability, the established seasonal declines in breeding performance may be somewhat buffered within more urbanized areas.

## METHODS

#### Study Site and Species

This study focused on the resident population of black sparrowhawks in Cape Town, Western Cape, South Africa (**Figure 1**). The study area covers various habitats over 595 km<sup>2</sup> (Martin et al., 2014b), including heavily urbanized areas, plantations of exotic tree stands, patches of Afromontane forest and indigenous fynbos vegetation. Cape Town experiences a

Mediterranean climate, with an average annual rainfall of 788 mm and average monthly temperatures ranging from 15 to 30◦C. This population breeds mainly during the winter months, which coincides with the rainy season (Amar et al., 2014; Martin et al., 2014b). The population has been actively monitored since 2000 (Amar et al., 2014).

Systematic monitoring by a team of experienced researchers occurred during the breeding season from March to November. Territories were located by surveying suitable stands of trees. Occupancy was confirmed by activity indicators such as calling, whitewash, prey remains or nest structures. Following confirmation of breeding attempts, pairs are then monitored approximately weekly. About 90% of the population is individually color ringed, allowing individual identification of many breeding pairs (see Martin et al., 2014a for further details). Adults were trapped on territories using a bal-chatri trap baited with a live white "racing" pigeon, Columba livia (Berger and Mueller, 1959; see Ethical Note in Martin et al., 2014a for details). Chicks were ringed when they were 3.5–4.5 weeks old. Age of chicks was estimated visually by the extent of wing and tail feather growth (using reference photographs from nest cameras). This study uses data collected between 2000 and 2015.

#### Defining the Urban Gradient

Quantification of the urban gradient was based on a circular buffer with a radius of 2,000 meters (m) assigned to each nest; the chosen scale was based on previous work in which adult males with global positioning system (GPS) tags were tracked and their home range sizes during the breeding season where determined (Sumasgutner et al., 2016b). Urban cover was calculated based on land cover classes from the 2013–2014 South African National land-cover dataset produced by GEOTERRA (Department of Environmental Affairs, 2015). This land cover dataset compiled 72 land types at a 30 m resolution, from which sealed, unproductive areas of land were classified as "urban." Percentage urban cover was derived for each nest buffer using the packages raster (Hijmans, 2015), sp (Pebesma and Bivand, 2005; Bivand et al., 2013), and rgdal (Bivand et al., 2016). **Figure 2** illustrates the binary urban/non-urban land covers as compared with aerial photographs.

## Statistical Analysis

The "lay month" (the only accurate measurement of the timing of breeding possible) for successful nests is backdated from the age of the chicks; we used the incubation behavior (female sitting low on nest) to time the onset of breeding for unsuccessful nests. "Breeding success" is a binomial variable distinguishing between successful and unsuccessful nests, "brood size" only includes successful nests (1-3 chicks that fledged) and "productivity" all active nests where 0–3 chicks fledged. All analyses were performed in relation to urbanization using Linear Mixed Models (LMMs) for the timing of breeding and Generalized Linear Mixed Models (GLMMs) for breeding performance, in R studio (R Core Team, 2016) with the packages nlme (Pinheiro et al., 2014), lme4 (Bates et al., 2015), MASS (Venables and Ripley, 2002), lattice (Deepayan, 2008), ggplots2 (Wickham, 2009) effects (Fox, 2003), and car (Fox and Weisberg, 2011). All

and 4. Data from Department of Department of Environmental Affairs (2015); Google Earth Pro (2016), and City of Cape Town (2016).

quantitative variables were scaled in order to bring the variables to comparable dimensions to interpret effect sizes for interaction terms. We tested the dispersion of our response variables using the "qcc.overdispersion.test" in the qcc package (Scrucca, 2004). Residual distributions of the models were inspected visually to assess model fit.

Unless otherwise stated (when using successful nests only), the sample size in all following analyses was 456 broods surveyed over 16 years starting in 2000. Year, territory ID, and nest ID were included as random terms to account for pseudoreplication arising from same nests sampled repeatedly within and between years (nest ID), and breeding pairs occupying territories over several years (territory ID), and multiple measures from different nests within the same year (year). All analyses were conducted at the nest level since some pairs build multiple nests in different locations within and between seasons, resulting in varying urban scores for some individuals. The lay month (Gaussian distribution) was modeled as the response variable with the urban gradient as the explanatory variable. For the other analyses examining breeding performance, we accounted for the timing of breeding (using lay month) as a quadratic term and explored the interaction of timing of breeding with the urban gradient. We included the lay month as linear and quadratic term to investigate a potential polynomial relationship. Error structures for the GLMMs were: negative binomial (productivity), Poisson (brood size) and binomial (breeding success). Least squares means (posthoc contrasts for mixed-models, Lenth and Hervé, 2015) are not yet implemented for interaction terms between continuous variables. Thus, to understand the nature of the relationship between the timing of breeding and levels of urbanization better, we performed additional tests whereby we selected the earlier 25% of nests (egg-laying Mar-May, n = 123) to see if there is a significant difference in the breeding performance in relation to the urban gradient. Then we repeated this analysis for the latest 25% of nests (egg-laying Sep-Nov, n = 120); see Tate et al. (2016) for a similar approach. Throughout, reported χ 2 and p-values are based on an ANOVA Table of Deviance using Type II Wald χ 2 tests for mixed models.

#### Ethical Note

The University of Cape Town's Science Faculty Animal Ethics Committee has approved all the procedures required for the longterm study of the black sparrowhawk population in Cape Town (permit number: 2012/V37/AA). Furthermore, all procedures were carried out under a Cape Nature Hunting Permit (No. 0035- AAA004-00428), a South African National Parks Permit and a SAFRING ringing permit (no. 1439).

#### RESULTS

#### Urban Gradient

The urban gradient around each nest ranged from 87% to 1.4%. See **Figure 2** for representations of (1) 1.4%, (2) 12.7%, (3) 47.1%, and (4) 87.0% urban cover, and the frequency of different urbanization scores for black sparrowhawk nests over the 16-year monitoring period.

## Timing of Breeding and Breeding Performance along the Urban Gradient

Timing of breeding did not differ in relation to the urban gradient (**Table 1**, **Figure 3**), nor was there a direct relationship between breeding performance and the urbanization gradient term. However, an interesting interaction was noted between timing of breeding and urbanization in relation to breeding performance (**Table 1**, **Figures 4A,C**). Firstly, for pairs with nests in the most urbanized areas (**Figure 4A**, blue dotted line), we found that productivity declined strongly as the season progressed, whereas this decline was less steep for pairs in less urban areas (**Figure 4A**, black solid line), indicating that the timing of breeding had greatest influence on productivity in more urbanized areas. This interaction term shifts after approximately one third of the breeding season (June/July). A very similar relationship was also seen for breeding success (**Figure 4C**). However, the interaction term was statistically not significant, but suggested a tendency for differential seasonal declines in relation to urbanization (productivity GLMM: χ <sup>2</sup> = 3.45, P = 0.063; breeding success GLMM: χ <sup>2</sup> = 3.20, P = 0.074). This apparent relationship was not observed for brood size (**Figure 4B**, successful nests only, 1–3 chicks) where we found slightly more chicks fledging in urban areas throughout the season and less chicks fledging in more rural areas. Thus, our results suggest that the interaction between timing of breeding and urban gradient was due to nest failures, rather than the number of fledglings produced in successful nests. When splitting the data for early nests vs. late nests these observations were confirmed: we found slightly higher productivity in early nests in more urbanized areas (χ <sup>2</sup> = 3.41, P = 0.065), but no such difference in late nests (χ <sup>2</sup> = 2.20, P = 0.138). Additionally, we found significantly higher probability of nest failure in more urbanized areas later in the season (χ <sup>2</sup> = 5.28, P = 0.022; vs. breeding success in early nests: χ <sup>2</sup> = 2.74, P = 0.098).

### DISCUSSION

The most fundamental result from our analysis was that the extent of urbanization surrounding nest sites did not seem to have a strong influence on breeding performance. Thus, despite many additional stressors that may come from breeding within a more urbanized environment (Isaksson, 2015), black sparrowhawks appear able to cope with these challenges and produced similar number of chicks irrespective of the levels of urbanization around their nest sites. This result therefore adds to the findings of Suri et al. (2017), which explored influences of urbanization on chick health for this same black sparrowhawk population, and found no negative effect on a range of health parameters. Suri et al. (2017) suggested that the main prey species (doves and pigeons, Streptopelia semitorquata, and Columba livia) were plentiful across all habitats, and this abundance of prey may explain why there were no negative impacts of urbanization either on chick health and ultimately on the population's productivity. Thus, from these two studies it does seem that the species is able to cope well with the urban environment within the Cape Town region.

Note that the interaction term between the urban gradient and the lay month accounted for a linear and quadratic relationship and that all quantitative variables were scaled. ":" indicating an interaction term, "ˆ2" indicating a quadratic term, significance codes: "\*\*\*" < 0.001, "\*" < 0.05, "•" < 0.1.

Contrary to our expectations, there was no direct relationship between the percentage of urban cover and timing of breeding. Many urban species are known to start breeding earlier compared to their rural counterparts (see reviews by Chamberlain et al., 2009), but these studies were located in the Northern hemisphere where the urban heat island effect might be beneficial (but see advanced incubation in Australian magpies Gymnorhina tibicen, Rollinson and Jones, 2002; and earlier first nest attempts for White-winged chough Corcorax melanorhamhos, Beck and Heinsohn, 2006). However, for black sparrowhawks in South Africa breeding coincides with the colder periods of the year, and within Cape Town with the rainy season (Martin et al., 2014a).

One of the most interesting findings from this study was the apparent difference in the degree of seasonal decline in breeding performance depending on the surrounding levels of urbanization. Declining productivity as breeding seasons progress is a well-known phenomenon, that has been documented in many bird species mostly in the Northern hemisphere (e.g., Both et al., 2004; Grüebler and Naef-Daenzer, 2010), but increasingly also in the Southern hemisphere (e.g., Garcia-Heras et al., 2016; Murgatroyd et al., 2016), including previously in our study system (Martin et al., 2014b). In

addition to this pattern, however, we also found a convincing indication that the level of this decline differed depending on the extent of urbanization surrounding the nests. Thus, it

appears that earlier on in the season productivity (the number of chicks produced) is highest for pairs breeding in more urban

habitats, but later on this advantage disappears. In fact, when focussing on breeding success, we found significantly higher

sample size. Note that all quantitative variables were scaled. Model details in

probability of nest failure in more urbanized areas later in the season.

Several other recent studies have documented differences in seasonal declines in breeding parameters between habitats, but usually on a larger geographical scale. For example, a similar result was noted for another South African raptor, the black harrier Circus maurus. Although the nests in that study were located in the different habitats, they were also often separated by large distances. The differences in seasonal declines in clutch size and to a lesser degree productivity that were found in that study were thought to be driven by differences in weather conditions between the areas (Garcia-Heras et al., 2016). Likewise Zárybnická et al. (2015) found different levels of seasonal declines between Tengmalm's owl Aegolius funereus breeding in either boreal or temperate forests. Here the differences were attributed to varying prey availability (small mammals) over the season influencing nestling mortality and failure rates. The only other study, as far as we know, to explore this issue in relation to urbanization, is the study by Wawrzyniak et al. (2015) on blue tits (Parus major). This study found that clutch size tended to decline during the breeding season in an urban setting but not within forest habitat. They also attributed this result largely to better prey availability within the forest habitat. Thus, in these other studies changes in either weather or prey were thought to be the drivers for differences in seasonal declines.

What might then be responsible for the relationship witnessed in our current study? It could be that food is more abundant in urban habitat earlier in the season. This could be the case if timing of breeding of avian prey species in urban areas follows the more usually trend seen for birds (Chamberlain et al., 2009). Thus prey may be more available, especially inexperienced juvenile prey, earlier on in the season, which may account for the higher productivity of earlier breeding black sparrowhawks within urban habitats compared to their rural conspecifics. Later on in the season, the situation may be reversed due to the later breeding of prey species in the more rural habitats. Unfortunately we have no information on the timing of breeding on any of the key prey species in the area in relation to urbanization nor are we aware of a seasonal variation in prey abundance (Suri et al., 2017). Additionally, we found that differences in productivity were principally driven by nest failures rather than differences in brood sizes of successful nests. Thus, the mechanism for the differential seasonal decline may not be food related, since prey abundance would be expected to impact brood sizes rather than to induce complete nest failures. Alternatively, we know from other studies that one of the main causes of nest failure in this black sparrowhawks is nest usurpation by Egyptian geese (Alopochen aegyptiaca, Curtis et al., 2007) and that this increases later in the breeding season (Sumasgutner et al., 2016a). Thus it may be that usurpation in more prevalent in more urbanized areas, since both species have recently expanded their South African range into the urban and suburban habitats of Cape Town (see Mangnall and Crowe, 2002 on Egyptian geese). This might further explain the significantly lower breeding success in more urbanized habitats later in the season, since the breeding season of Egyptian geese peaks later in the year (MacLean, 1997; Carboneras and Kirwan, 2016); when also the threat of nest usurpation increases (Sumasgutner et al., 2016a).

Table 1.

## CONCLUSION

In this study we found no evidence that black sparrowhawks suffer from a decrease in breeding performance as a result of living in an urban environment, but timing of breeding was important, specifically in more urbanized areas. To summarize, black sparrowhawks appear to have successfully navigated various steps outlined by Møller (2014) of becoming an urban raptor species, which was likely facilitated by a reliable food supply (Suri et al., 2017) and abundant nesting opportunities in the form of alien tree species (Malan and Robinson, 1999).

## AUTHOR CONTRIBUTIONS

SR and PS contributed equally to this manuscript (shared first authorship). The original idea and study design came from PS

### REFERENCES

and AA. AK performed most of the field work; help by others is accordingly acknowledged. SR and PS analyzed the data. The manuscript was prepared by SR, PS, and AA.

## FUNDING

The Black Sparrowhawk project is funded by the DST-NRF Centre of Excellence. SR was supported by the NRF and PS by the Claude Leon Foundation.

### ACKNOWLEDGMENTS

We are grateful to the Black Sparrowhawk team—Johan Koeslag, Mark Cowen, Odette Curtis, Gareth Tate, Margaret MacIver and Sharon Pryce—for the long-term population monitoring, data collection and extensive support in the field.

Newton, I. (1998). Population Limitation in Birds. Oxford: Academic Press.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Rose, Sumasgutner, Koeslag and Amar. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Mechanisms Associated with an Advance in the Timing of Seasonal Reproduction in an Urban Songbird

Adam M. Fudickar <sup>1</sup> \*, Timothy J. Greives <sup>2</sup> , Mikus Abolins-Abols <sup>1</sup> , Jonathan W. Atwell <sup>1</sup> , Simone L. Meddle<sup>3</sup> , Guillermo Friis <sup>4</sup> , Craig A. Stricker <sup>5</sup> and Ellen D. Ketterson<sup>1</sup>

<sup>1</sup> Department of Biology, Indiana University, Bloomington, IN, United States, <sup>2</sup> Department of Biological Sciences and Environmental and Conservation Sciences Program, North Dakota State University, Fargo, ND, United States, <sup>3</sup> The Roslin Institute, The Royal (Dick) School of Veterinary Studies, The University of Edinburgh, Midlothian, United Kingdom, <sup>4</sup> Museo Nacional de Ciencias Naturales (CSIC), Madrid, Spain, <sup>5</sup> Fort Collins Science Center, US Geological Survey, Fort Collins, CO, United States

#### Edited by:

Diego Gil, Museo Nacional de Ciencias Naturales (CSIC), Spain

#### Reviewed by:

Pierre J. Deviche, Arizona State University, United States Oscar Gordo, Estación Biológica de Doñana (CSIC), Spain

> \*Correspondence: Adam M. Fudickar<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Behavioral and Evolutionary Ecology, a section of the journal Frontiers in Ecology and Evolution

> Received: 31 January 2017 Accepted: 12 July 2017 Published: 02 August 2017

#### Citation:

Fudickar AM, Greives TJ, Abolins-Abols M, Atwell JW, Meddle SL, Friis G, Stricker CA and Ketterson ED (2017) Mechanisms Associated with an Advance in the Timing of Seasonal Reproduction in an Urban Songbird. Front. Ecol. Evol. 5:85. doi: 10.3389/fevo.2017.00085 The colonization of urban environments by animals is often accompanied by earlier breeding and associated changes in seasonal schedules. Accelerated timing of seasonal reproduction in derived urban populations is a potential cause of evolutionary divergence from ancestral populations if differences in physiological processes that regulate reproductive timing become fixed over time. We compared reproductive development in free-living and captive male dark-eyed juncos deriving from a population that recently colonized a city (∼35 years) and ceased migrating to that of conspecifics that live in sympatry with the urban population during winter and spring but migrate elsewhere to breed. We predicted that the earlier breeding sedentary urban birds would exhibit accelerated reproductive development in the spring along the hypothalamic-pituitary-gonadal (HPG) axis as compared to migrants. We found that free-living sedentary urban and migrant juncos differed at the level of the pituitary when measured as baseline luteinizing hormone (LH) levels, but not in increased LH when challenged with Gonadotropin-Releasing Hormone (GnRH). Among captives held in a common garden, and at the level of the gonad, we found that sedentary urban birds produced more testosterone in response to GnRH than migrants living in the same common environment, suggesting greater gonadal sensitivity in the derived urban population. Greater gonadal sensitivity could arise from greater upstream activation by LH or FSH or from reduced suppression of gonadal development by the adrenal axis. We compared abundance of gonadal transcripts for LH receptor (LHR), follicle stimulating hormone receptor (FSHR), glucocorticoid receptor (GR), and mineralocorticoid receptor (MR) in the common-garden, predicting either more abundant transcripts for LHR and FSHR or fewer transcripts for GR and MR in the earlier breeding sedentary urban breeders, as compared to the migrants. We found no difference in the expression of these genes. Together these data suggest that advanced timing of reproduction in a recently derived urban population is facilitated by earlier increase in upstream baseline activity of the HPG and earlier release from gonadal suppression by yet-to-be-discovered mechanisms. Evolutionarily, our results suggest that potential for gene flow between seasonally sympatric populations may be limited due to urban-induced advances in the timing of reproduction and resulting allochrony with ancestral forms.

Keywords: seasonal breeding, phenology, supplemental cues, photoperiod, migration, songbird, gonads, urbanization

#### INTRODUCTION

Over the past several centuries, urban landscapes have replaced native habitat in many parts of the world (Marzluff, 2016; Alberti et al., 2017). Urban environments can differ from the environments they replace in many ways, and while human activity in urban areas can pose novel challenges to animals (Shanahan et al., 2014), relatively mild urban microclimates and enhanced resource availability can also lead to increased opportunities to reproduce (Deviche and Davies, 2013).

A common observation is that urban bird populations begin their breeding season earlier compared to rural populations (Deviche and Davies, 2013). Seasonal fluctuations in climate and resource availability are often reduced in urban environments, which can lead to longer breeding seasons and year-round occupancy by formerly migratory lineages (Adriaensen and Dhondt, 1990; Partecke and Gwinner, 2007; Chamberlain et al., 2009; Atwell et al., 2014). If the physiological processes that lead to differences between urban and rural populations in reproductive timing and other seasonal traits become fixed over time, then gene flow and interbreeding could be limited (Evans et al., 2010; Fudickar et al., 2016a).

Though it is clear that urbanization can advance timing of reproduction, relatively little is known about the sources of variation in reproductive physiology that modulate the advancement or extension of breeding. The hypothalamicpituitary-gonadal (HPG) axis plays a central role in the seasonal regulation of reproduction in birds (Dawson, 2015; **Figure 1**). The timing and rate of the seasonal activation of the HPG is modulated by a combination of predictive (day length) and supplementary cues (e.g., food and temperature; Hahn et al., 2005; Schaper et al., 2012; Wingfield et al., 2012; Dawson, 2015; Watts et al., 2015). Advancement in breeding phenology in urban habitats, therefore, may result from a plastic response to the novel habitat by phenotypically flexible individuals and/or a genetic response to selection for earlier seasonal activation of the HPG. Whether or not differences in timing are fixed or flexible may have important evolutionary implications. For example, if advanced reproduction in urban populations is the result of selection for greater sensitivity of the HPG at shorter day lengths—either by an earlier seasonal response or responding at a faster rate—then the resulting reproductive allochrony (asynchronous reproduction) may limit gene flow between urban and rural populations, ultimately leading to selection against hybrids. Alternatively, if advanced timing in urban populations is modulated by plastic responses, then the scope for divergence

may be limited, as immigrants would be capable of adopting the timing of established urban populations. These alternatives also have important implications for the adaptability of both urban and rural populations to cope with future environmental change.

To date, there is limited evidence from studies that have compared the endocrine axes of urban vs. nearby rural populations in both field and common garden settings to support the importance of both plasticity and genetics in modulating earlier reproduction and HPG activation in urban habitats (Partecke et al., 2004, 2006; Schoech, 2009; Atwell et al., 2014; Davies et al., 2015). While these studies highlight the importance of differential HPG activity in facilitating earlier and extended breeding in urban habitats, much remains to be learned about the generality of those results, as well as the specific mechanistic sources of variation across the many "levels" of the HPG axis. Additionally, one interpretive challenge with such prior studies is that they typically sample putatively related populations that inhabit nearby yet radically different urban vs. rural habitats during the sampling period. In our study, we utilized both field and common garden settings to probe multiple levels of HPG activity in both urban-breeding and migratory nonresident populations during a critical life-history stage in which they co-occur in urban habitats, yet pursue divergent seasonal strategies.

Heteropatry—where closely related populations coexist during the non-breeding season (e.g., fall, winter, and /or early spring) yet pursue independent phenological and geographical trajectories during breeding—provides a unique opportunity to address the physiological mechanisms that underlie reproductive timing differences between populations (Winker, 2010; Ketterson et al., 2015; Bauer et al., 2016; Fudickar et al., 2016a,b; Greives et al., 2016; Ramenofsky et al., 2017). Broadly distributed avian species often have heteropatric distributions, in which some populations migrate while others do not (Winker, 2010). Despite experiencing the same photoperiodic and supplementary cues for much of the annual cycle (e.g., from the time that migrants arrive in the fall until they depart in the spring), there is often distinct phenological divergence in spring, when sedentary individuals transition into breeding, while migrants delay reproduction and prepare for and complete a migration before breeding. As a result, closely related sedentary and migratory birds are effectively sympatric during the nonbreeding season as well as the transitional periods between non-breeding and breeding, yet are entirely allopatric during the core breeding season. In the current study, we compared specific neuroendocrine mechanisms predicted to underlie reproductive timing differences between a recently established (∼35 years) urban population of juncos vs. heteropatric ancestral migrant juncos, under both free-living (field) and common-garden (captive) conditions.

This study focused on a population of dark-eyed juncos (J. hyemalis thurberi) that became established in urban San Diego CA USA in the early 1980s, likely as a result of overwintering migratory birds remaining to breed (Rasner et al., 2004; Yeh, 2004; Atwell et al., 2012, 2014). Since then, an isolated sedentary urban breeding population has persisted with limited subsequent immigration despite a sympatric distribution with migratory juncos between October and April (Yeh and Price, 2004; Unitt, 2005). The colonization event and current heteropatric distribution thus provides a unique opportunity to examine mechanisms associated with contemporary adaptation to an urban environment. Although the precise breeding location of the migrants is not known, previous studies have shown that first egg laying dates in the San Diego population are ∼2.5 months earlier than the closest non-urban populations (Yeh and Price, 2004; Atwell et al., 2014). A recent study, in which juncos from San Diego and the closest non-urban breeding population (∼50 km) were captured on their respective breeding grounds as juveniles and observed in a commongarden experiment, found that population differences in the seasonal timing of mean peak HPG activity (plasma testosterone and post Gonadotropin-Releasing Hormone (GnRH) challenge testosterone) observed in the wild did not persist in the common garden (Atwell et al., 2014), which suggests a degree of flexibility in reproductive timing in both populations.

Here, we asked whether differences in the timing of the seasonal onset of reproduction between heteropatric sedentary urban and migratory juncos result from differential activation of the HPG in early spring and whether any difference is linked to differences in sensitivity to day length. While Atwell et al. (2014) compared the duration and peak timing of HPG activity of sedentary urban juncos from San Diego with that of the closest non-urban population, in the current study we focused on a comparison of sedentary urban breeders with migrants that are sympatric during winter and early spring. Based on previous reports indicating that winter flocks of juncos consist of migrants from a range of breeding populations (Fudickar et al., 2016a; J. Atwell personal observations), we expected that migrant juncos in San Diego would originate from multiple breeding populations.

First, we compared free-living sedentary urban and migratory juncos on their shared overwintering grounds in February for responsiveness of the HPG endocrine axis—specifically the response of the pituitary gland to repeated GnRH stimulation. By repeatedly injecting birds with GnRH, our aim was to more accurately replicate the pulsatile nature of GnRH that occurs naturally in spring. By measuring luteinizing hormone (LH) following repeated injections of GnRH, we assessed the potential for variation in pituitary sensitivity to modulate differential HPG axis activity in resident vs. migrant cohorts during the period when their observed seasonal strategies diverge, despite exposure to identical environmental conditions. We have previously demonstrated in longer diverged (∼15,000 years) overwintering juncos that pituitary sensitivity to repeated GnRH injection may serve to functionally limit HPG axis activation in female, but not male migrants (Greives et al., 2016). The hypothesis to be tested here was whether migrant birds have a weaker response to GnRH as would be predicted if reduced pituitary sensitivity to repeated GnRH acts as a mechanism to delay reproduction in birds that will forego breeding in the urban environment and migrate prior to reproduction.

Next, we measured morphological and physiological correlates of reproductive development across 4 weeks in early spring in sedentary urban and migrant junco males held in a captive common-garden experiment that simulated natural photoperiod (during the same early spring period), and provided ad libitum food, mild temperatures, and reduced social interactions. If the seasonal timing of reproduction in sedentary urban juncos is advanced due to increased sensitivity to photoperiodic cues in early spring, we predicted that timing of gonadal development and endocrine profiles of urban-breeding and migrant juncos should remain divergent. In contrast, if the seasonal timing of reproduction differs in sedentary urban and migrant juncos due to flexible responses to the environment, we predicted convergence of seasonal timing of gonadal development and endocrine profiles in captivity. We limited our study to male juncos because, in captivity, females do not advance into reproduction in the spring as readily as males (E.K., personal observation).

Finally, we compared gonadal transcript abundance of LH receptor (LHR), follicle stimulating hormone receptor (FSHR), glucocorticoid receptor (GR), and mineralocorticoid receptor (MR) of sedentary urban and migrant juncos in early spring. LHR and FSHR are important for seasonal gonadal growth in birds, and GR and MR can suppress gonadal growth and activation in birds (McGuire et al., 2013; Blas, 2015; Vizcarra et al., 2015). Therefore, we predicted higher LHR and FSHR transcript abundance in sedentary urban juncos in early spring and higher GR and/or MR transcript abundance in migrant juncos in early spring.

We interpret our collective results in the context of both the proximate mechanisms underlying phenotypic divergence in reproductive timing, as well as the ultimate factors and evolutionary implications associated with the observed physiological divergences and convergences.

## METHODS

## Field Study

#### Capture and Morphological Measures

Thirty-seven male juncos were captured in the urban habitat of the University of California-San Diego (UCSD) campus in La Jolla, California, USA, using continuously monitored walk-in traps baited with seed. Birds in the field study were classified post hoc as sedentary urban breeders or migrants using a combination of morphological measures and stable isotopes in feather tissue (hydrogen; details below). We collected the outermost secondary feather from each bird's right wing during handling to examine stable hydrogen (δ <sup>2</sup>H) signatures. Using calipers, we measured the length of the right tarsus (± 0.1 mm). Using a ruler, we measured the length of the right wing (flattened) and the tail (± 0.5 mm). Previous studies have reported that juncos that breed in San Diego have reduced white on their tail feathers and black on their heads compared to rural migratory populations (Yeh, 2004; Atwell et al., 2014). We visually estimated the percentage of white in the four outermost right tail feathers of each bird at capture. Using calipers, we measured (a) the length of the entire hood from the bill to the ventral terminus and (b) the length from the bill to the ventral terminus of homogenous black feathers. Age class was determined based on wing plumage as juncos have a limited first prebasic molt, making it possible to distinguish between first-year and after-first-year birds (Nolan, 2002). Capture dates were from 13 February to 23 February 2014, ∼4 weeks prior to the historical first egg date in the resident population, and roughly 2–3 weeks prior to the apparent peak of migrant passage through southern California (J.W. Atwell personal observation). All individuals received a unique numbered metal band to avoid repeat sampling. All procedures were approved by the Indiana University, North Dakota State University, and University of California San Diego Institutional Animal Care and Use Committees and conducted under appropriate federal banding permits (permit #20261) and the California Department of Fish and Wildlife (permit #007852).

#### Blood Sample Collection—Field

Immediately after capture, a 50 µl blood sample was obtained from the alar wing vein for baseline measures of LH. The birds then received an intramuscular injection of 1.25 µg chicken GnRH (American Peptide, Sunnyvale, CA) in 50 µl of phosphatebuffered saline into the pectoralis muscle. This dose fully activates the HPG axis in this species (Jawor et al., 2006). Five minutes following the first GnRH injection, a second 50 µl blood sample was taken from the wing vein for LH. Previous work in this species and white-crowned sparrows (Zonotrichia leucophrys gambelii) indicated LH levels reach a peak 5 min following injection and then significantly decline by 20 min and return to baseline at 30 min (Wingfield et al., 1979; Bergeon Burns et al., 2014). The birds then received a second injection of GnRH (same dose as above) 30 min after the first injection, followed by a third injection of GnRH (same dose as above) 30 min following the second injection. A final 50 µl blood sample was collected 5 min following the third injection of GnRH (65 min after the first GnRH injection). All birds were held in an opaque paper bag between injections. Blood samples were stored on ice until later processing in the laboratory. Samples were centrifuged to separate red blood cells from plasma. Plasma was collected with a Hamilton syringe and stored at −20◦C until it was shipped on dry ice to The Roslin Institute, The University of Edinburgh for LH quantification.

#### Luteinizing Hormone Assay

To determine plasma LH, we used a micromodification of the radioimmunoassay described previously (Sharp et al., 1987). Briefly, the assay reaction volume was 60 µl, comprised of 20 µl of plasma sample or standard, 20 µl of primary rabbit LH antibody, and 20 µl of I125-labeled LH. The primary antibody was precipitated to separate free and bound I<sup>125</sup> label using 20 µl of donkey anti-rabbit precipitating serum and 20 µl of nonimmune rabbit serum. All samples were measured in duplicate in a single assay. The intra-assay coefficient of variation was 2.9% and the minimum detectable dose was 0.2 ng/ml. This LH radioimmunoassay has been used extensively in many avian species including dark-eyed juncos (Deviche et al., 2000; Meddle et al., 2002; Wingfield et al., 2012; Bergeon Burns et al., 2014; Greives et al., 2016).

#### Common Garden Study Bird Capture and Housing