Yolk androgens do not appear to mediate sexual conflict over parental investment in the collared flycatcher Ficedula albicollis.

Abstract

Males and females are in conflict over parental care, as it would be favourable for one parent to shift labour to the other. Yolk hormones may offer a mechanism through which female birds could influence offspring traits in ways that increase the relative investment by the male. We studied the role of yolk androgens in mediating sexual conflict over parental care in the collared flycatcher (Ficedula albicollis). In a cross-fostering experiment, the male's proportion of total feeding visits increased with increasing androgen levels in the foster eggs. This could suggest that sexual conflict over parental care may be influenced by the female's differential allocation of yolk androgens or a maternal effect associated with yolk androgens. However, when we experimentally elevated yolk androgen levels, male feeding rates did not differ between control and androgen-manipulated nests. This suggests that other egg components correlated with yolk androgen levels, rather than yolk androgen levels per se, may influence male parental effort. In conclusion, yolk androgens per se do not appear to mediate sexual conflict over parental investment in the collared flycatcher.

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Yolk androgens do not appear to mediate sexual conflict over parental investment in
the collared flycatcher Ficedula albicollis
Suvi Ruuskanena,⁎, Blandine Doligezb, Barbara Tschirrenc, Natalia Pitalad, Lars Gustafssone,
Ton G.G. Groothuisf, Toni Laaksonena
aSection of Ecology, Department of Biology, University of Turku, Finland
bCNRS; Université de Lyon, F-69000, Lyon; Université Lyon 1; Department of Biometry and Evolutionary Biology, LBBE UMR 5558, Bâtiment Gregor Mendel,
43 boulevard du 11 novembre 1918, 69622 Villeurbanne, France
cDepartment of Animal Ecology, Lund University, Sweden
dBird Ecology Unit, Department of Biological and Environmental Sciences, University of Helsinki, Finland
eDepartment of Animal Ecology/Ecology and Evolution, Evolutionary Biology Centre, Uppsala University, Sweden
fBehavioural Biology, University of Groningen, The Netherlands
a b s t r a c ta r t i c l e i n f o
Article history:
Received 1 December 2008
Revised 26 January 2009
Accepted 28 January 2009
Available online 5 February 2009
Keywords:
Hormones
Maternal effects
Parental care
Parental investment
Reproductive effort
Males and females are in conflict over parental care, as it would be favourable for one parent to shift labour to
the other. Yolk hormones may offer a mechanism through which female birds could influence offspring traits
in ways that increase the relative investment by the male. We studied the role of yolk androgens in mediating
sexual conflict over parental care in the collared flycatcher (Ficedula albicollis). In a cross-fostering
experiment, the male's proportion of total feeding visits increased with increasing androgen levels in the
foster eggs. This could suggest that sexual conflict over parental care may be influenced by the female's
differential allocation of yolk androgens or a maternal effect associated with yolk androgens. However, when
we experimentally elevated yolk androgen levels, male feeding rates did not differ between control and
androgen-manipulated nests. This suggests that other egg components correlated with yolk androgen levels,
rather than yolk androgen levels per se, may influence male parental effort. In conclusion, yolk androgens per
se do not appear to mediate sexual conflict over parental investment in the collared flycatcher.
© 2009 Elsevier Inc. All rights reserved.
Introduction
In species with biparental care, females and males are in conflict
over the amount of parental care they provide to their offspring
(Trivers, 1972; reviewed by Arnquist and Rowe, 2005; Houston et al.,
2005; Lessells, 2006; Hartley and Royle, 2007). For each parent,
increased investment reduces the parent's future reproductivesuccess
and survival prospects (e.g. Williams, 1966; Roff, 1992; Parker et al.,
2002), and therefore it would be favourable for one parent to shift
labour to the other (Parker et al., 2002; Houston et al., 2005). The
parents' decision about how much to invest in the offspring depends
onoffspringneedand/orbroodvalue, aswell asthefeeding behaviour
of the other parent (e.g. Hinde and Kilner, 2007). A parent could thus
try to manipulate the investment of its partner by changing either
offspring traits or his or her own behaviour (e.g. Slagsvold et al.,1995;
WestneatandSargent,1996;ArnquistandRowe,2005;Lessells,2006).
Offspring phenotype is affected by maternal effects (Mousseau and
Fox, 1998), for instance via egg composition (e.g. Groothuis et al.,
2005a). In some bird species, androgens deposited in the egg yolk
have been found to increase competitive ability of chicks (correlative
data in Schwabl,1993; experimental data in Eising et al., 2001), chick
begging (experimental data: Schwabl,1996; Schwabl and Lipar, 2002;
Eising and Groothuis, 2003; Boncoraglio et al., 2006; von Engelhardt
et al., 2006) and growth (experimental data: Schwabl,1996; Eising et
al., 2001; Pilz et al., 2004; Navara et al., 2005, 2006; Tschirren et al.,
2005). In other studies, however, no positive effect of elevated yolk
androgens on begging or growth was found (experimental data:
Sockman and Schwabl, 2000; Andersson et al., 2004; Pilz et al., 2004;
Uller et al., 2005; Saino et al., 2006), or the effect depended on
offspringsex (e.g.Sainoetal.,2006; Mülleretal.,2008;Sockman etal.,
2008). The effects of yolk androgens thus seem to vary, but there is
evidence that they can affect offspring traits. It has therefore been
proposed that by allocating more yolk androgens into the egg, a
female could affect offspring trait(s) to which males respond, and this
way increase the relative feeding effort of the male to her benefit
(Groothuis et al., 2005a; Michl et al., 2005; Gil et al., 2006; Lessells,
2006; Moreno-Rueda, 2007; Müller et al., 2007a).
This hypothesis relies on several assumptions. First, yolk androgen
levels should affect chick traits to which the parents respond by
changing their food provisioning. There is evidence from several
species for this assumption (reviewed e.g. by Müller et al., 2007a, see
Hormones and Behavior 55 (2009) 514–519
⁎ Corresponding author. Fax: +35823336550.
E-mail address: suvi.ruuskanen@utu.fi (S. Ruuskanen).
0018-506X/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.yhbeh.2009.01.010
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above). However, a direct link between yolk androgen levels and
parental feedingefforthasbeen investigated in onlyone studythusfar
(Tschirren and Richner, 2008). Second, increasing yolk androgen
levels should either impose a cost or be constrained, since otherwise
all females could afford to allocate high levels of androgens (e.g.
Groothuis et al., 2005a; Groothuis and Schwabl, 2008). Indeed,
elevated levels of androgens in the yolk can be costly for the chicks,
as an immunosuppressive effect of androgens has been reported in
some studies (e.g. Groothuis et al., 2005b; Müller et al., 2005; Navara
et al., 2005; but see Andersson et al. 2004; Tschirren et al., 2005;
Müller et al., 2007b). Moreover, such elevated levels may require
elevated circulating androgen levels in the female, which may, for
example, delay or inhibit egg laying (e.g. Clotfelter et al., 2004;
Rutkowska et al., 2005; discussed in Groothuis and Schwabl, 2008).
Finally, females should be able to respond differently to the chick trait
influenced by yolk androgens than males. This assumption is yet to be
explored. Two mechanisms are possible, depending on whether both
parents or only the male respond to the trait through which the
female attempts to manipulate the male (Müller et al., 2007a). 1) If
both parents respond to the androgen-mediated offspring trait, males
are expected to increase their investment in response to increasing
yolk androgen levels deposited in the eggs by the female; females,
however, should have lower responsiveness to androgen-mediated
chick traits when allocating high androgen levels into their eggs than
when allocating low levels. 2) If only males respond to the chick trait
(hereafter a male-specific trait), males should increase their feeding
rate with increasing yolk androgen levels while females should not
change their investment. Previous studies have shown that it is
possible that females and males respond to different components of
nestling behaviour and phenotype, for example colour (Jourdie et al.
2004, de Ayala et al. 2007; Ewen et al. 2008; Galvan et al. 2008; Tanner
and Richner, 2008), size (reviewed in Slagsvold,1997; Lessells, 2002),
condition (e.g. Christe et al. 1996) or visual vs. vocal cues of begging
(Kilner, 2002, reviewed in Müller et al., 2007a).
We studied whether yolk androgens could mediate sexual conflict
over parental care in a small passerine bird, the collared flycatcher
(Ficedula albicollis) by conducting two experiments. Firstly, we
examined whether naturally varying levels of yolk androgens affect
the relative work load of the parents. We did this by cross-fostering full
broods with naturally varying yolk androgen levels among nests and
monitoring the subsequent parental feeding rates. We predicted that if
females can manipulate male investment through differential yolk
androgendeposition,and theandrogen-mediatednestlingtraitismale-
specific, the proportion of male feeding visits should be positively
correlated with the yolk androgen levels of the foster eggs (hereafter
foster yolk androgens), but not of original eggs (hereafter original yolk
androgens). In contrast, if the manipulation is mediated through an
androgen-mediated trait towhichbothparentsrespond,wepredicted a
positive correlation between proportion of male feeding visits and
original androgen level, but no correlation with proportion of male
feeding visits and foster androgen level. This is because females
allocating high yolk androgen levels should have lower responsiveness
to androgen-mediated chick traits than females allocating low yolk
androgen levels. Secondly, we experimentally increased yolk androgen
levelsofwholeclutchesandmonitoredthesubsequentparentalfeeding
rates to test whether yolk androgensper se allow females to manipulate
males.Wepredictedthatifmalemanipulationbythefemaleismediated
byyolkandrogensperse,malefeedingrateshouldbehigherinbroodsof
the androgen-manipulated than the control group.
Methods
Study site and study species
The experiments were conducted in Spring 2007 on the island of
Gotland, Sweden (57°19' N, 18°29' E) in a nest box breeding
population of collared flycatchers monitored since 1980 (Gustafsson,
1989). The study area consists of several small and spatially discrete
forest patches. The collared flycatcher is a small (ca. 13 g), migratory
passerine, which breeds in Central and Eastern Europe and on the
islands of Gotland and Öland in Sweden. Average clutch size in this
population is six eggs, and average brood size four nestlings. Usually
both parents feed the young, but polygynous males (ca. 10%)
mainly feed their primary brood (Gustafsson, 1989; Gustafsson and
Qvarnström, 2006).
The cross-fostering experiment
Nest boxes were checked every other day starting at end of April to
monitortheprogressof nestbuildingandegglaying.Whenoneortwo
eggs were found in the nest, they were marked with non-toxic
permanent marker and the nest box was visited the in following day
(s) to collect the third egg of the clutch on the day of laying (N=31
nests). These eggs were taken to the laboratory, and yolks were
separated from albumen on the same day and stored frozen at −20 °C
until hormone analysis (see below). The collected egg was replaced by
a dummy egg in each nest. The average clutch size in this species and
population is six eggs, so the third egg is one of the middle eggs of a
clutch. In most species studied so far, including the collared flycatcher
and its sister species the pied flycatcher (Ficedula hypoleuca), the
between-clutch variation in yolk androgen levels is higher than the
within-clutch variation (e.g. Reed and Vleck, 2001; Groothuis and
Schwabl, 2002; Pilz et al., 2003; Tschirren et al., 2004; Michl et al.,
2005; Müller et al., 2007b, Tobler et al., 2007; T. Laaksonen, unpubl.
data). No within-clutch pattern of yolk androgens was found in a
Hungarian population of the collared flycatchers (Michl et al., 2005),
thus yolk androgen concentration in the middle egg should be a good
proxy for the mean hormone level of a clutch. Even if within-clutch
variation in hormone deposition would have occurred in some
individuals of our study population,the third egg would still represent
the medium concentration of the whole clutch, as long as the pattern
of within-clutch variation is linear (as found in many studied species
with significant within-clutch variation, e.g. Reed and Vleck, 2001;
Groothuis and Schwabl, 2002; Pilz et al., 2003; Tschirren et al., 2004).
Nests were monitored to record final clutch size and hatching date
as part of the population monitoring. Two days after hatching,
complete broods were cross-fostered between sampled nests
(matched for hatching date and brood size), so that in most cases
the yolk androgen levels of the foster brood (i.e. of the third egg of the
foster brood) was known. Both parents were caught in the nest box
using a swing-door-trap when feeding 6 to 10 day-old nestlings. They
wereweighedandmeasured andtheiragewasdeterminedas yearling
or older (see e.g. Pärt and Gustafsson, 1989).
Parental feeding frequency was measured at day 6 after hatching
by observing parental feeding behaviour from a hide ca.15 m distance
from the nest. After two feeding visits by the parents (to ensure that
they were feeding normally), 1 h of feeding visits by the parents was
observed. Peak of feeding activity in this species occurs between day 5
and day 10 of nestling rearing. Feeding rate was measured as the
number of feeding visits by each parent per hour. Feeding rate has
been shown to correlate positively with energy expenditure in this
population (Pärt et al., 1992), thus it can be used as a measure of
parental investment.Parentalfeedingratefurthercorrelates withboth
chick weight and condition at fledging and fledging success (Doligez
et al., 2004), and these variables are related to juvenile survival in this
species (Linden et al., 1992). Feeding rates were recorded mostly
between 8.00–12.00 am, and only in good weather conditions.
Yolk androgen analysis
Yolk testosterone (T) and androstenedione (A4) concentrations
were analysed by radioimmunoassay (RIA). Yolks were thawed and
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homogenised with 400 μl of distilled water. Aliquots of this yolk/
water emulsion (approximately 100 mg) were mixed with 150 μl of
distilledwaterand 50 μl of 3HTracer T (ca. 2000 counts per minute) to
assess extraction efficiency. The samples were extracted twice with
2.5 ml of 70% diethylether/30% petroleumether (vol: vol) and dried
under a stream of nitrogen. The extracts were then re-dissolved in
1 ml 70% methanol, centrifuged and decanted. The supernatant was
dried under a stream of nitrogen and re-dissolved in PBS. T and A4
were measured in duplicates using DSL (Diagnostic System Labora-
tories, USA) radioimmunoassay kits following the manufacturer's
protocol. The average recovery rate was 72%. The yolks were analysed
in two assays with an inter-assay coefficient of variation of 5.9% for A4
and 1.6% for Tand an intra-assaycoefficientof variation of 10.0% for A4
and 5.7% for T. Parallelism was confirmed, and concentrations of Tand
A4 were not affected by extraction efficiency (T concentration:
F1,21=0.01, p=0.94; A4 concentration: F1,21=0.0, p=0.99; range of
extraction efficiency: 66.7–76.6%), egg weight (T: F1,21=0.02,
p=0.89; A4: F1,21=0.01, p=0.92), yolk weight (T: F1,21=0.69,
p=0.42; A4: F1,21=0.06, p=0.80) or assay (T: F1,21=1.70, p=0.21;
A4: F1,21=3.73, p=0.07). There was no correlation between original
and foster yolk androgen levels (r=−0.31, N=19, p=0.19). Original
yolk androgen levels of eggs from 26 nests could be analysed, but due
to problems in cross-fostering, both original and foster androgen
levels (as estimated from the third egg) could be obtained for 19 nests
only. We decided to use the sum of the concentrations of A4 and T
(total androgen concentration, pg/mg) in the analyses as a measure of
total androgen available for binding to either androgen or oestrogen
receptors. This method was chosen because A4 is the total source
(precursor) available for hormone conversion to active androgens
[testosterone and dihydrotestosterone, (DHT)] and estradiol (E2), T is
also a source for DHT and E2, and DHT has a higher affinity to
androgen receptors than T (reviewed by e.g. Norris, 1996, Groothuis
and Schwabl, 2008). In addition, A4 and T levels are positively
correlated (r=0.50, N=26, p=0.01). However, whether the effects
of the two hormones differ is uncertain, and their affinity to receptors
is likely to differ (e.g. Groothuis and Schwabl, 2008). Therefore, we
also examined the effects of each hormone separately and report the
results when they clearly differ for the two hormones.
Yolk androgen manipulation experiment
This experiment was conducted on a different set of nests (N=66)
than the cross-fostering experiment. Nest boxes were checked every
other day to determine laying dates. On the estimated day of clutch
completion (i.e. day of laying of the sixth egg), each clutch was ran-
domly assigned to either the control or the androgen-manipulation
group. All the eggs of a clutch were treated in the same way. The eggs
were replaced with dummy eggs for the time of the injections. In the
androgen-manipulation group (N=36 nests), the eggs were injected
with 14.4 ng of testosterone (Fluka) and 50.8 ng of androstenedione
(Fluka) dissolved in 4 μl sesame oil. In the control group (N=30
nests), eggs were injected only with 4 μl of sesame oil. The amount of
injected androgens was calculated using previous data on natural yolk
androgen levels from the same population (T mean: 14.2 ng/yolk, SE
0.4 ng/yolk, maximum: 28.8 ng/yolk; A4 mean: 60.3 ng/yolk, SE
1.5 ng/yolk, maximum 111.1 ng/yolk; B. Doligez and B. Tschirren,
unpubl. data). The amount injected corresponds to the difference
between mean and maximum values of androgens per yolk, which
ensured that final yolk androgen level in the androgen-manipulated
clutches was on average at the upper limit of the natural range. The
original androgen concentration in the manipulated clutches was not
determined, as this would have required removing an egg and thus
changing brood size, which could have had a substantial effect on the
parental feeding rates. The position of the yolk was visualized using a
light source positioned beneath the egg. The surface of the egg was
first cleaned with 95% ethanol and a small hole was made using a
disposable 27 G needle. The oil-vehicle was injected into the yolk
using a 25 μl Hamilton syringe (702RN) and 26 G needle. After the
injection, the hole in the egg shell was sealed with a drop of tissue
adhesive (Vet-Seal, B. Braun Medical, Switzerland) or with a patch of a
flexible wound film (Opsite, Smith & Nephew, UK). The eggs were
returned to the nest immediately after the injections. Nests were
checked on the following day and the seventh egg was injected if
present, according to the clutch treatment. This method for mani-
pulating yolk androgen levels has been successfully used and
validated in previous studies (e.g. Tschirren et al., 2005). The hatching
success of injected eggs did not differ between the control group
(79.74%) and the androgen-manipulated group (79.77%, Wilcoxon
test: p=0.94, N=66). The natural hatching success of non-injected
clutches (excluding deserted and predated nests, where no egg
hatched) in this population is 92.0% (unmanipulated nests of years
1997–2002, N=1677, B. Doligez and L. Gustafsson, unpubl. data)
On day 9 after hatching, parental feeding rates were recorded with
digital video cameras for 2 h (SONY Handycam DCR-SR52). If parents
were giving frequent alarm calls at the beginning of the recording,
feeding rates were estimated from the time when parents stopped
alarming and made the first visit back to the nest. Video recordings
were watched blindly, i.e. the observers were unaware of nest
treatment. As for the cross-fosteringexperiment,parents werecaught,
weighed and their age was determined. Collared flycatchers are partly
polygynous (ca. 10% in this population, Gustafsson, 1989; Gustafsson
and Qvarnström, 2006), but none of the males in either experiment
were recorded feeding at a secondary nest, and thus polygyny does
not affect our results.
We tested whether feeding data from the two experiments
obtained at different nestling ages (6 and 9 days for the cross-
fostering and injection experiment, respectively) are comparable by
analysing both 6 days and 9 days post hatching feeding rates in a
sample of nests. There was a significant, positive correlation in the
male share of feedings between day 6 and day 9 (rs=0.67, p=0.0003,
N=25), and the average male share of feedings was equal at these
two measuring points (average male share of feedings: day 6=49.4%;
day9=49.0%), whichstronglyindicatesthatthedivisionof thelabour
within a pair is fairly constant over this 3-day feeding period.
All experiments were conducted under licences from the Swedish
National Board for Laboratory Animals and the Bird Ringing Centre of
the Swedish Museum of Natural History (Stockholm, Sweden). The
experimental protocols adhered to the standards of US National
Institute of Health.
Statistical analysis
All statistical analyses were performed with SAS 9.0. Nests where
only one parent was observed feeding, or where the age of one parent
was unknown (some males could not be caught) or uncertain, were
not used in the analyses, which reduces the sample size.
In the cross-fostering experiment, we analysed the male's propor-
tion of the total number of feeding visits in relation to yolk androgen
concentration (as estimated from the third egg) in original and foster
eggs, using a general linear model. We also included female and male
age as explanatory variables to control for possible age effects. Laying
date, brood size and time of the day when the observations were
started were added as covariates in this as well as in all subsequent
models. In a second step, we included egg mass and yolk mass in the
model to test whether they were associated with male share of
feeding activity. The interaction between female and male age could
not be tested due to small sample size in the yearling female–yearling
male category (sample sizes: yearling females=3, older females=13,
yearling males=2, older males=14).
In the androgen-manipulation experiment, we analysed parental
feeding rates in relation to androgen-manipulation treatment using a
general linear mixed model. We tested the influence of treatment
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(control or androgen-manipulated), sex, age of the parent and their
interactions on the number of feeding visits per hour (i.e. feeding
rate). Sample sizes were: yearling females=12, older females=32,
yearling males=14, older males=30. Nest identity (nested within
treatment) and forest patch were included as random effects to
control for the non-independence of parental responses within a pair
and for patch effects.
We used a backward model selection procedure, removing
interactions, covariates and main effects starting with the least
significant. To confirm the non-significance of the removed interac-
tion terms and main effects, each term was added to the final model
separately. Satterthwaite approximation was used to determine
denominator degrees of freedom (Littell et al., 1996) and normality
of the residuals was checked.
Results
Male feeding rate in the cross-fostering experiment
Average female feeding rate (±SD) was 12.0±5.2 feeding visits/
hour (N=31) and average male feeding rate was 11.4±4.5 (N=28).
The propor-tion of feeding visits by the male increased with increasing
yolkandro-genlevelsinthefostereggs(asmeasuredfromthethirdegg;
F1,12=8.98,p=0.011,Fig.1,averageproportionofmalefeedings(±SD):
49.4±17.9%), controlling for laying date (F1,12=10.17, p=0.008,
β±SE=−0.037±0.011), and time of the day (F1,12=4.38, p=0.058;
β±SE=0.031±0.015). The proportion of feeding visits by the male
was not related to the original yolk androgen levels (F1,11=0.24,
p=0.63). Separate analyses of both androgens revealed that male
proportion of feeding visits was related to yolk A4 (F1,12=12.33,
p=0.004) but not to yolk testosterone levels (F1,12=0.07, p=0.79). In
ordertofindoutwhethertheincreaseintheproportionofmale feeding
visits was explained byan increase in male feeding rate or a decrease in
female feeding rate, we analysed the absolute feeding rates of both
sexes. Male feeding rate increased with increasing foster yolk androgen
concentration (r=0.45, N=20, p=0.042), whereas no significant
correlation was observed in females (r=−0.24, N=20, p=0.30).
There was an interaction between parental sex and fos-ter yolk
androgens (F1,28=4.26, p=0.048; males: β±SE=0.033±0.028;
females: β±SE=−0.028±0.050). These results are in accordance
with the hypothesis that females manipulate males through a yolk
androgen-dependent nestling signal, to which only males react, and/or
a maternal effect that correlates with yolk androgen concentrations in
theeggs.Ageoftheparentsdidnotaffecttheproportionoffeedingvisits
by the male, and there were no significant interactions between female
or male age and original or foster yolk androgen concentration (all p-
valuesN0.08). Finally, the proportion of feeding visits by the male was
not related to egg mass (F1,11=0.41, p=0.53) or yolk mass (F1,11=0.32,
p=0.58).Thustherelationshipbetweenyolkandrogencontentinfoster
eggs and male feeding effort can not be explained by egg or yolk mass.
Feeding rates in the yolk androgen-manipulation experiment
There was no effect of the androgen-manipulation on parental
feeding rates and no interaction between treatment and sex
(interaction treatment×sex: F1,42=0.46, p=0.50; treatment: F1,41=
0.38, p=0.82; sex: F1,42=1.56, p=0.21, Fig. 2.). Average female
feeding rate (±SD) was 17.3±6.2 feeding visits/hour (N=44) and
average male feeding rate was 16.7±6.3 (N=44) (average propor-
tion of male feedings (±SD) being: 48.7±11.3%). Feeding rates of
yearling and older individuals did not differ between androgen-
manipulated and control group (interaction age×treatment:
F1,82.6=0.12, p=0.73; age: F1,82.3=0.02, p=0.89). Feeding rate
increased with brood size (controlled for in the above analyses;
F1,37=13.87, pb0.001, β±SE= 2.336±0.627). All other interactions
and covariates were non-significant (all p-valuesN0.12).
Discussion
Do females manipulate males through yolk androgens?
The results of the cross-fostering experiment suggest that, in the
collared flycatcher, sexual conflict over parental care may be
influenced by the female via differential allocation of yolk androgens
or other egg components (e.g. carotenoids, lipids) that are correlated
with the yolk androgen content of the egg. The correlation between
the proportion of feeding visits by the male and yolk androgens in the
foster eggs was explained byan increase in male absolute feeding rate,
which suggests that offspring traits associated with high yolk
androgen levels make specifically males to increase their effort.
However, when yolk androgen levels were experimentally elevated,
males feeding nestlings originating from androgen-manipulated eggs
Fig. 1. Male's proportion of feeding visits in relation to total androgen concentration in
eggs (as estimated from the third egg of the clutch) of fostered nestlings (N=16,
F1,12=8.98, p=0.011). Residuals are from a model in which the effects of laying date
and time were removed.
Fig. 2. Male and female feeding rates (mean, SD) in control and androgen manipulated
nests (interaction treatment×sex: F1,42=0.46, p=0.50). Sample size is indicated
above the bars.
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did not feed at a higher rate than those feeding control clutches. This
indicates that the androgen levels per se did not affect male effort.
It could be argued that the effects of androgens on the chicks may
depend on synergistic interactions with other egg components (e.g.
Groothuis and von Engelhardt, 2005), or that exogenous A4 and T in
the yolk could be differentially metabolized by steroidogenic enzymes
than endogenous steroids. In this case, experimental elevation of only
the androgen levels usingexogenous hormonesmight nothave had an
effect on the chicks. This is however unlikely, because experimental
elevation of yolk androgen levels following the same protocol as ours
had a sex-specific effect on chick growth in another set of nests in our
study population (Pitala et al., in press). This shows that exogenous
androgens do have an effect on the chicks. Thus the result of the
androgen-manipulation allows us to conclude that yolk androgens do
not appear to mediate sexual conflict over parental care in the collared
flycatcher (potential explanations for the association between yolk
androgens and male investment in the cross-fostering experiment are
discussed below).
To our knowledge, only one study thus far has examined whether
yolk androgens may play a role in mediating sexual conflict over
parental investment. In great tits, only females adjusted their parental
effort to androgen-mediated nestling traits, indicating that male
manipulation by the female via yolk androgens is also unlikely in this
species (Tschirren and Richner, 2008). Thus no empirical evidence
currently supports the hypothesis that females could successfully
manipulate male investment through yolk androgens. Selection may
have favoured males' reduced susceptibility to yolk androgen-mediated
nestling traits, allowing them to resist the manipulation by females
(Tschirren and Richner, 2008). In an evolutionary conflict of interests,
such as sexual conflict over parental investment, an “arms-race” is
expected, with each parent trying to minimise costs and maximise
benefits(e.g.Arnquistand Rowe, 2005).Thusmalesmaybeexpectedto
evolve responses to specific offspring signals that cannot be manipu-
lated by females and/or ignore signals that can be manipulated by
females (Müller et al., 2007a). Indeed, males and females are known to
usedifferentcues,forexamplenestlingsex,size,position,vocalorvisual
components of begging, in adjusting their feeding effort (e.g. reviewed
in Lessells, 2002, Müller et al., 2007a). The evolution of male counter-
responses to female manipulation will depend on the balance between
benefits (avoiding manipulation by the female) and costs (e.g. reduced
reproductive success) of ignoring the manipulated nestling traits.
Unfortunately, empirical testing of the hypothesis that past manipula-
tion by females has selected males to ignore certain offspring signals
may prove very difficult. However, Lessells (2006) pointed out that, in
general, manipulative behaviour in sexual conflict over parental
investment may be rare, as the benefits may be small and the costs
couldbesubstantial.Thus,sexualconflictoverparentalinvestmentmay
not generate rapid evolutionary change and antagonistic coevolution.
Relationship between male investment and yolk androgens in the
cross-fostering experiment
The results of the androgen manipulation experiment suggest that
yolk androgens per se do not mediate sexual conflict over parental
investment. Therefore, the correlation between male share of parental
careand yolk androgen level of fosteredclutches in the cross-fostering
experiment (Fig. 1) probably arises through a different pathway. For
instance, the amount of yolk androgens in the egg could be correlated
with another egg component that females could use to manipulate
male effort. The amount of yolk androgens has indeed been found to
correlate with, for example, the amounts of antioxidants and
immunoglobulins in the yolk (e.g. Royle et al., 2001; Groothuis et al.,
2006; Török et al., 2007). The role of egg components other than
androgens in mediating sexual conflict over parental investment
however remains to be investigated. The amount of yolk androgens
could also be positively correlated with the genetic quality or
condition of the parents and subsequently with the quality of the
offspring, to which the parents respond. It is yet possible that the
quality of the offspring depends on the quality of their natal territory.
A correlationwithyolk androgen levels could then arise for example if
higher quality territories are more fiercely defended, leading to higher
androgen levels in female circulation and subsequently to higher yolk
androgen levels in the eggs (e.g. Schwabl, 1997). Thus the positive
correlation between male parental care and yolk androgens could
arise from males responding to the overall quality of the offspring.
Conclusions
The results of our experiments indicate that female collared
flycatchers in our population do not differentially allocate yolk
androgens to manipulate males into higher parental investment.
However, they might be able to manipulate males via other maternal
effects correlated with yolk androgens. Identifying the egg compo-
nents or chick characteristics that appear to differentially influence
the effort of male and female parents would be an important next step
towards a better understanding of the potential of maternal effects in
mediating sexual conflict over parental investment.
Acknowledgments
This study was financially supported by Turku University founda-
tion (grant to S.R.), Emil Aaltonen foundation (grant to T.L.), the
French National Research Agency (ANR - ANR-06-JCJC-0082 to B.D.)
andthe French National Centre for Scientific Research (CNRS - PICSno.
3054 to B.D.), the Janggen-Pöhn Stiftung, the Association for the Study
of Ani-mal Behaviour (ASAB), the Basler Stiftung für Biologische
Forschung, and Swiss National Science Foundation (PA00A3-121466
to B.T.). We thank our field assistants (L. Puhakka, L. Baudot, T. Biteau,
G. Daniel, H. Girard, J. Haquet, S. Le Bastard, B. Hernout, A. Theillout, A.
Benard, L. Pomarède and M. Koivula) for precious help in the field and
organisation, and the landowners of Gotland who allowed us to use
their woodplots. We also thank Bonnie de Vries for help and advice
during the hormone analyses.
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