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Int J Primatol (2013) 34:246259

DOI 10.1007/s10764-013-9657-0

Travel Time Predicts Fecal Glucocorticoid Levels


in Free-Ranging Howlers (Alouatta palliata)
Jacob C. Dunn & Jurgi Cristbal-Azkarate &
Bjrn Schulte-Herbrggen & Roberto Chavira &
Joaqum J. Ve

Received: 5 August 2012 / Accepted: 20 December 2012 / Published online: 23 January 2013
# Springer Science+Business Media New York 2013

Abstract Environmental stressors impact physiology in many animal species.


Accordingly, the monitoring of fecal glucocorticoid metabolites (fGCM) has been
increasingly used to evaluate the physiological costs of habitat disturbance on wild
animal populations, providing a powerful tool for conservation and management.
Several studies have suggested that primates in forest fragments have higher fGCM
levels than those in continuous forests, yet the proximate causes of fGCM variation
remain to be identified. In previous studies of Mexican howlers (Alouatta palliata
mexicana) in Los Tuxtlas, Mexico, we found that individuals living in a smaller and
more disturbed forest fragment consumed significantly less fruit and had a significantly higher feeding effort than those living in a bigger, more conserved forest
fragment. Here, we aimed to examine the effects of fruit consumption and travel time
on fGCM levels in the same two groups of howlers, during three sampling sessions
Electronic supplementary material The online version of this article (doi:10.1007/s10764-013-9657-0)
contains supplementary material, which is available to authorized users.

J. C. Dunn (*) : J. Cristbal-Azkarate


Primate Immunogenetics and Molecular Ecology Research Group, Division of Biological
Anthropology, University of Cambridge, Cambridge( CB2 3QY( UK
e-mail: jcd54@cam.ac.uk

J. C. Dunn
Centro de Investigaciones Tropicales, Ex-Hacienda Lucas Martn, C.P. 91019, Xalapa, Veracruz,
Mxico
B. Schulte-Herbrggen
United Nations Environment Programme World Conservation Monitoring Centre, Ecosystem
Assessment Programme, Cambridge( CB3 0DL( UK
R. Chavira
Departamento de Biologa de la Reproduccin, Instituto de Ciencias Mdicas y Nutricin Salvador
Zubirn, C.P. 14000, Mexico, D.F., Mxico
J. J. Ve
Centre Especial de Recerca en Primats, Facultat de Psicologia, Universitat de Barcelona, Barcelona
08035 Spain

Fecal Glucocorticoid Levels in Free-Ranging Howlers

247

that differed markedly in fruit availability. We found that fGCM levels (N=202 fecal
samples) were higher in the howler group living in the smaller forest fragment and
varied seasonally in both focal groups, being lowest when fruit consumption was
highest. However, our results suggest that travel time is the main factor predicting
fGCM levels in howlers, and that although fruit consumption may be negatively
related to fGCM levels, this relationship is probably mediated by the strong effect that
fruit consumption has on travel time. Our results provide important insight into the
proximate causes of fGCM variation in primates in fragments and highlight the
potential conservation significance of studies showing that habitat loss and transformation can lead to increases in travel time in wild primates.
Keywords Activity . Alouatta palliata mexicana . Diet . Fecal glucocorticoids . Forest
Fragmentation . Fruit . Howler . Metabolic stress

Introduction
Animals living in the wild face stress through competition for food, mates, and
territories, as well as that imposed by the physical environment and exposure to
disease. Any of these stressors may disrupt physiological homeostasis, leading to
what is commonly termed the stress response (Sapolsky 2002). Although this
mechanism is adaptive in the short term, if a stressor persists too frequently, or
for too long, the resulting high levels of glucocorticoids (GCs), stress hormones
that function to optimize energy availability (Sapolsky 2002), can have deleterious effects on reproduction, immune function, and survival (Sapolsky 2002;
Sapolsky et al. 2000; Wingfield 2005; Wingfield and Romero 2001). Because
of this, GC monitoring has been increasingly used to evaluate the physiological
costs of habitat disturbance, thereby providing a powerful tool for the conservation and management of wild vertebrate populations (Busch and Hayward 2009).
Despite the enormous potential of this tool, determining the proximate causes
of GC variation is complicated by the presence of multiple potentially stressful
stimuli. Traditionally, endocrinological studies of wild primates have focused on
psychological stressors, such as dominance rank (Abbott et al. 2003; Gesquiere
et al. 2011a; Sapolsky 1993, 1994). However, ecological factors affecting the
availability of food resources, such as habitat characteristics (Behie et al. 2010;
Chapman et al. 2006; Jaimez et al. 2012; Martnez-Mota et al. 2007; RangelNegrn et al. 2009) and resource seasonality (Beehner and McCann 2008;
Carnegie et al. 2011; Gesquiere et al. 2008; 2011b; Pride 2005a), may represent
significant sources of stress in primates. For example, decreases in the availability (Behie et al. 2010; Foerster and Monfort 2010; Pride 2005a) and consumption (Champoux et al. 1993; Foerster et al. 2012; Muller and Wrangham 2004;
Pride 2005b) of ripe fruits, which are rich in usable energy, have been related to
increases in GC levels in primates. Further, increases in energetically costly
activities such as travel, which may be related to ecological factors, are also
reported to have positive effects on GC levels (Cavigelli 1999; Gesquiere et al.
2011b; Girard and Garland 2002; Kraemer et al. 2008; Muller and Wrangham
2004; Tharp 1975).

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In previous studies of Mexican howlers (Alouatta palliata mexicana), which are


listed as critically endangered by the IUCN (Cuarn et al. 2008), we found that
individuals living in a smaller and more disturbed forest fragment consumed significantly less fruit and exhibited a significantly higher feeding effort than those living in
a bigger, more conserved forest fragment (Dunn et al. 2009, 2010). Here, our aim was
to improve our understanding of the proximate mechanisms underlying GC variation
in primates in fragments by examining the effects of travel time and fruit consumption on fecal glucocorticoid metabolite (fGCM) levels in the same two groups of
howlers previously studied (Dunn et al. 2009, 2010), during three periods that
differed markedly in fruit availability.
Based on published evidence about the relationships between activity pattern,
fruit consumption, habitat characteristics, and fGCM levels in primates, we
proposed four hypotheses and used an information-theoretic approach to evaluate
the relative support for each of them: 1) fGCM levels are positively related to
travel time; 2) fGCM levels are negatively related to fruit consumption; 3) fGCM
levels are higher during periods of low fruit availability; and 4) fGCM levels are
higher in a group living in a smaller and more disturbed forest fragment than a
group living in a larger, more conserved forest fragment.

Materials and Methods


Study Site
The Los Tuxtlas Biosphere Reserve (1837 1835 N, 9508 -9505 W; 0400 m asl)
is the northernmost limit for populations of Alouatta palliata (Estrada and CoatesEstrada 1988) and represents the most northerly tropical rain forest distribution in the
American continent (Dirzo and Garcia 1992). The dominant vegetation in Los
Tuxtlas was originally tropical rain forest, but this region has been severely fragmented over the last 60 yr (Guevara et al. 2004). Therefore, the remaining populations in this region are isolated in an archipelago of forest fragments that vary in size,
isolation distance, and habitat quality (Arroyo-Rodrguez et al. 2008; CristbalAzkarate et al. 2005).
Local climate is warm and humid with a mean annual temperature of 25 C and
mean annual rainfall of 4900 mm (Soto and Gama 1997). There are three distinct
periods of fruit availability reported in the region: a primary peak at the end of the dry
season/beginning of the rainy season (AprilJune), a less intense secondary peak in
the wet season (AugustOctober), and a period of fruit scarcity from November to
March (Dunn et al. 2010).
Focal Groups
The RH group resided in a 244-ha forest fragment inhabited by five groups of
howlers and the total population density was 0.12 ind/ha (Cristbal-Azkarate et al.
2005). The RC3 group was the only group found in a 7.2-ha fragment and the total
howler population density was 1.11 ind/ha. The home range of the RH group was
markedly larger than that of the RC3 group (89.5 ha vs. 5.8 ha; Dunn et al. 2009) and

Fecal Glucocorticoid Levels in Free-Ranging Howlers

249

was more characteristic of an old-growth forest, with a greater availability of fruit and
big trees, whereas the home range of the RC3 group was more perturbed, with a
greater number of pioneer species (Dunn et al. 2009).
We selected the focal groups to control for variation in GC levels that may result
from differences in group size and composition. The RH group consisted of three
adult males, three adult females, and one infant at the start of the study, and one infant
was born during the study. The RC3 group also consisted of three adult males, three
adult females, and one infant, as well as one juvenile, and two infants were born
during the study. We collected data only from adult individuals. Both groups have
been observed almost continuously since 1999 (Asensio et al. 2009; CristbalAzkarate et al. 2005; Dunn et al. 2009, 2010, 2012) and the individuals were,
therefore, known to us and habituated to our presence.
Sampling Sessions
We collected data during three different sampling sessions between February
2006 and February 2007: Sampling Session 1 (SS1: February 4March 2, 2006
and January 20 February 9, 2007), during the annual period of fruit scarcity
reported for Los Tuxtlas; Sampling Session 2 (SS2: April 24May 20, 2006
and May 28June 14, 2006), during the primary peak in fruit production
reported for the region; and Sampling Session 3 (SS3: August 9August 27,
2006 and September 6September 26, 2006), during the secondary peak in fruit
production reported for the region (see Dunn et al. 2010 for a review of local
plant phenology).
Diet and Activity Pattern
The methods used to collect data on diet and activity pattern have been described in
detail elsewhere (Dunn et al. 2010). Briefly stated, we collected data from each group
on 16 nonconsecutive days in each sampling session, for a total of 48 d and 288
observation hours per group. We used focal observations of 2-h continuous sampling
on randomly selected adult individuals between 07:00 h and 15:00 h. We categorized
behavioral observations into resting (sleep or static without interaction), feeding
(inspection of food, bringing food to mouth, chewing and swallowing, moving while
feeding within a food patch), traveling (movement to a new area or tree), and other
behavior (remaining activities not categorized as resting, feeding, or traveling).
During feeding we also recorded the food item consumed: fruits, leaves, and other
items (flowers, petioles, or bark). We reported diet and activity as percentages of total
observation time (Fig. 1).
We used observational data and the birth dates of infants to evaluate the reproductive status of females, which included data from the biweekly monitoring of all
adult females during the 12 mo after the end of our study period (Cristbal-Azkarate
et al. 2011), basing our calculations on the mean howler gestation period of 6 mo
(Crockett and Rudran 1987; Glander 1980; Strier et al. 2001). We categorized
females into one of three reproductive categories: pregnant (defined as starting from
the day of estimated conception to the day before parturition), lactating (defined by
either observations of lactation, or, in case of uncertainty, as starting from the day of

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J.C. Dunn et al.


100%
90%

Observation Time

80%
70%

Leaf

60%
Fruit

50%
40%

Rest

30%

Travel

20%
10%
0%
RC3

RH
SS1

RC3

RH

RC3

SS2

RH
SS3

Sampling Session / Group


Fig. 1 The percentage of total observation time spent traveling, resting, and feeding (fruit and leaves) by
two groups of howlers in different forest fragments in Los Tuxtlas, Mexico, during three different sampling
sessions (SS1, SS2, and SS3).

parturition until 20 mo, when latest weaning occurs; Domingo-Balcells and Vea-Baro
2009), and other (neither pregnant nor lactating).
Fecal Collection and Glucocorticoid Assay
We collected the first defecation of the morning from each individual to control
for diurnal variations in fGCM excretion. Overall we analyzed 202 fecal samples
(N=112 RC3, 90 RH): 58 in SS1 (33 RC3, 25 RH), 81 in SS2 (43 RC3, 38 RH),
and 63 in SS3 (36 RC3, 27 RH), which were evenly distributed among the adult
individuals (meanSD samples/individual: RC3=18.62.5; RH=15.02.7) and
between sexes (94 female samples, 108 male samples). We collected samples
only if free of urine and other impurities (mud, dust, rain, etc.) in 10-ml
polypropylene tubes, which we labeled with the time and date of collection
and the identity of the individual, and immediately stored them in a cooler with
frozen gel packs. Once back at the field station each afternoon, we stored the
samples in a freezer at 20 C. Freezing samples is reported to have a minimal
effect on fGCM (Hunt and Wasser 2003; Khan et al. 2002) and we stored
samples for a maximum of 12 mo at 20 C. At the end of the study, we
lyophilized the samples to eliminate the water content. To be sure that all the
water was removed from the samples, we repeatedly weighed 30 of the biggest
samples during lyophilization until there was no variation in their weights.
We extracted fGCMs using a modified version of the method described in Wasser
et al. (2000), which has previously been used for Mexican howlers (CristbalAzkarate et al. 2007). Briefly stated, 0.7 g of pulverized dry feces was shaken for
20 h in 4.0 ml of methanol analytical grade reagent. We then centrifuged (3000 rpm)
extracts for 20 min and recovered and evaporated the supernatants containing the

Fecal Glucocorticoid Levels in Free-Ranging Howlers

251

steroids. This extraction technique has proven consistently to recover 84.32 % of


the steroids in the original sample (Wasser et al. 2000).
We performed glucocorticoid quantification by radioimmunoassay (RIA). We
reconstituted the extracts with a phosphate assay buffer free of steroids (DPC, Los
Angeles, CA) and used a corticosterone COAT-A-COUNT RIA kit (DPC, Los
Angeles, CA), which is a solid-phase I125 RIA method. The calibration range for
the assay was 202000 ngml1 (57.75772 nmoll1), with a 2-h incubation time at
room temperature (1528 C). Finally, we quantified fGCM levels in a gamma
counter for 1 min and report values in nanograms per gram of dry feces (ngg1).
Corticosterone antibodies have proven to be more effective than other antibodies for
measuring glucocorticoid metabolites in the feces of many different mammal species
(Wasser et al. 2000).
We were unable to conduct an adrenocorticotropic hormone (ACTH) challenge test to validate our method, as Mexican howlers are currently listed as
Critically Endangered by the IUCN (Cuarn et al. 2008) and their capture for
unwarranted invasive practices is prohibited. However, Aguilar-Cucurachi et al.
(2010) conducted biological validations with Alouatta palliata mexicana for the
same extraction and fGCM quantification method. They evaluated fGCM levels
before, during, and after an artificial stressor (capture and translocation) and
found a clear peak in corticosterone immediately after the stressor, indicating
that corticosterone assays provide a reliable adrenal stress signal in this
subspecies.
We ran the extracts in duplicate assays together with standards. We ran a
total of three assays of fGCM levels and the intra- and interassay coefficients
of variation were 2.4 % and 3.1 % respectively. Cross-reactivities of the
antibody with other steroids is <0.34 %. We validated hormone assays via
parallelism and accuracy tests. We performed parallelism by comparing the
slope of a serial dilution curve of pooled howler fecal extracts to the slope
of the standard curve using a Students t-test (Zar 2009). The difference was
not significant (t=0.25, P=0.94, N=10), indicating that the two slopes were
parallel. For accuracy, in addition to the standard curve, a second set of
standards was spiked with a constant amount of pooled howler fecal extract.
We examined accuracy by subtracting the value of the pooled extract from the
spiked samples (pooled extract+standard tubes) and by calculating the slope
that results from plotting the values of the standards and the spiked standards.
In our case, the slope that results from plotting these values was B=0.999,
indicating high accuracy.
Data Analyses
We used generalized linear mixed effect models (GLMMs) to analyze the effect of
independent variables (forest fragment, sampling session, travel time, and fruit
consumption) on a dependent continuous variable (fGCM levels; Table I). GLMMs
allow for the analysis of non-normally distributed data and the specification of a
random effect to account for the nonindependence of repeated measures. All statistical analyses were performed in the R environment, version 2.9.2 (R Development
Core Team 2011).

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J.C. Dunn et al.

Models included in the analyses excluded combinations containing both fruit


consumption and travel time, as these variables were not independent, leading to
collinearity (see Mundry 2010 for discussion of why this is problematic), and
excluded interaction terms, which were not relevant to our hypotheses. We specified
individuals within forest fragments as hierarchical random effects to control for the
repeated sampling of fecal samples from the same individual. Further, as pregnancy
and lactation have been found to significantly increase fGCM levels in female
primates (Engh et al. 2006; Foerster et al. 2012; Mastorakos and Ilias 2003;
Setchell et al. 2008), we evaluated fGCM levels among reproductive categories. As
pregnant and lactating females were found to have substantially higher fGCM levels
than nonreproducing females and males (Fig. S1), we controlled for this variable in
all models (Table I).
We based model evaluation on the information-theoretic approach using
Akaikes information criterion (AIC) to infer the relative support for alternative
models. This approach stems from the recognition that data rarely provide absolute
support for a single hypothesis; rather, data can influence only the extent to which
any given hypothesis is supported (relative to competing explanations). The
approach has become increasingly popular in behavioral ecology and is particularly well suited for studying ecological systems in which multiple hypotheses
could be proposed to explain observed phenomena (Lukacs et al. 2007; Richards et
al. 2010). We based the interpretation of GLMM results on model AICi, i.e., AIC
of respective model AIC of best model. Following the guidelines published by
Burnham and Anderson (2002), we considered models having AICi 2 to receive
substantial support, those having AICi within 47 of the best model to receive
considerably less support, and models with AICi >10 to have essentially no
support. We further examined all interpretations of relative support for individual
variables by assessing their respective effect sizes and standard errors. We tested

Table I Variables used in GLMMs


Variable

Definition

Dependent variables
fGCM level (ngg1)

Continuous variable (min=4.2; max=337.3; mean=117.1)

Independent variables (fixed effects)


Forest fragment

Factor variable: 2 levels (RC3, RH)

Sampling session

Factor variable: 3 levels (SS1, SS2, SS3)

Travel (% total observation time)

Continuous variable (min=0.6; max=31; mean=12)

Fruit consumption (% total observation time) Continuous variable (min=0; max=100; mean=56)
Forest fragment

Factor variable: 2 levels (RC3, RH)

Independent variables (random effects)


Individual

Factor variable: 12 levels (12 individuals)

Independent variables (control variables)


Reproductive status

Factor variable: binary response (a) lactating or


reproducing females, (b) neither lactating nor
reproducing females, and males

Fecal Glucocorticoid Levels in Free-Ranging Howlers

253

the validity of models regarding the assumed normal distribution of intragroup


errors and randomly distributed random effects, qualitatively by plotting intragroup
residuals, which provide a good surrogate for intragroup errors, and inspection of
fitted versus residual plots (Zuur et al. 2009).

Results
Travel time was present in all four of the best-supported models and was found
to have a strong positive effect on fGCM levels (Tables II and III). For
example, an increase in daily travel time from 10 % to 20 % of observation
time (total range: 1 %31 %), equated to a ca. 50 % increase in fGCM levels
(Fig. 2). The effect of fruit consumption on fGCM levels received very little
support compared to the alternative models. This was confirmed by a low effect
size compared to its standard error and less support for the single parameter
model than the null model (Table II).
There was some support for an effect of sampling session on fGCM levels,
based on the fact that sampling session was present in two of the three bestperforming models (Table II). The effect sizes of the best-supported models
show that fGCM levels were higher in SS1 than in SS2 or SS3 (Table III).
However, the single parameter model of sampling session received very little
support compared to travel time (Table II). Similarly, forest fragment was
present in the third best performing model (i =2.6), providing some support
for an effect of forest fragment on fGCM levels (Table II). The effect sizes of
this model suggest that fGCM levels might have been higher in the smaller
forest fragment (RC3) than in the larger one (RH); however, the effect size was

Table II Results of generalized linear mixed-effect models investigating the effects of travel time and fruit
consumption on fGCM levels in two groups of howlers in different forest fragments (FF) in Los Tuxtlas,
Mexico, during each of three different sampling sessions (SS)
Model

Delta AIC
(i)

Log likelihood
(Li)

Relative likelihood
(li)

Akaike weight
(wi)

Evidence ratio
(wi/wj)

Travel+SS

1073.7

1.00

0.51

Travel

1076.2

0.61

0.31

1.65

Travel+SS+FF

2.6

1074.0

0.27

0.14

3.67

Travel+FF

4.9

1077.1

0.09

0.04

11.59

FF+SS

12

1079.7

<0.01

0.00

403.43

SS

12.5

1080.9

<0.01

0.00

518.01

Fruit+FF+SS

13.2

1079.2

<0.01

0.00

735.10

Fruit+SS

13.3

1080.3

<0.01

0.00

772.78

FF

32.9

1092.1

<0.01

0.00

1.39E+07

Null model

33.2

1093.2

<0.01

0.00

1.62E+07

Fruit+FF

34.5

1091.9

<0.01

0.00

3.10E+07

Fruit

34.9

1093.1

<0.01

0.00

3.79E+07

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J.C. Dunn et al.

Table III Effect size and standard error (SE) of individual variables in the three best-supported
models from Table II

Model

Effect size

SE

(Intercept)

94.1

18.4

Travel

3.6

1.0

Sampling session (SS2)

20.2

10.5

Sampling session (SS3)

22.2

9.6

Reproductive status (pregnant)

24.3

8.3

(Intercept)

69.6

7.5

Travel

3.1

0.5

Reproductive status (pregnant)

29.0

8.1

(Intercept)

102.3

17.2

Travel

3.0

0.7

Forest fragment (RH)

9.4

13.7

Sampling session (SS2)

21.3

10.5

Sampling session (SS3)

24.1

9.6

Reproductive status (pregnant)

25.5

7.6

Travel+sampling session

Travel

Travel+fragment+sampling session

low compared to its standard error (Table III) and the single-parameter model
of forest fragment received little support (Table II). Data on mean ( SE)
fGCM levels for each group in each of the three sampling sessions is given
in Fig. 3, and data on mean ( SE) fGCM levels, fruit consumption, and travel
time for all individuals is given in Table SI.

400

fGCM (ngg - 1 dry mass)

350
300
250
200
150
100
50
0

10

15

20

25

30

35

Travel Time (%)


Fig. 2 The relationship between fGCM levels and travel time in two groups of howlers in different forest
fragments in Los Tuxtlas, Mexico.

Fecal Glucocorticoid Levels in Free-Ranging Howlers

255

200

RC3

RH

fGCM (ngg-1 dry mass)

180
160
140
120
100
80
60
40
20
0
1

Sampling Session
Fig. 3 Mean ( SE) fGCM levels for two groups of howlers in different forest fragments in Los Tuxtlas,
Mexico, during each of three different sampling sessions (SS1, SS2, and SS3).

Discussion
The results of this study strongly suggest that increases in travel time result in
increases in fGCM levels in free-ranging howlers. Minimizing energy expenditure
is thought to be fundamental to the foraging strategy of howlers as a result of their
highly folivorous diet (Milton 1980). However, to our knowledge, this is the first time
that an indicator of energy expenditure has been related to any measure of physiological stress in this primate genus. Our results agree with the observations of other
authors who have suggested that increases in travel can lead to increases in stress in
wild primates (Cavigelli 1999; Foerster and Monfort 2010; Muller and Wrangham
2004) and bring insight into the proximate mechanisms underlying increases in GC
levels in primates in fragments (Chapman et al. 2006; Martnez-Mota et al. 2007;
Rangel-Negrn et al. 2009). Moreover, they highlight the potential conservation
significance of studies showing that habitat loss and transformation can lead to
increases in travel time in wild primates (Donati et al. 2011; Dunn et al. 2009,
2010; Gonzalez-Zamora et al. 2011; Hardus et al. 2012).
Fecal glucocorticoid metabolite levels were highest in both groups during SS1
(Fig. 3), the period of lowest fruit consumption (Fig. 1). This supports the findings of
Behie et al. (2010), who proposed that fruit availability is an important factor predicting
stress in howlers, as well as the growing recognition (Behie et al. 2010; Dunn et al.
2009, 2010) that howlers, traditionally described as primarily folivorous, may be more
reliant on fruit than previously believed. However, our models showed no support for an
effect of fruit consumption per se on fGCM levels compared to alternative hypotheses
(Table II). We argue that the relationship between fruit availability and glucocorticoid
levels previously reported for howlers (Behie et al. 2010) is probably mediated by the
negative effect that fruit consumption has on travel time (present study; cf. Asensio et al.
2007; Dunn et al. 2009, 2010; Rodrguez-Luna et al. 2003).
The mean fGCM levels of the RH group, in a bigger, more conserved forest
fragment, were lower than those of the RC3 group, in a smaller and more disturbed
forest fragment, particularly during SS1 (Fig. 3). This is consistent with previous
studies showing that howlers inhabiting smaller forest fragments have higher fGCM
levels (Martnez-Mota et al. 2007). However, the direct effect of forest fragment on

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J.C. Dunn et al.

fGCM levels received little support in our models, suggesting that the difference in
stress levels between the two focal groups result mainly from the effect of habitat
characteristics on travel time. Indeed, howlers inhabiting smaller and more degraded
forest fragments have been reported to consume less fruit (Asensio et al. 2007; Dunn
et al. 2009, 2010; Juan et al. 2000), feed from more secondary food resources (Dunn
et al. 2012), and feed from smaller trees (Dunn et al. 2009) than groups in bigger
fragments, all of which result in increases in travel time (Dunn et al. 2009, 2010,
2012). As the availabilities of fruit (Dunn et al. 2009, 2010) and big trees (Laurance
et al. 2000) are negatively affected by habitat loss and fragmentation, the results of
our study have important implications for the conservation and management of
primates in fragments and highlight the importance of protecting large, undisturbed
forested areas. Our results also suggest that researchers examining stress levels
among primate populations across different habitats should carry out their fieldwork
during periods of resource scarcity, when they would be likely to obtain the clearest
difference among groups. This is important, as field endocrinology is costly in terms
of both time and resources.
Other factors, including exposure to tourists (Behie et al. 2010) and parasitization
(Chapman et al. 2006), may also affect the stress response in primates. However,
neither of our study groups is exposed to tourists or high levels of human contact.
Further studies are needed to determine how parasite infection may relate to fGCM
levels in our focal groups.
Finally, it is important to note that a year-long study in two forest fragments may not
be sufficient for deriving general conclusions about the responses of howlers to forest
fragmentation. Therefore, our results should be interpreted as preliminary. However, our
results suggest that forest fragmentation, by limiting the availability of fruit and big
trees, and forcing howlers to increase travel time, may lead to increases in stress and
limit the long-term viability of populations. GC levels have been related to survivorship
in studies of several vertebrate species (Bonier et al. 2009; Romero and Wikelski 2001).
However, long-term studies showing a direct causeeffect relationship between fGCM
levels and survival in primates are lacking. Therefore, determining how relevant our
results are for the survivorship and/or fitness of howlers in forest fragments will require
long-term studies of demographic change and fGCM levels.
Acknowledgments We are thank L. Mendoza and B. Gomez for their help in the field; G. Garca-Lopez,
R. Valenzuela, L. Boeck, and E. O. Ameca for their assistance in the laboratory; C. McOwen for statistical
advice; C. Huber and the Palacios family for access to their land; and R. Coates for logistical support. We
also thank the Fundacin BBVA, which provided a studentship for J. Dunn and financial support for the
project and E. Rodrguez-Luna and the Universidad Veracruzana for the use of their facilities. Finally, we
thank three anonymous reviewers and the Associate Editor, Oliver Schlke, for their very helpful and
constructive comments on a previous version of this manuscript.

References
Abbott, D. H., Keverne, E. B., Bercovitch, F. B., Shively, C. A., Mendoza, S. P., Saltzman, W., Snowdon,
C. T., Ziegler, T. E., Banjevic, M., Garland, T., & Sapolsky, R. M. (2003). Are subordinates always
stressed? A comparative analysis of rank differences in cortisol levels among primates. Hormones and
Behavior, 43, 6782.

Fecal Glucocorticoid Levels in Free-Ranging Howlers

257

Aguilar-Cucurachi, M. S., Dias, P. A., Rangel-Negrn, A., Chavira, R., Boeck, L., & Canales-Espinosa, D.
(2010). Preliminary evidence of accumulation of stress during translocation in howlers. American
Journal of Primatology, 71, 16.
Arroyo-Rodrguez, V., Mandujano, S., & Bentez-Malvido, J. (2008). Landscape attributes affecting patch
occupancy by howler monkeys (Alouatta palliata mexicana) in fragmented landscapes at Los Tuxtlas,
Mexico. American Journal of Primatology, 70, 6977.
Asensio, N., Arroyo-Rodrguez, V., Dunn, J. C., & Cristbal-Azkarate, J. (2009). Conservation value of
landscape supplementation for howler monkeys living in forest patches. Biotropica, 41, 768773.
Asensio, N., Cristbal-Azkarate, J., Dias, P. A. D., Vea-Baro, J. J., & Rodrguez-Luna, E. (2007). Foraging
habits of Alouatta palliata mexicana in three forest fragments. Folia Primatologica, 78, 141153.
Beehner, J. C., & McCann, C. (2008). Seasonal and altitudinal effects on glucocorticoid metabolites in a
wild primate (Theropithecus gelada). Physiology & Behavior, 95, 508514.
Behie, A. M., Pavelka, M. S., & Chapman, C. A. (2010). Sources of variation in faecal cortisol levels in
howler monkeys in Belize. American Journal of Primatology, 72, 600606.
Bonier, F., Martin, P. R., Moore, I. T., & Wingfield, J. C. (2009). Do baseline glucocorticoids predict
fitness? TREE, 24, 634642.
Burnham, K. P., & Anderson, D. R. (2002). Model selection and multimodel inference: A practical
information-theoretic approach (2nd ed.). New York: Springer.
Busch, D. S., & Hayward, L. S. (2009). Stress in a conservation context: A discussion of glucocorticoid
actions and how levels change with conservation-relevant variables. Biological Conservation, 12,
28442853.
Carnegie, S., Fedigan, L. M., & Ziegler, T. E. (2011). Social and environmental factors affecting fecal
glucocorticoids in wild, female white-faced capuchins (Cebus capucinus). American Journal of
Primatology, 869, 861869.
Cavigelli, S. A. (1999). Behavioural patterns associated with faecal cortisol levels in free-ranging female
ring-tailed lemurs, Lemur catta. Animal Behaviour, 57, 935944.
Champoux, M., Zanker, D., & Levine, S. (1993). Food search demand effort effects on behavior and
cortisol in adult female squirrel monkeys. Physiology and Behavior, 54, 10911097.
Chapman, C. A., Wassermann, M. D., Gillespie, T. R., Speirs, M. L., Lawes, M. J., Saj, T. L., & Ziegler, T.
E. (2006). Do food availability, parasitism, and stress have synergistic effects on red colobus populations living in forest fragments? American Journal of Physical Anthropology, 131, 525534.
Cristbal-Azkarate, J., Chavira, R., Boeck, L., Rodrguez-Luna, R., & Ve, J. J. (2007). Glucocorticoid
levels in free ranging resident howlers: A study of coping strategies. American Journal of Primatology,
69, 111.
Cristbal-Azkarate, J., Domingo-Balcells, C., Dunn, J. C., & Vea-Baro, J. (2011). Evolucin demogrfica
de monos aulladores en fragmentos de selva en Los Tuxtlas. In P. A. Dias, A. R. Negrin, & D. Canales
(Eds.), La conservacin de los primates en Mxico (pp. 2743). Veracruz: Consejo Veracruzano de
Investigacin Cientfica y Desarrollo Tecnolgico.
Cristbal-Azkarate, J., Ve, J. J., Asensio, N., & Rodrguez-Luna, E. (2005). Biogeographical and floristic
predictors of the presence and abundance of howler monkeys (Alouatta palliata mexicana) in the
rainforest fragments of Los Tuxtlas, Mexico. American Journal of Primatology, 67, 209222.
Crockett, C., & Rudran, R. (1987). Red howler monkey birth data I: Seasonal variation. American Journal
of Primatology, 13, 347368.
Cuarn, A. D., Shedden, A., Rodrguez-Luna, E., de Grammont, P. C., & Link, A. (2008). Alouatta palliata
ssp. mexicana. In 2008 IUCN Red List of Threatened Species. Retrieved from: www.iucnredlist.org
(Accessed February 4, 2012).
Dirzo, R., & Garcia, M. C. (1992). Rates of deforestation in Los Tuxtlas, a neotropical area in southeast
Mexico. Conservation Biology, 6, 8594.
Domingo-Balcells, C., & Vea-Baro, J. (2009). Developmental stages in the howler monkey, subspecies
Alouatta palliata mexicana: A new classification using age-sex categories. Neotropical Primates, 16, 18.
Donati, G., Kesch, K., Ndremifidy, K., Schmidt, S. L., Ramanamanjato, J.-B., Borgognini-Tarli, S. M., &
Ganzhorn, J. U. (2011). Better few than hungry: Flexible feeding ecology of collared lemurs Eulemur
collaris in littoral forest fragments. PloS One, 6(5), e19807. doi:10.1371/journal.pone.0019807.
Dunn, J. C., Asensio, N., Arroyo-Rodrguez, V., Schnitzer, S., & Cristbal-Azkarate, J. (2012). The ranging
costs of a fallback food: liana consumption supplements diet but increases foraging effort in howler
monkeys. Biotropica. doi:10.1111/j.1744-7429.2012.00856.x.
Dunn, J. C., Cristbal-Azkarate, J., & Ve, J. (2009). Differences in diet and activity pattern between two
groups of Alouatta palliata associated with the availability of big trees and fruit of top food taxa.
American Journal of Primatology, 71, 654662.

258

J.C. Dunn et al.

Dunn, J. C., Cristbal-Azkarate, J., & Ve, J. (2010). Seasonal variations in the diet and feeding effort of
two groups of howler monkeys in different sized forest fragments. International Journal of
Primatology, 31, 887903.
Engh, A., Beehner, J. C., Bergman, T., Whitten, P., Hoffmeier, R. R., Seyfarth, R. M., & Cheney, D. L.
(2006). Female hierarchy instability, male immigration and infanticide increase glucocorticoid levels in
female chacma baboons. Animal Behaviour, 71, 12271237.
Estrada, A., & Coates-Estrada, R. (1988). Tropical rain forest conversion and perspectives in the conservation of wild primates (Alouatta and Ateles) in Mexico. International Journal of Primatology, 14,
315327.
Foerster, S., Cords, M., & Monfort, S. L. (2012). Seasonal energetic stress in a tropical forest primate:
Proximate causes and evolutionary implications. PLoS One, 7, e50108.
Foerster, S., & Monfort, S. L. (2010). Fecal glucocorticoids as indicators of metabolic stress in female
Sykes' monkeys (Cercopithecus mitis albogularis). Hormones and Behavior, 58, 685697.
Gesquiere, L. R., Khan, M., Shek, L., Wango, T. L., Wango, E. O., Alberts, S. C., & Altmann, J. (2008).
Coping with a challenging environment: Effects of seasonal variability and reproductive status on
glucocorticoid concentrations of female baboons (Papio cynocephalus). Hormones and Behavior, 54,
410416.
Gesquiere, L. R., Learn, N. H., Simao, M. C. M., Onyango, P. O., Alberts, S. C., & Altmann, J. (2011a).
Life at the top: Rank and stress in wild male baboons. Science, 333, 357360.
Gesquiere, L. R., Onyango, P. O., Alberts, S. C., & Altmann, J. (2011b). Endocrinology of year-round
reproduction in a highly seasonal habitat: Environmental variability in testosterone and glucocorticoids
in baboon males. American Journal of Physical Anthropology, 176, 169176.
Girard, I., & Garland, T. (2002). Plasma corticosterone response to acute and chronic voluntary exercise in
female house mice. Journal of Applied Physiology, 92, 15531561.
Glander, K. (1980). Reproduction and population growth in free-ranging howler monkeys. American
Journal of Physical Anthropology, 53, 2536.
Gonzalez-Zamora, A., Arroyo-Rodriguez, V., Chaves, O., Sanchez-Lopez, S., Aureli, F., & Stoner, K.
(2011). Influence of climatic variables, forest type, and condition on activity patterns of Geoffroyis
spider monkeys throughout Mesoamerica. American Journal of Primatology, 73, 11891198.
Guevara, S., Laborde, J., & Snchez-Rios, G. (2004). Los Tuxtlas: El paisaje de la sierra. Xalapa: Instituto
de Ecologa, A.C./European Union.
Hardus, M. E., Lameira, A. R., Menken, S. B. J., & Wich, S. A. (2012). Effects of logging on orangutan
behavior. Biological Conservation, 146, 177187.
Hunt, K. E., & Wasser, S. K. (2003). Effect of long-term preservation methods on fecal glucocorticoid
concentrations of grizzly bear and African elephant. Physiological and Biochemical Zoology, 76, 918928.
Jaimez, N., Bribiescas, R. G., Aronsen, G. P., Anestis, S., & Watts, D. P. (2012). Urinary cortisol levels of
gray-cheeked mangabeys are higher in disturbed compared to undisturbed forest areas in Kibale
National Park, Uganda. Animal Conservation, 15, 242247.
Juan, S., Estrada, A., & Coates-Estrada, R. (2000). Contrastes y similitudes en el uso de recursos y patrn
general de actividades en tropas de monos aulladores (Alouatta palliata) en fragmentos de selva de Los
Tuxtlas, Mxico. Neotropical Primates, 8, 131135.
Khan, M. Z., Altmann, J., Isani, S. S., & Yu, J. (2002). A matter of time: Evaluating the storage of fecal
samples for steroid analysis. General and Comparative Endocrinology, 128, 5764.
Kraemer, W.J., Fragala, M.S., Watson, G., Volek, J.S., Rubin, M.R., French, D.N., Maresh, C.M., Vingren,
J.L., Spiering, B.A., Yu-Ho, J., Hughes, S.L., Case, H.S., Stuempfle, K.J., Lehmann, D.R., Bailey, S.,
& Evans, D.S. (2008). Hormonal responses to a 160km race across frozen Alaska. British Journal of
Sports Medicine, 42, 116120.
Laurance, W. F., Delamnica, P., Laurance, S. G., Vasconcelos, H. L., & Lovejoy, T. E. (2000). Rainforest
fragmentation kills big trees. Nature, 404, 836.
Lukacs, P. M., Thompson, W. L., Kendall, W. L., Gould, W. R., Doherty, P. F., Burnham, K. P., &
Anderson, D. R. (2007). Concerns regarding a call for pluralism of information theory and hypothesis
testing. Journal of Applied Ecology, 44(2), 456460.
Martnez-Mota, R., Valdespino, C., Snchez-Ramos, M. A., & Serio-Silva, J. C. (2007). Effects of forest
fragmentation on the physiological stress response of black howler monkeys. Animal Conservation, 10,
374379.
Mastorakos, G., & Ilias, I. (2003). Maternal and fetal hypothalamicpituitaryadrenal axes during pregnancy and postpartum. Annals of the New York Academy of Science, 997, 136149.
Milton, K. (1980). The foraging strategy of howler monkeys: A study of primate economics. New York:
Columbia University Press.

Fecal Glucocorticoid Levels in Free-Ranging Howlers

259

Muller, M. N., & Wrangham, R. W. (2004). Dominance, cortisol and stress in wild chimpanzees (Pan
troglodytes schweinfurthii). Behavioral Ecology and Sociobiology, 55, 332340.
Mundry, R. (2010). Issues in information theory-based statistical inferencea commentary from a frequentists perspective. Behavioral Ecology and Sociobiology, 65, 5768.
Pride, R. E. (2005a). Optimal group size and seasonal stress in ring-tailed lemurs (Lemur catta). Behavioral
Ecology, 16, 550560.
Pride, R. E. (2005b). Foraging success, agonism and predator alarms: Behavioural predictors of cortisol in
Lemur catta. International Journal of Primatology, 26, 295319.
R Development Core Team. (2011). R: A language and environment for statistical computing. Vienna: R
Foundation for Statistical Computing.
Rangel-Negrn, A., Alfaro, J. L., Valdez, R. A., Romano, M. C., & Serio-Silva, J. C. (2009). Stress in
Yucatan spider monkeys: Effects of environmental conditions on fecal cortisol levels in wild and
captive populations. Animal Conservation, 12, 496502.
Richards, S. A., Whittingham, M. J., & Stephens, P. A. (2010). Model selection and model averaging in
behavioural ecology: The utility of the IT-AIC framework. Behavioral Ecology and Sociobiology, 65, 7789.
Rodrguez-Luna, E., Domnguez-Domnguez, L. E., Morales-Mvil, J. E., & Martnez-Morales, M. (2003).
Foraging strategy changes in a Alouatta palliata mexicana troop released on an island. In L. K. Marsh
(Ed.), Primates in fragments: Ecology and conservation (pp. 229250). New York: Kluwer Academic/
Plenum.
Romero, L. M., & Wikelski, M. (2001). Corticosterone levels predict survival probabilities of Galapagos
marine iguanas during el Nio events. Proceedings of the National Academy of Sciences of the USA,
98, 73667370.
Sapolsky, R. M. (1993). The physiology of dominance in stable versus unstable social hierarchies. In W. A.
Mason & S. P. Mendoza (Eds.), Primate social conflict (pp. 171204). Albany: State University of
New York Press.
Sapolsky, R. M. (1994). Individual differences and the stress response. Seminars in Neuroscience, 6, 261269.
Sapolsky, R. M. (2002). Endocrinology of the stress response. In J. B. Becker, S. M. Breedlove, & D.
Crews (Eds.), Behavioural endocrinology (pp. 409450). Cambridge, MA: MIT Press.
Sapolsky, R. M., Romero, L. M., & Munck, A. U. (2000). How do glucocorticoids influence stress
responses? Integrating permissive, suppressive, stimulatory and preparative actions. Endocrine
Reviews, 21, 5589.
Setchell, J., Smith, T., Wickings, E. J., & Knapp, L. A. (2008). Factors affecting fecal glucocorticoid levels in
semi-free-ranging female mandrills (Mandrillus sphinx). American Journal of Primatology, 70, 110.
Soto, M., & Gama, L. (1997). Climas. In E. Gonzlez Soriano, R. Dirzo, & R. Vogt (Eds.), Historia natural
de Los Tuxtlas (pp. 723). Mxico City: UNAM and CONABIO.
Strier, K. B., Mendes, S. L., & Santos, R. R. (2001). Timing of births in sympatric brown howler monkeys
(Alouatta fusca clamitans) and northern muriquis (Brachyteles arachnoids hypoxanthus). American
Journal of Primatology, 55, 87100.
Tharp, G. D. (1975). The role of glucocorticoids in exercise. Medicine & Science in Sports & Exercise, 7, 611.
Wasser, S. K., Hunt, K. E., Brown, J. L., Cooper, K., Crockett, C. M., Bechert, U., Millspaugh, J. J., Larson,
S., & Monfort, S. L. (2000). A generalized fecal glucocorticoid assay for use in a diverse array of
nondomestic mammalian and avian species. General and Comparative Endocrinology, 120, 260275.
Wingfield, J. C. (2005). The concept of allostasis: Coping with a capricious environment. Journal of
Mammalogy, 86, 248254.
Wingfield, J. C., & Romero, L. M. (2001). Adrenocortical responses to stress and their modulation in freeliving vertebrates. In B. S. McEwan (Ed.), Handbook of physiology. Section 7: The endocrine system.
Vol. 4: Coping with the environment: neural and endocrine mechanisms (pp. 211236). Oxford:
Oxford University Press.
Zar, J. (2009). Biostatistical analysis (5th ed.). Upper Saddle River, NJ: Prentice-Hall.
Zuur, A. F., Ieno, E. N., Walker, N. J., Saveliev, A. A., & Smith, G. M. (2009). Mixed effects models and
extensions in ecology with R. New York: Springer.

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