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Effects of Social Environment on Baseline Glucocorticoid Levels in a Com-munally Breeding Rodent, the Colonial Tuco-tuco (Ctenomys sociabilis)

Julie A. Woodruff, Eileen A. Lacey, George E. Bentley, Lance J. Kriegsfeld

PII: S0018-506X(13)00146-3DOI: doi: 10.1016/j.yhbeh.2013.07.008Reference: YHBEH 3596

To appear in: Hormones and Behavior

Received date: 9 March 2013Revised date: 28 July 2013Accepted date: 29 July 2013

Please cite this article as: Woodruff, Julie A., Lacey, Eileen A., Bentley, George E.,Kriegsfeld, Lance J., Effects of Social Environment on Baseline Glucocorticoid Levels ina Communally Breeding Rodent, the Colonial Tuco-tuco (Ctenomys sociabilis), Hormonesand Behavior (2013), doi: 10.1016/j.yhbeh.2013.07.008

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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Effects of Social Environment on Baseline Glucocorticoid Levels

in a Communally Breeding Rodent, the Colonial Tuco-tuco

(Ctenomys sociabilis)

Julie A. Woodruff 1,2

, Eileen A. Lacey 1,2

, George E. Bentley 2 , and Lance J. Kriegsfeld

3

1 Museum of Vertebrate Zoology, University of California, Berkeley, CA 94720

2 Department of Integrative Biology, University of California, Berkeley, CA 94720

3 Department of Psychology, University of California, Berkeley, CA 94720

Corresponding author:

Julie A. Woodruff

Email: woodjace@gmail.com

Key words:

Glucocorticoids, social environment, plural breeding, tuco-tucos, Ctenomys

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ABSTRACT

The social environment in which an animal lives can profoundly impact its physiology, including

glucocorticoid (GC) responses to external stressors. In social, group-living species, individuals

may face stressors arising from regular interactions with conspecifics as well as those associated

with basic life history needs such as acquiring food or shelter. To explore the relative

contributions of these two types of stressors on glucocorticoid physiology in a communally

breeding mammal, we characterized baseline GC levels in female colonial tuco-tucos (Ctenomys

sociabilis), which are subterranean rodents endemic to southwestern Argentina. Long-term field

studies have revealed that while about half of all yearling female C. sociabilis live and breed

alone, the remainder live and breed within their natal group. We assessed the effects of this

intraspecific variation in social environment on GC physiology by comparing concentrations of

baseline fecal corticosterone metabolite (fCM) for (1) lone and group-living yearling females in

a free-living population of C. sociabilis and (2) captive yearling female C. sociabilis that had

been experimentally assigned to living alone or with conspecifics. In both cases, lone females

displayed significantly higher mean baseline fCM concentrations. Data from free-living animals

indicated that this outcome arose from differences in circadian patterns of GC production. fCM

concentrations for group-living animals declined in the afternoon while fCM in lone individuals

did not. These findings suggest that for C. sociabilis, stressors associated with basic life history

functions present greater challenges than those arising from interactions with conspecifics. Our

study is one of the first to examine GC levels in a plural-breeding mammal in which the effects

of group living are not confounded by differences in reproductive or dominance status, thereby

generating important insights into the endocrine consequences of group living.

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INTRODUCTION

The social environment – specifically, whether an animal lives alone or within a group of

conspecifics – is a powerful force that affects numerous aspects of an individual’s biology.

Solitary versus group life imposes distinct sets of challenges on an animal, reflecting different

combinations of social and physical stressors (Alexander, 1974; Ebensperger et al., 2011). For

example, animals that live alone should experience fewer challenges associated with interactions

among conspecifics. At the same time, however, living alone may entail greater exposure to

physical stressors because basic life history demands of foraging, predator defense, and offspring

care are not shared with conspecifics. The balance of such social and physical stressors

determines whether, on average, individuals experience greater physiological stress when living

alone or in a group.

While the adaptive consequences of solitary versus group life have been explored for

multiple mammal species (Hayes, 2000; Komdeur, 1992; Randall et al., 2005; Silk, 2007;

Solomon and French, 1997), the physiological implications of such differences in social setting

have been less thoroughly investigated. Glucocorticoids (GCs) provide a particularly appropriate

means of assessing the physiological consequences of variation in social environment due to the

critical role that these steroid hormones play in mediating homeostasis and allostasis (Goymann

and Wingfield, 2004; McEwen and Wingfield, 2003; Wingfield and Kitaysky, 2002).

Specifically, because GCs influence a diverse array of metabolic and other bodily processes

(e.g., gluconeogenesis, reproduction), they provide a functionally relevant and biologically

important measure of physiological response to environmental variables. Such responses may be

triggered by unexpected environmental conditions, including short-term challenges (e.g., severe

storms) that induce acute changes in GC levels as well as more enduring challenges (e.g., limited

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food availability throughout a breeding season) that produce chronic, potentially harmful

changes in GC levels (McEwen and Wingfield, 2003; Sapolsky et al., 2000; Wingfield 2003). In

contrast, baseline GC levels play a critical role in response to predictable changes such as

circadian or seasonal variation in environmental conditions (Breuner et al., 1999; Landys et al.,

2006; Romero 2002). As a result, baseline GC levels provide an important indication of

individual response to routine variation in environmental stressors, including variations from

persistent differences in the social environment (Bonier et al., 2009; Korte et al., 2005; McEwen

and Wingfield, 2003; Wasser et al., 1997).

Studies of free-living mammals that have examined the relationship between social

environment and baseline GC levels have typically focused on singular breeding societies in

which a pronounced reproductive hierarchy exists between dominant, breeding and subordinate,

non-breeding individuals (Creel, 2001; Hackländer et al., 2003; Pride, 2005; Raouf et al., 2005).

In contrast, few field studies have investigated the effects of the social environment on GCs in

plural breeding systems in which most or all group members reproduce (but see Ebensperger et

al., 2011; Schoepf and Schradin, 2013; Schradin, 2008). Singular and plural breeding represent

substantially different social systems (Lacey and Sherman, 2007; Rubenstein et al., in prep) that

may impose distinct stressors on group members. In particular, while comparisons of individuals

in singular breeding groups are typically confounded by differences in dominance as well as

reproductive status, the same confounds are either absent or are greatly reduced in plural

breeding groups. As a result, studies of plural breeding systems provide an important opportunity

to explore the effects of group living on baseline GC levels.

This study investigates the relationship between variation in social environment and baseline

GC levels in the colonial tuco-tuco (Ctenomys sociabilis), a plural-breeding species of

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subterranean rodent that is endemic to the Andean precordillera in southwestern Argentina

(Lacey et al., 1997; Lacey and Wieczorek, 2003). Unlike most members of the genus Ctenomys,

C. sociabilis is social, with burrow systems routinely shared by 2-6 adult females and, in many

cases, a single adult male (Lacey et al., 1997; Lacey and Wieczorek, 2004). All members of a

social group participate in foraging, excavating burrows, and alarm calling in response to

predators, and all female group mates rear their young in a single, shared nest. Each female

produces a single litter of young per year, beginning in her yearling season. As yearlings,

females that are philopatric (i.e., remain in their natal burrow system) live and rear their young

communally with close female kin. In contrast, females that disperse from their natal burrow

systems as juveniles live and rear their young alone as yearlings (Lacey and Wieczorek, 2004).

This pronounced variation in yearling social environment is associated with significant

differences in per capita direct fitness and probability of survival to a second breeding season

(Lacey, 2001; Lacey and Wieczorek, 2004), and it provides an important opportunity to assess

the physiological (GC) consequences of living alone versus within a group.

To explore relationships between social environment and physiological response to stressors,

we compared baseline GC levels in lone versus group-living yearling female C. sociabilis. We

predicted that frequent social interactions among group members may represent significant

stressors that cause baseline levels of GC to be higher among group-living yearling females than

in lone females. In contrast, if sharing basic life history demands such as foraging and burrow

construction among group members helps to reduce stress on individuals, baseline levels of GC

should be higher among lone yearling females than in group-living females. We tested these

predictions by comparing levels of fecal glucocorticoid metabolite (fGCM) for lone and group-

living yearling females from a free-living and a captive population of colonial tuco-tucos. This

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integration of field and lab data provides a particularly powerful means of assessing the effects

of social environment on baseline GCs because it combines information gathered in “real world”

environments with experimental manipulations conducted in a controlled laboratory setting

(Calisi and Bentley, 2009). Because this study is one of the first to explore these relationships in

a plural breeding mammal, our analyses yield important new insights into the effects of group

living on physiological responses to external stressors.

METHODS

Field studies

Study population. We sampled a free-living population of colonial tuco-tucos (Ctenomys

sociabilis) located on Estancia Rincon Grande, Neuquén Province, Argentina (40°57’S,

71°03’W). The study site consisted of ca 20-ha area of open meadow dominated by seasonal

grasses and sedges and containing several species of woody shrubs (Tammone et al., 2012). All

members of the study population had been live-trapped annually since 1992 as part of an

intensive investigation of the behavioral ecology of this species. Trapping was conducted during

the austral spring (October-December), which corresponds to the portion of the year between the

birth and weaning of young.

Colonial tuco-tucos are subterranean, with individuals typically emerging only half a body

length from their burrows to feed on surface vegetation. We captured individuals as they

emerged to forage using hand-held nooses placed just inside the rim of an active burrow entrance

(Lacey et al., 1997; Lacey and Wieczorek, 2004). Upon first capture, we individually marked

each animal with a magnetically coded bead (IMI-1000 Implantable Transponders, BioMedic

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Data Systems, Seaford, DE) inserted beneath the skin at the nape of the neck; implanted

transponders were read using a hand-held scanner (DAS 4001 Pocket Scanner, BioMedic Data

Systems).

Characterizing social environment. To characterize the social environments of individual

females, we determined both the sizes and age structures of the social groups present on the

study site during 2005 to 2009. Identifying distinct burrow systems based on surface evidence of

activity (e.g., locations of burrow entrances) is difficult and thus we used radiotelemetry to

determine burrow system boundaries (Lacey et al., 1997; Lacey and Wieczorek, 2004). At least

one adult per putative burrow system was captured and fitted with a small (< 7 g) radio-collar

consisting of an acrylic-encased transmitter (GV-13 transmitters, AVM Instruments, Inc.,

Colfax, CA) attached to a plastic cable tie. Radio-collared adults were released at the point of

capture, after which we monitored their locations using hand-held Yagi antennas and CE-12

receivers (AVM Instruments, Inc.). Previous research (Lacey et al., 1997) had revealed that

individual adult C. sociabilis use effectively all of the burrow system in which they live during a

24-hr period and thus by recording the localities of radio-collared individuals multiple times per

day (> 1 hr between successive radio fixes) over a period of at least 2 weeks, we were able to

collect precise data regarding burrow system boundaries. Animals captured within the

boundaries of a given burrow system were considered to be members of the same social group

(Lacey et al., 1997; Lacey and Wieczorek, 2004).

We quantified group size by capturing all animals resident in a burrow system during a given

field season. Lacey et al. (1997) describe the methods used to ensure that all residents were

captured. In brief, captured animals were temporarily (< 12 hrs) held in cages, until monitoring

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of burrow entrances revealed no further evidence of animal activity in the target system. Group

age structure was determined from capture records for members of the study population.

Typically, residents of a burrow system were captured annually when juveniles in the system

first began emerging above ground to forage for themselves (age ca. 4 weeks). Because we

captured and marked the vast majority of females in the study population (ca. 84%) as juveniles,

we knew the exact ages of most of these animals.

Quantifying fitness. Baseline GC levels may also be influenced by reproductive effort (Bonier et

al., 2009; Ebensperger et al., 2011). Although all adult females in our study population were

lactating (and thus of comparable reproductive status), the number of pups reared varied among

social groups. To explore the effects of differences in reproductive success on baseline GC

levels, we quantified per capita direct fitness for females by capturing all juveniles in each

burrow system. For burrow systems containing a single adult female, direct fitness equaled the

number of pups weaned. Due to low genetic variability in the study population (Lacey, 2001), we

were unable to determine the maternity of pups reared in burrow systems containing more than

one adult female. As a result, fitness for these females was determined by dividing the number of

pups weaned by the number of lactating adult female residents in the burrow system to yield a

per capita estimate of individual reproductive success (Lacey, 2004).

Laboratory studies

Study population. The laboratory population of C. sociabilis studied consisted of ~ 35 captive-

born individuals, all descended from an initial set of 12 animals captured in Neuquén Province,

Argentina, in January 1996. Captive animals were housed in artificial burrow systems comprised

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of three clear acrylic boxes connected by ~ 10 m of acrylic tunnels. Two of the boxes (30 x 30 x

15 cm) served as nest chambers and latrines while the third box (30 x 50 x 40 cm), which was

slightly elevated (~ 5 cm) above the rest of the burrow system, was used to introduce food to

animals. The floor of each box was covered with ~ 2 cm layer of aspen bedding. Rooms housing

the burrow systems were maintained at 20°C under a light:dark cycle that imitated seasonal

changes in day length at 41°S, the latitude at which the original members of the lab population

were captured. The animals were fed ad-libitum quantities of commercially available rat chow

(Simonsen’s Inc., Gilroy, CA) and were provided daily with fresh produce (corn, carrots and

lettuce).

To test experimentally the effects of social environment on baseline GC levels, in May 2011,

we randomly assigned each of 10 yearling females to be housed either alone or with one of her

recently weaned female offspring. The experiment was conducted when offspring were ~ 4

months old, which corresponds to the time of the year when the young disperse from their

burrow system. While all juvenile male C. sociabilis disperse at ca. 4 months of age, many

juvenile females are philopatric (Lacey and Wieczorek, 2004), and thus mothers and daughters

typically share the same burrow system. Females from both treatment groups were subject to

identical husbandry procedures, which were the same as those applied to the rest of the captive

study population. Females were housed alone or with a daughter for 30 days, at the end of which

we collected fecal samples from all yearling females for analyses of baseline GC levels.

All field and lab procedures involving live animals were approved by the University of

California, Berkeley, Animal Care and Use Committee and followed guidelines established by

the American Society of Mammalogists (Sikes and Gannon, 2011).

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Fecal sample collection

Baseline GC levels were assessed by measuring levels of GC metabolites in fecal samples

collected from the study animals. Fecal samples provide a temporally more comprehensive

picture of baseline GC concentrations than blood samples (Harper and Austad, 2000; Millspaugh

and Washburn, 2004; Touma and Palme, 2005) and can be obtained non-invasively without

altering GC levels through the act of sample collection. They have been used to examine GC

concentrations in multiple taxa, including several species of rodents (Bauer et al., 2008; Palme et

al., 2005; Ponzio et al., 2004; Soto-Gamboa et al., 2009; Touma and Palme, 2005; Wasser et al.

2000) and have been shown to be a reliable proxy for circulating GC concentrations in colonial

tuco-tucos (Woodruff et al., 2010).

Field sample collection. Immediately upon capture (see above), each animal was placed in a

cloth bag and held until a minimum of 5 fecal pellets had been deposited (typically 10-15 min).

Colonial tuco-tucos naturally deposit fecal pellets during routine marking, weighing, and

handling. To allow for post-hoc analyses of potential circadian variation in baseline GC

concentrations, we recorded the time of sample collection for each individual. Fecal pellets were

transferred to cryogenic vials and immediately (≤ 10 minutes after collection) flash frozen in

liquid nitrogen. All samples were shipped on dry ice to the Berkeley campus and stored at -80° C

until assayed. Analyses comparing fGCMs for lone versus group-living female colonial tuco-

tucos were restricted to lactating yearlings to control for breeding status and age; because most

lone females in the field population are yearlings (Lacey and Wieczorek, 2004), this age class

was most appropriate for comparisons of fGCMs as a function of social environment. We also

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collected and analyzed samples for a subset of older females to assess the effects of age and

group structure on baseline fGCMs.

Laboratory sample collection. To collect fecal samples, females were temporarily removed from

their artificial burrow systems and housed individually in standard polycarbonate rodent cages (1

m x 0.5 m x 0.25 m), the floors of which were lined with approximately 2 cm of aspen bedding.

Based on preliminary data (see below) regarding circadian changes in fGCM production in free-

living colonial tuco-tucos, we restricted collection of fecal samples from captive females to the

afternoon. We checked cages every 2 h (up to a total of 6 h) and collected feces directly from the

cage bottom, with care taken to avoid fecal pellets lying in bedding that was soaked with urine.

Individual samples were placed in cryogenic vials and stored in a -20° C freezer until assayed.

Steroid extractions and GC assays

Following the methods of Mateo and Cavigelli (2005), fecal samples were thawed and dried at

95°C for 4 h. We then crushed samples using a mortar and pestle, and placed 0.2 g of fecal

powder in a microcentrifuge tube. We added a 1.5 ml aliquot of 100% ethanol to each sample,

vortexed it for 8-10 sec, followed by centrifuging at 2500 g for 45 min to precipitate all solid

matter. The supernatant was collected and frozen at -20°C until assayed.

To quantify fGCMs, we used a commercially available corticosterone enzyme immunoassay

kit (Cayman Chemical Co., Ann Arbor, MI), which had previously been validated for colonial

tuco-tucos (Woodruff et al., 2010). All samples assayed yielded fecal corticosterone metabolite

(fCM) concentrations that were above the manufacturer’s reported limit of detection for

corticosterone (38 pg/ml at 80% binding). Sensitivity of the assay for corticosterone at 80%

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binding was 232 pg/ml. Fecal samples were assayed in duplicate and reanalyzed when the

coefficient of variation exceeded 20%. Intra- and inter- assay coefficients of variation for fCMs

were 11% (N = 7) and 14% (N = 6), respectively.

Data analyses

Two-sample statistical comparisons were performed using two-tailed t-tests unless the

distribution of data points indicated that non-parametric tests were required. We used an

ANOVA to examine differences in fCMs between social groups, time of day, group size and age

structure. We used an ANCOVA to determine the relationship between baseline fCMs and the

per capita number of pups in single or multiple female social systems. We calculated effect sizes

using partial eta-squared for ANOVA results and Cohen’s d for pair-wise comparisons.

Statistical analyses were performed using Statistica 6.0 (StatSoft, Inc. 1984-2008). All values are

reported as 1± SEM.

RESULTS

A total of 175 free-living C. sociabilis from 29 social groups were captured as part of this study.

Of these, 59 (33.7%) were yearling females from which fecal samples were collected for fCM

analysis. Twenty-five (42.4%) of these yearlings were lone females; these females were the only

adults resident in their respective burrow systems. The remaining 34 yearlings lived in groups

containing a mean of 3.7 (± 1.9; range = 2-6) adult females; for groups that contained more than

1 yearling female, fecal samples were analyzed for only a single, randomly selected yearling per

group. We found no effect of year on fCM concentrations (ANOVA: F4, 22 = 2.2, p = 0.12, p2 =

0.23) and thus we pooled data from all years for subsequent analyses. Six (26%) of the multi-

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female groups sampled also included an adult male. The presence of a male had no effect on

fCM concentrations (male: 656.16 ± 579.71 vs. no male: 464.49 ± 372.96; t = 2.04, df = 32, p =

0.31, Cohen’s d = 0.39) and thus data from all multi-female groups were used in our analyses.

Circadian variation in fCM concentrations

To determine if fCM production by members of our free-living study population varied with time

of day, we examined fCM concentrations as a function of the capture times for the yearling

females sampled. Colonial tuco-tucos are diurnal (Lacey et al. 1997), and our observations of the

free-living study population suggested that the animals were most active in the morning and

evening, with less surface evidence of activity (e.g., burrow excavation, foraging) during the

afternoon. Accordingly, we compared fCM concentrations for samples collected before 1300

hours to those collected after 1300 hours. For samples collected from different females (i.e., one

sample per individual), there was a significant effect of time of day (Two-way ANOVA: F1, 56 =

4.1, p = 0.047, p2 = 0.07) but no effect of social system (F1, 56 = 0.19, p = 0.66, p

2 = 0.003) on

fCM levels (Fig. 1). Subsequent inspection of these data revealed a significant decrease in fCM

levels from morning to afternoon for group-living but not for lone females (MWU; group-living:

U = 66.0, Z = 2.65, N = 19, 15, p = 0.008, Cohen’s d = 1.02; lone: U = 74.0, Z = -0.22, N = 13,

12, p = 0.83, Cohen’s d = 0.014). Consistent with this result, while fCM levels for lone and

group living females did not differ for samples collected during the morning (MWU: U = 100.0,

Z = 0.90, N = 19, 13, p = 0.37, Cohen’s d = 0.29), a significant difference between lone and

group living animals was evident in samples collected during the afternoon (t = -2.14, df = 25, p

= 0.042, Cohen’s d = 0.8), providing an initial indication that social environment may influence

baseline GC levels. Small effect size in non-significant results does not confirm the null

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hypothesis, though there is nothing to indicate that anything was overlooked for non-significant

effects. Consequently, to control for circadian variation in GCs, we restricted our comparisons of

the effects of social environment, reproductive effort and group structure on fCM levels to

samples that had been collected after 1300 hrs.

Effects of social environment

As reported above, afternoon fCM concentrations were significantly higher for yearling females

living alone versus those living in groups (lone: 541.8 ± 405.34 vs. group: 287.42 ± 197.41; t = -

2.14, df = 25, p = 0.042, Cohen’s d = 0.8, Fig. 2a). Data from experimental manipulations of

captive females were similar: captive yearlings housed alone had significantly higher fCM levels

than did yearling females housed with a daughter (lone: 157.82 ± 89.27 vs. group: 58.65 ± 24.29;

t = 2.4, df = 8, p = 0.043, Cohen’s d = 1.51; Fig. 2b).

Effects of reproductive effort

To determine if differences in baseline GCM levels were influenced by socially-mediated

differences in reproductive success, we examined the effect of reproductive output on fCMs

among members of the free-living study population. Neither the absolute number of pups in a

burrow system (ANOVA: F1, 40 = 0.92, p = 0.34, p2 = 0.02) nor the per capita number of pups in

a burrow system (ANCOVA: F1, 39 = 1.3, p = 0.26, p2 = 0.03) were associated with differences

in fCMs for either lone or group-living females (F1, 39 = 0.07, p = 0.79, p2 = 0.002).

Effects of group structure

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To explore potentially more subtle impacts of social setting on baseline GC levels, we

investigated how group size and age structure influenced fCMs in group-living individuals.

Analyses of data from free-living yearling females revealed no significant relationship between

fCM concentrations and the total number of adult females per burrow system (ANOVA: F3, 11 =

1.5, p = 0.26, p2 = 0.29). Thus, group size did not appear to affect baseline GC levels for group-

living yearlings in our study population.

Baseline GC levels, however, appeared to be influenced by the age structure of social groups.

To examine the effects of age structure, we partitioned groups into 3 types: groups composed

only of yearling females, groups with only 1 older (> 1 year) female, and groups with > 1 older

female. While baseline fCM levels for yearling females did not vary across these categories

(one-way ANOVA: F2, 12 = 3.0, p = 0.087, p2 = 0.33), visual inspection of these data suggested

that groups composed of only a single older female were characterized by considerably higher

fCM levels.

Building upon this finding, a more detailed evaluation of the data indicated that 4 of the 5

groups containing only a single older female consisted of 2 yearlings and the older female; the

fifth group consisted of one yearling and one older female. When only data from the groups

containing 2 yearlings and 1 older female were included in these analyses, fCMs for yearlings

varied significantly with group structure (one-way ANOVA: F2, 11 = 11.4, p = 0.002, p2 = 0.68;

Fig. 3). Tukey HSD post-hoc tests indicated that fCM levels were significantly greater for groups

containing a single older female than for either of the other group structures considered (versus

all yearling females: Tukey HSD: df = 11, p = 0.02; versus multiple older females: Tukey HSD:

df = 11, p = 0.002). No significant difference was found between yearling females living in

groups composed of only yearlings and those living in groups with more than one older female

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(Tukey HSD: df = 11, p = 0.55). Thus, groups composed of 2 yearlings and 1 older female were

characterized by particularly high fCM levels.

DISCUSSION

The social environment can be a powerful influence on multiple aspects of an individual’s

physiology. In this study, we found a significant effect of social setting on baseline fGC

concentrations. Yearling female C. sociabilis living alone exhibited higher fCM levels than

yearling females living in groups in both free-living and captive populations of colonial tuco-

tucos. The results of our laboratory experiment suggest a causal relationship between social

environment and baseline fCM levels. We cannot rule out the possibility that dispersal decisions

are influenced by juvenile GC levels, but we note that both philopatric and dispersing juveniles

occur within a single litter of young, thereby reducing the chances that these animals are

characterized by substantially different (e.g., maternally influenced) GC levels. In the lab, we

assigned animals randomly to each housing condition and thus any pre-existing differences in

GC levels should not have led to consistently higher baseline GCs in lone animals. Animals pre-

disposed to disperse should have been randomized with respect to treatment. Similarly, animals

more pre-disposed to respond strongly to isolation (females that would “choose” to be

philopatric) should have been randomized with respect to housing treatment.

The difference between lone and group-living yearlings appeared to result from differences in

diurnal patterns of fCM production. While fCM levels for group-living yearlings declined

significantly during the afternoon, we did not detect a comparable result for lone females. A

major feature of circulating baseline GCs is circadian variation in production, and, among

mammals, GCs tend to peak at the onset of daily activity (Chung et al., 2011; Son et al., 2011).

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The observation that fCM levels did not decline from morning to afternoon in lone female C.

sociabilis suggests that higher GC levels resulted from the failure of these animals to follow the

same pattern of circadian GC production as group living females.

Other potential influences on baseline GC levels

The social environment is not the only factor that may influence fCM concentrations. Baseline

GC levels have also been shown to vary with reproductive parameters, including breeding status

(Boswell et al., 1994; Kenagy et al., 1990, 1999). Woodruff et al. (2010) found no significant

difference in fCMs between lactating and non-breeding female C. sociabilis, suggesting that

differences in breeding condition did not appear to act as a stressor in this species. Further, all

samples analyzed in this study were collected from reproductive females shortly after they

weaned their litters (Lacey and Wieczorek, 2004) and thus variation in fCM levels due to

differences in reproductive status should have been minimal. With regard to reproductive

success, Bonier et al. (2009) have argued that baseline GCs may increase with reproductive

effort due to the additional physical challenges associated with producing and rearing offspring.

Although Ebensperger et al. (2011) found that baseline cortisol levels increased with the per

capita number of pups produced by free-living degus (Octodon degus), we found no relationship

between baseline fCM levels and either the total number or the per capita number of pups

weaned. Thus, reproductive success does not appear to influence baseline GC levels in C.

sociabilis. In sum, the differences in fCM levels reported here did not appear to be influenced by

differences in standard life history factors such as age or reproductive condition.

Group structure and baseline GC levels

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Although all group living females in our sample shared their burrow systems with adult

conspecifics, the social environments of these animals differed with respect to both group size

and age structure. With regard to group size, effects on GC levels appear to vary among species.

For example, Pride (2005) found that ring-tailed lemurs (Lemur catta) exhibited high fecal

cortisol concentrations in groups that were atypically large or small, resulting in a U-shaped

relationship between group size and GC levels. In contrast, Ebensperger et al. (2011) found no

effect of group size on mean cortisol levels in adult female degus (O. degus). Similarly, we

found no relationship between group size and fCMs in yearling female colonial tuco-tucos. Thus,

group size does not appear to be a consistent predictor of baseline GC levels in group living

mammals.

With regard to group structure, most analyses of social mammals have focused on kinship

among group mates (Silk, 2007), although differences in sex ratio have also been considered

(McGuire et al., 2002). Given that groups of C. sociabilis, are composed of multiple, closely

related adult females and, at most, a single adult male (Lacey and Wieczorek, 2004), differences

in sex ratio and kin structure were not salient features of our study population. In contrast, the

age structure of social groups did vary markedly, ranging from groups composed only of

yearling females to groups composed of several generations of adult females. Although no

conspicuous age-related dominance hierarchy is evident among female C. sociabilis, age has

been found to influence dominance interactions and GC responses in other species of social

mammals (e.g., Marmota marmota: Hackländer et al., 2003). Among our study animals, females

in groups composed of two yearlings and a single older female had significantly higher fCMs

than females in groups containing either no or more than two older females. The resulting

inverted U-shaped relationship between number of older females and fCM levels suggests that

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not all within-group relationships are the same. Future studies that examine group structure in

greater detail may help to elucidate how the number of older females per group influences

baseline GC levels in C. sociabilis.

Baseline GC levels and plural breeding

Most studies of GC responses in free-living social mammals have been limited to singular

breeding species in which a distinct reproductive hierarchy exists between dominant and

subordinate individuals (Creel, 2001; Hackländer et al., 2003; Raouf et al., 2005; Young et al.,

2006). In contrast, few studies have examined GC levels in plural breeding mammals in which

most or all females in a group reproduce. Plural breeding species provide an important

opportunity to examine the physiological correlates of group living without the confounding

reproductive hierarchy observed in singular breeding species. To the best of our knowledge, only

Ebensperger et al. (2011) have addressed the relationship between social environment and

baseline GC levels in a free-living, plural-breeding mammal. These authors report that baseline

GC levels in O. degus increased with the number of pups reared in a social group but not with

the number of adult females per group, suggesting that reproductive effort is a more important

determinant of baseline GC levels than group size. Because Ebensperger et al. (2010) did not

explicitly contrast GC levels for lone versus group living females, direct comparisons of their

data with ours are challenging. Nevertheless, our findings differ in that GC levels in C. sociabilis

did not vary with reproductive effort but were influenced by the distinction of living alone versus

living in a group. Collectively, these data suggest that relationships among social structure,

reproduction, and baseline GC levels vary among species of plural breeding mammals. Ideally,

as studies of plural-breeding species increase in number, it will become possible to relate

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differences in the factors affecting baseline GC levels to variation in the adaptive benefits of

group living.

Social versus physical challenges

Because GCs mediate responses to a variety of challenges, it is difficult to distinguish the effects

of the social environment from those of the physical environment, especially in free-living

animals. For example, wild female chimpanzees display increased cortisol response to both

aggressive social interactions with conspecifics and the energetic demands of breeding

(Thompson et al., 2010). In our study population, females that live alone must perform all

activities associated with maintaining a burrow system, finding food, and caring for offspring; in

contrast, group-living females share these tasks with conspecifics, potentially resulting in a per

capita decrease in the effort expended on these activities (Ebensperger and Bozinovic, 2000;

Ebensperger et al., 2007). In C. sociabilis, lone females also have higher per capita direct fitness

than group-living females (Lacey, 2004), indicating that in addition to facing potentially greater

challenges associated with survival, lone females are likely to be investing more in offspring

care. Given that one function of GCs is to maintain homeostasis by altering glucose availability

in response to energetic challenges imposed by the environment (Goymann and Wingfield, 2004;

Sapolsky et al., 2000), it seems likely that lone females must sustain higher levels of GCs as an

adaptive means of meeting the demands associated with living alone.

Balanced against these physical challenges are the challenges associated with increased

social contacts with conspecifics (Creel, 2001; Marchlewska, 1997; Thompson et al., 2010).

Although interactions with conspecifics likely influence GC levels in C. sociabilis, our

experimental manipulation of the social conditions under which captive females were housed

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provides compelling evidence that when physical demands and variation in individual

phenotypes are controlled, interactions with conspecifics do not result in higher baseline GC

levels. Woodruff et al. (2010) demonstrated that baseline GC levels are significantly higher for

free-living versus captive colonial tuco-tucos, suggesting that the former face greater physical

challenges associated with living in “real” environments. How free-living individuals respond to

those increased physical challenges may be mediated by the social environment. Specifically,

baseline GC levels may be elevated in lone females because the physical demands of living and

rearing young alone outweigh any increase in GC levels among group living females due to

regular interactions with conspecifics.

Conclusion

This study is one of the first to examine the effects of the social environment on baseline GC

levels in a plural-breeding species in which individuals naturally live alone or in groups.

Multiple laboratory studies have examined relationships between social setting and GC

concentrations, with emphasis on the effects of overcrowding or social isolation (Marchlewska-

Koj, 1997; Serra et al., 2000; Viveros et al., 1988). Laboratory studies have also explored the GC

consequences of naturally occurring phenotypic variation among group-living individuals (e.g.,

maternal effects: Liu et al., 1997; aggression: Castro and Matt, 1997). Few studies, however,

have considered the effects of group living on baseline GC levels. Our integration of data from

free-living and captive colonial tuco-tucos provides compelling evidence that whether a female

lives alone or in a group significantly affects her baseline GC response to external challenges.

More generally, our data suggest that the social environment is an important mediator of

response to physical as well as social challenges that impact glucocorticoid physiology.

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ACKNOWLEDGMENTS

For permission to work on Estancia Rincon Grande, we thank Alain Thouyaret. For permission

to work in La Reserva Nacional Nahuel Huapi, we thank the Delegación Tecnica de la

Administracion de Parques Nacionales Argentinas, in particular, Claudio Chehebar and Hernan

Pastore. We especially thank J. Wieczorek, who made field captures over successive seasons

possible. For assistance in the lab, we thank the numerous UC Berkeley undergraduates (aka the

“Tuco Wranglers”) who contributed to animal care. Logistic support was provided by UC

Berkeley veterinarians and animal care technicians, especially K. Jensen. We thank the two

reviewers of this journal for constructive comments to improve this manuscript. Financial

support was provided by the American Society of Mammalogists, the Museum of Vertebrate

Zoology and the Department of Integrative Biology at the University of California, Berkeley.

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FIGURES

Figure 1. Comparison of fecal corticosterone metabolite concentrations (fCMs) from samples

collected in the morning versus the afternoon. All samples were from field-caught yearling

female colonial tuco-tucos living alone or in groups; sample size is given in each bar. Horizontal

line with asterisk denotes p < 0.05.

Figure 2. Comparisons of fecal corticosterone metabolite concentrations (fCMs) from lone and

group-living female colonial tuco-tucos. Data in (a) are from free-living yearling females

captured during 2005-2009. Data in (b) are from captive yearling females experimentally housed

alone or in female-female pairs during 2011. Sample size is given in each bar. Horizontal line

with asterisk denotes p < 0.05.

Figure 3. Fecal corticosterone metabolite concentrations (fCMs) from free-living yearling female

colonial tuco-tucos living in groups consisting of (1) yearlings only, (2) 2 yearlings and 1 older

female, or (3) yearlings and > 2 older females. Sample size is given in each bar. Horizontal line

with asterisk denotes p < 0.05.

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Highlights

We examine fecal corticosterone metabolites in female Ctenomys sociabilis.

We compared yearling females living alone with females living in groups.

Free-living and captive females living alone exhibit higher corticosterone.

Differences appear to result from differing patterns of corticosterone production.

Differences in group structure also appear to be a factor in corticosterone levels.