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General and Comparative Endocrinology 165 (2010) 97–103
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General and Comparative Endocrinology
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Androgens during development in a bird species with extremely sexually
dimorphic growth, the brown songlark, Cinclorhamphus cruralis
C. Isaksson a,b,*, M.J.L. Magrath a,c, T.G.G. Groothuis b, J. Komdeur a
a
Animal Ecology Group, Centre for Ecological and Evolutionary Studies, University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands
Behaviour Biology, University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands
c
Department of Zoology, University of Melbourne, Vic. 3010, Australia
b
a r t i c l e
i n f o
Article history:
Received 12 February 2009
Revised 6 June 2009
Accepted 12 June 2009
Available online 17 June 2009
Keywords:
Testosterone
Sexual size dimorphism
Differential allocation
Maternal effects
Sex ratio
a b s t r a c t
In birds, early exposure to androgens has been shown to influence offspring growth and begging behaviour, and has been proposed as a mechanism for the development of sexual size dimorphism (SSD). Sex
specific effects during development can occur due to sex-specific allocation of maternal androgens, sensitivity to, or synthesis of, androgens. In addition, maternal hormones have been suggested as a mechanism to skew brood sex ratio. This study uses one of the world’s most extreme SSD species, the brown
songlark Cinclorhamphus cruralis, to investigate (1) sex-specific differences of androgens in yolk and chick
plasma and (2) the relationship between androgens and sex ratio bias. The study reveals no indication of
sex-specific maternal allocation, but a modest sex effect during the later stages of incubation when the
embryo starts to produce its own androgens. Moreover, there was a strong seasonal sex ratio bias:
female-biased early and male-biased later in the season, but yolk testosterone (T) did not show a seasonal
trend. Taken together these results suggest that if androgens, from any source, have a significant role in
development of SSD in this species it is most likely via sex-specific sensitivity or synthesis rather than
differential maternal transfer to the egg.
Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction
Sexual size dimorphism (SSD) is very common in animals but
the underlying mechanisms remain poorly understood (Badyaev,
2002; Fairbairn et al., 2007). In birds and mammals, the male is often the larger sex, whereas the opposite is often found in snakes,
fishes and insects (Fairbairn et al., 2007). At the ultimate level,
sex differences can often be explained by the breeding system of
the species. In some polygynous species for example, male–male
competition over high quality territories tends to favor large males
over small males (Andersson, 1994). At the proximate level, SSD
should be constrained by a shared genome and similarity in environmental conditions during development. For example, even
though male and female birds and mammals have different sex
chromosomes, the genes that underlie traits that show SSD are
not generally linked to these chromosomes (Badyaev, 2002, and
references therein). Instead the sex chromosomes are translated
in a complex process via hormone production and growth factors
that give rise to phenotypic variation. This opens the possibility
for hormones from embryonic and extra-embryonic source to
influence SSD development.
* Corresponding author. Present address: University of Oxford, Edward Grey
Institute for Field Ornithology, Oxford OX1 3PS, UK.
E-mail address: [email protected] (C. Isaksson).
0016-6480/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.ygcen.2009.06.015
Indeed, both maternal and offspring androgens have been suggested to be of particular importance for growth (e.g., Schwabl,
1996; von Engelhardt et al., 2006; but see also Andersson et al.,
2004). In oviparous species, females transfer steroid hormones to
the growing oocytes (e.g., Surai, 2002; Groothuis et al., 2005a),
which have been suggested to act as growth factors of embryos
and mediators of subsequent sensitivity to endogenous hormones.
Yolk androgens, such as testosterone (T) and androstenedione (A4)
have been shown to influence both embryonic and nestling
growth, and begging behaviours (e.g., Eising et al., 2001, 2003;
Cox and John-Alder, 2005; Groothuis et al., 2005b; von Engelhardt
et al., 2006; Gil et al., 2007; but see also Andersson et al., 2004;
Rubolini et al., 2006; Tobler et al., 2007). In addition, embryos start
to produce their own androgens in the course of incubation and
plasma concentrations in chicks have been related to sibling competition which can also affect growth rates (Sasvari et al., 1999;
Goodship and Buchanan, 2006). Consequently, androgens may
mediate SSD in three ways: first, by sex-specific transfer of maternal androgens to the egg yolks (e.g., Müller et al., 2002; Badyaev,
2005; Rutstein et al., 2005; Gilbert et al., 2005; but see Pilz et al.,
2005; Saino et al., 2006; Loyau et al., 2007 for negative results);
second, by sex-specific sensitivity to androgens (e.g., Saino et al.,
2006; von Engelhardt et al., 2006; Sockman et al., 2008); and third,
by sex-specific synthesis of androgens during development (e.g.,
Adkins-Regan et al., 1990).
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C. Isaksson et al. / General and Comparative Endocrinology 165 (2010) 97–103
Most research on androgens during development in birds has
been conducted on species with little or no SSD. However, if androgens play a role in SSD and all else is equal, greater SSD should be
accompanied by greater sex differences in maternal or endogenously produced androgen levels (Badyaev, 2002; Uller and Badyaev, 2009), with potential consequences for the joint evolution
of sex bias and sex-specific maternal effects (e.g., Carranza, 2004;
Uller, 2006; Uller and Badyaev, 2009).
Maternal androgens may also play a role in influencing the sex
ratio of the brood. In birds, females are the heterogametic sex and
therefore seems to be able to adjust the clutch sex ratio to suit the
prevailing environment, though the mechanism remains poorly
understood (for a review see Pike and Petrie, 2003; Carere and Balthazart, 2007). Interestingly, maternal yolk hormones have recently been advanced as a mechanism for sex ratio bias either by
affecting meiosis and thereby sex determination (Rutkowska and
Badyaev, 2008; Uller and Badyaev, 2009), or by affecting resorption
or growth of the follicle of the wrong sex (for a review see AlonsoAlvarez, 2006). In several studies, elevation of maternal testosterone induced a male-biased sex ratio (Veiga et al., 2004; Rutkowska
and Cichon, 2006).
An ideal model species to investigate the influence of androgens
on the development of SSD is the brown songlark, C. cruralis. This
species exhibits one of the world’s most extreme examples of
SSD among birds (Andersson, 1994). Females hatch from marginally larger eggs (on average 3%), and are initially heavier but not
structurally larger (Magrath et al., 2003a). However, at fledging
(11.5 days) males weigh over 60% more than their sisters and as
adults this mass difference has increased to well over 100% (Amadon, 1977; Magrath et al., 2003a,b, 2007). As nestlings, brothers receive both larger quantity and higher quality food compared to
their sisters (Magrath et al., 2004, 2007), and these disparities
could result from post-hatching differences in androgen mediated
competitive ability.
Thus, as a first step towards addressing the endocrine basis of
SSD in brown songlarks (and size dimorphic birds more generally)
the present study aims to investigate sex differences in (1) yolk T
and A4 concentrations during embryonic development (i.e., maternal and subsequent embryonic synthesis); (2) plasma T concentrations during the nestling period. Finally, we explore; (3) the
relationship between yolk androgens and primary sex ratio and
whether the sex ratio differs from parity. We predict that if androgens are part of the proximate mechanism underlying SSD in the
brown songlark, androgen concentrations in egg yolk and/or in
nestling plasma should be higher in males than in females. Finally,
we predict that if yolk androgens influence the primary sex ratio in
this species, high androgen concentrations should be associated
with a male-biased sex ratio.
2. Methods
2.1. Field site and data collection
The study was carried out between late August and early October (2007), in south-western New South Wales, Australia, in a area
of mixed grassland and shrubland adjacent to the Cobb Highway
between Hay and Deniliquin (35°070 S, 144°480 E). This study area
was selected because it had a relatively high density of territorial
males in late August, compared to other sites in the region that
were visited during the same period. Songlarks are somewhat nomadic and breed opportunistically (Magrath et al., 2003b), but
males generally arrive in this region in winter (June–August) and
breeding occurs during the spring months (September–November).
Average clutch size is 3.2, with eggs laid every 24 h, and incubation
time is on average 12.1 days (Magrath et al., 2003b).
Nests were found by observing females with binoculars from a
stationary vehicle or by systematic walking through the study area
to flush incubating females from their nest (more details in Magrath et al. (2003b)). Nests were marked by placing two sticks opposite each other and 5 m on either side of the nest. Nests found
during construction or laying were checked every day until the
clutch was complete (no new eggs), after which the clutch was collected following 2 more days of incubation for embryonic development to allow molecular sexing. Most nests were found after egg
completion and therefore we were unable to determine the egg position in the laying sequence. Development of the embryo was
checked in the field by illuminating the egg with the light from a
small torch. If embryonic development was evident the clutch
was collected, otherwise the clutch was left in the nest for 2 further
days before collection. Additionally, three clutches were incubated
for 48 h in an incubator (maintained at about 37 °C), to avoid the
risk of predation. Upon collection, all eggs were weighed to the
nearest 0.1 g using a Pesola spring balance, and egg length and
width measured to the nearest 0.1 mm using callipers. The eggs
were then stored at 20 °C until analysis.
When nests were found already with nestlings, we measured
tarsus length (to the nearest 0.1 mm) and wing length (to the nearest 0.1 mm) with callipers and body mass (to the nearest 0.05 g)
with a Pesola spring balance. A small blood sample (approximately
100 ll) was taken from the jugular vein with a heparinised syringe
for sexing and hormone analysis (see below). Based on feather
development and weight, the approximate age of these broods
could be estimated on the basis of previously collected data on
the growth curves of known aged nestlings (Magrath, unpublished
data). The ages of the sampled chicks were between 8 and 14 days
old (more details in Table 1b).
2.2. Androgen extractions and radioimmunoassay (RIA)
Embryos were dissected from the frozen yolk at the time of
androgen extraction and, based on the developmental stages detailed in Starck and Ricklefs (1998), assigned into one of five developmental categories (stage 14–18 (up to 5 mm in length), stage
19–26 (5–10 mm), stage 27–33 (10–15 mm), 34–39 (15–20 mm),
stage 40–42 (20–25 mm)). Embryos in the 5 mm category had a
developed budding of allantois, while those in the last category
(25 mm) had developed feathers, and were only a day or 2 from
hatching. Precocial and altricial embryos have the same sequence
of 42 structurally defined developmental stages, with almost
invariant patterns of development (Starck and Ricklefs, 1998).
The only difference is in the later stages of development, i.e., just
before hatching, where altricial species develop faster than precocial species. Consequently, the developmental stages of the precocial chicken can be directly compared to the brown songlark.
Hormonal synthesis is known to start at stage 29 (Starck and Ricklefs, 1998).
From 44 eggs (n = 17 clutches), the frozen yolks were separated
from the albumen, and the embryo dissected. Yolks were weighed
and homogenized with water (1 ml/g yolk). Around 200 mg of pure
yolk was used from each egg. We followed a standard procedure
according to Kingma et al. (2009), with minor modifications. Both
androstenedione (A4) and testosterone (T) were extracted from all
yolks. To remove proteins and lipids that may interfere with the assay samples were extracted twice using diethyl-/petroleum ether
(70:30 v/v), dried under a stream of nitrogen, and then reconstituted in 70% methanol and stored at 20 °C over night. The following day these were spun down, and the supernatants decanted and
dried under a stream of nitrogen.
Plasma extractions (n = 20 chicks) followed the same procedure
as for yolks, but due to low concentrations and small volume of
plasma we were only able to extract T. All available plasma was
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C. Isaksson et al. / General and Comparative Endocrinology 165 (2010) 97–103
Table 1
(a) Mean yolk concentrations (pg/mg yolk) of testosterone (T) and androstenedione (A4) in female and male eggs of different developmental stages (see Section 2). (b) Mean
plasma concentrations (ng/ml plasma) of T in female and male chicks from ages between 8 and 14 days old.
Embr. develop. stage
14–18
(a) Yolk hormones
Female T
Male T
Female A4
Male A4
Chick age (days)
19–26
(b) Plasma hormones
Female T
–
Male T
0.66
40–42
±SD
n
Mean
±SD
n
Mean
±SD
n
Mean
±SD
n
0.506
0.521
10.062
8.701
0.095
0.236
3.026
4.648
2
11
2
11
0.164
0.150
1.487
0.974
0.083
0.020
1.317
0.510
6
5
6
5
0.176
0.276
0.923
2.330
0.051
0.083
0.835
2.314
10
3
10
3
0.218
0.292
0.719
1.556
0.068
0.091
0.214
0.072
5
2
5
2
8
Mean
34–39
Mean
9
10
11
12
13
14
±SD
n
Mean
n
Mean
±SD
n
Mean
±SD
n
Mean
n
Mean
n
Mean
n
–
0.01
–
0.42
1
0.81
0.57
0.44
–
4
1
0.95
0.58
0.38
0.13
6
2
0.55
0.50
1
1
–
0.39
1
0.76
–
1
2
used during extraction (37–125 mg). Recovery for both yolk and
plasma samples was, based on added tritiated T, 74%.
A competitive-binding radioimmunoassay (RIA) was used to
determine the androgen concentrations in both yolks and plasma
(Diagnostic Systems Laboratories, Texas, USA). Standard curves
for both yolk and plasma were performed to validate the assay
and antibody specificity for this species. Cross-reactivity of T with
DHT is 5.8%, with A4 2.3%, with E2 zero. Intra-assay coefficient of
variation was 4.1% and 3.4%, respectively.
2.3. Molecular sexing
Table 2
Model summaries examining yolk testosterone (T) concentration in brown songlark
eggs in relation to a number of potential explanatory variables. Due to differences in
androgen origin, two dependent variables was used in two separate GLMMs (1)
Maternal origin, between developmental stage 14–26 and (2) maternal + embryonic
leakage to yolk, between developmental stage 34–42. Significant effects are indicated
by bold text.
Yolk T
Maternal origin
Source
ndf,
ddf
Sex
1,
15.7
1, 7.2
Embryo size
DNA was extracted from embryos and nestling blood samples
using a Chelex extraction procedure (Walsh et al., 1991). Sex of
the offspring was determined following Griffiths et al. (1998).
The primer pair P2 and P8 amplifies homologous sections of the
avian genes CHD-Z (present in both sexes) and CHD-W (only present in females). The amplified products were separated by electrophoresis in a 2% agarose gel and visualized with ethidium bromide
staining under UV light.
2.4. Statistics
Yolk T concentration was log transformed to normalize the distribution of these data, whereas yolk A4 could not be transformed
to fit the model assumptions of parametric tests. Due to a strong
correlation between T and A4 (see Section 3), small sample size,
and the non-normal distribution for A4 concentrations, statistical
analyses are only presented for T, the most biological active hormone of the two, although we give means and standard errors
for both hormones where appropriate. Analyses were performed
using Generalized Linear Mixed Modeling (GLMM) with backward
elimination (JMP 8, SAS Institute, 2007, North Carolina). Nest identity was included as a random factor in all models in order to control for the non-independence of eggs/chicks from the same clutch/
brood and this was also applied in models using sex ratio as
dependent variable. Egg mass and laying date were included as
covariates in the model explaining variation in yolk T
concentrations (see Table 2). Egg volume was calculated as
(0.6057 0.0018B)LB2 (B = egg breadth, L = egg length, Narushin,
2005). Egg mass and volume were highly correlated (r = 0.793,
p < 0.0001), so we used only egg mass for our analyses. Embryo
development and nestling age are known to affect steroid concentrations (Bowden et al., 2002; Elf and Fivizzani, 2002; Lovern and
Wade, 2003), and was incorporated as predictors in the models
(see Tables 2 and 3 for full models). Two models were performed
for yolk T; one using data at stages when only maternal T is present
in the yolk (stages 14–26) and a second model when the embryo
Egg mass
Laying date
Sex embryo
size
Sex egg mass
Sex laying
date
1,
20.9
1, 6.3
1,
17.2
1,
15.2
1,
12.1
Maternal + embryo origin
p
Ordera
ndf,
ddf
F
p
Ordera
0.54
0.474
5
5.63
0.029
7
16.53
0.005
7
0.16
0.269
6
1.15
0.297
6
1,
17.9
1,
4.76
1, 9.4
0.00
0.971
5
0.02
0.49
0.882
0.494
2
3
0.16
0.35
0.707
0.562
3
2
0.26
0.620
4
2.84
0.116
4
0.01
0.929
1
1, 5.8
1,
12.9
1,
13.1
1, 9.2
1.41
0.265
1
F
a
Backward elimination is used and the order of exclusion is shown in table. The
results are shown before eliminated from the model. Clutch identity was retained as
a random factor in all models.
Table 3
Model summaries examining plasma testosterone (T) concentration in brown
songlark chicks in relation to a number of potential explanatory variables. Final
model is indicated by bold text. The results for the excluded parameters are shown
before elimination from the model. Clutch identity was retained in all models.
Plasma T
ndf, ddf
F
p
Sex
Age
Body mass
Sex body mass
Sex age
1,
1,
1,
1,
1,
5.50
0.23
12.57
7.68
0.39
0.035
0.646
0.003
0.018
0.542
13.2
8.19
13.6
11
13.9
has started with its own synthesis of hormones (stages 34–42).
To specifically compare yolk T concentrations between brothers
and sisters, we fitted a mixed model using only mixed sex clutches,
regardless of hormonal origin. However, in this case we used the
deviation in T from the clutch means, rather than the actual T concentration, to exclude any between clutch variation in hormone
concentrations. The sample size of mixed-sex broods was too small
(four broods) to allow a similar analysis on plasma T concentration.
Finally, GLMM was also used for examining sex ratio bias in rela-
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C. Isaksson et al. / General and Comparative Endocrinology 165 (2010) 97–103
Fig. 1. Proportion of sons in brown songlark clutches over the season. The first clutch (zero on the x-axis) was laid on the 25th of August ±1 day.
tion to mean clutch yolk T and laying date. Clutch identity was included as a random factor.
3. Results
Mean clutch size was 2.77 ± 0.06 (n = 17 clutches, 21 male and
23 female eggs). There was no general sex difference in egg weight
(mean egg weight (g); male eggs: 3.07 ± 0.37, female eggs:
2.99 ± 0.40, F1,26 = 0.55, p = 0.465) or egg volume (mean egg volume; male eggs: 8.22 108 ± 1.24 108, female eggs:
7.80 108 ± 1.22 108, F1,26 = 1.97, p = 0.173). Mean clutch sex ratio did not differ from parity (n = 15, mean clutch sex ratio
0.466 ± 0.10; one sample t-test = 4.843, p = 0.999, two clutches
were excluded because the sex ratio of the complete clutch was
not known). However, there was a strong seasonal sex ratio bias,
with predominately female biased clutches early in the season
and male biased clutches later in the season (n = 15, F1,13 = 15.20,
p = 0.002; Fig. 1). Yolk testosterone concentration was not associated with sex ratio, and therefore removed from the above model
(F1,12 = 2.43, p = 0.145). However, a positive trend between sex ratio and yolk T was revealed if only yolk T was used in the model
(F1,13 = 4.17, p = 0.062).
Overall means of yolk T concentrations for females and males
were 0.210 ± 0.024 (nfemales = 23) and 0.376 ± 0.051 (nmales = 21)
pg/mg yolk, and 0.854 ± 0.364 (nfemales = 12), and 0.670 ± 0.381
(nmales = 8) ng/ml plasma, respectively. A detailed summary of
mean T and A4 concentrations of yolks are shown in Table 1a
and b and Fig. 2. T and A4 concentrations were highly correlated
in both female (rs = 0.882, n = 23, p < 0.0001) and male egg yolks
(rs = 0.930, n = 21, p < 0.0001). Full initial models for yolk T are
shown in Table 2. There was no sex difference in maternal transfer
of T to the yolk (model on stages 14–26: p = 0.474, Table 2), but later during incubation when embryos have started to synthesize
Fig. 2. Yolk testosterone (T) (a) and androstenedione (A4) (b) in yolks of male and female brown songlarks during four different developmental stages ranging from 2 to
12 days of incubation (see Section 2 for more details).
C. Isaksson et al. / General and Comparative Endocrinology 165 (2010) 97–103
101
Fig. 3. Plasma testosterone (T) in relation to chick body mass (males = non-filled and females = filled).
their own T a slight significant difference between males and females was revealed (p = 0.029, Tables 1a and 2, Fig. 2). As a response to embryonic development and metabolism the maternal
yolk T concentrations decrease rapidly between the two early
phases of development (p = 0.005, Table 2 and see Fig. 2). In a model comparing yolk T concentrations between brothers and sisters,
there was a tendency for brothers to have a higher yolk T concentration (eight clutches, n (females) = 11, n (males) = 11, F1,20 = 4.14,
p = 0.055), and this difference was not significantly related to the
developmental stage of the embryo (embryo size; F3,14 = 0.25,
p = 0.857, sex embryo size; F3,14 = 1.90, p = 0.176).
Furthermore, when using plasma T as the response variable a
significant interaction between sex and body mass was revealed
(F1,11 = 7.68, p = 0.018, see Table 3 for full model and Fig. 3), with
a steeper decrease of plasma T with body mass for females compared to males (Fig. 3). However, since there is limited overlap in
mass between the sexes it is hard to disentangle the effects of body
mass from those of sex.
4. Discussion
The brown songlark C. cruralis shows an extreme sexual size
dimorphism (SSD), which starts to develop during early ontogeny
(Magrath et al., 2007). We explored whether these sexual differences in growth patterns could be mediated by maternal or endogenous androgens, specifically A4 and its active metabolite T.
In the first days of development, embryos are only exposed to
hormones of maternal origin. Our data suggest that male and female eggs do not accumulate different amounts of androgens, indicating that differential exposure to maternal androgens is not
responsible for SSD development, contrary to what would be predicted based on recent models of the evolution of environmental
effects on offspring sex in birds (Uller and Badyaev, 2009).
Although several authors have reported sex differences in maternal
androgens in avian eggs, all these findings do not concern overall
sex differences, as we have tested in our study, but sex differences
in interaction with a diversity of other factors, such as laying sequence (Badyaev et al., 2006), dominance (Müller et al., 2002) or
treatment and laying sequence (Rutstein et al., 2005; Gilbert
et al., 2005). It is clear from our data that during the first week
of development maternal T concentrations in the yolk rapidly decrease. These results are in accordance with other studies of yolk
androgens during embryonic development (e.g., Bowden et al.,
2002; Elf and Fivizzani, 2002; Lovern and Wade, 2003). Studies
suggest that this may be due to dilution of yolk by albumin (Gilbert
et al., 2005), and the conversion of steroid hormones to conjugated
forms (Bowden et al., 2002).
After the age at which embryos probably start to produce their
own androgens (based on extrapolation of chicken data, see meth-
ods) yolk of male eggs have higher androgen concentrations than
that of female eggs. Since male embryos produce somewhat higher
amounts of androgens than female embryos (see Müller et al., 2005
for references) this indicates leakage of their endogenously synthesized T to the yolk, a phenomenon that has only been described for
estradiol (Elf and Fivizzani, 2002). Whether a sex specific androgen
production generates a disparity in the behaviours of male and female siblings needs further investigations by injecting female eggs
with androgens.
There was a strong seasonal sex ratio bias, with female biased
clutches early in the season and male bias later. The seasonal sex
ratio bias could confound effects of season and sex on maternal T
allocation, however, neither was significant in the model with
maternal T as dependent variable. Seasonal sex ratio shifts in either
direction have previously been shown in several species of birds
and other taxons (e.g., female bias early in the season in Daan et
al. (1996) and Ristow and Wink (2004); male-biased early in the
season in Andersson et al. (2003); reviewed in Komdeur and Pen
(2002)), and may be mediated by physiological integration of hormonal regulation of onset of breeding, incubation and oocyte
growth and maturation (Badyaev, 2005; Badyaev et al., 2006; Carere and Balthazart, 2007; Sockman et al., 2008; Uller and Badyaev,
2009). This may have adaptive significance either because it generates a match between sex and hatching date, for example, if one
sex benefits from a longer growth period before migration or
hibernation, or because it mediates within-brood sibling competition under different environmental conditions (Daan et al., 1996;
Uller, 2006; Uller and Badyaev, 2009). Indeed, the seasonal population sex ratio shifts was not seen during earlier breeding seasons
(1998–2000, Magrath, unpublished data), suggesting that such
mechanisms may be influenced by between-year variation in environmental conditions. The year of the present study was extremely
dry and the number of breeding attempts relatively low (compared
with Magrath et al., 2003b). Therefore, the overproduction of
cheaper daughters early in season may have been an adaptive response to low food availability. However, an explanation for the
shift to male-biased broods later in the season is unclear. Possibly,
there was a change in the abundance, size or composition of prey
species that favoured the production of sons, but this possibility remains unknown. The highly nomadic nature of brown songlarks
makes it, unfortunately, impossible to relate birth date with chance
of recruiting to the population the following season (e.g., female
higher recruiting chance if they are born first).
Finally, given that the growth difference appears when nestlings are 4 days old, post-hatching hormones could also be important in triggering the SSD. The data suggests that T is negatively
related to body mass in females, but not in males. Perhaps competition with their larger brother induces an up-regulation of T production, especially in lighter females. However, since all blood
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C. Isaksson et al. / General and Comparative Endocrinology 165 (2010) 97–103
samples were taken when the size difference between males and
females was already established (between 8 and 14 days old), we
were unable to separate the effects of sex from body mass. Other
species with less pronounced SSD reveal that there is no clear pattern. In some species, such as the great tit (Parus major), the
slightly larger nestling males have higher T but only during a short
time window, i.e., second day after hatching (Silverin and Sharp,
1996). However, other species have no detectable difference, while
others show the opposite pattern, i.e., females have higher T concentrations (reviewed in Fargallo et al., 2007). Clearly, this need
to be investigated in more detail, since even in young birds, like
in adults, plasma T concentrations are highly flexible and dependent on the nature and level of social stimulation (Ros et al.,
2002). In any case, our current data suggest that the extreme sexual difference in growth (regardless of brood identity) is probably
more likely an effect of sex-specific sensitivity to T via its effect on
secretion of growth hormone (see Badyaev, 2002). Potentially,
other hormones or non-hormonal factors, such as estrogens or protein content in diet, are regulating growth rates of this species.
In conclusion, although the brown songlark is the most SSD bird
species in the world, and males grow much faster than females
post-hatching, this does not seem to be regulated by maternal allocation of androgens, but potentially by sex-specific synthesis prehatching or sensitivity to androgens. Non-hormonal regulation or
hormones other than androgens may be more important in generating the dramatic growth difference between male and female
brown songlarks, however, this needs further investigation.
Acknowledgments
We would like to thank Per Flodin, Jeroen van Dijk, Erica van
Rooij, Maria von Post, and Iain Woxvold for field assistance. We
are grateful to Ken and Mary McCrabb (Avenel/Wangenella), and
Derek McFarland (McFarlands St F/Hay) for allowing us to work
on their properties, and Bill and Mary-Anne Butcher of Elmsleigh
Station for letting us stay on their property and for logistical support. We would also like to thank the NPWS and Rural Lands and
Protection Board in Hay for advice. In the Netherlands we would
like to thank, Marco van der Velde for molecular sex determination, Bonnie de Vries and Jelle Boonekamp for lab assistance and
advice, and Bonnie also for help with calculations of the hormone
concentrations. Tobias Uller kindly commented on the paper. This
work was conducted with the approval of the Animal Experimentation Ethics Committee of the Australian National University
and the NSW NPWS. The project was funded by a NWO Rubicon
fellowship to C.I. (project number 825.07.004).
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