Behavioral Ecology

doi:10.1093/beheco/arr169
Advance Access publication 11 October 2011

Original Article

Stress-induced sex ratios in ground squirrels:

support for a mechanistic hypothesis
Calen P. Ryan,a,b W. Gary Anderson,a Laura E. Gardiner,a,c and James F. Harea
Department of Biological Sciences, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada,
b
Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, BritishColumbia V5A 1S6, Canada, and cDepartment of Biology, University of Regina, 3737 Wascana Parkway,
Regina, Saskatchewan S4S 0A2, Canada

Systematic deviations in sex ratio may be adaptive in the face of the prevailing environmental or physiological cues experienced
by the mother; yet some theoretical and mechanistic hypotheses remain at odds and are rarely examined noninvasively under
natural conditions. Conventional interpretations of the Trivers and Willard (Trivers RL, Willard DE. 1973. Natural selection of
parental ability to vary the sex ratio of offspring. Science 179:9092) hypothesis (TWH) predict that higher stress should be
associated with female-biased litters; yet, several mechanistic hypotheses predict the opposite. We tested the predictions of the
TWH and 2 stress-related mechanistic hypotheses in a free-living polygynandrous sciurid, Richardsons ground squirrel, Urocitellus
richardsonii. We examined the relationship between litter sex ratio and indicators of maternal condition and investment. These
included litter size, juvenile mass at emergence, as well as maternal age, changes in maternal body mass, and maternal fecal
glucocorticoid (cortisol and corticosterone) levels during gestation, lactation, and post-weaning. Males born of small litters were
significantly heavier at emergence than males from larger litters, which were female biased, whereas females showed no significant change in mass with litter size. Mothers with higher fecal cortisol levels during the gestation period (but not during
lactation or post-weaning) were more likely to produce male-biased litters, whereas females producing larger litters showed
significantly higher cortisol levels during lactation (but not gestation or post-weaning) than mothers producing smaller litters.
Our results provide support for both the proximate glucose-mediated mechanistic model of Cameron (Cameron EZ. 2004.
Facultative adjustment of mammalian sex ratios in support of the Trivers-Willard hypothesis: evidence for a mechanism. Proc R
Soc Ser B Biol Sci. 271:17231728) and the broad-sense TWH, where social and life-history characteristics play an integral role.
Key words: advantaged matriline, glucocorticoids, litter size, maternal condition, sex ratio, stress, Urocitellus richardsonii. [Behav
Ecol 23:160167 (2012)]

INTRODUCTION
kewed sex ratios at birth, weaning, or fledging in vertebrates
have been attributed to the differential costs of successfully
rearing offspring of a given sex and to condition-dependent
differences in the relative reproductive value of each sex
(Hardy 1997). In polygynous mammals, the fitness returns
associated with high-quality male offspring may exceed those
derived from producing high-quality female offspring because
of the huge variability in male reproductive success. As a result,
females with the greatest capacity for investment are predicted
to bear more male offspring, whereas the opposite should be
true for poorer quality females (Trivers and Willard 1973).
Among mammals, the costs for producing male versus female
offspring in any given species are often difficult to quantify
(Frank 1990), but the immediate energetic investment into
male offspring is ordinarily greater because of their higher
developmental rates, larger size, and greater nutritional demands (Gomendio et al. 1990; Redondo et al. 1992; but see
Koskela et al. 2009).
Despite evidence for sex ratio manipulation in mammals,
the study of adaptive sex allocation (ASA) is plagued by incon-

Address correspondence to C.P. Ryan. E-mail: [email protected].

Received 26 February 2011; revised 4 August 2011; accepted 12
September 2011.

The Author 2011. Published by Oxford University Press on behalf of
the International Society for Behavioral Ecology. All rights reserved.
For permissions, please e-mail: [email protected]

sistent results (Charnov 1982; Clutton-Brock and Iason 1986;

Frank 1990; Hardy 1997). Because the nature or timing of
measurements used in sex allocation studies appear critical
to the outcome, resolving these inconsistencies may hinge
on elucidating the proximate physiological mechanisms for
ASA, in addition to the ultimate factors responsible for them
(Linklater 2007). Indeed, numerous potential mechanisms
have been proposed but remain a source of contention and
constitute the major limitation of arguments in support of
ASA (Krackow 1995). Understanding the mechanisms by
which sex ratio is manipulated in mammals is therefore an
important goal for interpreting published work and for effectively designing future experiments to study ASA.
Endocrine systems play an essential role in the translation
of external events into physiologically relevant responses
(Sapolsky et al. 2000), providing an interface between environmental stimuli (e.g., resource abundance, predation pressure, social interactions) and behavior and life-history traits,
such as growth, development, and reproduction (Boonstra
et al. 2007). As such, hormones may be of critical importance
to the study of litter size and sex ratio manipulation in mammals, by coupling maternal state and parameters of offspring
production (Kaiser and Sachser 2009). The hypothalamic
pituitaryadrenal axis (HPA or stress axis) is of particular
interest as a candidate system providing the mechanistic
underpinnings of ASA because it is profoundly linked to reproductive physiology and behavior (Wingfield and Sapolsky

Ryan et al.

Stress-induced sex ratios in ground squirrels

2003) and can orchestrate entire physiological systems, influencing the secretion and activity of other hormones (Ketterson
et al. 1991).
Baseline glucocorticoid (or GC, cortisol, and corticosterone) levels can provide an indication of individual state or
condition by quantifying the physiological effort to sustain
homeostatic balance in the face of social or environmental
perturbations (Palme et al. 2005). Stressors associated with
increases in baseline GC levels (such as overcrowding, resource limitation, or intraspecific competition) also match
the conditions predicted to lead to the adaptive manipulation
of offspring sex ratio (Cameron 2004). Furthermore, GCs
demonstrate marked inhibitory effects on reproductive physiology and behavior, as well as parental investment (Wingfield
and Sapolsky 2003), providing a critical link between the conditions favoring ASA and the physiological and behavioral
mechanism for accomplishing sex ratio manipulation. Indeed, measures of social or environmental stress have been
linked to ASA (Pratt and Lisk 1989; Gotz et al. 2008; Love and
Williams 2008; Ideta et al. 2009); however, the actual role of
GCs themselves in sex ratio manipulation remains poorly understood, particularly in free-living mammalian populations.
Among the proposed mechanisms for sex ratio manipulation in mammals (likely to differ from those operating in birds
due to fundamental differences in sex determination), 2 in
particular suggest an indirect role for the stress axis and GCs
(Cameron 2004; Grant 2007). Both models make predictions
that appear to conflict with those most commonly associated
with the Trivers and Willard hypothesis (TWH; i.e., that
stressed females in poor condition with high GC levels should
produce a greater proportion of females). The question that
remains, therefore, is to what extent these mechanistic models
are empirically supported and if they can be reconciled in the
face of what appear to be the contrary predictions of conventional sex ratio theory.
To address these questions, we quantified the relationships
between measures of maternal quality (age, spring mass, and
gain in mass over active season), maternal investment (litter
size, sex ratio, and offspring size), and stress or allostatic
load (in the form of fecal GC levels) in free-living Richardsons ground squirrels (Urocitellus richardsonii). Traditional
interpretations of sex ratio manipulation (so called narrowsense TWH; Cockburn et al. 2002) predict that poor quality
females with the lowest capacity to invest (i.e., the lightest,
most stressed, or the youngest) should produce female-biased litters. Under these conditions, high GC levels would also
be predicted to be associated with female-biased litters and
potentially with other aspects of female condition (e.g., age,
mass; Clutton-Brock and Iason 1986).
On the other hand, support for both mechanistic hypotheses
requires that GC levels be negatively associated with sex ratio
(i.e., the ratio of males to the total number of offspring produced within a litter). The mechanism proposed by Cameron
(2004) predicts that sex ratio will be positively associated with
litter size, due to the differential mortality of developing female embryos, whereas the model of Grant and Irwin (2005)
does not predict this relationship. The novelty of this study is
that it tests the predictions of both mechanistic (proximate)
and adaptive (ultimate) hypotheses using noninvasive techniques under ecologically relevant conditions.
Our study species, Richardsons ground squirrels, are semifossorial, hibernating colonial rodents found in the northern
plains and intermontane valleys of North America (Michener
and Koeppl 1985). Following emergence from hibernation,
males compete intensely, often fatally, for mating opportunities (Michener 1983b). Although all females are typically inseminated in a given year, many males do not successfully
reproduce (Michener and Locklear 1990). This single annual

161

reproductive event, coupled with the relatively low interyear

recovery rates of U. richardsonii, results in enormous selective
pressure for males to maximize their reproductive output during this time. The intense competition between males, and
extreme variability in male reproductive success, fits criteria
outlined by Trivers and Willard (1973) when discussing conditions that would lead to differential fitness consequences for
high-quality males versus high-quality females in a population
and thus selection for sex ratio manipulation. By carrying out
this study using numerous, noninvasive fecal GC measurements throughout the active season, in an environment similar to that in which the behavior would have evolved, we
investigate the proximate mechanisms of sex ratio manipulation without sacrificing the ultimate ecological relevance of
the results.
MATERIALS AND METHODS
Study site and data collection
Our research focused on individuals belonging to a population
of Richardsons ground squirrels, U. richardsonii, residing on
mowed fields and berms within the grounds of the Assiniboine Park Zoo in Winnipeg, Manitoba (lat 4952#N, long
9714#W). This free-living population has been extensively
studied since 2003, and detailed genealogical information
and data regarding the social behavior in this colony are available (e.g., Hare et al. 2004; Wilson and Hare 2004).
We commenced trapping on 18 March 2009, when male
squirrels began to emerge from hibernation, and continued
trapping until early July when most adults and yearlings had
descended into their burrows for hibernation. Females
emerged from hibernation after the males and were trapped,
on average, twice weekly beginning in early April by placing
National or Tomahawk live traps (Tomahawk Live Trap Co.,
Tomahawk, WI) baited with peanut butter (No Name Smooth
Peanut Butter; Loblaws Inc., Toronto, Ontario, Canada) at or
near burrow entrances. Using Pesola spring balances, we
weighed females to the nearest 5 g at each capture and collected feces from beneath the traps for GC assays. We applied
commercially available hair dye (Clairol Hydrience 52S; Pearl
Black, Stamford, CT) to the dorsal pelage of the animals in
unique patterns to facilitate individual identification and inserted numbered metal ear tags (National Band and Tag Company, Monel no. 1, Newport, KY) through the right pinna of
each squirrel for permanent identification.
Because U. richardsonii females utilize and defend small core
areas with little overlap with the core areas of other females
during pregnancy and lactation (Michener 1979), the offspring belonging to a given mother were determined by which
burrow the litter emerged from and when. We trapped young
of the year as soon as possible, typically within 2 days of their
initial emergence from their natal burrow. The juvenile squirrels were then sexed, marked, and released until all pups in
the litter were accounted for. We recorded litter sex ratios
for each mother, which are henceforth described in the
conventional manner, as a ratio of males to total offspring
produced (Charnov 1982). We carried out all trapping,
marking, and handling of animals in accordance with the
guidelines set forth by the Canadian Council on Animal
Care as approved under protocol F08-012 of the University
of Manitoba, Fort Garry Campus Protocol Management and
Review Committee.
We determined maternal stress levels using a noninvasive
measure of GCs (cortisol and corticosterone) in the feces.
Fresh feces were collected from females during trapping,
which we carried out between 1100 and 1600 h Central Daylight Time to minimize circadian fluctuations in GC hormones

162

(Boonstra et al. 2007). We then transferred fecal pellets to

20-ml polycarbonate sample vials using disposable wooden
sticks (Puritan, Guilford, ME), carefully discarding fecal
pellets contaminated with urine, another excretory route
for circulating GCs (see Mateo and Cavigelli 2005). We
numbered vials sequentially to allow cross-referencing to
field notes providing the time of sample collection, date,
and female identity and then stored them temporarily at
24 C in a freezer located at the Assiniboine Park Zoo.
Samples were later transferred to a 220 C freezer for
storage until required for GC assays.
GC assays
We used radioimmunoassay (RIA) to ascertain fecal cortisol
and corticosterone levels in our focal individuals, based on
methods described by Mateo and Cavigelli (2005) but modified and validated for U. richardsonii (Hare JF, Skyner L,
Enright C, and Anderson WG, unpublished). Briefly, we dried
fecal samples overnight at 60 C before combining approximately 0.2 g of the dried fecal matter with 1.5 ml 95% ethanol.
We then vortexed each sample vigorously before centrifugation at 4 C for 10 min at 13 000 3 g. After centrifugation, we
drew off the resulting supernatant, which was then stored at
280 C until measurement in the RIA, at which time we removed 100 ll of supernatant and evaporated the ethanol in
a sample concentrator (Savant speed vac; Thermo Scientific,
Waltham, MA). The resultant pellet was then resuspended in
RIA buffer (0.1 M phosphate buffer, 0.9% NaCl [w/v], and
0.5% bovine serum albumin [w/v]), and each sample was
vortexed to ensure complete dissolution.
For the cortisol RIA, we combined 100 ll of a cortisol-specific
antibody (1:16 000 dilution; Fitzgerald Industries, North Acton,
MA) in an assay tube with 5000 disintegrations per minute/100
ml (dpm) of tritiated cortisol (GE Healthcare, Piscataway, NJ)
and 100 ll of either sample or a known concentration of cortisol (Steraloids, Newport, RI). We then incubated the assay at
room temperature for 1 h and then at 4 C overnight, after
which time we added dextran (0.5% w/v)-coated charcoal (5%
w/v) (100 ll) to the solution to terminate the reaction. After 15
min on ice, we centrifuged assay tubes for 30 min at 4 C (2500
3 g) and decanted the resulting supernatant into a 7-ml scintillation vial. To each vial, we added 4 ml of Ultima Gold scintillation fluid (Perkin Elmer) and measured radioactivity using
a liquid scintillation counter (LS6500; Beckman Coulter, Brea,
CA). By interpolating from a standard curve made from known
concentrations of cold cortisol, we determined cortisol concentration (nanograms per gram of dry mass of fecal matter) for
each sample. For accuracy, we processed all samples in duplicate and standards in triplicate. We carried out corticosterone
assays using identical techniques but with corticosterone antibody (Antibodies-online, Aachen, Germany), standards (Steraloids, Newport, RI, USA), and labelled hormone (GE
Healthcare) in place of cortisol-specific analogs. Cortisol inter-assay and intra-assay variations were 10.9 6 6% and 15.3%,
respectively, and the minimum detectable level was 0.051 ng/
ml. Inter-assay and intra-assay variations for corticosterone were
14.7 6 8.9% and 8.8%, respectively, and the minimum detectable level for this hormone was 0.005 ng/ml. Samples demonstrated good parallelism for both hormones (data not shown),
and extraction efficiency was 90% and 91% for cortisol and
corticosterone, respectively.
Data analysis
Prior to statistical analyses, we assessed the distribution of each
variable independently and ranked models according to

Behavioral Ecology

Akaikes Information Criterion, corrected for small sample

sizes (AICc). We ultimately chose models with the lowest AICc
value and least number of parameters, as in Servanty et al.
(2007). Based on the AICc values for distribution goodnessof-fit, most of our data did not depart significantly from those
expected to come from a population following a Gaussian
distribution, and so we did not transform data prior to analysis
using standard parametric tests. Our GC assay values were not
normally distributed but did approximate both log- and generalized log-normal distributions. Based on the nature of the
data (derived from a biological assay and possessing a nonconstant variance; SAS Institute Inc. 2007), as well as the high
P value for goodness-of-fit (P . 0.80 for both cortisol and
corticosterone), we used generalized logarithm transformed
(Glog) values for all GC analyses. We then analyzed the transformed hormone data using standard parametric tests, including Pearsons correlation for the relationships between stress
and sex ratio and litter size and one-way analysis of variance
for categorical variables.
We partitioned stress hormone data into 3, biologically relevant time periods, termed gestation, lactation, and
post-weaning. We defined the gestation period, roughly
23 days (Michener 1980b), as the time immediately prior to
conception and ending with the inferred date of parturition.
Because females give birth to their litter in their burrows, we
inferred parturition date based on the date of juvenile emergence, which is on average 29.2 days following parturition
(Michener 1985). The lactation period was the time period
between parturition and litter emergence, whereas the postweaning period consisted of the remainder of the summer
following litter emergence, ending with female immergence
into hibernation.
Early season mass was female mass on 18 or 19 April, dates on
which the most females (15/21) were trapped and which corresponded to the time prior to conception for the majority
(.85%) of females, based on the inferred parturition described above. We determined summer weight gain by subtracting the early season mass from the prehibernation mass.
Prehibernation mass was the mass on 2 July, a date on which
masses for most (18/21) females were recorded and which
was within 1 week of immergence into hibernation for all
but 1 female. We were able to trap and weigh most (.90%)
juveniles within 2 days of their first emergence from the natal
burrow. If more than 3 days passed between the first and the
last juvenile trapping from a given litter, data from those litters (2/21 litters) were not used for these analyses as juveniles
gain mass rapidly within the first 2 weeks of emergence (Hare
JF, unpublished data).
We tested sex ratios for deviations from parity for individual
litters and for all juveniles combined using cumulative distribution binomial statistics, whereas sex differences in offspring
mass at emergence were analyzed using an unpaired t-test.
Age-specific differences in sex ratio, litter size, GC levels,
and offspring mass were analyzed using one-way analysis of
variance, where females were categorized into yearlings,
2-year-olds, and 3 years and older. We excluded 1 immigrant
female of unknown age from all age comparisons. Pearsons
productmoment correlation was used to examine the relationships between all continuous variables, including the relationship between litter size and sex ratio, dam mass gain
over summer, and juvenile masses at emergence. All pairwise
correlations were based on the residual maximum likelihood
method, with significance set to 5% (a 0.05). Analyses were
carried out using JMP, Version 8.0.2.2 (SAS Institute Inc.,
Cary, NC) with significance set to a 0.05, and figures were
created using Graphpad Prism Software, version 5.0 (San
Diego, CA, www.graphpad.com) or R (version 2.12.2 1.36
GUI (5691); R Development Core Team, 2011).

Ryan et al.

Stress-induced sex ratios in ground squirrels

163

RESULTS
Age-effects on maternal condition and offspring production
To examine the potential for age-related effects on maternal
condition, we compared age classes separately for spring emergence mass and weight gain over the summer. We were also interested if another measure of maternal condition, GC levels
are affected by the age of the female. Yearling females
emerged significantly lighter (259.5 6 12.5 g) than two 2year-olds or females 3 years and older (325.0 6 15.3 g and
329.9 6 11.5 g, respectively; F2,14 9.85, P 0.002), but there
were no age-related differences in weight gain from spring to
prehibernation (F2,12 0.481, P 0.629).
We assessed potential age-related effects on investment in
the form of litter size, sex ratio, and the mean of juvenile
masses. Neither litter size (F2,18 0.974, P 0.397), sex ratio
(F2,18 0.0145, P 0.986) nor mean juvenile mass (F2,17
0.0747, P 0.928) differed significantly among the 3 age
classes. Furthermore, age class had no significant effect on
cortisol or corticosterone levels for any of the 3 periods (gestation, lactation, or post-weaning; P . 0.190 in all cases).
Age-independent maternal condition and offspring
production
We considered the associations between parameters of maternal investment, including litter size and sex ratio, juvenile mass,
and maternal seasonal changes in mass, to examine the effects
of maternal condition on litter production and vice versa.
Independent of age class, spring emergence mass was significantly correlated with subsequent litter size, with heavier mothers tending to produce larger litters (P 0.019; Table 1).
Although mothers producing larger litters exhibited neither
greater nor lesser gains in mass over the active season (P
0.276; Table 1), weight gain was significantly related to litter
sex ratio; mothers that produced more males gained more
mass over the summer (P 0.038; Table 1).
Individual litter sex ratio varied considerably and was inversely correlated with the number of offspring in the litter
(P 0.0496; Table 1). A pooled analysis of juvenile masses at
emergence (2931 days old) revealed significant differences
between males and females (MannWhitney U 1543, nm
73, nf 60, P 0.003), with males significantly heavier than
juvenile females at emergence (96.5 6 2.2 g versus 88.9 6
2.2 g). Male mass decreased significantly (Spearmans
q 20.359, df 54, P 0.007) with increases in litter size,
whereas female mass did not (Figure 1).
GCs, maternal condition, and offspring production
To test the predictions of both the mechanistic and the theoretical hypotheses, we quantified the association between ma-

Figure 1
Relationship between litter size and emergence mass for Urocitellus
richardsonii juveniles. Emergence mass was not related to litter size for
females (degrees of freedom [df] 64, P 0.366; grey circles, solid
grey line), but males from larger litters were significantly lighter than
those from smaller litters (Spearmans q 20.359, df 54, P
0.007; black diamonds, broken black line). Number of observations
for each sex represented by symbol size, between 1 and 5
observations. Least-squares regression lines are based on parametric
correlations (not used due to the data distribution) but are provided
for illustrative purposes.

ternal GC levels and measures of condition, as well as

parameters of investment during 3 distinct reproductive periods. We observed a significant relationship between cortisol
levels during gestation and the subsequent litter sex ratio of
the gestating mother (Figure 2). Mothers with a high mean
cortisol level during gestation were significantly more likely to
produce male-biased litters (P 0.049; Figure 2). We did not
observe a relationship between corticosterone and sex ratio in
gestating mothers (P 0.450; Table 2) nor did we find a significant correlation between either corticosteone or cortisol
and litter sex ratio during the lactation or post-weaning periods (P . 0.326 for all; Table 2).
Neither cortisol nor corticosterone levels during gestation
were significantly related to litter size (P . 0.424 for both;
Table 2). This was also true of post-weaning GC levels (P .
0.188 for both; Table 2); however, higher cortisol levels during
lactation were associated with the production of larger litters
(P 0.014; Figure 3). Corticosterone levels during lactation
were not significantly correlated with litter size (P . 0.118;

Table 1
Relationship between indicators of maternal condition and litter composition in Urocitellus richardsonii
Variable

By variable

Correlation (r)

Lower 95%

Upper 95%

Probability

Mass gain
Litter size
Litter size
Sex ratio
Sex ratio
Sex ratio

Spring mass
Spring mass
Mass gain
Spring mass
Mass gain
Litter size

16
18
16
18
16
22

20.290
0.548
20.286
20.339
0.522
20.423

20.687
0.109
20.685
20.696
0.036
20.717

0.240
0.808
0.244
0.152
0.809
20.002

0.2756
*0.0186
0.2822
0.1690
*0.0381
*0.0496

Measures of maternal condition approximate the time of conception (spring mass) and the gain in mass between spring mass and descent into
hibernation (mass gain). Correlations estimated by residual maximum likelihood method, with significance (P , 0.05) indicated by an asterisk (*).

Behavioral Ecology

164

Figure 2
Relationship between fecal cortisol levels of gestating Urocitellus
richardsonii females (n 21) and the sex ratio of the subsequent litter
emerging from the maternal burrow. Generalized log-transformed
(Glog) data fit a normal distribution and showed a significant
relationship with litter sex ratio (r2 0.19, P 0.049; y 0.16[GlogMeanCortisol] 1 0.56).

Table 2). Cortisol and corticosterone levels showed a strong

positive correlation with each other for all 3 reproductive
periods (P , 0.010 for all; Table 2).
DISCUSSION
Support for the narrow-sense TWH led to the prediction that
females in poorer body condition would have higher GC levels
and fewer sons. Conversely, both mechanistic hypotheses sug-

Figure 3
Relationship between fecal cortisol levels of female Urocitellus
richardsonii (n 22) during lactation and the number of offspring in
the litter at juvenile emergence. Generalized log-transformed (Glog)
cortisol values showed a significant relationship with litter size (r2
0.27, P 0.014; y 1.84[Glog-MeanCortisol] 1 6.00).

gested that females with high GC levels should have more

sons, and the mechanistic model of Cameron (2004) led us
to predict that mothers with proportionally more sons would
also tend to have smaller litters. We found substantial evidence supporting litter size and sex-ratio optimization with
respect to maternal condition and fecal cortisol levels, supporting trade-offs in investment and a mechanism associated
with sex-differential mortality. Increases in litter size were accompanied by a smaller proportion of males in the litter, but

Table 2
Relationship between cortisol and corticosterone levels during distinct reproductive phases (gestation, lactation, and post-weaning) and
reproductive allocation (sex ratio and litter size) in Urocitellus richardsonii
Variable
Gestation
Mean (Glog CORT)
Sex ratio
Sex ratio
Litter size
Litter size
Lactation
Mean (Glog CORT)
Sex ratio
Sex ratio
Litter size
Litter size
Post-weaning
Mean (Glog CORT)
Sex ratio
Sex ratio
Litter size
Litter size

By variable

Correlation (r)

Lower 95%

Upper 95%

Probability

Mean
Mean
Mean
Mean
Mean

(Glog
(Glog
(Glog
(Glog
(Glog

Cortisol)
Cortisol)
CORT)
Cortisol)
CORT)

21
21
21
21
21

0.607
0.435
0.174
20.184
0.132

0.238
0.004
20.278
20.570
20.318

0.823
0.729
0.564
0.269
0.534

*0.0035
*0.0490
0.4502
0.4249
0.5678

Mean
Mean
Mean
Mean
Mean

(Glog
(Glog
(Glog
(Glog
(Glog

Cortisol)
Cortisol)
CORT)
Cortisol)
CORT)

22
22
22
22
22

0.845
20.200
20.226
0.549
0.266

0.657
20.574
20.592
0.166
20.175

0.934
0.242
0.216
0.788
0.619

*,.0001
0.4339
0.3269
*0.0143
0.1186

Mean
Mean
Mean
Mean
Mean

(Glog
(Glog
(Glog
(Glog
(Glog

Cortisol)
Cortisol)
CORT)
Cortisol)
CORT)

21
21
21
21
21

0.555
20.179
0.064
0.299
20.098

0.162
20.567
20.378
20.153
20.508

0.796
0.274
0.482
0.647
0.348

*0.0090
0.4367
0.7832
0.1884
0.6719

Correlations were determined by residual maximum likelihood method using generalized log-transformed (Glog) hormonal data (Cortisol
cortisol and CORT corticosterone) from fecal GC extractions. Significance (P , 0.050) is indicated by an asterisk (*).

Ryan et al.

Stress-induced sex ratios in ground squirrels

males born of small litters were heavier at emergence than

males from larger litters (Figure 1). Mothers with higher fecal
cortisol levels during the gestation period (but not during the
lactation or post-weaning periods) were more likely to produce male-biased litters (Figure 2), whereas females that
produced larger litters showed significantly higher cortisol
levels during lactation (but not gestation or post-weaning)
than mothers producing smaller litters (Figure 3).
The positive correlation we observed between the mass of
the mother shortly after emergence from hibernation and subsequent litter size (Table 1) corresponds to the time of ovulation, when reproductive allocation in response to maternal
condition would prove most advantageous. In Richardsons
ground squirrels and other ground-dwelling sciurids with
low infant mortality, larger litters result in a greater number
of surviving young and have not been associated with a decrease in future survivorship or reproductive output for dams
(Michener and Locklear 1990; Hare and Murie 1992). Consistent with the predictions made under the individual optimization hypothesis (Perrins and Moss 1975; Pettifor et al.
1988), U. richardsonii mothers may produce optimally sized
litters that match their capacity for rearing them, obscuring
the apparent costs of producing larger litters (Risch et al.
2007). Accordingly, we observed no significant correlation between the size of litter a dam produced and her capacity to
gain weight over the active season (Table 1). The fact that
litter size was not significantly different for dams belonging
to the different age classeseven though mass following
spring emergence, around the time of mating wassuggests
that individual condition overrides age-related effects in this
population (see also Broussard et al. 2005, 2006).
Although individual females may produce litters that complement their capacity to raise them, demonstrating an effect
of quality or condition on litter production, evidence for the
cost of litter size and offspring sex is still apparent. We found
a significant relationship between the number of offspring
and the sex ratio of the emerging litter, with larger litters
becoming more female biased (Table 1). Michener (1980a)
found similar relationships among captive U. richardsonii litters, and these results are supported by a growing body of
evidence for other polytocous species, which gestate and give
birth to multiple young (Krackow and Hoeck 1989; Krackow
1993; Servanty et al. 2007; Shibata and Kawamichi 2009). In
our study, juvenile males were significantly heavier than females at the time of emergence, and their mass declined significantly with increases in litter size, whereas female mass did
not (Figure 1). These data strongly imply a greater cost to
mothers for producing male offspring, a greater cost to males
for being born of larger litters, and a trade-off between offspring sex and the total number of young.
We also observed a significant positive relationship between
mean cortisol levels during gestation and the sex ratio of the litter a mother subsequently produced (Figure 2). Mothers with
high gestational cortisol levels produced proportionally more
males, even though GC levels during the rest of the summer
showed no significant relationship to sex ratio (Table 2). The
relationships between sex ratio and both litter size and cortisol
levels support 2 key predictions of the glucose metabolism
hypothesis of Cameron (2004) as a mechanism for sex ratio
manipulation, which can be summarized as follows.
Weight gain in gestating and lactating mammals is in part
orchestrated by sustained moderate elevation of circulating
GC levels, which stimulate foraging behavior and resource
mobilization and are often accompanied by an elevation in
circulating glucose (Sapolsky et al. 2000). In mice, experimentally reduced (Cameron et al. 2008) and elevated (Machado
et al. 2001) glucose levels corresponded to female and malebiased litters, respectively, and naturally high serum glucose

165

levels were related to smaller male-biased litters in field voles

Microtus agrestis (Helle et al. 2008). Moreover, elevated glucose
has been shown to inhibit the development of femalebut
not maleblastocysts in vitro (Larson et al. 2001), by way of Xchromosomelinked increases in glucose metabolism accompanied by the build up of toxic by-products from the pyruvate
pentose phosphate pathway (Kimura et al. 2005). Additional
support for sex differential glucose-mediated mortality comes
from supplementary feeding of high fat diets (Austad and
Sunquist 1986; Rosenfeld 2004; Dama et al. 2011), as well as
the effect of timing of conception on sex ratio (Sheldon and
West 2004; James 2008), which relate to variation in circulating glucose levels and its availability to the developing conceptus (Cameron 2004). In U. richardsonii, the number of
follicles ovulated determines the maximum litter size, and
so restricts any mechanisms for sex-ratio and litter-size optimization to differential embryo mortality.
We also identified a significant positive correlation between
the size of litter a dam produced and her fecal cortisol levels
during lactation (Figure 3). This relationship was absent earlier in the summer during the period corresponding to gestation and later in the summer post-weaning period,
suggesting that cortisol plays an important role in sustaining
a key parameter of maternal offspring investment. Indeed,
lactation itself has been described as the most energetically
taxing component of reproduction, particularly in smaller
animals, which are less able to rely on fat reserves (Gittleman
and Thompson 1988). Compared with other sciurids, the
costs of lactation in U. richardsonii are thought to be especially
high and vary directly with litter size (Michener 1989) and are
compounded by other maternal behaviors expressed during
lactation (e.g., nesting, huddling, licking). Because the stress
response is typically attenuated during lactation (Bauman and
Currie 1980), it is unlikely that the higher cortisol levels that
we observed during lactation for larger litters correspond to
acute stressors but rather should be interpreted in the context
of their effect on foraging and the mobilization of internal
energy stores (Hill et al. 2003). Therefore, females with larger
litters endure greater costs during lactation, and the allostatic load of nursing larger litters is evident in the dams
HPA and cortisol response.
Finally, we found a significant relationship between sex ratio
and summer mass gain, with dams who produced more males
gaining significantly more weight overall (Table 1). Interestingly, there was no significant relationship between gain in
mass and litter size (Table 1). In the matrilineal societies of
U. richardsonii, daughters tend to remain philopatric, whereas
males often disperse, and dispersal is usually prior to hibernation in the first summer (Michener and Michener 1977). Postweaning investment by dams in female offspring is likely to be
greater than into dispersing male offspring (Michener
1980a). Mothers of males who disperse may therefore devote
less time to vigilance and establishing juveniles within the
colony and more time to foraging, making the production
of small male-biased litters a strategy that favors recouping
the costs of reproduction later in the summer.
Thus, GCs were positively related to sex ratio, which was
negatively associated with litter size, but GCs showed no relationship to age or other predictors of maternal condition.
These results support the glucose metabolism hypothesis, but
conflict with conventional predictions of the narrow-sense
TWH. Still, we believe that rejecting the TWH entirely based
on support for the mechanistic hypotheses alone is premature
for several reasons.
First, original predictions that poor condition mothers
should invest more in females were made in species which typically give birth to 1 offspring in a given reproductive bout
(Trivers and Willard 1973). Uncovering ASA is complicated

Behavioral Ecology

166

for iteroparous, polytocous species, where sex-differential

costs and potential reproductive pay-offs interact with offspring number and a dams future reproductive potential
(Williams 1979). Thus, the production of male-biased litters,
when in conjunction with a reduction in litter size, may still be
a conservative strategy for small poor condition mothers
(Myers 1978; McGinley 1984). Second, the shift in our general
understanding of the role of GCs and stress from one of
disease to one of allostasis and protective measures
(McEwen 1998) has important implications in how these types
of data are interpreted. For example, females nursing large
litters required the energy mobilization and behaviors necessary to feed those litters (accompanied by high cortisol), but
did not gain less mass, or have higher GC levels during the
post-weaning period, demonstrating the transient effect of
this hormonal response. Thus, GC levels are not always indicative of individual quality in an absolute sense but rather of
the mediation of environmental and physiological challenges
(Sterling and Eyer 1988). The fact that age and mass were not
related to GC levels supports this conclusion. Finally, the
ultimate adaptive function of sex ratio manipulation is not
necessarily exclusive of the mechanistic hypotheses. Our predictions were aimed at testing the narrow-sense TWH, where
condition plays the predominant role, but a number of predictions arise depending on a host of other variables
(Cockburn et al. 2002). Based on the social structure and life
history of Richardsons ground squirrels, we propose an ultimate explanation for how male-biased litters produced by
stressed mothers with high cortisol levels could prove adaptive.
The establishment and maintenance of matrilineal kin clusters allow for the control of access to burrow systems essential
for hibernation, predator avoidance, and rearing young
(Michener 1983a; Hare and Murie 2007). Female members
of such clusters benefit from an inclusive fitness payoff by
producing daughters that become integrated into their existing kin cluster, potentially enhancing the overall competitive
ability of their matriline relative to others (Michener 1980a).
Females living outside the context of an existing matriline, on
the other hand, would presumably have access to a poorer
resource base, be subject to an increased risk of predation,
and would be subject to more aggressive interactions with
conspecifics (Michener 1983a; Hare and Murie 2007). The
production of males, which disperse from the natal area to
pursue breeding opportunities with females residing in existing matrilines elsewhere (Michener and Michener 1977),
would be selectively favored for struggling females lacking
an established matriline. This advantaged matriline hypothesis has seen theoretical (Altmann M and Altmann J 1991)
and mixed empirical support derived from research involving
nonhuman primates (Simpson MJA and Simpson AE 1982;
Altmann et al. 1988), ungulates (Clutton-Brock et al. 1984),
and canids (Sidorovich et al. 2007), where daughters often
inherit maternal rank.
Our study provides evidence for a GC-mediated mechanism
driving sex ratio manipulation. Due to interactions between
litter size and sex ratio, our data best support the glucose
metabolism model of Cameron (2004). Fine tuning of investment such that sex ratio is modified in conjunction with other
factors, such as litter size, and in response to other variables,
including those which are social or ecological in nature,
means that the expression of ASA may be nearly as diverse
as the species and environments responsible for shaping this
behavior. Manipulative experiments testing presumptive
mechanisms of sex ratio manipulation and their potentially
adaptive role in free-living populations (sensu Ketterson et al.
1996) will prove particularly informative but should continue
to be considered with respect to the social and life-history
context in which they evolved.

FUNDING
Discovery Grants from the Natural Sciences and Engineering
Research Council of Canada (#311909 to W.G.A., #154271 to
J.F.H.).
The authors thank Ffion Cassidy and Lindsay Skyner for their contributions in the field and lab, and Steve Whyard, Spencer Sealy, and Tony
Williams for helpful comments on the manuscript. This manuscript was
also greatly improved by the insightful comments of the editor, Candy
Rowe, and one anonymous reviewer. Graphical assistance from Kristen
B. Gorman and Sebastian A. Pardo were greatly appreciated. We also
thank the management and staff of the Assiniboine Park Zoo for allowing access to the squirrels, and Mr Scott Gray of the Zoological Society
of Manitoba for providing access to a freezer and space for equipment
storage at the Zoo.

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