Boldness behavior and stress physiology in a novel urban environment suggest rapid correlated evolutionary adaptation

Abstract

Novel or changing environments expose animals to diverse stressors that likely require coordinated hormonal and behavioral adaptations. Predicted adaptations to urban environments include attenuated physiological responses to stressors and bolder exploratory behaviors, but few studies to date have evaluated the impact of urban life on codivergence of these hormonal and behavioral traits in natural systems. Here, we demonstrate rapid adaptive shifts in both stress physiology and correlated boldness behaviors in a songbird, the dark-eyed junco, following its colonization of a novel urban environment. We compared elevation in corticosterone (CORT) in response to handling and flight initiation distances in birds from a recently established urban population in San Diego, California to birds from a nearby wildland population in the species' ancestral montane breeding range. We also measured CORT and exploratory behavior in birds raised from early life in a captive common garden study. We found persistent population differences for both reduced CORT responses and bolder exploratory behavior in birds from the colonist population, as well as significant negative covariation between maximum CORT and exploratory behavior. Although early developmental effects cannot be ruled out, these results suggest contemporary adaptive evolution of correlated hormonal and behavioral traits associated with colonization of an urban habitat.

Full text

Behavioral Ecology
doi:10.1093/beheco/ars059
Advance Access publication 4 May 2012
May 4
Behavioral Ecology
doi:10.1093/beheco/ars059
Original Article
Boldness behavior and stress physiology
in a novel urban environment suggest rapid
correlated evolutionary adaptation
Jonathan W. Atwell,
a
Goncxalo C. Cardoso,
b
Danielle J. Whittaker,
a,c
Samuel Campbell-Nelson,
a
Kyle W. Robertson,
a
and Ellen D. Ketterson
a
a
Department of Biology, Indiana University, 1001 E. 3rd Street, Bloomington, IN 47405, USA,
b
CIBIO—Centro de Investigacxa
˜o em Biodiversidade e Recursos Gene´ticos, R. Padre Armando Quintas,
Universidade do Porto, Campus Agra´rio de Vaira
˜o, 4485-661 Vaira
˜o, Portugal, and
c
BEACON Center for
the Study of Evolution in Action, Michigan State University, 1441 Biomedical and Physical Sciences, 567
Wilson Road, East Lansing, MI 48824, USA
Novel or changing environments expose animals to diverse stressors that likely require coordinated hormonal and behavioral
adaptations. Predicted adaptations to urban environments include attenuated physiological responses to stressors and bolder
exploratory behaviors, but few studies to date have evaluated the impact of urban life on codivergence of these hormonal and
behavioral traits in natural systems. Here, we demonstrate rapid adaptive shifts in both stress physiology and correlated boldness
behaviors in a songbird, the dark-eyed junco, following its colonization of a novel urban environment. We compared elevation in
corticosterone (CORT) in response to handling and flight initiation distances in birds from a recently established urban
population in San Diego, California to birds from a nearby wildland population in the species’ ancestral montane breeding
range. We also measured CORT and exploratory behavior in birds raised from early life in a captive common garden study. We
found persistent population differences for both reduced CORT responses and bolder exploratory behavior in birds from the
colonist population, as well as significant negative covariation between maximum CORT and exploratory behavior. Although
early developmental effects cannot be ruled out, these results suggest contemporary adaptive evolution of correlated hormonal
and behavioral traits associated with colonization of an urban habitat. Key words: adaptation, boldness, corticosterone, evolution,
junco, urbanization. [Behav Ecol]
INTRODUCTION
Understanding how correlated behavioral and physiologi-
cal traits respond to new or changing environmental con-
ditions has implications for both the study of basic evolutionary
processes and for predicting and managing biological
responses to anthropogenic global change (Cockrem 2005;
van Oers et al. 2011). For example, exposure to urbanization
or climate change likely requires rapid changes in multiple
traits in order for populations to persist (Gaston 2010; Møller
et al. 2010), yet much remains to be learned about whether
preexisting trait correlations act to constrain or facilitate the
developmental or genetic responses that enable persistence
(Agrawal and Stinchcombe 2009; Ketterson et al. 2009). In
particular, few studies have simultaneously evaluated how cor-
related characters, such as behavioral traits and associated
hormonal mechanisms, respond to new environments.
Furthermore, it is often unclear the degree to which pheno-
typic plasticity or genetic evolution may underlie observed
differences in behavior or physiology among divergent popu-
lations (Diamond 1986; Møller 2008; Angelier et al. 2011),
and the interplay of both processes is likely to be significant.
In this study, we examined changes in tameness and boldness
behaviors associated with the recent establishment of a song-
bird population in a novel urban environment, as well as
changes in the endocrine stress response, a physiological
mechanism hypothesized to underlie behavioral variation in
personality traits.
Recent research in animal behavior and neuroendocrinol-
ogy has demonstrated that individual animals vary consistently
in suites of behavioral traits called ‘‘animal personality’’
(Wilson et al. 1994; Sih et al. 2004), including those described
as ‘‘boldness’’ or ‘‘neophobia’’ behaviors. Boldness can be gen-
erally defined as the tendency of individuals to be exploratory
and take risks, particularly in novel contexts (Wilson et al.
1994), and there is evidence that underlying hormonal mech-
anisms play a key role in modulating such behavioral differ-
ences (Koolhaas et al. 1999; van Oers et al. 2011). One such
personality trait, described as ‘‘early exploratory behavior,’’
generally measures how quickly and/or extensively individuals
move through and examine a novel environment. Studies of
early exploratory behavior in birds have demonstrated that
this trait can be repeatable among individuals (Verbeek
et al. 1994), heritable in parent–offspring and sibling studies
(Dingemanse et al. 2002), and responsive to artificial selection
(Drent et al. 2003; van Oers, Derent, de Goede, et al. 2004).
Exploratory behavior has also been shown to be a target of both
natural and sexual selection within free-living populations
Address correspondence to J.W. Atwell. E-mail: jwatwell@indiana.
edu.
Received 10 November 2011; revised 19 March 2012; accepted 22
March 2012.
The Author 2012. Published by Oxford University Press on behalf of
the International Society for Behavioral Ecology. All rights reserved.
For permissions, please e-mail: journals.permissions@oup.com
(Dingemanse et al. 2004; van Oers et al. 2008), and its expres-
sion can be influenced by both genetic and environmental fac-
tors (van Oers, Derent, de Jong, et al. 2004; van Oers et al. 2005).
Collectively, these prior studies indicate that boldness behav-
iors, such as exploratory and risk-taking behaviors, should
diverge among populations occupying environments that favor
shy or bold individuals (Fidler et al. 2007), such as wildland
versus urban habitats, respectively, but few studies have
reported evidence for adaptive evolution of personality traits
across populations (but for examples in fish, see Dingemanse
et al. 2007; Herczeg et al. 2009). Several studies have provided
evidence for divergence of behavioral phenotypes in association
with urbanization (e.g., Møller 2008; Evans et al. 2010), but
typically not evaluating whether phenotypic plasticity or genetic
differences likely underlie it (Diamond 1986; Møller 2008). In
this paper, we focus on early exploratory behavior and another
behavioral trait, ‘‘flight initiation distance’’ (FID) or ‘‘flush
distance,’’ which can also be interpreted in relation to boldness
and tameness (Møller 2008). FID also shows consistent individ-
ual variation in avian species (Carrete and Tella 2010) and has
been associated with adaptation to urbanization in many
animals (Møller 2008). We refer to ‘‘boldness’’ generally in
our discussion of behavior, as it pertains to both FID and early
exploratory behavior (EEB) assays, which were conducted in
our field and common garden studies, respectively.
A primary physiological mechanism underlying individual dif-
ferences in boldness behaviors is the endocrine stress response,
particularly that of the hypothalamic–pituitary–adrenal (HPA)
axis (Koolhaas et al. 1999; van Oers et al. 2011). Acute eleva-
tions in corticosterone (CORT) levels are often associated with
adaptive short-term behavioral and physiological survival
responses such as self-maintenance behaviors and mobilization
of energy reserves, while chronically elevated CORT levels may
reduce survival (Breuner et al. 2008). Generally, shyer or more
passive behavioral phenotypes are predicted to be associated
with more acute HPA axis activity and greater release of gluco-
corticoids in response to short-term stressors and vice versa for
bolder or more assertive behavioral phenotypes (Koolhaas et al.
1999). Results from artificial selection and observational studies
support these predictions in birds and mammals (Korte et al.
1997; Lendvai et al. 2011) (but see Martins et al. 2007).
Comparing populations, CORT levels appear to be repeat-
able, to have a genetic basis, and thus to have the potential
to evolve rapidly (Evans et al. 2006; Partecke et al. 2006;
Rensel and Schoech 2011). CORT levels vary among subspe-
cies that differ in their life-history (e.g., Angelier et al. 2011)
and they have been associated with adaptation to urban
environments (e.g., Partecke et al. 2006; Fokidis et al. 2009)
and to ecotourism (e.g., Romero and Wikelski 2002).
The research presented here is unique in simultaneously
examining population divergence in both a behavioral trait
and an associated hormonal mechanism, and in evaluating
plasticity versus genetic evolution as possible causes. We
sampled behavior and plasma CORT levels in free-living
individuals from two recently diverged songbird populations,
one montane and the other a costal urban colonist, and in
captives from these populations raised in a common garden
experiment. These populations diverged following a recent
colonization event (;1983), in which a historically montane
forest–breeding songbird species, the dark-eyed junco (Junco
hyemalis), colonized a novel urban environment in Southern
California, USA. Since establishment, the colonist population
has persisted as a small (’80 pairs) but stable population, and
it exists as an effective biogeographic island in the species-
atypical urban and coastal habitat, isolated from the ancestral
range mountain populations ’70 km inland to the east.
Details of the colonization event and the study system, includ-
ing previously characterized behavioral and morphological
differences in this system, are described below (see MATERI-
ALS AND METHODS).
We tested 1) whether tameness and boldness behaviors and
glucocorticoid levels differed in the novel urban habitat when
compared with a nearby ancestral range montane forest–
breeding population, 2) the degree to which these differences
likely represent phenotypic plasticity versus genetic evolution us-
ing a common garden approach, and 3) whether boldness
behaviors and glucocorticoid levels covary within and among
populations. The latter goal allows assessing if the evolutionary
responses of these traits are integrated or independent. Because
either selection post-colonization or genetic drift could both ac-
count for apparent genetic differences inferred between popu-
lations, we also further analyzed the possibility that drift alone
could explain the observed population divergences in this sys-
tem. Finally, we examined one possible molecular mechanism
underlying behavioral variation (a single-nucleotide
polymorphism [SNP 830] in the Drd4 dopamine receptor gene,
which has been previously associated with exploratory behavior
in a songbird Fidler et al. 2007; Korsten et al. 2010).
MATERIALS AND METHODS
Study system
We collected behavioral data and blood samples, and captured
birds for a common garden study from 2 recently diverged
dark-eyed junco populations in San Diego County, CA, USA.
One of the populations was recently (ca. 1983) established
in the novel urban and coastal habitat on the University of
California–San Diego (UCSD) campus (elevation 30 m,
3240#N, 11710#W), and the other is a nearby ancestral range
montane forest–breeding population near Mt Laguna, CA
(elevation 1700 m, 3252#N, 11625#W).
In far southern California, the breeding range of dark-eyed
juncos (J. hyemalis thurberi) is confined to higher elevation
(e.g., .1500 m) forests and wooded canyons inland from
the coast (Miller 1941; Unitt 2005). However, in the early
1980s, a population of juncos became established on the coast
of the Pacific Ocean in an urban environment on the campus
of UCSD (Walens S, personal communication), most likely
established by a flock of wintering migrants staying to breed
(Yeh 2004; Yeh and Price 2004). Microsatellite DNA analyses
indicate genetic isolation of the San Diego juncos relative to
those of the montane breeding range (Rasner et al. 2004),
and they suggest that a sizeable number of individuals
founded the urban population (e.g., minimum n8–20
birds), such that phenotypic divergence due to a founder
effect is unlikely (Rasner et al. 2004; Yeh 2004). The San
Diego colonist population has remained small but stable at
about 80 breeding pairs over the past decade, and it consti-
tutes an effective biogeographic island, since the closest
montane forest–breeding ranges lies ’70 km inland to the
east, separated by unsuitable habitat (Unitt 2005).
The environment experienced by the urban colonist popu-
lation differs in many ways from that in the ancestral range,
owing to both its mild coastal climate and urban setting:
reduced variation in temperature and rainfall, increased noise
and light levels, constant human disturbances (e.g., vehicles
and foot traffic), altered acoustic transmission (Slabbekoorn
et al. 2007), novel predator communities (Suarez et al. 2005),
abundant ornamental vegetation and watered lawns, and
novel food and nesting resources (Yeh et al. 2007). Numerous
behavioral differences between the colonist and an ancestral
range population have been documented, including earlier
onset of reproduction (Yeh and Price 2004), loss of migratory
behavior (Yeh 2004), reduced male territorial responses (New-
man et al. 2006), altered song frequencies (Slabbekoorn et al.,
2Behavioral Ecology
at Michigan State University on September 4, 2012http://beheco.oxfordjournals.org/Downloaded from

Atwell et al. • Rapid divergence in boldness and corticosterone 961
Behavioral Ecology
doi:10.1093/beheco/ars059
Original Article
Boldness behavior and stress physiology
in a novel urban environment suggest rapid
correlated evolutionary adaptation
Jonathan W. Atwell,
a
Goncxalo C. Cardoso,
b
Danielle J. Whittaker,
a,c
Samuel Campbell-Nelson,
a
Kyle W. Robertson,
a
and Ellen D. Ketterson
a
a
Department of Biology, Indiana University, 1001 E. 3rd Street, Bloomington, IN 47405, USA,
b
CIBIO—Centro de Investigacxa
˜o em Biodiversidade e Recursos Gene´ticos, R. Padre Armando Quintas,
Universidade do Porto, Campus Agra´rio de Vaira
˜o, 4485-661 Vaira
˜o, Portugal, and
c
BEACON Center for
the Study of Evolution in Action, Michigan State University, 1441 Biomedical and Physical Sciences, 567
Wilson Road, East Lansing, MI 48824, USA
Novel or changing environments expose animals to diverse stressors that likely require coordinated hormonal and behavioral
adaptations. Predicted adaptations to urban environments include attenuated physiological responses to stressors and bolder
exploratory behaviors, but few studies to date have evaluated the impact of urban life on codivergence of these hormonal and
behavioral traits in natural systems. Here, we demonstrate rapid adaptive shifts in both stress physiology and correlated boldness
behaviors in a songbird, the dark-eyed junco, following its colonization of a novel urban environment. We compared elevation in
corticosterone (CORT) in response to handling and flight initiation distances in birds from a recently established urban
population in San Diego, California to birds from a nearby wildland population in the species’ ancestral montane breeding
range. We also measured CORT and exploratory behavior in birds raised from early life in a captive common garden study. We
found persistent population differences for both reduced CORT responses and bolder exploratory behavior in birds from the
colonist population, as well as significant negative covariation between maximum CORT and exploratory behavior. Although
early developmental effects cannot be ruled out, these results suggest contemporary adaptive evolution of correlated hormonal
and behavioral traits associated with colonization of an urban habitat. Key words: adaptation, boldness, corticosterone, evolution,
junco, urbanization. [Behav Ecol]
INTRODUCTION
Understanding how correlated behavioral and physiologi-
cal traits respond to new or changing environmental con-
ditions has implications for both the study of basic evolutionary
processes and for predicting and managing biological
responses to anthropogenic global change (Cockrem 2005;
van Oers et al. 2011). For example, exposure to urbanization
or climate change likely requires rapid changes in multiple
traits in order for populations to persist (Gaston 2010; Møller
et al. 2010), yet much remains to be learned about whether
preexisting trait correlations act to constrain or facilitate the
developmental or genetic responses that enable persistence
(Agrawal and Stinchcombe 2009; Ketterson et al. 2009). In
particular, few studies have simultaneously evaluated how cor-
related characters, such as behavioral traits and associated
hormonal mechanisms, respond to new environments.
Furthermore, it is often unclear the degree to which pheno-
typic plasticity or genetic evolution may underlie observed
differences in behavior or physiology among divergent popu-
lations (Diamond 1986; Møller 2008; Angelier et al. 2011),
and the interplay of both processes is likely to be significant.
In this study, we examined changes in tameness and boldness
behaviors associated with the recent establishment of a song-
bird population in a novel urban environment, as well as
changes in the endocrine stress response, a physiological
mechanism hypothesized to underlie behavioral variation in
personality traits.
Recent research in animal behavior and neuroendocrinol-
ogy has demonstrated that individual animals vary consistently
in suites of behavioral traits called ‘‘animal personality’’
(Wilson et al. 1994; Sih et al. 2004), including those described
as ‘‘boldness’’ or ‘‘neophobia’’ behaviors. Boldness can be gen-
erally defined as the tendency of individuals to be exploratory
and take risks, particularly in novel contexts (Wilson et al.
1994), and there is evidence that underlying hormonal mech-
anisms play a key role in modulating such behavioral differ-
ences (Koolhaas et al. 1999; van Oers et al. 2011). One such
personality trait, described as ‘‘early exploratory behavior,’’
generally measures how quickly and/or extensively individuals
move through and examine a novel environment. Studies of
early exploratory behavior in birds have demonstrated that
this trait can be repeatable among individuals (Verbeek
et al. 1994), heritable in parent–offspring and sibling studies
(Dingemanse et al. 2002), and responsive to artificial selection
(Drent et al. 2003; van Oers, Derent, de Goede, et al. 2004).
Exploratory behavior has also been shown to be a target of both
natural and sexual selection within free-living populations
Address correspondence to J.W. Atwell. E-mail: jwatwell@indiana.
edu.
Received 10 November 2011; revised 19 March 2012; accepted 22
March 2012.
The Author 2012. Published by Oxford University Press on behalf of
the International Society for Behavioral Ecology. All rights reserved.
For permissions, please e-mail: journals.permissions@oup.com
(Dingemanse et al. 2004; van Oers et al. 2008), and its expres-
sion can be influenced by both genetic and environmental fac-
tors (van Oers, Derent, de Jong, et al. 2004; van Oers et al. 2005).
Collectively, these prior studies indicate that boldness behav-
iors, such as exploratory and risk-taking behaviors, should
diverge among populations occupying environments that favor
shy or bold individuals (Fidler et al. 2007), such as wildland
versus urban habitats, respectively, but few studies have
reported evidence for adaptive evolution of personality traits
across populations (but for examples in fish, see Dingemanse
et al. 2007; Herczeg et al. 2009). Several studies have provided
evidence for divergence of behavioral phenotypes in association
with urbanization (e.g., Møller 2008; Evans et al. 2010), but
typically not evaluating whether phenotypic plasticity or genetic
differences likely underlie it (Diamond 1986; Møller 2008). In
this paper, we focus on early exploratory behavior and another
behavioral trait, ‘‘flight initiation distance’’ (FID) or ‘‘flush
distance,’’ which can also be interpreted in relation to boldness
and tameness (Møller 2008). FID also shows consistent individ-
ual variation in avian species (Carrete and Tella 2010) and has
been associated with adaptation to urbanization in many
animals (Møller 2008). We refer to ‘‘boldness’’ generally in
our discussion of behavior, as it pertains to both FID and early
exploratory behavior (EEB) assays, which were conducted in
our field and common garden studies, respectively.
A primary physiological mechanism underlying individual dif-
ferences in boldness behaviors is the endocrine stress response,
particularly that of the hypothalamic–pituitary–adrenal (HPA)
axis (Koolhaas et al. 1999; van Oers et al. 2011). Acute eleva-
tions in corticosterone (CORT) levels are often associated with
adaptive short-term behavioral and physiological survival
responses such as self-maintenance behaviors and mobilization
of energy reserves, while chronically elevated CORT levels may
reduce survival (Breuner et al. 2008). Generally, shyer or more
passive behavioral phenotypes are predicted to be associated
with more acute HPA axis activity and greater release of gluco-
corticoids in response to short-term stressors and vice versa for
bolder or more assertive behavioral phenotypes (Koolhaas et al.
1999). Results from artificial selection and observational studies
support these predictions in birds and mammals (Korte et al.
1997; Lendvai et al. 2011) (but see Martins et al. 2007).
Comparing populations, CORT levels appear to be repeat-
able, to have a genetic basis, and thus to have the potential
to evolve rapidly (Evans et al. 2006; Partecke et al. 2006;
Rensel and Schoech 2011). CORT levels vary among subspe-
cies that differ in their life-history (e.g., Angelier et al. 2011)
and they have been associated with adaptation to urban
environments (e.g., Partecke et al. 2006; Fokidis et al. 2009)
and to ecotourism (e.g., Romero and Wikelski 2002).
The research presented here is unique in simultaneously
examining population divergence in both a behavioral trait
and an associated hormonal mechanism, and in evaluating
plasticity versus genetic evolution as possible causes. We
sampled behavior and plasma CORT levels in free-living
individuals from two recently diverged songbird populations,
one montane and the other a costal urban colonist, and in
captives from these populations raised in a common garden
experiment. These populations diverged following a recent
colonization event (;1983), in which a historically montane
forest–breeding songbird species, the dark-eyed junco (Junco
hyemalis), colonized a novel urban environment in Southern
California, USA. Since establishment, the colonist population
has persisted as a small (’80 pairs) but stable population, and
it exists as an effective biogeographic island in the species-
atypical urban and coastal habitat, isolated from the ancestral
range mountain populations ’70 km inland to the east.
Details of the colonization event and the study system, includ-
ing previously characterized behavioral and morphological
differences in this system, are described below (see MATERI-
ALS AND METHODS).
We tested 1) whether tameness and boldness behaviors and
glucocorticoid levels differed in the novel urban habitat when
compared with a nearby ancestral range montane forest–
breeding population, 2) the degree to which these differences
likely represent phenotypic plasticity versus genetic evolution us-
ing a common garden approach, and 3) whether boldness
behaviors and glucocorticoid levels covary within and among
populations. The latter goal allows assessing if the evolutionary
responses of these traits are integrated or independent. Because
either selection post-colonization or genetic drift could both ac-
count for apparent genetic differences inferred between popu-
lations, we also further analyzed the possibility that drift alone
could explain the observed population divergences in this sys-
tem. Finally, we examined one possible molecular mechanism
underlying behavioral variation (a single-nucleotide
polymorphism [SNP 830] in the Drd4 dopamine receptor gene,
which has been previously associated with exploratory behavior
in a songbird Fidler et al. 2007; Korsten et al. 2010).
MATERIALS AND METHODS
Study system
We collected behavioral data and blood samples, and captured
birds for a common garden study from 2 recently diverged
dark-eyed junco populations in San Diego County, CA, USA.
One of the populations was recently (ca. 1983) established
in the novel urban and coastal habitat on the University of
California–San Diego (UCSD) campus (elevation 30 m,
3240#N, 11710#W), and the other is a nearby ancestral range
montane forest–breeding population near Mt Laguna, CA
(elevation 1700 m, 3252#N, 11625#W).
In far southern California, the breeding range of dark-eyed
juncos (J. hyemalis thurberi) is confined to higher elevation
(e.g., .1500 m) forests and wooded canyons inland from
the coast (Miller 1941; Unitt 2005). However, in the early
1980s, a population of juncos became established on the coast
of the Pacific Ocean in an urban environment on the campus
of UCSD (Walens S, personal communication), most likely
established by a flock of wintering migrants staying to breed
(Yeh 2004; Yeh and Price 2004). Microsatellite DNA analyses
indicate genetic isolation of the San Diego juncos relative to
those of the montane breeding range (Rasner et al. 2004),
and they suggest that a sizeable number of individuals
founded the urban population (e.g., minimum n8–20
birds), such that phenotypic divergence due to a founder
effect is unlikely (Rasner et al. 2004; Yeh 2004). The San
Diego colonist population has remained small but stable at
about 80 breeding pairs over the past decade, and it consti-
tutes an effective biogeographic island, since the closest
montane forest–breeding ranges lies ’70 km inland to the
east, separated by unsuitable habitat (Unitt 2005).
The environment experienced by the urban colonist popu-
lation differs in many ways from that in the ancestral range,
owing to both its mild coastal climate and urban setting:
reduced variation in temperature and rainfall, increased noise
and light levels, constant human disturbances (e.g., vehicles
and foot traffic), altered acoustic transmission (Slabbekoorn
et al. 2007), novel predator communities (Suarez et al. 2005),
abundant ornamental vegetation and watered lawns, and
novel food and nesting resources (Yeh et al. 2007). Numerous
behavioral differences between the colonist and an ancestral
range population have been documented, including earlier
onset of reproduction (Yeh and Price 2004), loss of migratory
behavior (Yeh 2004), reduced male territorial responses (New-
man et al. 2006), altered song frequencies (Slabbekoorn et al.,
2Behavioral Ecology
at Michigan State University on September 4, 2012http://beheco.oxfordjournals.org/Downloaded from

Behavioral Ecology962
2007; Cardoso and Atwell 2011), and reduced plumage orna-
mentation (a difference which was shown to be genetic; Yeh
2004). We have also observed that colonist birds are quicker to
approach novel objects, such as food bait, walk-in traps, or
mist nets (J.W.A., anecdotal observation).
Field and common garden methods
In both populations during 2006–2007, breeding pairs were in-
dividually marked with color bands (San Diego: n’50 pairs; Mt
Laguna: n’30 pairs), and we monitored prebreeding and
breeding activities during 1 February–30 July (San Diego) and
15 March–15 July (Mt Laguna). In 2007, we conducted behav-
ioral assays of FID of foraging birds and incubating females,
and collected serial blood samples to assess the endocrine stress
response (plasma CORT) from nesting females. We also
captured juveniles from early life for common garden study
that included tests of exploratory behavior, endocrine assays,
and genotyping a candidate gene polymorphism for behavioral
differences. These are described in turn below.
Flight initiation distance (field)
FID was recorded for both foraging birds and incubating
females in the field, using methods similar to prior studies
of this measure (Møller 2008). After marking the starting
location of the observer by dropping a small weight, the single
observer (S.C.N., who wore the same clothes for all trials)
directly approached the focal bird or the focal nest, walking
at a constant speed of approximately 1.5 m/s. The observer
dropped a second small weight to mark the distance at the
precise moment when the focal bird took flight and, lastly,
marked the exact preflight location of the focal bird or nest.
FID and starting distances to the focal bird were then
measured with a tape to the nearest centimeter.
For foraging birds, focal individuals (San Diego: n= 30;
Mt Laguna: n= 24) were opportunistically chosen throughout
both study sites from 7 June to 26 June 2007 between the
hours of 0730 and 1400, and we recorded FID, starting dis-
tance (range: 5.93–55.55 m; mean: 22.7 m), and flock size
(range: 1–6 birds; mean: 1.5 birds). Individuals were identi-
fied from color bands when possible (n= 15), and we spatially
segregated sampling to avoid testing the same individuals
(juncos are territorial during the breeding season).
For incubating females, the observer approached each nest
(juncos are ground nesters) from a randomly chosen direction
that allowed a path to the nest that was not obviously visually
obscured (i.e., to avoid not being perceived by the female). For
nests with obvious visual obstructions (e.g., next to a bush), the
nest would be approached from a direction of least obstruc-
tion. In order to control for possible population differences
in nest concealment, we took a digital photograph of each nest
from 0.5 m above it and another from 0.5 m in the direction
that the nest was most visible, and quantified the exposure of
the nest cup or contents in each picture (sum of pixels showing
visible parts of the nest or contents using Scion image software,
such that higher pixel counts indicated more exposure). We
sampled FID from incubating females (San Diego: n= 11;
Mt Laguna: n= 17) between 21 May and 14 June 2007 during
the hours of 0730 to 1230 and also recorded starting distance
(range: 7.00–17.80 m; mean: 13.0 m). Time of day and nest
concealment measures did not differ between populations (all
jtj,1.8, all P.0.1), and these variables did not correlate with
FID within either population (all jrj,0.22, all P.0.25) and
were thus not included in the statistical models described below.
CORT in response to handling (field)
We measured initial plasma CORT levels and the short-term
increases in response to handling stress of females during
the nesting stage (incubating eggs or feeding nestlings),
according to a standardized technique (e.g., see Zysling et al.
2006). Females were caught near their nests with mist nets,
and an initial 100 ul blood sample was immediately collected
within 0–3 min postcapture. Individuals were placed in a paper
holding bag, and 2 additional 50-ul samples were taken at 15
and 30 min postcapture. Samples were stored on ice until
plasma was separated by centrifugation and frozen at 220 �C.
We sampled females across the respective breeding season in
both populations, which was 8 March–27 June 2007 at San Die-
go (n= 31) and 10 May–25 June 2007 (n= 27) at Mt Laguna.
Common garden, general methods
During June and July 2007, we captured 40 juveniles from both
San Diego and Mt Laguna, using mist nets and walk-in traps.
We targeted juveniles that had recently become nutritionally
independent. The age of juveniles was confirmed through field
observations of families and/or measurements of wing and tail
length (Nolan et al. 2002). In some cases (n= 12 at
Mt Laguna; n= 19 at San Diego), we knew the exact age of
the captured juveniles because they were banded as nestlings
(mean 6standard error of the mean; San Diego: 40.9 64.3
days; Mt Laguna: 38.2 61.7 days; t
29
= 0.59, P= 0.56). Because
junco nestlings fledge at ’12 days posthatch and remain with
and dependent on their parents until ’25–30 days posthatch
(Nolan et al. 2002), the juveniles that we captured had limited
early exposure to their natal habitats (on average ,15–20 days
of life outside the nest).
Capture locations were distributed spatially throughout the
study areas to avoid capturing closely related individuals (i.e.,
siblings), with juveniles captured from more than 13 different
locations and on 15 different capture days within each study
population across a period of 30 days. Of the subset of captured
juveniles that were banded in the nest and thus had known
parents (n= 31; see above), we only had 2 siblings from each
population that were sampled for this study (San Diego: n=2
of 31; Mt Laguna: n= 2 of 23).
Juveniles were housed in flocks in temporary outdoor aviar-
ies (2.4 m L 31.8 m W 32.4 m H) in a fenced lawn in suburban
San Diego, CA until 15 July 2007, when they were shipped via
air cargo to the Kent Farm Bird Observatory indoor aviaries at
Indiana University. From July 2007 onwards, birds from each
population were housed in mixed sex flocks (’50:50 male:fe-
male) in both large (6.4 m L 33.2 m W 32.4 m H) and small
(2.5 m L 32.1 m W 32.4 m H) aviary rooms (henceforth
‘‘home aviaries’’) with equivalent densities (’1 bird/m
2
)
and identical housing conditions. Birds were segregated by
population, and all aviary rooms had identical exposure to
human researchers and animal care staff.
Early exploratory behavior (common garden)
From 25 March to 13 April 2008, we measured how rapidly and
how extensively the birds from the common garden explored
a novel aviary room, following methods adapted from Verbeek
et al. (1994). The test room (2.5 m L 32.1 m W 32.4 m H)
had not previously been inhabited by any of the birds in this
study. There was a small (10 cm 310 cm) cardboard loading
door that allowed us to introduce the bird into the test room
with minimal disturbance, and behaviors were observed
through a one-way glass window.
The floor of the test room was visually divided by tape mark-
ings into 4 quadrants, and 5 plastic food containers were
positioned around the floor. The small (10-cm) plastic food
dishes were identical to the ones used in the home aviaries
but contained no food. Instead, we placed wood shavings
(which covered the floors of the home aviaries) inside the food
dishes, which thus provided no incentive for the birds to
remain with the first dish they visited. We also positioned
Atwell et al. •Rapid divergence in boldness and corticosterone 3
5 artificial trees made of wooden rods in the test room to pro-
vide perches and additional spaces that birds could explore.
The target individual was captured from its home aviary after
darkening the lights and then immediately (,60 s) introduced
into the darkened test room. The lights of the test room were
then turned on and a single observer (K.W.R.), blind to the
bird’s population of origin, recorded 8 behaviors for a period
of 20 min: latency to make first movement (0–1200 s), number
of floor quadrants visited (1–4), latency to visit 4/4 floor quad-
rants (0–1200 s), number of food dishes visited (0–5), latency to
visit 4/5 food dishes (0–1200 s), number of trees visited (0–5),
latency to visit 4/5 trees (0–1200 s), and total number of move-
ments (0). Movements were defined as any flights or hops in
which a bird crossed quadrant boundaries.
CORT in response to handling (common garden)
We collected blood samples to measure both initial CORT
(0 min) and stress-induced CORTat 15 and 30 min postcapture
for a subset of individuals (n= 10 per population per sex; n=
40 total) in the common garden using methods analogous to
those described above for field studies. Focal birds were cap-
tured from home aviary rooms within 30 s of dimming the
aviary lights, in order to minimize stress during capture. This
was conducted during 11–13 July 2008 for females and 24–27
July 2009 for males, which were 12 and 24 months following
establishment of the common garden study, respectively.
Radioimmunoassay for CORT
Plasma CORT concentrations were measured using direct radio-
immunoassay (RIA) methods that are described elsewhere (e.g.,
Schoech et al. 1998). In brief, ’20-ul samples were allowed to
equilibrate overnight with 2000 cpm of radiolabeled CORT.
Samples were then extracted with 4.0 ml of diethyl ether
anhydrous and reconstituted with phosphate buffered saline
with gelatin. The competitive binding assay was run in duplicate
and calculations corrected for variation in plasma volume and
individual recoveries. We ran a total of 6 assays (4 for field
studies and 2 for common garden studies), and individual
samples were randomized across assays. We included 3 standards
of known concentration in each assay, and these were used to
calculate intraassay variation (4–28%), interassay variation
(25%), and assay correction factors (assay standard mean/global
standard mean) that were multiplied to standardize values across
assays.
Statistical analyses
We tested for population differences in FID, exploratory behav-
iors and plasma CORT levels with General Linear Models
(GLMs) that included factors and covariates associated with
our a priori hypotheses. The full GLM models, including b,
F, and Pvalues for all factors and covariates, are reported in
Supplementary Tables S1–S5; test statistics most relevant to
our hypotheses appear in RESULTS and figures of the main
text. We used principal components analysis to reduce
correlated behavioral variables into composite boldness scores
for the early exploratory behavior assay (Table 1). We also
compared populations (by sex) for the individual behavioral
variables in the exploratory behavior assay using Mann–
Whitney U-tests (Supplementary Table S6). With respect to
CORT, we analyzed 1) Initial (baseline) CORT (‘‘0 min’’), 2)
maximum CORT (at 15 or 30 min postcapture), and 3) CORT
Response, which was determined by subtracting the Initial
CORT from all 3 time points and calculating the area under
the resulting curve. This integrated approach considers both
the CORT increase and clearance over the 30 min of restraint
(Breuner et al. 1999). All analyses were made with SPSS v.18
(SPSS 2010), and all reported Pvalues are two-tailed.
Evaluating drift
Using methods similar to Yeh (2004), we examined whether
drift alone could explain the observed population differences
reported here. We used the following equation from Lande
(1976):N¼hð1:96Þ2h2ti.½ðx12x2Þ=r2;where N* is the
maximum population size that would allow drift to account
for an observed difference in trait values between 2 popula-
tions over time (P,0.05) and in the total absence of selec-
tion. Observed effective population sizes greater than N*
indicate that drift alone is unlikely to account for population
differences in trait values. h
2
denotes heritability of the trait,
tis time in number of generations, x
1
is the trait value at time
1, x
2
is the trait value at time 2, and ris the standard deviation
of the trait (Lande 1976; Yeh 2004).
For exploratory behavior and plasma CORT levels, we used
published heritability estimates from prior parent–offspring,
sibling, or artificial selection studies of other songbird
species: 0.22–0.54 reported range for exploratory behavior
(Dingemanse et al. 2002; Drent et al. 2003) and 0.08–0.27
for CORT (Evans et al. 2006). We were unable to find re-
ported heritability values for FIDs, but we did find a published
estimate of individual repeatability (0.88, see Carrete and
Tella 2010), which can be interpreted as a reasonable upper
bound of heritability (Falconer and Mackay 1996). For esti-
mating the number of generations (t) since colonization, we
used methods following Yeh (2004), based on the time since
population establishment (’27 years) divided by the average
age of adults in the San Diego population (2.17) to arrive at
a conservative estimate of 12 generations.
Sequencing of SNP 830 region
We sequenced the SNP 830 region of the Drd4 dopamine
receptor gene, which has been found to correlate with explor-
atory behavior in both captive and free-living populations of
another passerine species, Parus major (Fidler et al. 2007;
Korsten et al. 2010). Genomic DNA was isolated from blood
samples using standard phenol–chloroform extraction proce-
dures. We amplified a 152-bp region of the Drd4 gene using
primers designed for P. major: SNP830 forward, 5#-AAGCTGA-
GAGGCTGCATCTATGG-3#and SNP830 reverse, 5#-ATCC-
CACTGTTCATCCCACACTC-3#(Fidler et al. 2007). This
region was amplified in a 20-ul reaction containing 2 mM
Table 1
Trait loadings and eigenvalues in principal components analysis
(n554)
Variable
a
PC1 PC2
Floor quadrants visited (of 4) 0.63 0.39
Latency to visit 4 quadrants 20.87 20.24
Food dishes visited (of 5) 0.89 0.33
Latency to visit 4/5 dishes 20.66 20.41
Trees visited (of 5) 0.58 20.70
Latency to visit 4/5 trees 20.40 0.83
Number of hops 1flights 0.79 20.25
Latency to first hop/flight 20.64 0.14
Eigenvalue 3.93 1.73
Percentage of variance 49.1 21.7
a
All eight variables loaded significantly for PC1 and PC2
4Behavioral Ecology
at Michigan State University on September 4, 2012http://beheco.oxfordjournals.org/Downloaded from

Atwell et al. • Rapid divergence in boldness and corticosterone 963
2007; Cardoso and Atwell 2011), and reduced plumage orna-
mentation (a difference which was shown to be genetic; Yeh
2004). We have also observed that colonist birds are quicker to
approach novel objects, such as food bait, walk-in traps, or
mist nets (J.W.A., anecdotal observation).
Field and common garden methods
In both populations during 2006–2007, breeding pairs were in-
dividually marked with color bands (San Diego: n’50 pairs; Mt
Laguna: n’30 pairs), and we monitored prebreeding and
breeding activities during 1 February–30 July (San Diego) and
15 March–15 July (Mt Laguna). In 2007, we conducted behav-
ioral assays of FID of foraging birds and incubating females,
and collected serial blood samples to assess the endocrine stress
response (plasma CORT) from nesting females. We also
captured juveniles from early life for common garden study
that included tests of exploratory behavior, endocrine assays,
and genotyping a candidate gene polymorphism for behavioral
differences. These are described in turn below.
Flight initiation distance (field)
FID was recorded for both foraging birds and incubating
females in the field, using methods similar to prior studies
of this measure (Møller 2008). After marking the starting
location of the observer by dropping a small weight, the single
observer (S.C.N., who wore the same clothes for all trials)
directly approached the focal bird or the focal nest, walking
at a constant speed of approximately 1.5 m/s. The observer
dropped a second small weight to mark the distance at the
precise moment when the focal bird took flight and, lastly,
marked the exact preflight location of the focal bird or nest.
FID and starting distances to the focal bird were then
measured with a tape to the nearest centimeter.
For foraging birds, focal individuals (San Diego: n= 30;
Mt Laguna: n= 24) were opportunistically chosen throughout
both study sites from 7 June to 26 June 2007 between the
hours of 0730 and 1400, and we recorded FID, starting dis-
tance (range: 5.93–55.55 m; mean: 22.7 m), and flock size
(range: 1–6 birds; mean: 1.5 birds). Individuals were identi-
fied from color bands when possible (n= 15), and we spatially
segregated sampling to avoid testing the same individuals
(juncos are territorial during the breeding season).
For incubating females, the observer approached each nest
(juncos are ground nesters) from a randomly chosen direction
that allowed a path to the nest that was not obviously visually
obscured (i.e., to avoid not being perceived by the female). For
nests with obvious visual obstructions (e.g., next to a bush), the
nest would be approached from a direction of least obstruc-
tion. In order to control for possible population differences
in nest concealment, we took a digital photograph of each nest
from 0.5 m above it and another from 0.5 m in the direction
that the nest was most visible, and quantified the exposure of
the nest cup or contents in each picture (sum of pixels showing
visible parts of the nest or contents using Scion image software,
such that higher pixel counts indicated more exposure). We
sampled FID from incubating females (San Diego: n= 11;
Mt Laguna: n= 17) between 21 May and 14 June 2007 during
the hours of 0730 to 1230 and also recorded starting distance
(range: 7.00–17.80 m; mean: 13.0 m). Time of day and nest
concealment measures did not differ between populations (all
jtj,1.8, all P.0.1), and these variables did not correlate with
FID within either population (all jrj,0.22, all P.0.25) and
were thus not included in the statistical models described below.
CORT in response to handling (field)
We measured initial plasma CORT levels and the short-term
increases in response to handling stress of females during
the nesting stage (incubating eggs or feeding nestlings),
according to a standardized technique (e.g., see Zysling et al.
2006). Females were caught near their nests with mist nets,
and an initial 100 ul blood sample was immediately collected
within 0–3 min postcapture. Individuals were placed in a paper
holding bag, and 2 additional 50-ul samples were taken at 15
and 30 min postcapture. Samples were stored on ice until
plasma was separated by centrifugation and frozen at 220 �C.
We sampled females across the respective breeding season in
both populations, which was 8 March–27 June 2007 at San Die-
go (n= 31) and 10 May–25 June 2007 (n= 27) at Mt Laguna.
Common garden, general methods
During June and July 2007, we captured 40 juveniles from both
San Diego and Mt Laguna, using mist nets and walk-in traps.
We targeted juveniles that had recently become nutritionally
independent. The age of juveniles was confirmed through field
observations of families and/or measurements of wing and tail
length (Nolan et al. 2002). In some cases (n= 12 at
Mt Laguna; n= 19 at San Diego), we knew the exact age of
the captured juveniles because they were banded as nestlings
(mean 6standard error of the mean; San Diego: 40.9 64.3
days; Mt Laguna: 38.2 61.7 days; t
29
= 0.59, P= 0.56). Because
junco nestlings fledge at ’12 days posthatch and remain with
and dependent on their parents until ’25–30 days posthatch
(Nolan et al. 2002), the juveniles that we captured had limited
early exposure to their natal habitats (on average ,15–20 days
of life outside the nest).
Capture locations were distributed spatially throughout the
study areas to avoid capturing closely related individuals (i.e.,
siblings), with juveniles captured from more than 13 different
locations and on 15 different capture days within each study
population across a period of 30 days. Of the subset of captured
juveniles that were banded in the nest and thus had known
parents (n= 31; see above), we only had 2 siblings from each
population that were sampled for this study (San Diego: n=2
of 31; Mt Laguna: n= 2 of 23).
Juveniles were housed in flocks in temporary outdoor aviar-
ies (2.4 m L 31.8 m W 32.4 m H) in a fenced lawn in suburban
San Diego, CA until 15 July 2007, when they were shipped via
air cargo to the Kent Farm Bird Observatory indoor aviaries at
Indiana University. From July 2007 onwards, birds from each
population were housed in mixed sex flocks (’50:50 male:fe-
male) in both large (6.4 m L 33.2 m W 32.4 m H) and small
(2.5 m L 32.1 m W 32.4 m H) aviary rooms (henceforth
‘‘home aviaries’’) with equivalent densities (’1 bird/m
2
)
and identical housing conditions. Birds were segregated by
population, and all aviary rooms had identical exposure to
human researchers and animal care staff.
Early exploratory behavior (common garden)
From 25 March to 13 April 2008, we measured how rapidly and
how extensively the birds from the common garden explored
a novel aviary room, following methods adapted from Verbeek
et al. (1994). The test room (2.5 m L 32.1 m W 32.4 m H)
had not previously been inhabited by any of the birds in this
study. There was a small (10 cm 310 cm) cardboard loading
door that allowed us to introduce the bird into the test room
with minimal disturbance, and behaviors were observed
through a one-way glass window.
The floor of the test room was visually divided by tape mark-
ings into 4 quadrants, and 5 plastic food containers were
positioned around the floor. The small (10-cm) plastic food
dishes were identical to the ones used in the home aviaries
but contained no food. Instead, we placed wood shavings
(which covered the floors of the home aviaries) inside the food
dishes, which thus provided no incentive for the birds to
remain with the first dish they visited. We also positioned
Atwell et al. •Rapid divergence in boldness and corticosterone 3
5 artificial trees made of wooden rods in the test room to pro-
vide perches and additional spaces that birds could explore.
The target individual was captured from its home aviary after
darkening the lights and then immediately (,60 s) introduced
into the darkened test room. The lights of the test room were
then turned on and a single observer (K.W.R.), blind to the
bird’s population of origin, recorded 8 behaviors for a period
of 20 min: latency to make first movement (0–1200 s), number
of floor quadrants visited (1–4), latency to visit 4/4 floor quad-
rants (0–1200 s), number of food dishes visited (0–5), latency to
visit 4/5 food dishes (0–1200 s), number of trees visited (0–5),
latency to visit 4/5 trees (0–1200 s), and total number of move-
ments (0). Movements were defined as any flights or hops in
which a bird crossed quadrant boundaries.
CORT in response to handling (common garden)
We collected blood samples to measure both initial CORT
(0 min) and stress-induced CORTat 15 and 30 min postcapture
for a subset of individuals (n= 10 per population per sex; n=
40 total) in the common garden using methods analogous to
those described above for field studies. Focal birds were cap-
tured from home aviary rooms within 30 s of dimming the
aviary lights, in order to minimize stress during capture. This
was conducted during 11–13 July 2008 for females and 24–27
July 2009 for males, which were 12 and 24 months following
establishment of the common garden study, respectively.
Radioimmunoassay for CORT
Plasma CORT concentrations were measured using direct radio-
immunoassay (RIA) methods that are described elsewhere (e.g.,
Schoech et al. 1998). In brief, ’20-ul samples were allowed to
equilibrate overnight with 2000 cpm of radiolabeled CORT.
Samples were then extracted with 4.0 ml of diethyl ether
anhydrous and reconstituted with phosphate buffered saline
with gelatin. The competitive binding assay was run in duplicate
and calculations corrected for variation in plasma volume and
individual recoveries. We ran a total of 6 assays (4 for field
studies and 2 for common garden studies), and individual
samples were randomized across assays. We included 3 standards
of known concentration in each assay, and these were used to
calculate intraassay variation (4–28%), interassay variation
(25%), and assay correction factors (assay standard mean/global
standard mean) that were multiplied to standardize values across
assays.
Statistical analyses
We tested for population differences in FID, exploratory behav-
iors and plasma CORT levels with General Linear Models
(GLMs) that included factors and covariates associated with
our a priori hypotheses. The full GLM models, including b,
F, and Pvalues for all factors and covariates, are reported in
Supplementary Tables S1–S5; test statistics most relevant to
our hypotheses appear in RESULTS and figures of the main
text. We used principal components analysis to reduce
correlated behavioral variables into composite boldness scores
for the early exploratory behavior assay (Table 1). We also
compared populations (by sex) for the individual behavioral
variables in the exploratory behavior assay using Mann–
Whitney U-tests (Supplementary Table S6). With respect to
CORT, we analyzed 1) Initial (baseline) CORT (‘‘0 min’’), 2)
maximum CORT (at 15 or 30 min postcapture), and 3) CORT
Response, which was determined by subtracting the Initial
CORT from all 3 time points and calculating the area under
the resulting curve. This integrated approach considers both
the CORT increase and clearance over the 30 min of restraint
(Breuner et al. 1999). All analyses were made with SPSS v.18
(SPSS 2010), and all reported Pvalues are two-tailed.
Evaluating drift
Using methods similar to Yeh (2004), we examined whether
drift alone could explain the observed population differences
reported here. We used the following equation from Lande
(1976):N¼hð1:96Þ2h2ti.½ðx12x2Þ=r2;where N* is the
maximum population size that would allow drift to account
for an observed difference in trait values between 2 popula-
tions over time (P,0.05) and in the total absence of selec-
tion. Observed effective population sizes greater than N*
indicate that drift alone is unlikely to account for population
differences in trait values. h
2
denotes heritability of the trait,
tis time in number of generations, x
1
is the trait value at time
1, x
2
is the trait value at time 2, and ris the standard deviation
of the trait (Lande 1976; Yeh 2004).
For exploratory behavior and plasma CORT levels, we used
published heritability estimates from prior parent–offspring,
sibling, or artificial selection studies of other songbird
species: 0.22–0.54 reported range for exploratory behavior
(Dingemanse et al. 2002; Drent et al. 2003) and 0.08–0.27
for CORT (Evans et al. 2006). We were unable to find re-
ported heritability values for FIDs, but we did find a published
estimate of individual repeatability (0.88, see Carrete and
Tella 2010), which can be interpreted as a reasonable upper
bound of heritability (Falconer and Mackay 1996). For esti-
mating the number of generations (t) since colonization, we
used methods following Yeh (2004), based on the time since
population establishment (’27 years) divided by the average
age of adults in the San Diego population (2.17) to arrive at
a conservative estimate of 12 generations.
Sequencing of SNP 830 region
We sequenced the SNP 830 region of the Drd4 dopamine
receptor gene, which has been found to correlate with explor-
atory behavior in both captive and free-living populations of
another passerine species, Parus major (Fidler et al. 2007;
Korsten et al. 2010). Genomic DNA was isolated from blood
samples using standard phenol–chloroform extraction proce-
dures. We amplified a 152-bp region of the Drd4 gene using
primers designed for P. major: SNP830 forward, 5#-AAGCTGA-
GAGGCTGCATCTATGG-3#and SNP830 reverse, 5#-ATCC-
CACTGTTCATCCCACACTC-3#(Fidler et al. 2007). This
region was amplified in a 20-ul reaction containing 2 mM
Table 1
Trait loadings and eigenvalues in principal components analysis
(n554)
Variable
a
PC1 PC2
Floor quadrants visited (of 4) 0.63 0.39
Latency to visit 4 quadrants 20.87 20.24
Food dishes visited (of 5) 0.89 0.33
Latency to visit 4/5 dishes 20.66 20.41
Trees visited (of 5) 0.58 20.70
Latency to visit 4/5 trees 20.40 0.83
Number of hops 1flights 0.79 20.25
Latency to first hop/flight 20.64 0.14
Eigenvalue 3.93 1.73
Percentage of variance 49.1 21.7
a
All eight variables loaded significantly for PC1 and PC2
4Behavioral Ecology
at Michigan State University on September 4, 2012http://beheco.oxfordjournals.org/Downloaded from

Behavioral Ecology964
MgCl
2
and 1 U Taq, using standard thermocycling conditions
with an annealing temperature of 61 �C.
To test whether these primers amplify the same region in
J. hyemalis as in P. major, we first sequenced 12 juncos and
compared the sequences to a published P. major dopamine
receptor D4 mRNA sequence (Genbank accession DQ006801;
Figure 1a). All of these individuals but one were homozogyous
for a single allele; one individual was a heterozygote with an
allele that differed at a single site (basepair 119 in the align-
ment in Supplementary Figure S1a). These sequences are very
similar to a region of the P. major sequence, with a 3-bp deletion
corresponding to a single amino acid at basepairs 100–102 and
2 substitutions, both transversions, at sites 39 (synonymous)
and 93 (nonsynonymous) (Supplementary Figures S1a,b).
The J. hyemalis sequences did not show the same polymor-
phism as in P. major (SNP830C/T); however, another
polymorphism appeared to affect a neighboring amino acid.
G is replaced with A, a nonsynonymous substitution changing
glycine to aspartic acid. This polymorphism also affects a NaeI
restriction enzyme site, as in P. major, by resulting in the pres-
ence (5#-GCCGGC-3#) or the absence (5#-GCCGAC-3#) of the
restriction enzyme cleavage site (also see Fidler et al. 2007).
We used the NaeI restriction enzyme to screen the captive
birds for this polymorphism. We amplified the region with a flu-
orescently labeled forward SNP830 primer, incubated the prod-
uct with the NaeI restriction enzyme following standard
protocols, and analyzed the resulting fragments with the ABI
3730XL automated sequencer and the Genescan 4.0 software.
The ‘‘normal’’ allele was cut by the restriction enzyme and the
resulting fragment was 110-bp long; the ‘‘mutant’’ allele was not
cut and the fragment was 147 bp. Individuals that were found to
have one or two 147 bp alleles were then sequenced to verify the
presence of the mutant allele, and thus to exclude the possibility
of incomplete digestion by the restriction enzyme reaction.
RESULTS
Flight initiation distances—field study
For both foraging birds and incubating females, FID were sig-
nificantly shorter in the San Diego population (foraging birds:
F
1,53
= 30.1, P,0.001; incubating females: F
1,27
= 10.4, P=
0.003) (Figure 1). For foraging birds, the flock size and the
starting distance of the observer both had significant effects in
the GLM model and were positively correlated with FID (flock
size: F
1,53
= 6.1, P= 0.017; starting distance: F
1,53
= 9.4, P= 0.004).
We also tested for interactions between starting distance and
population and flock size and population for the foraging birds,
however, neither interaction term was significant and both
were removed from the final model (population 3flock size:
F
1,53
= 1.05, P= 0.31; population 3starting distance: F
1,53
= 0.95,
P= 0.36). There was no significant effect of starting distance on
FID for incubating females (F
1,27
= 0.1, P= 0.75).
CORT—field study
For initial (baseline) CORT levels, we found no significant dif-
ference between females from the San Diego versus Mt Laguna
populations (F
1,52
= 0.42, P= 0.518), and there was no signif-
icant effect of body mass (F
1,27
= 0.1, P= 0.75) or date (F
1,27
=
0.1, P= 0.75) (Figure 2a). However, birds from the San Diego
population exhibited attenuated plasma CORT elevations in
response to handling, as both maximum CORT and overall
CORT response (area) were significantly lower in the
San Diego population (max CORT: F
1,52
= 10.8, P= 0.002;
CORT response: F
1,52
= 4.4, P= 0.042) (Figure 2a).
Exploratory behavior—common garden study
The 8 behavioral variables of the exploratory behavior assay
were highly correlated with one another, and a PCA returned
2 principal components with eigenvalues larger than 1
(Table 1). The first component (PC1, eigenvalue = 3.93,
49.1% variation) was clearly interpretable as positively associ-
ated with faster and more extensive (i.e., bolder) exploratory
behavior (Table 1). Specifically, birds that scored highly for
PC1 made their initial movement sooner, visited more floor
quadrants, food dishes, and trees more quickly, and made
more total movements (Table 1). PC2 (eigenvalue = 1.73,
21.7% of variance) was less readily interpretable; it mainly
predicted whether individuals, regardless of their level of ex-
ploration, tended to remain on the ground or visit trees
(strongest trait loadings were fewer trees visited and higher
latency to visit trees, Table 1). PC1 is the focus of our sub-
sequent analysis, because it quantifies the speed and extent of
exploratory behavior, but we also analyzed PC2 because it
explained a considerable proportion of variation in behavior.
Figure 1
FID (means 6standard error of the mean) were shorter in field
studies of both foraging birds (GLM, P,0.001) and incubating
females (GLM, P= 0.003) in colonist (San Diego) versus ancestral
range (Mt Laguna) populations.
Figure 2
Initial (baseline) and stress-induced plasma corticosterone (CORT;
means 6 standard error of the mean) in colonist (San Diego) and
ancestral range populations are shown from (a) field study of
freeliving nesting females and (b) a captive common garden study of
birds raised from early life under identical aviary conditions. We
found significant population differences in both maximum CORT
and CORT responsiveness (area) in both field and common garden
studies, and there were also sex differences in the common garden
(see RESULTS).
Atwell et al. •Rapid divergence in boldness and corticosterone 5
Birds from the San Diego population had significantly higher
boldness (PC1) scores than birds from Mt Laguna (F
1,53
= 13.3,
P= 0.001, Figure 3). The effect of sex was also highly significant
(F
1,53
= 7.3, P= 0.009), with males showing higher PC1 scores
than females in both populations (Figure 3). For PC2, there
were no significant effects of population, sex, or body mass (all
P.0.25), but we did detect a significant interaction of pop-
ulation 3sex (P= 0.05) (Supplementary Table S3). Population
comparisons (by sex) for individual behavioral variables are
summarized in Supplementary Table S1 and are consistent with
the conclusions of the main test of PC1, with faster and more
extensive exploratory behavior observed in birds originating
from the urban colonist population (Supplementary Table S1).
CORT—common garden study
In the common garden study, we found that males had lower
initial (baseline) CORT than females (F
1,39
= 4.42, P= 0.043),
but we did not detect significant population differences for
initial CORT (F
1,39
= 0.3, P= 0.59) (Figure 2b). For maximum
CORT, there was a trend toward lower values in the San Diego
population (F
1,39
= 3.10, P= 0.089), and males also had lower
maximum CORTcompared with females (F
1,39
= 6.63, P= 0.017)
(Figure 2b). For CORT responsiveness (area), a measure that
controls for initial CORT levels, we found a marginally signifi-
cant effect of population (F
1,39
= 4.10, P= 0.052), but not for sex
(F
1,39
= 1.99, P= 0.17), with birds originating from San Diego
exhibiting an attenuated CORT response (Figure 2b). There
were no significant effects of body mass or the interaction of
sex 3population on any of the CORT measures in the common
garden study (all P.0.1; Supplementary Table S4).
Covariation between CORT and exploratory
behavior—common garden study
In the common garden study, the same individuals were
sampled for exploratory behavior and plasma CORT, allowing
us to test for covariation between behavioral and hormonal
measures. While controlling for variation attributed to popu-
lation, sex, and body mass differences in exploratory behavior
(Supplementary Table S5; Figure 4), we found that maximum
plasma CORT negatively predicted exploratory behavior (F
1,39
= 5.84, P= 0.021) (Figure 4). Negative covariation between
maximum CORT and exploratory behavior was similar for
both sexes within both populations (Figure 4), and thus there
was no detectable interaction effect of population 3maxi-
mum CORT (F
1,39
= 2.11, P= 0.16). Neither initial (baseline)
CORT nor CORT response (area) predicted exploratory
behavior (P.0.1; Supplementary Table S5).
Drift
We estimated the maximum effective population size (N*) at
which drift alone, in the absence of selection, could likely
explain the observed population differences in the field and
the common garden, given the time since population estab-
lishment (’12 generations) and heritability estimates from
the literature. These N* values ranged from 1 to 23 (average
3–13) and are shown in Table 2. Since 1998, when systematic
observations were begun, the population has remained stable
at ’160 birds (Yeh and Price 2004; Atwell J, unpublished
data). Thus, given typical models of population growth, it is
unlikely that the observed population differences could be
due to drift alone in the absence of selection. Similarly, an
explanation based on a founder effect would require that the
maximum contributing number of founders was limited to
fewer than ’8 individuals. Microsatellite diversity in the col-
onist population suggests a minimum of ’8–20 individuals
established the population (Rasner et al. 2004).
Variation at Drd4 locus
As described above (see MATERIALS AND METHODS), the
J. hyemalis sequences did not show the same polymorphism
as in P. major (SNP830C/T); however, another polymorphism
appeared to affect a neighboring amino acid. Only 3 individ-
uals (of 59 screened) were found to be heterozygous at the
new polymorphic locus, and none was homozygous for the
mutant allele. Two of the heterozygotes were from the Mt
Laguna population, and one from San Diego. Thus, the lack
of variation found at these loci precluded any further anal-
yses of the relationships between sequence variation and
behavior.
DISCUSSION
Birds colonizing an urban environment are required to use
novel food, water, and nesting resources, and they must inter-
act with constant anthropogenic disturbances and highly
diverse stimuli and stressors, including vehicles, humans, pets,
lights, and noises associated with their urban environment. We
found that free-living birds in a recently established (;1983)
urban colonist population exhibited reduced FID and attenu-
ated endocrine (CORT) responses to handling. In a common
garden study, in which birds from both populations were raised
from early life under identical aviary conditions, we found
faster and more extensive exploratory behavior in the colonist
urban population, as well as persistent population differences
in CORT levels. Importantly, in the common garden study,
birds with higher maximum CORT levels were less bold, even
after controlling for variation attributed to sex and population.
This may shed light on both the hormonal basis of boldness
and the potential that hormones may facilitate rapid evolution
of correlated traits. Together, to the extent that common garden
data can be used to draw inferences about genetic divergence,
these findings provide unique evidence for rapid adaptive ge-
netic evolution of behavior and a correlated hormonal mecha-
nism after colonization of an urban environment.
Although we did not study natural or sexual selection
gradients in the colonist versus ancestral range habitats, the
differences in boldness behaviors and CORT profiles in
Figure 3
Exploratory behavior (PC1, means 6 standard error of the mean)
scores from a common garden study are shown by population of
origin and sex. The effects of both population (GLM, P= 0.001) and
sex (GLM, P= 0.009) were significant.
6Behavioral Ecology
at Michigan State University on September 4, 2012http://beheco.oxfordjournals.org/Downloaded from

Atwell et al. • Rapid divergence in boldness and corticosterone 965
MgCl
2
and 1 U Taq, using standard thermocycling conditions
with an annealing temperature of 61 �C.
To test whether these primers amplify the same region in
J. hyemalis as in P. major, we first sequenced 12 juncos and
compared the sequences to a published P. major dopamine
receptor D4 mRNA sequence (Genbank accession DQ006801;
Figure 1a). All of these individuals but one were homozogyous
for a single allele; one individual was a heterozygote with an
allele that differed at a single site (basepair 119 in the align-
ment in Supplementary Figure S1a). These sequences are very
similar to a region of the P. major sequence, with a 3-bp deletion
corresponding to a single amino acid at basepairs 100–102 and
2 substitutions, both transversions, at sites 39 (synonymous)
and 93 (nonsynonymous) (Supplementary Figures S1a,b).
The J. hyemalis sequences did not show the same polymor-
phism as in P. major (SNP830C/T); however, another
polymorphism appeared to affect a neighboring amino acid.
G is replaced with A, a nonsynonymous substitution changing
glycine to aspartic acid. This polymorphism also affects a NaeI
restriction enzyme site, as in P. major, by resulting in the pres-
ence (5#-GCCGGC-3#) or the absence (5#-GCCGAC-3#) of the
restriction enzyme cleavage site (also see Fidler et al. 2007).
We used the NaeI restriction enzyme to screen the captive
birds for this polymorphism. We amplified the region with a flu-
orescently labeled forward SNP830 primer, incubated the prod-
uct with the NaeI restriction enzyme following standard
protocols, and analyzed the resulting fragments with the ABI
3730XL automated sequencer and the Genescan 4.0 software.
The ‘‘normal’’ allele was cut by the restriction enzyme and the
resulting fragment was 110-bp long; the ‘‘mutant’’ allele was not
cut and the fragment was 147 bp. Individuals that were found to
have one or two 147 bp alleles were then sequenced to verify the
presence of the mutant allele, and thus to exclude the possibility
of incomplete digestion by the restriction enzyme reaction.
RESULTS
Flight initiation distances—field study
For both foraging birds and incubating females, FID were sig-
nificantly shorter in the San Diego population (foraging birds:
F
1,53
= 30.1, P,0.001; incubating females: F
1,27
= 10.4, P=
0.003) (Figure 1). For foraging birds, the flock size and the
starting distance of the observer both had significant effects in
the GLM model and were positively correlated with FID (flock
size: F
1,53
= 6.1, P= 0.017; starting distance: F
1,53
= 9.4, P= 0.004).
We also tested for interactions between starting distance and
population and flock size and population for the foraging birds,
however, neither interaction term was significant and both
were removed from the final model (population 3flock size:
F
1,53
= 1.05, P= 0.31; population 3starting distance: F
1,53
= 0.95,
P= 0.36). There was no significant effect of starting distance on
FID for incubating females (F
1,27
= 0.1, P= 0.75).
CORT—field study
For initial (baseline) CORT levels, we found no significant dif-
ference between females from the San Diego versus Mt Laguna
populations (F
1,52
= 0.42, P= 0.518), and there was no signif-
icant effect of body mass (F
1,27
= 0.1, P= 0.75) or date (F
1,27
=
0.1, P= 0.75) (Figure 2a). However, birds from the San Diego
population exhibited attenuated plasma CORT elevations in
response to handling, as both maximum CORT and overall
CORT response (area) were significantly lower in the
San Diego population (max CORT: F
1,52
= 10.8, P= 0.002;
CORT response: F
1,52
= 4.4, P= 0.042) (Figure 2a).
Exploratory behavior—common garden study
The 8 behavioral variables of the exploratory behavior assay
were highly correlated with one another, and a PCA returned
2 principal components with eigenvalues larger than 1
(Table 1). The first component (PC1, eigenvalue = 3.93,
49.1% variation) was clearly interpretable as positively associ-
ated with faster and more extensive (i.e., bolder) exploratory
behavior (Table 1). Specifically, birds that scored highly for
PC1 made their initial movement sooner, visited more floor
quadrants, food dishes, and trees more quickly, and made
more total movements (Table 1). PC2 (eigenvalue = 1.73,
21.7% of variance) was less readily interpretable; it mainly
predicted whether individuals, regardless of their level of ex-
ploration, tended to remain on the ground or visit trees
(strongest trait loadings were fewer trees visited and higher
latency to visit trees, Table 1). PC1 is the focus of our sub-
sequent analysis, because it quantifies the speed and extent of
exploratory behavior, but we also analyzed PC2 because it
explained a considerable proportion of variation in behavior.
Figure 1
FID (means 6standard error of the mean) were shorter in field
studies of both foraging birds (GLM, P,0.001) and incubating
females (GLM, P= 0.003) in colonist (San Diego) versus ancestral
range (Mt Laguna) populations.
Figure 2
Initial (baseline) and stress-induced plasma corticosterone (CORT;
means 6 standard error of the mean) in colonist (San Diego) and
ancestral range populations are shown from (a) field study of
freeliving nesting females and (b) a captive common garden study of
birds raised from early life under identical aviary conditions. We
found significant population differences in both maximum CORT
and CORT responsiveness (area) in both field and common garden
studies, and there were also sex differences in the common garden
(see RESULTS).
Atwell et al. •Rapid divergence in boldness and corticosterone 5
Birds from the San Diego population had significantly higher
boldness (PC1) scores than birds from Mt Laguna (F
1,53
= 13.3,
P= 0.001, Figure 3). The effect of sex was also highly significant
(F
1,53
= 7.3, P= 0.009), with males showing higher PC1 scores
than females in both populations (Figure 3). For PC2, there
were no significant effects of population, sex, or body mass (all
P.0.25), but we did detect a significant interaction of pop-
ulation 3sex (P= 0.05) (Supplementary Table S3). Population
comparisons (by sex) for individual behavioral variables are
summarized in Supplementary Table S1 and are consistent with
the conclusions of the main test of PC1, with faster and more
extensive exploratory behavior observed in birds originating
from the urban colonist population (Supplementary Table S1).
CORT—common garden study
In the common garden study, we found that males had lower
initial (baseline) CORT than females (F
1,39
= 4.42, P= 0.043),
but we did not detect significant population differences for
initial CORT (F
1,39
= 0.3, P= 0.59) (Figure 2b). For maximum
CORT, there was a trend toward lower values in the San Diego
population (F
1,39
= 3.10, P= 0.089), and males also had lower
maximum CORTcompared with females (F
1,39
= 6.63, P= 0.017)
(Figure 2b). For CORT responsiveness (area), a measure that
controls for initial CORT levels, we found a marginally signifi-
cant effect of population (F
1,39
= 4.10, P= 0.052), but not for sex
(F
1,39
= 1.99, P= 0.17), with birds originating from San Diego
exhibiting an attenuated CORT response (Figure 2b). There
were no significant effects of body mass or the interaction of
sex 3population on any of the CORT measures in the common
garden study (all P.0.1; Supplementary Table S4).
Covariation between CORT and exploratory
behavior—common garden study
In the common garden study, the same individuals were
sampled for exploratory behavior and plasma CORT, allowing
us to test for covariation between behavioral and hormonal
measures. While controlling for variation attributed to popu-
lation, sex, and body mass differences in exploratory behavior
(Supplementary Table S5; Figure 4), we found that maximum
plasma CORT negatively predicted exploratory behavior (F
1,39
= 5.84, P= 0.021) (Figure 4). Negative covariation between
maximum CORT and exploratory behavior was similar for
both sexes within both populations (Figure 4), and thus there
was no detectable interaction effect of population 3maxi-
mum CORT (F
1,39
= 2.11, P= 0.16). Neither initial (baseline)
CORT nor CORT response (area) predicted exploratory
behavior (P.0.1; Supplementary Table S5).
Drift
We estimated the maximum effective population size (N*) at
which drift alone, in the absence of selection, could likely
explain the observed population differences in the field and
the common garden, given the time since population estab-
lishment (’12 generations) and heritability estimates from
the literature. These N* values ranged from 1 to 23 (average
3–13) and are shown in Table 2. Since 1998, when systematic
observations were begun, the population has remained stable
at ’160 birds (Yeh and Price 2004; Atwell J, unpublished
data). Thus, given typical models of population growth, it is
unlikely that the observed population differences could be
due to drift alone in the absence of selection. Similarly, an
explanation based on a founder effect would require that the
maximum contributing number of founders was limited to
fewer than ’8 individuals. Microsatellite diversity in the col-
onist population suggests a minimum of ’8–20 individuals
established the population (Rasner et al. 2004).
Variation at Drd4 locus
As described above (see MATERIALS AND METHODS), the
J. hyemalis sequences did not show the same polymorphism
as in P. major (SNP830C/T); however, another polymorphism
appeared to affect a neighboring amino acid. Only 3 individ-
uals (of 59 screened) were found to be heterozygous at the
new polymorphic locus, and none was homozygous for the
mutant allele. Two of the heterozygotes were from the Mt
Laguna population, and one from San Diego. Thus, the lack
of variation found at these loci precluded any further anal-
yses of the relationships between sequence variation and
behavior.
DISCUSSION
Birds colonizing an urban environment are required to use
novel food, water, and nesting resources, and they must inter-
act with constant anthropogenic disturbances and highly
diverse stimuli and stressors, including vehicles, humans, pets,
lights, and noises associated with their urban environment. We
found that free-living birds in a recently established (;1983)
urban colonist population exhibited reduced FID and attenu-
ated endocrine (CORT) responses to handling. In a common
garden study, in which birds from both populations were raised
from early life under identical aviary conditions, we found
faster and more extensive exploratory behavior in the colonist
urban population, as well as persistent population differences
in CORT levels. Importantly, in the common garden study,
birds with higher maximum CORT levels were less bold, even
after controlling for variation attributed to sex and population.
This may shed light on both the hormonal basis of boldness
and the potential that hormones may facilitate rapid evolution
of correlated traits. Together, to the extent that common garden
data can be used to draw inferences about genetic divergence,
these findings provide unique evidence for rapid adaptive ge-
netic evolution of behavior and a correlated hormonal mecha-
nism after colonization of an urban environment.
Although we did not study natural or sexual selection
gradients in the colonist versus ancestral range habitats, the
differences in boldness behaviors and CORT profiles in
Figure 3
Exploratory behavior (PC1, means 6 standard error of the mean)
scores from a common garden study are shown by population of
origin and sex. The effects of both population (GLM, P= 0.001) and
sex (GLM, P= 0.009) were significant.
6Behavioral Ecology
at Michigan State University on September 4, 2012http://beheco.oxfordjournals.org/Downloaded from

Behavioral Ecology966
the colonist population likely represent adaptive phenotypic
changes that have allowed the colonist population to persist
in this species-atypical urban environment. We acknowledge
that one drawback of our study system is that by comparing
only two populations there are limitations on the inferences
that can be made—both with respect to the generality of the
observed phenomena as well as the causality of which specific
ecological factors may underlie population differences
(Garland and Adolph 1994). However, our interpretation
of the joint differences in behavior and CORT levels of
the urban population are based on a priori predictions sup-
ported by studies in other systems that found differences
either in one or the other trait (behavior: Møller 2008;
Evans et al. 2010; CORT levels: Romero and Wikelski 2002;
Partecke et al. 2006) associated with human-disturbed hab-
itats. Colonization events such as that presented here pro-
vide rare opportunities to study evolution, and it is only by
building up results from multiple studies that we can assess
the generality of findings.
In most similar studies, it is unclear whether population dif-
ferences are underlain by genetic change or phenotypic plas-
ticity (e.g., habituation to frequent human disturbance).
Persistence of character differences in a common garden pro-
vides strong evidence that the likely explanation for the cur-
rent population differences in behavior and CORT is not
plasticity. Accordingly, earlier molecular studies confirmed
that the colonist population is genetically distinct from ances-
tral range montane populations at microsatellite loci (Rasner
et al. 2004), and a previous common garden study also found
persistent differences between these 2 populations in mor-
phological characters (Rasner et al. 2004; Yeh 2004).
Based on our results, we propose that novel selective forces
associated with the urban environment have likely led to rapid
evolution of increased boldness and an attenuated endocrine
stress response postcolonization, and that the population differ-
ences we found reflect divergent behavioral and physiological
fitness optima in the colonist versus ancestral range habitats.
Given the negative correlation between maximum CORT and
exploratory behavior, it is plausible that these traits have evolved
in concert, either in response to selection on boldness behavior
or selection on CORTresponsiveness via its modulation of other
traits. It is also possible that correlational selection acted directly
on hormone–phenotype coexpression. To test these hypotheses,
future work should focus on the shape of natural or social selec-
tion gradients, and there is evidence from other systems that
both exploratory behavior (Dingemanse et al. 2004) and CORT
profiles (Breuner et al. 2008) are subject to selection in free-
living songbird populations.
An alternative explanation is that the apparent genetic dif-
ferences reported here are the result of drift. For example, it is
possible that a founder population of individuals with tamer or
bolder behavioral phenotypes and corresponding attenuations
of the endocrine stress response may have been ‘‘preadapted’’
to establish an urban colonist population. Consistent with this
alternative hypothesis, some studies on other species have
found bolder or more exploratory individuals to disperse more
or over longer distances (e.g., Dingemanse et al. 2003), but
negative correlations between boldness and dispersal have also
been reported (reviewed in Cote et al. 2010). We calculated
that in our study system, the effective population size would
have had to remain unrealistically small for several generations
for any type of drift (including a founder effect) to explain the
observed divergences in boldness behaviors and CORT ob-
served in our studies (Lande 1976; Yeh 2004). Nevertheless,
a nonrandom founder event in which only a small proportion
of preadapted individuals became the sole genetic contributors
to the small and relatively isolated colonist population cannot
be ruled out—a scenario that invokes aspects of both selection
and drift and has been described previously as ‘‘immigrant
selection’’ (Brown and Lomolino 1998, p. 436).
Whether via selection or drift, this study provides some of the
first evidence for rapid adaptive evolution of boldness behavior
across natural populations. We are aware of atleast 2studies pro-
viding evidence for adaptive evolution of personality traits across
populations of stickleback fish, apparently in response to varying
predation pressures, but on much longer time scales than
reported here (e.g. Dingemanse et al. 2007; and Herczeg
et al. 2009). Another common garden study of birds reported
that house sparrows (Passer domesticus) approached and con-
sumed novel foods more quickly if they were members of an
actively invading 28-year-old population as compared with
members of a 150-year-old resident population, but the spar-
rows were adults when captured and the behavioral assays were
substantially different from the ones employed here (Martin
and Fitzgerald 2005).
Table 2
Estimates of effective population above which drift would be
unlikely to explain divergence
Population difference Heritabilty
c
Generations Max N*
Exploratory behavior $0.22–0.54 12 4–23
Exploratory behavior #0.22–0.54 12 1–8
Max corticosterone
a
$0.08–0.24 12 1–16
CORT response
a
$0.08–0.24 12 2–23
Max corticosterone
a
#0.08–0.24 12 1–7
CORT reponse (area)
a
#0.08–0.24 12 1–4
Flight distance (foraging)
b
0.88 12 9
Flight distance (incubating)
b
$0.88 12 15
Max CORT
b
$0.08–0.24 12 1–15
CORT response (area)
b
$0.08–0.24 12 1–15
a
Measured in common garden study.
b
Measured in field study.
c
Estimated from published studies, see MATERIALS AND METHODS.
Figure 4
Individual variation for maximum CORT and exploratory behavior
from the common garden study. Max CORT predicted individual
exploratory behavior scores (P= 0.021) in a GLM model the
also included significant effects of population (P= 0.015) and sex
(P= 0.049).
Atwell et al. •Rapid divergence in boldness and corticosterone 7
Similarly, studies of population or subspecific variation in hor-
monal systems are rare, particularly those that evaluate develop-
mental versus genetic underpinnings. In an exception and
a system similar to ours, Partecke et al. (2006) found that urban
blackbirds raised in a common garden showed attenuated
CORT response to handling stress when compared with their
wildland counterparts. Thus, our findings further extend the
generalization that attenuated HPA responsiveness and reduced
neophobia are associated with adaptation to urbanization.
Another common garden study also recently concluded that
variation in the CORT response among closely related songbird
species with varying life histories also likely has a genetic basis
(Angelier et al. 2011). One particularly relevant life-history dif-
ference that has been hypothesized to underlie variation in glu-
cocorticoid responses is the value of current versus future
reproduction and survival opportunities, and comparative data
across species support the idea that breeding season length
correlates positively with HPA responsiveness to short-term
stressors (Bokony et al. 2009). This would predict weaker not
stronger response to stressors in the urban junco population
which has an unusually long breeding season length
(February–August) as compared with the species-typical May–
July breeding season observed at Mt Laguna. This contrasting
result to the usual among-species pattern suggests that in
our case, the selective factors associated with urbanization are
likely more important than those associated with length of the
breeding season.
Although our findings suggest a genetic basis for behavioral
and hormonal differences, environmental effects or early de-
velopmental effects could also play a role. The magnitude of
the difference in CORT profiles between populations was
not as strong in the common garden as in the field (although
sample sizes were also smaller and absolute levels were gener-
ally lower in captivity), and reported heritabilities in other sys-
tems for exploratory behavior and CORT levels are moderate
(’0.2–0.4) (Dingemanse et al. 2002; van Oers et al. 2005;
Evans et al. 2006), which leaves room for a substantial envi-
ronmental contribution to the behavioral and hormonal phe-
notype. For example, further reduced CORT response in the
urban population due to habituation to frequent stressors
may be a likely explanation for the larger difference in CORT
profiles in the field than in the common garden study.
Maternal effects are another type of developmental effects
that could influence behavior in adulthood. For example, dif-
ferential maternal allocation of egg yolk hormones can alter
behavioral development in ways that persist into adulthood
(Groothuis et al. 2005). For example, 9-month-old zebra
finches (Taeniopygia gutatta) hatched from testosterone-
treated eggs habituated faster in a test of neophobia (Tobler
and Sandell 2007). Therefore, if junco females in the urban
population deposited more androgens into their egg yolks,
this might contribute to the faster exploratory behavior of
San Diego birds. But most of the phenotypic differences be-
tween the San Diego and Laguna Mountain populations (e.g.,
breeding season length, male territoriality, plumage ornamen-
tation) have been associated with lower testosterone not high-
er, which would predict lower androgen levels in the San
Diego population during development (Yeh 2004; Newman
et al. 2006). Thus differences in yolk androgens are unlikely
to explain the population differences in behavior reported
here. Exposure to elevated maternal and postnatal CORT
and developmental stress treatments is also known to alter
boldness behaviors and glucocorticoid levels even into adult-
hood (reviewed in Schoech et al. 2011), but the direction of
the effects and diverse methodologies make generalizing the
results of these studies challenging.
We also cannot rule out the possibility that early learning
of exploratory behaviors could have taken place between
hatching and capture for the common garden study. The pe-
riod during which the juveniles were foraging independently
(10–15 days on average) was far briefer than the time spent
developing in the common environment prior to behavioral
testing (.8 months), but little is known about critical periods
for development of exploratory behavior. Notwithstanding that
maternal or early developmental effects cannot be completely
ruled out, we suggest that genetic evolution is the most likely
cause for the persistent behavioral and hormonal differences
in the colonist population.
Differences in exploratory behavior were not explained by
allelic variation at the SNP830 region of the dopamine recep-
tor gene (Drd4), as this region was found to be mostly invari-
ant in our sample. A polymorphism at the SNP830 locus
correlates with exploratory behavior in some populations of
great tits (P. major), both captive and free-living, and this poly-
morphism responded to artificial selection for fast or slow
exploratory behavior (Fidler et al. 2007; Korsten et al.
2010). However, many other loci with small effects are also
expected to contribute to differences in boldness among in-
dividuals (van Oers, Drent, de Jong, et al. 2004; van Oers et al.
2005), especially since the allelic variation at SNP830 only
explained 4.5–6.0% of the variation in behavior in the P. major
study (Fidler et al. 2007; Korsten et al. 2010). The lack of
variation at the Drd4 SNP830 in the junco suggests that the
divergence in the urban population may have been achieved
via other genes or alternative mutations within the larger Drd4
gene. This is unsurprising given that studies of 3 additional
P. major populations found no clear association between
SNP830 and behavior, suggesting this mutation was popula-
tion specific (Korsten et al. 2010). The SNP830 region of the
Drd4 gene was highly conserved in dark-eyed juncos, which
enhances the prospects for future sequencing of other re-
gions of the Drd4 gene, and across avian taxa, several other
indels and SNP within Drd4 are being found (Abe et al. 2011).
In sum, our data show an integrated pattern of adaptive pop-
ulation divergence for a behavioral trait and a putative causal
hormonal mechanism that persisted in a common environ-
ment, suggesting genetic evolution. The differences between
populations lie in the direction predicted by within-population
hormone-phenotype relationships, suggesting correlated evo-
lution along lines of least resistance (Schluter 1996), rather
than changes in reactions norms to adjust behavior indepen-
dently of hormonal levels. Maintenance of individual covaria-
tion among CORT and boldness behaviors within divergent
populations underscores the point that there is likely no sin-
gle optimum hormone–behavior phenotypic strategy in a pop-
ulation, but rather a range of successful individual strategies
maintained by functional trade-offs or opposing or variable
selective factors (Koolhaas et al. 1999; Schoech et al. 2011).
Although evolutionary conservation of trait correlations, in-
cluding hormone–phenotype associations, has most generally
been considered evidence of a likely constraint on evolutionary
diversification (Schluter 1996; Hau 2007), functionally sensible
trait correlations could also facilitate adaptation to novel envi-
ronments (Agrawal and Stinchcombe 2009; Ketterson et al.
2009). This may be the case in our study system, where changes
in an integrating hormonal mechanism (the HPA endocrine
axis) may underlie divergence for a suite of adaptive behavioral
and physiological characteristics, allowing for more rapid adap-
tation (evolutionary or developmental) to novel environments.
Additional studies of the neuroendocrine and genetic mecha-
nisms linking hormonal and behavioral phenotypes are needed
to assess the evolutionary significance of these trait associations.
Future research should thus continue to focus both on identi-
fying the mechanistic sources of variance underlying trait
correlations and evaluating how integrated phenotypes
respond to novel and changing environments.
8Behavioral Ecology
at Michigan State University on September 4, 2012http://beheco.oxfordjournals.org/Downloaded from

Atwell et al. • Rapid divergence in boldness and corticosterone 967
the colonist population likely represent adaptive phenotypic
changes that have allowed the colonist population to persist
in this species-atypical urban environment. We acknowledge
that one drawback of our study system is that by comparing
only two populations there are limitations on the inferences
that can be made—both with respect to the generality of the
observed phenomena as well as the causality of which specific
ecological factors may underlie population differences
(Garland and Adolph 1994). However, our interpretation
of the joint differences in behavior and CORT levels of
the urban population are based on a priori predictions sup-
ported by studies in other systems that found differences
either in one or the other trait (behavior: Møller 2008;
Evans et al. 2010; CORT levels: Romero and Wikelski 2002;
Partecke et al. 2006) associated with human-disturbed hab-
itats. Colonization events such as that presented here pro-
vide rare opportunities to study evolution, and it is only by
building up results from multiple studies that we can assess
the generality of findings.
In most similar studies, it is unclear whether population dif-
ferences are underlain by genetic change or phenotypic plas-
ticity (e.g., habituation to frequent human disturbance).
Persistence of character differences in a common garden pro-
vides strong evidence that the likely explanation for the cur-
rent population differences in behavior and CORT is not
plasticity. Accordingly, earlier molecular studies confirmed
that the colonist population is genetically distinct from ances-
tral range montane populations at microsatellite loci (Rasner
et al. 2004), and a previous common garden study also found
persistent differences between these 2 populations in mor-
phological characters (Rasner et al. 2004; Yeh 2004).
Based on our results, we propose that novel selective forces
associated with the urban environment have likely led to rapid
evolution of increased boldness and an attenuated endocrine
stress response postcolonization, and that the population differ-
ences we found reflect divergent behavioral and physiological
fitness optima in the colonist versus ancestral range habitats.
Given the negative correlation between maximum CORT and
exploratory behavior, it is plausible that these traits have evolved
in concert, either in response to selection on boldness behavior
or selection on CORTresponsiveness via its modulation of other
traits. It is also possible that correlational selection acted directly
on hormone–phenotype coexpression. To test these hypotheses,
future work should focus on the shape of natural or social selec-
tion gradients, and there is evidence from other systems that
both exploratory behavior (Dingemanse et al. 2004) and CORT
profiles (Breuner et al. 2008) are subject to selection in free-
living songbird populations.
An alternative explanation is that the apparent genetic dif-
ferences reported here are the result of drift. For example, it is
possible that a founder population of individuals with tamer or
bolder behavioral phenotypes and corresponding attenuations
of the endocrine stress response may have been ‘‘preadapted’’
to establish an urban colonist population. Consistent with this
alternative hypothesis, some studies on other species have
found bolder or more exploratory individuals to disperse more
or over longer distances (e.g., Dingemanse et al. 2003), but
negative correlations between boldness and dispersal have also
been reported (reviewed in Cote et al. 2010). We calculated
that in our study system, the effective population size would
have had to remain unrealistically small for several generations
for any type of drift (including a founder effect) to explain the
observed divergences in boldness behaviors and CORT ob-
served in our studies (Lande 1976; Yeh 2004). Nevertheless,
a nonrandom founder event in which only a small proportion
of preadapted individuals became the sole genetic contributors
to the small and relatively isolated colonist population cannot
be ruled out—a scenario that invokes aspects of both selection
and drift and has been described previously as ‘‘immigrant
selection’’ (Brown and Lomolino 1998, p. 436).
Whether via selection or drift, this study provides some of the
first evidence for rapid adaptive evolution of boldness behavior
across natural populations. We are aware of atleast 2studies pro-
viding evidence for adaptive evolution of personality traits across
populations of stickleback fish, apparently in response to varying
predation pressures, but on much longer time scales than
reported here (e.g. Dingemanse et al. 2007; and Herczeg
et al. 2009). Another common garden study of birds reported
that house sparrows (Passer domesticus) approached and con-
sumed novel foods more quickly if they were members of an
actively invading 28-year-old population as compared with
members of a 150-year-old resident population, but the spar-
rows were adults when captured and the behavioral assays were
substantially different from the ones employed here (Martin
and Fitzgerald 2005).
Table 2
Estimates of effective population above which drift would be
unlikely to explain divergence
Population difference Heritabilty
c
Generations Max N*
Exploratory behavior $0.22–0.54 12 4–23
Exploratory behavior #0.22–0.54 12 1–8
Max corticosterone
a
$0.08–0.24 12 1–16
CORT response
a
$0.08–0.24 12 2–23
Max corticosterone
a
#0.08–0.24 12 1–7
CORT reponse (area)
a
#0.08–0.24 12 1–4
Flight distance (foraging)
b
0.88 12 9
Flight distance (incubating)
b
$0.88 12 15
Max CORT
b
$0.08–0.24 12 1–15
CORT response (area)
b
$0.08–0.24 12 1–15
a
Measured in common garden study.
b
Measured in field study.
c
Estimated from published studies, see MATERIALS AND METHODS.
Figure 4
Individual variation for maximum CORT and exploratory behavior
from the common garden study. Max CORT predicted individual
exploratory behavior scores (P= 0.021) in a GLM model the
also included significant effects of population (P= 0.015) and sex
(P= 0.049).
Atwell et al. •Rapid divergence in boldness and corticosterone 7
Similarly, studies of population or subspecific variation in hor-
monal systems are rare, particularly those that evaluate develop-
mental versus genetic underpinnings. In an exception and
a system similar to ours, Partecke et al. (2006) found that urban
blackbirds raised in a common garden showed attenuated
CORT response to handling stress when compared with their
wildland counterparts. Thus, our findings further extend the
generalization that attenuated HPA responsiveness and reduced
neophobia are associated with adaptation to urbanization.
Another common garden study also recently concluded that
variation in the CORT response among closely related songbird
species with varying life histories also likely has a genetic basis
(Angelier et al. 2011). One particularly relevant life-history dif-
ference that has been hypothesized to underlie variation in glu-
cocorticoid responses is the value of current versus future
reproduction and survival opportunities, and comparative data
across species support the idea that breeding season length
correlates positively with HPA responsiveness to short-term
stressors (Bokony et al. 2009). This would predict weaker not
stronger response to stressors in the urban junco population
which has an unusually long breeding season length
(February–August) as compared with the species-typical May–
July breeding season observed at Mt Laguna. This contrasting
result to the usual among-species pattern suggests that in
our case, the selective factors associated with urbanization are
likely more important than those associated with length of the
breeding season.
Although our findings suggest a genetic basis for behavioral
and hormonal differences, environmental effects or early de-
velopmental effects could also play a role. The magnitude of
the difference in CORT profiles between populations was
not as strong in the common garden as in the field (although
sample sizes were also smaller and absolute levels were gener-
ally lower in captivity), and reported heritabilities in other sys-
tems for exploratory behavior and CORT levels are moderate
(’0.2–0.4) (Dingemanse et al. 2002; van Oers et al. 2005;
Evans et al. 2006), which leaves room for a substantial envi-
ronmental contribution to the behavioral and hormonal phe-
notype. For example, further reduced CORT response in the
urban population due to habituation to frequent stressors
may be a likely explanation for the larger difference in CORT
profiles in the field than in the common garden study.
Maternal effects are another type of developmental effects
that could influence behavior in adulthood. For example, dif-
ferential maternal allocation of egg yolk hormones can alter
behavioral development in ways that persist into adulthood
(Groothuis et al. 2005). For example, 9-month-old zebra
finches (Taeniopygia gutatta) hatched from testosterone-
treated eggs habituated faster in a test of neophobia (Tobler
and Sandell 2007). Therefore, if junco females in the urban
population deposited more androgens into their egg yolks,
this might contribute to the faster exploratory behavior of
San Diego birds. But most of the phenotypic differences be-
tween the San Diego and Laguna Mountain populations (e.g.,
breeding season length, male territoriality, plumage ornamen-
tation) have been associated with lower testosterone not high-
er, which would predict lower androgen levels in the San
Diego population during development (Yeh 2004; Newman
et al. 2006). Thus differences in yolk androgens are unlikely
to explain the population differences in behavior reported
here. Exposure to elevated maternal and postnatal CORT
and developmental stress treatments is also known to alter
boldness behaviors and glucocorticoid levels even into adult-
hood (reviewed in Schoech et al. 2011), but the direction of
the effects and diverse methodologies make generalizing the
results of these studies challenging.
We also cannot rule out the possibility that early learning
of exploratory behaviors could have taken place between
hatching and capture for the common garden study. The pe-
riod during which the juveniles were foraging independently
(10–15 days on average) was far briefer than the time spent
developing in the common environment prior to behavioral
testing (.8 months), but little is known about critical periods
for development of exploratory behavior. Notwithstanding that
maternal or early developmental effects cannot be completely
ruled out, we suggest that genetic evolution is the most likely
cause for the persistent behavioral and hormonal differences
in the colonist population.
Differences in exploratory behavior were not explained by
allelic variation at the SNP830 region of the dopamine recep-
tor gene (Drd4), as this region was found to be mostly invari-
ant in our sample. A polymorphism at the SNP830 locus
correlates with exploratory behavior in some populations of
great tits (P. major), both captive and free-living, and this poly-
morphism responded to artificial selection for fast or slow
exploratory behavior (Fidler et al. 2007; Korsten et al.
2010). However, many other loci with small effects are also
expected to contribute to differences in boldness among in-
dividuals (van Oers, Drent, de Jong, et al. 2004; van Oers et al.
2005), especially since the allelic variation at SNP830 only
explained 4.5–6.0% of the variation in behavior in the P. major
study (Fidler et al. 2007; Korsten et al. 2010). The lack of
variation at the Drd4 SNP830 in the junco suggests that the
divergence in the urban population may have been achieved
via other genes or alternative mutations within the larger Drd4
gene. This is unsurprising given that studies of 3 additional
P. major populations found no clear association between
SNP830 and behavior, suggesting this mutation was popula-
tion specific (Korsten et al. 2010). The SNP830 region of the
Drd4 gene was highly conserved in dark-eyed juncos, which
enhances the prospects for future sequencing of other re-
gions of the Drd4 gene, and across avian taxa, several other
indels and SNP within Drd4 are being found (Abe et al. 2011).
In sum, our data show an integrated pattern of adaptive pop-
ulation divergence for a behavioral trait and a putative causal
hormonal mechanism that persisted in a common environ-
ment, suggesting genetic evolution. The differences between
populations lie in the direction predicted by within-population
hormone-phenotype relationships, suggesting correlated evo-
lution along lines of least resistance (Schluter 1996), rather
than changes in reactions norms to adjust behavior indepen-
dently of hormonal levels. Maintenance of individual covaria-
tion among CORT and boldness behaviors within divergent
populations underscores the point that there is likely no sin-
gle optimum hormone–behavior phenotypic strategy in a pop-
ulation, but rather a range of successful individual strategies
maintained by functional trade-offs or opposing or variable
selective factors (Koolhaas et al. 1999; Schoech et al. 2011).
Although evolutionary conservation of trait correlations, in-
cluding hormone–phenotype associations, has most generally
been considered evidence of a likely constraint on evolutionary
diversification (Schluter 1996; Hau 2007), functionally sensible
trait correlations could also facilitate adaptation to novel envi-
ronments (Agrawal and Stinchcombe 2009; Ketterson et al.
2009). This may be the case in our study system, where changes
in an integrating hormonal mechanism (the HPA endocrine
axis) may underlie divergence for a suite of adaptive behavioral
and physiological characteristics, allowing for more rapid adap-
tation (evolutionary or developmental) to novel environments.
Additional studies of the neuroendocrine and genetic mecha-
nisms linking hormonal and behavioral phenotypes are needed
to assess the evolutionary significance of these trait associations.
Future research should thus continue to focus both on identi-
fying the mechanistic sources of variance underlying trait
correlations and evaluating how integrated phenotypes
respond to novel and changing environments.
8Behavioral Ecology
at Michigan State University on September 4, 2012http://beheco.oxfordjournals.org/Downloaded from

Behavioral Ecology968
SUPPLEMENTARY MATERIAL
Supplementary material can be found at http://www.beheco.
oxfordjournals.org/.
FUNDING
National Science Foundation (Grant #BSC05-19211 to E.D.K.;
0808284 to J.W.A. and E.D.K.); Indiana University Faculty Re-
search Support Program and Graduate Student and Professional
Organization; the Center for the Integrative Study of Animal
Behaviour at Indiana University, the Indiana Academy of Scien-
ces, the Explorer’s Club, Sigma Xi, the Society for Integrative
and Comparative Biology, and the Animal Behaviour Society.
We would like to thank the Descanso Ranger District of the Cleveland
National Forest and the Ecology, Behavior & Evolution Division of Bi-
ology at University of California–San Diego (UCSD) for logistical sup-
port and permission to work in the study areas. K. Marchetti and R.
Lande at UCSD, T. Price at University of Chicago, and the Al Bahr
Shrine Camp on Mt Laguna also provided logistical support. Amanda
Brothers, Eric Snajdr, Kim Roth, Sarah Puckett, Angela Kemsley, Alli-
son Miller, and Russell Nichols assisted with fieldwork. Ediri Metitiri
assisted with lab work. J.W.A. was supported by a Graduate Research
Fellowship from the National Science Foundation and the Training
Grant from National Institute of Health (NICHD: T32HD049336) and
G.C.C. by a postdoctoral fellowship from the Fundacxa
˜o para a Cieˆncia
e a Tecnologia. This research was conducted with the approval of the
Indiana University Animal Care and Use Committee (Study #06-242)
and with permits from the US Fish and Wildlife Service, the California
Department of Fish and Game, and the US Forest Service.
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10 Behavioral Ecology
at Michigan State University on September 4, 2012http://beheco.oxfordjournals.org/Downloaded from

Atwell et al. • Rapid divergence in boldness and corticosterone 969
SUPPLEMENTARY MATERIAL
Supplementary material can be found at http://www.beheco.
oxfordjournals.org/.
FUNDING
National Science Foundation (Grant #BSC05-19211 to E.D.K.;
0808284 to J.W.A. and E.D.K.); Indiana University Faculty Re-
search Support Program and Graduate Student and Professional
Organization; the Center for the Integrative Study of Animal
Behaviour at Indiana University, the Indiana Academy of Scien-
ces, the Explorer’s Club, Sigma Xi, the Society for Integrative
and Comparative Biology, and the Animal Behaviour Society.
We would like to thank the Descanso Ranger District of the Cleveland
National Forest and the Ecology, Behavior & Evolution Division of Bi-
ology at University of California–San Diego (UCSD) for logistical sup-
port and permission to work in the study areas. K. Marchetti and R.
Lande at UCSD, T. Price at University of Chicago, and the Al Bahr
Shrine Camp on Mt Laguna also provided logistical support. Amanda
Brothers, Eric Snajdr, Kim Roth, Sarah Puckett, Angela Kemsley, Alli-
son Miller, and Russell Nichols assisted with fieldwork. Ediri Metitiri
assisted with lab work. J.W.A. was supported by a Graduate Research
Fellowship from the National Science Foundation and the Training
Grant from National Institute of Health (NICHD: T32HD049336) and
G.C.C. by a postdoctoral fellowship from the Fundacxa
˜o para a Cieˆncia
e a Tecnologia. This research was conducted with the approval of the
Indiana University Animal Care and Use Committee (Study #06-242)
and with permits from the US Fish and Wildlife Service, the California
Department of Fish and Game, and the US Forest Service.
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