Journal of Animal Ecology 2017, 86, 227–238 doi: 10.1111/1365-2656.

12621

Repeatability, heritability, and age-dependence of

seasonal plasticity in aggressiveness in a wild
passerine bird
Yimen G. Araya-Ajoy*,1,2 and Niels J. Dingemanse1,3
1
Research Group Evolutionary Ecology of Variation, Max Planck Institute for Ornithology, Seewiesen, Germany;
2
Center for Biodiversity Dynamics, Norwegian University of Science and Technology, Trondheim, Norway; and
3
Behavioural Ecology, Department of Biology, Ludwig-Maximilians University of Munich, Planegg-Martinsried,
Germany

Summary
1. Labile characters allow individuals to flexibly adjust their phenotype to changes in envi-
ronmental conditions. There is growing evidence that individuals can differ both in average
expression and level of plasticity in this type of character. Both of these aspects are studied in
conjunction within a reaction norm framework.
2. Theoreticians have investigated the factors promoting variation in reaction norm intercepts
(average phenotype) and slopes (level of plasticity) of a key labile character: behaviour. A
general prediction from their work is that selection will favour the evolution of repeatable
individual variation in level of plasticity only under certain ecological conditions. While fac-
tors promoting individual repeatability of plasticity have thus been identified, empirical esti-
mates of this phenomenon are largely lacking for wild populations.
3. We assayed aggressiveness of individual male great tits (Parus major) twice during their
egg-laying stage and twice during their egg-incubation stage to quantify each male’s level of
seasonal plasticity. This procedure was applied during six consecutive years; all males breed-
ing in our plots during those years were assayed, resulting in repeated measures of individual
reaction norms for any individual breeding in multiple years. We quantified among- and
within-individual variation in reaction norm components, allowing us to estimate repeatability
of seasonal plasticity. Using social pedigree information, we further partitioned reaction norm
components into their additive genetic and permanent environmental counterparts.
4. Cross-year individual repeatability for the intercepts (average aggressiveness) and slopes
(level of seasonal plasticity) of the aggressiveness reaction norms were 0
574 and 0
516 respec-
tively. The mean of the posterior distributions suggested modest heritabilities (h2 = 0
260 for
intercepts; h2 = 0
266 for slopes), but these estimates were relatively uncertain. Males
behaved more aggressively in areas with higher breeding densities, and became less aggressive
and less plastic with increasing age; plasticity thus varied within individuals and was multidi-
mensional in nature.
5. This empirical study quantified cross-year individual repeatability, heritability and age-
related reversible plasticity in behaviour. Acknowledging such patterns of multi-level variation
is important not only for testing behavioural ecology theory concerning the evolution of
repeatable differences in behavioural plasticity but also for predicting how reversible plasticity
may evolve in natural populations.
Key-words: aggression, animal personality, multi-level variation, plasticity, random regres-
sion, reaction norm, repeatability

*Correspondence author. E-mail: yimencr@gmail.com

© 2016 The Authors. Journal of Animal Ecology © 2016 British Ecological Society
228 Y. G. Araya-Ajoy & N. J. Dingemanse
Introduction
Certain phenotypic characters, like behaviour and physiol-
ogy, are expressed repeatedly within the same individual.
Like most phenotypic characters, such ‘labile’ characters
can be both heritable and be affected by early-environ-
mental factors (i.e. harbour developmental plasticity)
(Stirling, R
eale & Roff 2002; Dochtermann, Schwab &
Sih 2015). What uniquely defines them, however, is that
they can also be adjusted from one expression to the next
by means of reversible plasticity. Labile characters thereby
represent key mediators between organisms and their
environment because they allow individuals to adaptively
match their phenotype to environmental factors that vary
within their lifetime (Gabriel et al. 2005; Duckworth
2008; Botero et al. 2014).
The level of reversible plasticity may also vary among
individuals (Nussey, Wilson & Brommer 2007), with some
individuals responding to environmental changes, whereas
others do not; this variation may also be heritable (e.g.
Brommer et al. 2005; Nussey et al. 2005), implying that
reversible plasticity can evolve. Individual variation in
reversible plasticity is commonly studied within the reac-
tion norm framework (Schlichting & Pigliucci 1998): ‘indi-
vidual’ reaction norms are (linear or nonlinear) functions
describing the dependency of an individual’s phenotypic
expressions on a particular environmental gradient (Nus-
sey, Wilson & Brommer 2007; Dingemanse et al. 2010).
In their simplest form, the variation in reaction norms
that is present within a population can be described by
estimating three key variance components: (i) variation
among-individuals in reaction norm intercept, where an
individual’s intercept is typically modelled to represent its
average phenotypic expression in the population-average
environment, (ii) variation among-individuals in reaction
norm slope, where an individual’s slope represents its
degree of (reversible) plasticity in response to a focal envi-
ronmental gradient and (iii) the covariance between these
two components, representing a measure of how an indi-
vidual’s phenotype in the average environment relates to
its level of plasticity. The concept of reaction norms in
combination with variance decomposition approaches has
provided evolutionary ecologists with a conceptual and
methodological framework by which the causes and con-
sequences of variation in average behaviour and level of
reversible plasticity can be studied in conjunction (Nussey,
Wilson & Brommer 2007; van de Pol 2012; Dingemanse
& Dochtermann 2013).
Over the past decade, behavioural research has increas-
ingly focussed on documenting repeatable individual dif-
ferences in behavioural characters, such as aggressiveness,
feeding specializations and cooperative behaviour (re-
viewed by Bolnick et al. 2003; Dall, Houston & McNa-
mara 2004; R
eale et al. 2007; Bell, Hankison &
Laskowski 2009). At the same time, a suite of conceptual
and theoretical models have been developed to explain
why individual differences in average behaviour (i.e. the
© 2016 The Authors. Journal of Animal Ecology © 2016 British Ecological Society, Journal of Animal Ecology, 86, 227–238
among-individual variation in reaction norm intercepts
described above) might evolve (e.g. Wolf et al. 2007;
McNamara et al. 2009; Wolf & Weissing 2010). While
empiricists are putting their model predictions and
assumptions to the test, e.g. (Nicolaus et al. 2012; Las-
kowski & Pruitt 2014), theoreticians have also started to
explore the ecological conditions that favour the evolution
of repeatability in reversible plasticity (Wolf, van Doorn
& Weissing 2008; Wolf, Van Doorn & Weissing 2011;
reviewed by Dingemanse & Wolf 2013). An important
insight of these modelling studies is that selection may
favour the evolution of either among-individual or within-
individual variation in reversible plasticity. The occur-
rence of among-individual variation would imply that
individuals are repeatable in level of reversible plasticity,
whereas within-individual variation would imply that indi-
viduals adjust their level of reversible plasticity across
time depending on socio-ecological conditions (Fig. 1). To
date, the question of whether reversible plasticity is or is
not repeatable has not been addressed for wild popula-
tions. We thus require empirical estimates of the repeata-
bility of reaction norm components in wild populations
such that productive feedback between theoreticians and
empiricists can help further research on the evolutionary
ecology of reversible plasticity in behaviour and other
labile characters.
The quantification of the repeatability of reaction norm
components is not only important for testing predictions
of theoretical models developed in behavioural ecology; it
also provides important clues on whether reversible plas-
ticity might evolve in response to selection. This would
require reversible plasticity to be heritable (Brommer
et al. 2005; Nussey et al. 2005), where repeatability esti-
mates are useful as they enable the quantification of the
upper limit to heritability (Lynch & Walsh 1998). More-
over, information on within-individual sources of varia-
tion in reversible plasticity will provide important further
insights in whether individuals are potentially able to
directly, and adaptively, adjust their level of reversible
plasticity when faced with environmental change. This
would facilitate responses on an ecological rather than
evolutionary time-scale, and require that plastic within-
individual responses towards one environmental gradient
are a function of another environmental factor. In birds,
for example parents increase nestling provisioning rate
with nestling age but the magnitude of this plastic adjust-
ment is a function of the number of offspring in the nest
(Westneat et al. 2011); this flexibility allows parents to
adaptively adjust their provisioning reaction norm to eco-
logical conditions that determine brood size in a focal
year. Therefore, determining whether reaction norms are
repeatable, and studying the genetic and environmental
sources of variation in reaction norms, is of key impor-
tance to further our understanding of the evolution of
labile characters.
In this paper, we focus on an important labile character
(territorial aggressiveness) that male passerine birds

Fig. 1. Graphical representation showing
that reaction norms can vary both within
and among individuals, here illustrated for
two males (colours) assayed twice in each
of two breeding stages (egg-laying and
incubation) in each of 2 years. In panel
(a), reaction norms show cross-year indi-
vidual repeatability, in panel (b) this is
not the case. [Colour figure can be viewed
at wileyonlinelibrary.com]
express when faced with a territorial intrusion, and that
affects key fitness components (Duckworth 2006) and
population level processes (Mougeot et al. 2003). We
tested for the existence of cross-year repeatability in
aggressiveness reaction norms of male great tits (Parus
major) using a 6-year data set collected in 12 nest box
plots. We focused on cross-year repeatability of within-
year patterns of seasonal plasticity as we have previously
shown that great tits decrease their territorial aggressive-
ness between egg laying and incubation (Araya-Ajoy &
Dingemanse 2014). We measured this seasonal adjustment
each year in which a focal male reproduced in our popu-
lation. This enabled us to statistically quantify the total
phenotypic variation in male aggressiveness reaction
norms in response to breeding stage during the whole
6-year period. Following Araya-Ajoy, Mathot & Dinge-
manse (2015), we partitioned this variation into its long
term (i.e. among-individual) and short term (i.e. within-
individual-among-year) components to test whether the
individual-level intercepts and slopes of these aggressive-
ness reaction norms showed long-term individual repeata-
bility. We then proceeded to study key sources of
variation that might underpin long-term repeatability, and
used social pedigree information to determine how much
of the among-individual variation in reaction norm inter-
cepts and slopes was underpinned by additive genetic vari-
ation vs. permanent environmental effects.
We further studied whether these seasonal changes in
aggressiveness varied within individuals across years as a
function of other environmental factors; such effects may
be expected as the balance between the costs and bene-
fits of an aggressive interaction typically depend on
© 2016 The Authors. Journal of Animal Ecology © 2016 British Ecological Society, Journal of Animal Ecology, 86, 227–238
Repeatability of seasonal plasticity 229
ecological conditions that vary across years such as
breeding density. We thus considered that while heritable
variation in reaction norm components would result in
long-term (i.e. among-year) individual repeatability, envi-
ronmental sources of variation may either cause long-
term or short-term variation in reaction norm compo-
nents (Araya-Ajoy, Mathot & Dingemanse 2015; Senner,
Conklin & Piersma 2015). For instance the optimal level
of aggressiveness of a male great tit, and how much it
should down-regulate its aggressiveness over the breeding
season, should vary as a function of breeding density as
this socioecological factor affects the costs and benefits
associated with territorial aggressiveness (Fig. 2).
Among-year variation in density should thus affect both
the individual’s year-specific average level of aggressive-
ness as well as its level of within-year seasonal plasticity.
Among-year changes in density may thus cause among-
year variation in an individual’s reaction norm intercept
(i.e. average level of aggressiveness) and slope (i.e. level
of seasonal plasticity). Importantly, breeding densities
show substantial spatial variation in great tits (e.g. Nico-
laus et al. 2013, 2016). Hence, individuals are likely
exposed to similar competitive regimes across multiple
years, which may thus cause cross-year repeatability in
aggressiveness reaction norms. We therefore further
tested whether variation in reaction norms was attributa-
ble to spatial and temporal variation in breeding density.
Finally, we investigated how age affected the aggressive-
ness reaction norms as this intrinsic factor should affect
the costs and benefits of average aggressiveness and its
seasonal plasticity (Stearns & Koella 1986; Kindlmann,
Dixon & Dost
alkov

a 2001).
230 Y. G. Araya-Ajoy & N. J. Dingemanse
Materials and methods
population and study site
We studied 12 nest box plots of great tits that were established in
2009 in Southern Germany (Bavarian Landkreis Starnberg;
47°550 14″ N–48°010 15″ N, 11°090 24″ E–11°200 27″ E). Each plot
consisted of a grid of 50 boxes with 50 m between adjacent
boxes. From April onwards, boxes were checked twice a week,
and lay date (back-calculated assuming that one egg was laid per
day) and the onset of incubation (inferred from the presence of
an incubating female or warm eggs) determined. When the nest-
lings were 7 days old, parents were caught with a spring trap set
in the box, weighed, measured and marked with an aluminium
ring and unique colour ring combination, if not previously
marked.
experimental protocol
Aggressiveness data were collected over a 6-year period (2010–
2015) for all first broods produced by great tit pairs in our study
plots. The aggression test started when a taxidermic mount of a
male great tit was presented on a 1
2 m wooden pole with a play-
back song 1 m away from the subject’s nest box. We subse-
quently recorded the behaviour of the focal male for a period of
3 min after it had entered a 15-m radius around the box. Simu-
lated territorial intrusions were performed twice during the egg-
Fig. 2. An illustration of a hypothetical population-level reaction
norm plane where the phenotype is a function of two environ-
mental gradients. We show here a reaction norm plane for a situ-
ation where there is multi-dimensional plasticity due to
interactive effects of two environmental covariates causing a
‘warped’ reaction norm plane. Three individuals (colours) are
depicted; each individual was assayed in each of 2 years. Within
males, density varied only across the 2 years. Variation in density
causes among- and within-individual variation in the intercept
and the slope of the seasonal aggressiveness reaction norms.
[Colour figure can be viewed at wileyonlinelibrary.com]
© 2016 The Authors. Journal of Animal Ecology © 2016 British Ecological Society, Journal of Animal Ecology, 86, 227–238
laying stage and twice during the egg-incubation stage of each
focal nest. Details of the experimental setup, and essayed beha-
viours, are provided in Araya-Ajoy & Dingemanse (2014). In
short, an aggressive response was characterized by intensive
alarm calling, approach to the stimulus, and, in the most extreme
case, jumping and pecking of the cage that protected the mount.
For the purpose of this paper we used the subject’s minimum dis-
tance to the mount as a measure of aggressiveness because previ-
ous work implied that this behaviour represented a reliable
predictor of the intensity of an aggressive response for both
breeding stages (Araya-Ajoy & Dingemanse 2014).
statistical analyses
Estimating variation in reaction norms
We used a suite of multi-level random regression mixed-effects
models to quantify variation in reaction norm intercepts and
slopes within and among individual males. Aggressiveness was
modelled as a function of the stage of the focal male’s nest.
Breeding stage was fitted as an environmental covariate consist-
ing of two levels (egg-laying vs. egg-incubation stage). These two
levels were first coded as zero and one, respectively, and subse-
quently mean centred and standardized to two standard deviation
units; for a discussion about the usefulness of this transformation
see Gelman (2008) and Araya-Ajoy, Mathot & Dingemanse
(2015). All models presented estimated breeding stage-specific
residual variances (i.e. heterogeneous residuals; Cleasby & Naka-
gawa 2011). Note that the reaction norm approach that we use
here (i.e. modelling reaction norm variation with two discrete
environments while considering heterogeneous residuals) is math-
ematically equivalent to applying a character state approach (Via,
Gomulkiewicz & De Jong 1995). Indeed, re-analyses of our data
using this alternative approach (Table S2, Supporting Informa-
tion) corroborated the findings based on reaction norm analyses
(see Results).
Following Araya-Ajoy, Mathot & Dingemanse (2015), we used
various parameterizations of random regression models to quan-
tify the existence of variation in reaction norms at various hierar-
chical levels. The initial model (#1) included random intercepts
and slopes for breeding attempt identity (i.e. the unique combina-
tion of male and year; 1042 levels). Breeding attempt represents
the temporal unit within which we collected repeated measures of
an individual in response to the focal environmental gradient
(breeding stage). We obtained up to two measurements of aggres-
siveness for each of the two breeding stages (egg-laying and egg-
incubation) for each male in each year. This study design allowed
us to model random intercepts and slopes for breeding attempt
identity, and enabled us to quantify the total phenotypic variance
in reaction norm intercepts and slopes in our population during
the entire study period. The second model (#2) included not only
random intercepts and slopes for each breeding attempt (see
above) but also for individual identity (679 levels). This model
thus allowed us to partition the phenotypic variation in reaction
norm intercepts and slopes into its long-term (i.e. among-
individual) vs. short-term (i.e. within-individual-among-year)
components.
As a next step, we tested whether breeding density explained
either within- or among-individual variation in reaction norm com-
ponents (see Introduction). We addressed this question by fitting
the interaction between plot-average density (i.e. the plot’s mean
 Repeatability of seasonal plasticity 231

value over the years 2010–2015) and breeding stage, as well as the
interaction between breeding stage and the yearly deviations in
density from each plot mean, to evaluate spatial and temporal
effects of density respectively (Nicolaus et al. 2016). We then deter-
mined whether these fixed-effects explained within- and/or among-
individual variation in reaction norm intercepts and slopes. Finally,
to test whether reaction norm characteristics changed with age, we
ran a model where we fitted the interaction between male age and
breeding stage. Age was categorized as first year vs. older breeders
coded as zero vs. one respectively. We also standardized these
values to two standard deviation units prior to analyses.
As a final step, we determined how much of the among-indivi-
dual variance in intercepts and slopes was due to additive genetic
vs. permanent environmental effects. We used the social pedigree
information available for our population to transform our mixed-
effects model into an animal model (Wilson et al. 2010). We use
social pedigrees as genetic paternity was not determined for all
years of study. Moreover, simulation studies imply that extra-pair
reproduction typically does not substantially bias quantitative
genetic parameters (Charmantier & R
eale 2005; but see Firth
et al. 2015). Social pedigree information is provided in Figs. S1,
S2 and Table S1.
Repeatability and heritability estimates
Repeatability represents a standardized metric that allows com-
parison across traits and studies (Nakagawa & Schielzeth 2010).
Reaction norm intercept and slope repeatabilities were calculated
from parameter estimates extracted from our multi-level random
regression model (see Table 1) following Araya-Ajoy, Mathot &
Dingemanse (2015). Intercept repeatability was estimated as the
amount of among-individual variance in intercepts divided by the
total phenotypic variance in intercepts (i.e. the sum of the
among-individual and among-breeding attempt variances in inter-
cept). Slope repeatability was estimated as the amount of among-
individual variance in slopes divided by the total phenotypic
variation in slopes (i.e. the sum of the among-individual and
among-breeding attempt variances in slope). Similarly, intercept
and slope heritabilities were estimated as the amount of additive
genetic variance in intercepts and slopes extracted from the ani-
mal model divided by their respective total phenotypic variance
(i.e. the sum of the additive genetic, permanent environment and
among-breeding attempt variances).
General modelling procedures
We fitted the (multi-level) random regression models detailed
above using a Bayesian framework implemented in R v3.3 (R
Core Team 2015) with the package MCMCglmm (Hadfield
2010). We ran 3 005 000 iterations per model, from which we dis-
carded the initial 5000 (burn in period). Each chain was sampled
at an interval of 3000 iterations, which resulted in a low autocor-
relation among thinned samples. Posterior means and 95% credi-
ble intervals were estimated across the thinned samples for the
mean effects (fixed effects), (co)variances and variance ratios (i.e.
repeatabilites and heritabilities). Fixed effect priors were normally
distributed and diffuse with a mean of zero and a large variance
(100). Different priors were used for the variance-covariance
matrices; the results presented in the paper correspond to param-
eter-expanded priors for the random regression models (see
Appendix S1); mean values of the posterior distributions were
robust to different relatively un-informative prior settings, and

Table 1. Results from random regression models estimating fixed and random effects explaining variation in aggressiveness and its sea-
sonal plasticity. Model 1 quantifies the total amount of variation in reaction norms intercepts and slopes (with respect to breeding stage:
egg laying vs. incubation) among all breeding attempts. Model 2 partitions this variation into among- and within-individual effects.
Models 3 and 4 ask how much of the among- and within-individual variation can be captured by effects of breeding density and age
respectively. We provide mean values of the posterior distribution of parameter estimates and their 95% credible intervals (CI)

Model 1 Model 2 Model 3 Model 4

Fixed effects b (95% CI) b (95% CI) b (95% CI) b (95% CI)
Intercept 2
892 (2
834, 2
951) 2
855 (2
788, 2
920) 2
834 (2
746, 2
916) 2
777 (2
694, 2
858)
Breeding stage 0
579 (0
491. 0
661) 0
602 (0
508. 0
696) 0
492 (0
257, 0
713) 0
602 (0
486, 0
725)
Spatial density 0
155 (0
234, 0
086)
Temporal density 0
003 (0
020, 0
015)
Breeding stage * spatial 0
018 (0
097, 0
124)
density
Breeding stage * temporal 0
011 (0
038, 0
016)
density
Age 0
261 (0
140, 9
389)
Breeding stage * age 0
197 (0
396, 0
001)
Random effects r2 (95% CI) r2 (95% CI) r2 (95% CI) r2 (95% CI)
Among-male variance
Vintercepts 0
210 (0
110, 0
326) 0
186 (0
087, 0
293) 0
275 (0
182, 0
380)
Vslopes 0
179 (0
002, 0
444) 0
176 (0
003, 0
406) 0
201 (0
004, 0
439)
Covintercepts-slopes 0
065 (0
183, 0
027) 0
067 (0
190, 0
027) 0
074 (0
179, 0
024)
Among-breeding attempt variance
Vintercepts 0
495 (0
408, 0
585) 0
283 (0
177, 0
401) 0
281 (0
181, 0
349) 0
167 (0
077, 0
275)
Vslopes 0
385 (0
160, 0
602) 0
205 (0
002, 0
497) 0
195 (0
001, 0
494) 0
176 (0
001, 0
478)
Covintercepts-slopes 0
162 (0
273, 0
054) 0
086 (0
221, 0
023) 0
075 (0
219, 0
038) 0
053 (0
183, 0
046)
Residual within-individual variance
Vlaying 1
341 (1
171, 1
524) 1
349 (1
194, 1
530) 1
370 (1
202, 1
556) 1
364 (1
205, 1
552)
Vincubation 0
870 (0
783, 0
966) 0
867 (0
782, 0
961) 0
866 (0
782, 0
955) 0
871 (0
782, 0
968)

© 2016 The Authors. Journal of Animal Ecology © 2016 British Ecological Society, Journal of Animal Ecology, 86, 227–238
232 Y. G. Araya-Ajoy & N. J. Dingemanse
were quantitatively similar when a restricted maximum likelihood
framework was used instead (results not shown).
Statistical support for fixed and random effects
We considered estimates of fixed effects and covariances to be
significantly different from zero (i.e. in the frequentist’s sense)
when their associated 95% credible intervals did not overlap zero.
The statistical support for a non-zero value of a variance compo-
nent was assessed differently because variance components are
bound to be positive, and because prior choice may influence the
credible intervals derived from the posterior distribution. For
variances we therefore determined the probability that an esti-
mated variance was different from a null expectation based on
permutation tests (Good 2000). During each permutation, the
aggressiveness data were randomly re-allocated to different obser-
vations. This resulted in a new data set with the same mean, vari-
ance and level of replication as our observed data set; the only
difference was that the aggressiveness measurements were reshuf-
fled randomly across the data set. We then proceeded to perform
the mixed-effects models (detailed above) to this new data set,
and estimated, for each permutation, a posterior mean value for
each variance component of interest. This procedure was repeated
100 times to generate a ‘null’ distribution of posterior mean esti-
mates. We then calculate the probability (permutation.p) that the
observed posterior mean value of a focal variance component
was greater than any value expected from this permutation-based
null distribution.
Results
We analysed 2854 aggression tests performed to 1042
breeding attempts of 679 individuals. The average number
of years for which we obtained an individual’s reaction
norm was 1
4, with 513, 142, 44, 8, 8 and 1 individual(s)
sampled for one, two, three, four, five or six breeding
attempt(s) (years) respectively. On average, we acquired
2
8 (out of 4) data points for male aggressiveness per
breeding attempt (i.e. year), because males did not always
respond to the territorial intrusion experiment (see also
Araya-Ajoy & Dingemanse 2014). The average male in
the average environmental condition (breeding stage)
approached our visual stimulus to a distance of 10
28 m
(standard deviation: 7
45); males approached the model
2
86 (1
37) m closer during the egg-laying compared to
the egg-incubation stage.
reaction norm analyses
As expected based on our previous analyses (Araya-Ajoy
& Dingemanse 2014), aggressiveness was down-regulated
from the egg-laying to the egg-incubation stage owing to
a positive effect of breeding stage on approach distance
(Table 1). We found strong support for intercept (mean:
0
495; 95% CI: 0
408–0
585) and slope variance (0
385;
0
160–0
602) among breeding attempts (permutation.p
was <0
001 for both estimates). Furthermore, we found
strong support for a negative correlation between inter-
cepts and slopes (0
371; 95% CI: 0
602, 0
141).
© 2016 The Authors. Journal of Animal Ecology © 2016 British Ecological Society, Journal of Animal Ecology, 86, 227–238
Altogether, those three patterns of (co)variation implied
that breeding attempts characterized by relatively high
average levels of aggressiveness were also characterized by
a substantial down-regulation of aggressiveness from the
egg-laying to the incubation stage.
Next, we partitioned this phenotypic variation in
reaction norm characteristics into its within- and among-
individual components. We found strong support for both
among-individual and within-individual-among-year vari-
ance in intercepts (permutation.p was <0
001 for both
estimates). Both levels contributed similar to the total
phenotypic variation in reaction norm intercepts
(Table 2); individual cross-year repeatability of reaction
norm intercept was 0
574 (95% CI: 0
372, 0
777). We also
found support for non-zero within- and among-individual
variation in reaction norm slopes (permutation.p = 0
03
and <0
001 respectively); we note that the credible inter-
vals for both variance estimates were very broad (Table 1)
and therefore it is difficult to make confident inferences
about their exact values. Individual cross-year repeatabil-
ity in slope was 0
516 (95% CI: 0
004, 0
996). Finally, we
found a negative intercept-slope covariance at both the
among- and within-individual levels, although the credible
intervals for both estimates overlapped zero (Table 1).
Altogether, these findings implied (i) that great tits
showed long-term (i.e. among-year) individual repeatabil-
ity in their average aggressiveness and level of seasonal
down-regulation and (ii) that their aggressiveness was
simultaneously plastically adjusted to (unknown) year-spe-
cific environmental conditions.
As a next step, we asked whether patterns of among- and
within-individual variation in these aggressiveness reaction
norms could be attributed to repeatable among-plot differ-
ences in density or within-plot-among-year (i.e. temporal)
fluctuations in density. The average level of aggressiveness
of male great tits was higher in plots with higher densities
(Fig. 3). This result implies that some of the among-indivi-
dual variation in reaction norm intercepts could be
Table 2. Proportions of phenotypic variance in components of
aggressiveness reaction norms (with respect to breeding stage)
explained by among-individual and among-brood (i.e. among-
year-within-individual) variance. Using an animal model, we fur-
ther partitioned the among-individual variance into additive
genetic and permanent environmental variance. We provide mean
values of the posterior distribution of the proportions (i.e. repeata-
bility and heritability) and their 95% credible intervals (CI)
Proportions Intercept (95% CI) Slope (95% CI)
Among-individual 0
574 (0
372, 0
772) 0
516 (0
004, 0
996)
Among-broods 0
426 (0
228, 0
628) 0
484 (0
004, 0
996)
Permanent 0
184 (0
000, 0
507) 0
302 (0
001, 0
888)
environmental
effects
Additive genetic 0
260 (0
005, 0
548) 0
266 (0
000, 0
853)
variance*
*This proportion represent the narrow-sense heritability (h2) of
this reaction norm component.

Fig. 3. The relationship between aggressiveness and the average
breeding density of each of our 12 study plots. Aggressiveness
was measured as the distance approached to the taxidermic
mount used during the simulated territorial intrusion; higher val-
ues therefore represent a lower aggressive response. Each plot
was fitted with 50 nest boxes; breeding density represents the
number of boxes occupied. Plot density was estimated as the
number of breeding pairs per plot (mean value over the years
2010–2015). The black line represents the predicted point estimate
across the range of densities and the grey-shaded area is the 95%
credible range.
attributed to among-population differences in density
(Table 1); because credible intervals associated with this
variance component were very broad, it is, however, diffi-
cult to assess how much variation was explained by density.
In contrast, temporal fluctuations in density did not affect
the expression of aggressiveness reaction norm intercepts or
slopes (Table 1) and therefore did not explained variation
in reaction norm intercepts or slopes at any level. Age sig-
nificantly affected the average level of aggression, as well as
the magnitude of the plastic adjustment to changes in
breeding stage (Table 1): as males aged, they became less
aggressive and showed less seasonal plasticity (Fig. 4).
These results, therefore, imply that age explained within-
individual variation in reaction norm intercepts and slopes.
As a final step, we proceeded to partition the detected
among-individual variation in reaction norm characteris-
tics into additive genetic and permanent environment
components using a random regression animal model
(Table 3). We found support for non-zero additive genetic
variation in intercepts (permutation.p < 0
001) and also
for non-zero permanent environmental effects (permuta-
tion.p < 0
001). The credible intervals associated with
both variance components were, notably, very broad
(Table 3), implying that we are uncertain about the exact
amount of variance explained by each. The proportion of
variation in reaction norm intercepts underpinned by
additive genetic variance (heritability) was 0
26 (95% CI:
0
005–0
548), and the proportion explained by permanent
environmental effects was 0
184 (0
000–0
507). With
regards to reaction norm slopes, we found evidence for
significant non-zero additive genetic and permanent envi-
ronmental variance, though support for non-zero effects
were modest (permutation.p = 0
03 and 0
01 respectively).
The heritability of reaction norm slope was 0
266 (0
000,
© 2016 The Authors. Journal of Animal Ecology © 2016 British Ecological Society, Journal of Animal Ecology, 86, 227–238
Repeatability of seasonal plasticity 233
Fig. 4. Aggressiveness reaction norms for two age groups. The
solid vs. dotted lines represent the predicted average reaction
norm for first-year vs. older males. Aggressiveness was measured
as the distance approached to the taxidermic mount used in the
simulated territorial intrusion; higher values therefore represent
a lower aggressive response. Shaded areas represent the 95%
credible range.
0
853), whereas the proportion of variation in slopes
explained by permanent environmental effects was 0
302
(0
001, 0
888). As above, the credible intervals were very
broad for both for of these proportions, and we therefore
cannot draw firm conclusions about their exact magni-
tudes (Table 2).
Discussion
We quantified the sources of variation in aggressiveness
reaction norms of male great tits in the wild. We detected
substantial cross-year individual repeatability in both
average level of aggressiveness and level of seasonal plas-
ticity. Quantitative genetics analyses provided support for
nonzero heritability in average behaviour (reaction norm
intercept) and (weaker evidence for) level of seasonal plas-
ticity (reaction norm slope), but uncertainty in these esti-
mates was very high and therefore we cannot draw firm
conclusions about their magnitude. Repeatable differences
in aggressiveness were partly explained by spatial varia-
tion in breeding density: males breeding in plots with high
breeding densities were also more aggressive overall.
Finally, aggressiveness reaction norms harboured patterns
of multidimensional plasticity: males became less aggres-
sive and less responsive when they were older.
repeatability of aggressiveness reaction
norms
Theoretical models predict that repeatable reaction norms
should evolve in situations where there is spatiotemporal
heterogeneity in resources causing frequency-dependent
selection pressures acting on socially expressed traits for
which the cost of plasticity decrease with experience
(Wolf, van Doorn & Weissing 2008; Wolf, Van Doorn &
Weissing 2011). Aggressiveness mediates competition for
spatiotemporally varying resources (Johnson, Grant &
234 Y. G. Araya-Ajoy & N. J. Dingemanse
Table 3. Results from an animal model that partitions among-
individual variation in aggressiveness reaction norm intercepts
and slopes (with respect to breeding stage) into additive genetic
and permanent environmental components. We provide mean
values of the posterior distribution of (co)variance estimates and
their 95% credible intervals (CI)
Random effects r2 (95% CI)
Additive genetic variance
Vintercepts 0
127 (0
002, 0
263)
Vslopes 0
102 (0
000, 0
321)
Covintercepts-slopes 0
036 (0
149, 0
030)
Permanent environmental variance
Vintercepts 0
091 (0
000, 0
252)
Vslopes 0
115 (0
000, 0
351)
Covintercepts-slopes 0
030 (0
149, 0
030)
Among-breeding attempts variance
Vintercepts 0
273 (0
173, 0
389)
Vslopes 0
176 (0
002, 0
441)
Covintercepts-slopes 0
082 (0
212, 0
020)
Residual within-individual variance
Vlaying 1
351 (1
183, 1
537)
Vincubation 0
867 (0
781, 0
953)
Giraldeau 2004; Duckworth & Badyaev 2007) and is com-
monly thought to be under frequency-dependent selection
(Sinervo & Lively 1996; Kokko, Lopez-Sepulcre & Mor-
rell 2006). Aggressiveness also mediates social interac-
tions, as individuals adjust their aggressiveness in
response to the behaviour expressed by conspecifics in
both mammals (Wilson et al. 2009) and insects (Santoste-
fano et al. 2016). This form of phenotypic plasticity is
also predicted to be under frequency-dependent selection
(Wolf, van Doorn & Weissing 2008; Wolf, Van Doorn &
Weissing 2011). Moreover, individuals adjust their aggres-
siveness throughout their reproductive cycle in response
to seasonal changes in costs and benefits. Importantly, the
benefits associated with this form of plasticity likely vary
between individuals depending on characteristics of their
social neighbourhood (e.g. breeding density), further eco-
logical conditions and predictability of the environment.
As many of the mentioned ecological conditions hypothe-
sised to promote repeatability of reversible plasticity may
be met in territorial passerine species, we expected long-
term individual repeatability in components of aggressive-
ness reaction norms expressed by our great tits, which we
indeed detected. Nevertheless, whether the evolution of
repeatability in reversible plasticity of this particular beha-
viour in this particular species can be explained by the
hypothesized mechanisms requires explicit testing of
model assumptions. For example, is territorial aggressive-
ness in great tits under negative frequency-dependent
selection? And do the costs associated with aggressiveness
decrease with experience?
Individual differences in how males down-regulate their
aggressiveness over the reproductive season may be
related to a male’s paternity strategy (Araya-Ajoy et al.
2016). Male great tits can increase paternity either by
investing in seeking extra-pair copulations (leading to
© 2016 The Authors. Journal of Animal Ecology © 2016 British Ecological Society, Journal of Animal Ecology, 86, 227–238
increased extra-pair paternity) or by aggressively defend-
ing their territory (leading to decreased within-pair pater-
nity loss). The costs associated with paternity loss should,
importantly, change between the egg-laying and egg-incu-
bation stages because males can only lose within-pair
paternity before their mate lays the last egg and starts
incubating. Consequently, males that increase their invest-
ment in aggressiveness when their female is in the laying
stage but subsequently decrease it when she starts clutch
incubation, should be able to avoid paternity loss while
simultaneously freeing up time to invest in searching for
extra-pair mates. Such plastic adjustments should, how-
ever, be costly if males misjudge the risk of a territorial
intrusion or the date of their mate’s onset of incubation.
Therefore, optimal average levels, as well plastic seasonal
adjustments, of a male’s territorial aggressiveness should
depend on the aggressive strategies played by other males
in the population. Importantly, the benefits associated
with this form of plasticity likely vary between males
depending on their attractiveness, characteristics of their
social mate (e.g. her level of promiscuity; van Oers et al.
2008; Patrick et al. 2012; Araya-Ajoy et al. 2016), or
characteristics of the social neighbourhood (Araya-Ajoy,
Dingemanse & Kempenaers 2016). Such differences
between individuals may be reinforced if the costs associ-
ated with plasticity decrease with experience, leading to
repeatable individual differences in plasticity (Wolf, van
Doorn & Weissing 2008; Wolf, Van Doorn & Weissing
2011).
Adaptive theory predicts that repeatability of plasticity
may evolve in situations where the costs of plasticity
decrease with experience (Wolf, van Doorn & Weissing
2008; Wolf, Van Doorn & Weissing 2011). We found that
aggressiveness reaction norms of male great tits changed
with age, which may represent a proxy for their experi-
ence. In great tits, costs of seasonal plasticity in aggres-
siveness might reasonably be expected to decrease with
age because older males should be more experienced in
assessing the threat associated with an aggressive intru-
sion in terms of risk of paternity or territory loss. We
therefore expected that males would become more plastic
with age. Contrary to this idea, we found that first-year
breeders were more plastic than older males, but also
more aggressive (Fig. 3). This observed pattern may be
caused by first-year breeders needing to establish stable
territory boundaries. Great tits generally occupy the same
territory across years; new males thus need to either
‘squeeze in’ or occupy vacant territories. Given that older
males typically already possess a territory, selection may
not favour them to be so aggressive in the beginning of
the breeding season; consequently, older males may not
need to down-regulate their aggressiveness over the season
as much as first-year males. This may explain why our
great tits became less rather than more plastic with age.
Alternatively, our findings may imply that is not the costs
associated with plasticity that decrease with experience
but rather the costs of associated with each strategy. That

is, if individuals become better with experience in exploit-
ing aggressive, plastic or non-plastic strategies, repeatable
differences in average behaviour and level of plasticity
may still arise.
estimating levels of variation in behavioural
reaction norms
Our estimates of individual repeatability of seasonal plas-
ticity in aggressiveness had very broad confidence inter-
vals. Our comparisons with permutation-based null
distributions revealed—despite these broad confidence
intervals—significant non-zero variance for both levels
(see Results). In other words, we were able to detect sig-
nificant effects while our estimates were simultaneously
very uncertain. Reassuringly, comparison of parameters
derived from reaction norm (Table 1) vs. character state
approaches (Table S2) further corroborated this conclu-
sion: crossing reaction norms causing variance in reaction
norm slopes equate to character state-based cross-context
correlations below one (Roff 1997). The posterior mean
interval of the character-state based cross-context correla-
tion between the laying and incubation stage indeed devi-
ated from one at both the among-individual (r = 0
746;
95% CI: 0
255, 0
997) and within-individual level
(r = 0
816; 95% CI: 0
378, 0
998), implying that there was
indeed variation in seasonal plasticity at both of these
levels (though we note that the upper credible interval
was relatively close to one, implying that the evidence for
crossing reaction norms was not strong). We therefore
conclude that great tit males showed both repeatability
and multidimensional plasticity in seasonal plasticity, but
we are not able to assess their respective magnitudes.
Repeatable individual differences in reaction norm
characteristics can be underpinned by additive genetic
variation and by permanent environmental effects, where
the latter effect may either be mediated by developmental
plasticity or by reversible plasticity in response to repeat-
able individual differences in environmental conditions.
Using an animal model, we estimated the relative contri-
butions of additive genetic and permanent environmental
effects. In line with other behavioural genetics studies (re-
viewed by Dochtermann, Schwab & Sih 2015), we found
that about half of the repeatable variation was caused by
additive genetic variation, whereas the other half was
caused by permanent environmental effects. We found
strong support for non-zero additive genetic variance in
average level of aggressiveness (i.e. reaction norm inter-
cept), and weaker (though significant) support for additive
genetic variance in seasonal plasticity (i.e. reaction norm
slopes), though we note that associated confidence inter-
vals were very broad for both. We further note that a re-
analysis of the data using a character state approach (for
rational, see above; Table S1) confirmed the presence of
genetic variation in plasticity as the cross-context genetic
correlation between aggressiveness during laying vs. incu-
bation (r = 0
491; 95% CI: 0
540, 0
938) was
© 2016 The Authors. Journal of Animal Ecology © 2016 British Ecological Society, Journal of Animal Ecology, 86, 227–238
Repeatability of seasonal plasticity 235
substantially below one. Heritability is a key parameter in
evolutionary theory that has not been estimated for reac-
tion norms slopes in a wild population (see Dochtermann,
Schwab & Sih 2015 for the heritability of intercepts of
behavioural reaction norms). Unfortunately, we found
much uncertainty in the additive genetic and permanent
environmental variance of our estimates of seasonal plas-
ticity, which in turn caused the confidence interval of the
heritability estimate to be extremely broad. Firm conclu-
sions about the exact magnitude of heritable variation in
reaction norm slopes can therefore not be drawn. Impor-
tantly, data requirements for accurately and precisely esti-
mating the heritability of reaction norm slopes have not
been explored systematically. What is known is that rela-
tively large sample sizes are required to accurately and
precisely estimate the relative magnitudes of among- ver-
sus within-individual variation in reaction norms, and that
this requires studies designs with sufficient replication at
each hierarchical level (Araya-Ajoy, Mathot & Dinge-
manse 2015). The estimation of the heritability of plasti-
city in repeatedly expressed reaction norms will therefore
represent an extremely difficult task, and may be possible
in few study systems that enable ample replication at mul-
tiple hierarchical levels. The increased availability of tools
enabling genome-wide sequencing and automated data
collecting may facilitate the study of the quantitative
genetic underpinning of multi-level reaction norms in
future studies.
The repeatable differences that we classified as perma-
nent environmental effects could partly be caused by
reversible plasticity in response to repeatable individual
differences in environmental conditions. For instance, we
showed that great tits are more aggressive in plots with
higher densities (Fig. 3), which corroborates the idea that
aggressiveness is related to spatial variation in competi-
tion for resources. Importantly, it may also suggest that
some of the repeatability in reaction norm intercepts may
have been caused by environmental effects. We offer sev-
eral explanations for this pattern of density-dependent
behaviour. First, individuals that are more aggressive
might be more likely to settle in dense plots, regardless of
whether their behavioural differences were underpinned
genetically or shaped by developmental plasticity. Second,
individuals might plastically adjust their aggressiveness in
response to density by means of reversible plasticity.
Importantly, individuals did not adjust their behaviour in
response to year-to-year changes in density which suggests
that the birds were not responding to density per se. We
therefore conclude that these plot-level patterns of density-
dependent aggressiveness were either caused by differential
settlement of aggressiveness ‘types’ or by individuals
responding to specific factors that were associated with spa-
tial—not temporal—variation in breeding density.
We further explored whether some of the individual dif-
ferences in aggressiveness, and its seasonal plasticity, were
caused by other general characteristics of the study plot
by expanding our models to include random intercepts
236 Y. G. Araya-Ajoy & N. J. Dingemanse
and slopes for the identity of the study plot. Study plot
characteristics indeed explained some variation in aggres-
siveness (i.e. reaction norm intercepts) but not in its sea-
sonal plasticity (i.e. reaction norm slopes; Table S3). We
therefore conclude that some of the non-genetic individual
variation in average behaviour and most of the non-
genetic individual variation in seasonal plasticity was
caused by reversible plasticity in response to repeatable
small-scale characteristics of male territories that we were
unable to quantify.
within-individual variation in reaction norms
Conceptually, we can view repeatedly expressed reaction
norms themselves as labile phenotypic characters that can
vary among- and within-individuals. Within-individual
variation in this type of reaction norm would arise if an
individual’s phenotypic response to changes in one envi-
ronmental gradient is a function of another environmental
gradient (Westneat, Wright & Dingemanse 2015). Our
analyses show that male great tits adjusted the intercept
of their aggressiveness reaction norms as a function of
environmental gradients that changed from year to year:
we detected within-individual-among-year variance in
reaction norms when controlling for male identity
(Table 1). This implies that the reaction norms were ‘mul-
tidimensional’ (sensu Westneat, Stewart & Hatch 2009)
because aggressiveness responded to multiple environmen-
tal axes. The existence of this level of variation is not sur-
prising because selection should often favour plasticity in
reaction norms in response to multiple environmental
axes. We have shown that age is such an internal environ-
mental gradient that causes within-individual variation in
reaction norms, as previously suggested (Kindlmann,
Dixon & Dost
alkov
a 2001). The effects of age, notably,
explained a relatively minor proportion of the within-indi-
vidual variation (Table 1). We therefore also asked
whether among-year-within-individual variation in aggres-
siveness and its seasonal plasticity was underpinned by
year-to-year changes in unknown environmental effects
(Table S4). Some of the within-individual variation in
reaction norm intercepts was indeed explained by ‘year’
though this factor explained little variation; reaction norm
slopes did not vary between years. We therefore conclude
that a major portion of the within-individual variation in
aggressiveness and its seasonal plasticity must have been
caused by individual- and year-specific changes in envi-
ronmental factors. Habituation towards our simulated ter-
ritorial intrusion across years may also have caused such
within-individual variation in aggressiveness reaction
norms. We explored this possibility by fitting the number
of years an individual had been exposed to a test across
the entire study period in an interaction with breeding
stage (laying vs. incubation). We found the same pattern
as for age (Table S5), likely because age and repeated
exposure are highly correlated so we cannot statistically
tease these effects apart.
© 2016 The Authors. Journal of Animal Ecology © 2016 British Ecological Society, Journal of Animal Ecology, 86, 227–238
Conclusions
Aggressiveness and its seasonal plasticity harboured long-
term individual repeatability in a wild passerine bird. Our
study thereby demonstrated that such within-population
variation in reaction norms can be caused both by
among- and within-individual level processes. Impor-
tantly, our findings highlight that individuals can adjust
their plastic response to a particular environmental gradi-
ent over their lifetime, whereas individuals may simultane-
ously be repeatable in level of plasticity. Acknowledging
such forms of multi-level variation is important not only
for testing behavioural ecology theory regarding the evo-
lution of repeatable differences in plasticity, but also for
predicting how individuals may cope with rapidly chang-
ing environments. Aggressiveness in particular represents
a key labile character as it may affect survival and com-
petitive regimes, and in turn population dynamics, breed-
ing density and population growth. It is therefore
important to acknowledge the various processes at vari-
ous hierarchical levels that generate variation in aggres-
siveness, if only because repeatable vs. unrepeatable
variation in plasticity may affect eco-evolutionary dynam-
ics of populations very differently.
Authors’ contributions
Y.G.A.-A. analysed the data; both authors contributed equally to all other
aspects of the paper.
Acknowledgements
We thank members of the research group Evolutionary Ecology of Varia-
tion, and the Department of Behavioural Ecology and Evolutionary Genet-
ics, for support and help in collecting the data, and members of the
working group SQuID for insightful discussion. We also gratefully
acknowledge feedback from Martin van de Pol, Alecia Carter, Julien Mar-
tin and an anonymous reviewer. The study was supported by funding of
the Max Planck Society. Y.A.-A. was supported by the German Academic
Exchange Service (DAAD), the International Max Planck Research School
for Organismal Biology and the Research Council of Norway through its
Centres of Excellence funding scheme (project number 223257).
Data accessibility
Data available from the Dryad Digital Repository http://dx.doi.org/10.
5061/dryad.7p160 (Araya-Ajoy & Dingemanse 2016).
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Received 22 December 2015; accepted 24 November 2016
Handling Editor: Martijn van de Pol
Supporting Information
Details of electronic Supporting Information are provided below.
Fig. S1. Full pedigree of our population. Red lines represent
maternal links; blue lines represent paternal links.
Fig. S2. Pedigree with the individuals informative for the animal
model analysis of aggressiveness highlighted in colour. Red lines
represent maternal links; blue lines represent paternal links.
© 2016 The Authors. Journal of Animal Ecology © 2016 British Ecological Society, Journal of Animal Ecology, 86, 227–238
Table S1. Pedigree summary statistics. Values are for the pruned
pedigree, with only individuals informative for our analysis
(Figure S2).
Table S2. Results from (a) a bivariate mixed-effects model per-
formed to quantify variation in male aggressiveness among indi-
viduals, among breeding attempts (i.e. within-individuals among-
years), and within breeding attempts (within-years) for each of
two breeding stages and (b) a model that further partitioned the
among-individual (co)variation in both contexts in to its additive
genetic and permanent environment components. We provide
mean values of the posterior distribution of (co)variance esti-
mates and their 95% credible intervals.
Table S3. Results from two multi-level random regression models
performed to study the sources of variation in aggressiveness
reaction norms in response to the breeding stage of their partners in
wild male great tits.
Table S4. Results from two multi-level random regression models
performed to study the sources of variation in aggressiveness
reaction norms in response to the breeding stage of their partners in
wild male great tits.
Table S5. Results from multi-level random regression models
performed to study the sources of variation in aggressiveness
reaction norms in response to the breeding stage of their partners in
wild male great tits.
Appendix S1. Prior settings.