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Social correlates of testosterone and ornamentation in male mandrills

Joanna M. Setchell a,⁎, Tessa Smith b, E. Jean Wickings c, Leslie A. Knapp d

a Evolutionary Anthropology Research Group, Department of Anthropology, Durham University, UKb Biology Department, Chester University, Chester, UKc Centre International de Recherches Médicales, BP 769, Franceville, Gabond Department of Biological Anthropology, University of Cambridge, Downing Street, Cambridge CB2 3DZ, UK

A B S T R A C TA R T I C L E I N F O

Article history:Received 26 February 2008Revised 22 April 2008Accepted 11 May 2008Available online 22 May 2008

Keywords:Dynamic traitSignalsChallenge hypothesisMandrillus sphinxColorationCondition dependent traits

We investigated relationships between fecal androgen concentrations, facial coloration and behaviour insemi-free-ranging male mandrills. We found that fecal androgen levels were significantly positively relatedto dominance rank, independent of rank stability and the mating period, suggesting that male mandrills livein a permanently aggressive context in which they must actively maintain their dominance status. Facial redcoloration was also significantly related to both fecal androgen levels and rank, with high ranking maleshaving both higher androgen levels and redder faces, although dominant males did not always have thehighest androgen levels or the reddest faces. Predictive relationships between androgen levels, colorationand rank were short-term. Androgen concentrations and facial redness both increased in the presence ofreceptive females, as did the former during periods of rank instability. We conclude that male facial rednessis likely to represent an honest signal (to other males) of current androgen status, competitive ability andwillingness to engage in fights and that females may also use this to assess male condition. Further, ourfindings provide support for the “challenge hypothesis” as originally proposed for birds by Wingfield.

© 2008 Elsevier Inc. All rights reserved.

Introduction

The steroid hormone testosterone is closely linked to reproductionin vertebrates, and influences multiple morphological, physiologicaland behavioral traits, including the expression ofmale sexual behavior,aggression, behavioral displays and the development of secondarysexual characters (Dixson, 1998, Fig. 3). Among primates that formmulti-male, multi-female groups, males typically compete for highrank, which leads to increased access to receptive females, and highreproductive success (Cowlishaw and Dunbar, 1991; Kutsukake andNunn, 2006; van Noordwijk and van Schaik, 2004). Dominant malesmight therefore be expected to have higher levels of testosterone,and some studies have found that high testosterone levels are indeedassociated with high rank. For example, a significant positive asso-ciation between testosterone levels and dominance rank has beenreported for captive rhesus macaques (Macaca mulatta, Bercovitch,1993; Rose et al., 1971), and wild chimpanzees (Pan troglodytes,Muehlenbein et al., 2004;Muller andWrangham, 2004). However, thisis not always the case, and studies of captive rhesusmacaques (Gordonet al.,1976), captive vervetmonkeys (Chlorocebus aethiops, Steklis et al.,1985), wild and captive Japanese macaques (Macaca fuscata, Barrett

et al., 2002; Eaton, 1978), wild tufted capuchin monkeys (Cebus apellanigritus, Lynch et al., 2002) andwild chacmababoons (Papio hamadryasursinus, Beehner et al., 2006) have found no significant relationshipbetween male dominance rank and testosterone levels.

The lack of a consistent relationship between testosterone anddominance rank in male primates may be due to the evolutionary“costs” of high circulating levels of testosterone (Wingfield et al.,1997).In addition to promoting costly display behaviors (Dixson, 1998), highlevels of testosterone may impair immune function (Folstad andKarter, 1992), increase stress levels (Braude et al., 1999) and increasethe risk of mortality (Marler and Moore, 1988). The necessity ofbalancing the costs and benefits of high testosterone levels underliesthe “challenge hypothesis” (Wingfield et al., 1990). This hypothesisproposes that testosterone-driven increases in the frequency or inten-sity of aggression are strongest under conditions of social instability,such as during the development of dominance relationships or whenmales challenge one another for access to mates (Wingfield et al.,1990). Originally formulated for birds, the challenge hypothesis hasnow received support from studies of a wide range of vertebratespecies (Hirschenhauser and Oliveira, 2006; Oliveira, 2004).

Studies of testosterone levels in male primates provide broadsupport for the challenge hypothesis. First, increases in male testos-terone accompany seasonal sexual activity in many species, a timewhen males challenge one another for access to mates (Dixson, 1998;but see Strier et al., 1999). Second, social factors such as aggression ordominance status also influence testosterone secretion (Whitten,

Hormones and Behavior 54 (2008) 365–372

⁎ Corresponding author. Evolutionary Anthropology Research Group, Department ofAnthropology, Durham University, 43 Old Elvet Road, Durham DH1 3HN, UK. Fax: +44191 334 6101.

E-mail address: joanna.setchell@durham.ac.uk (J.M. Setchell).

0018-506X/$ – see front matter © 2008 Elsevier Inc. All rights reserved.doi:10.1016/j.yhbeh.2008.05.004

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2000). Third, where conditions are stable, testosterone levels are oftenun-correlated with either dominance rank or aggressive behavior inmales (Barrett et al., 2002; Cavigelli and Pereira, 2000; Gordon et al.,1976; Lynch et al., 2002; Nieuwenhuijsen et al., 1987; Steklis et al.,1985). Where social hierarchies are disrupted, however, and ranks areactively disputed, positive relationships between testosterone anddominance rank do occur (Beehner et al., 2006; Brockman et al., 2001;Cavigelli and Pereira, 2000; Sapolsky, 1983). Fourth, increasedaggressive competition for access to females is associated with anincrease in testosterone in both seasonal and non-seasonally breedingspecies, including ring-tailed lemurs (Lemur catta, Cavigelli and Pereira,2000), rhesus macaques (Higley et al., 1996), mantled howler monkeys(Allouatta palliata, Cristóbal-Azkarate et al., 2006), red-fronted lemurs(Eulemur fulvus rufus, Ostner et al., 2002) and chimpanzees (Muller andWrangham, 2004). However, no such relationship is found in specieswhere direct male competition is less important, such as capuchins(Lynch et al., 2002), muriquis (Brachyteles arachnoides, Strier et al.,1999) and moustached tamarins (Saguinus mystax, Huck et al., 2005).Finally, in wild chacma baboons, testosterone levels are positivelycorrelated with future rank (Beehner et al., 2006). The authors of thisstudy interpreted this finding as consistent with the challengehypothesis, with elevations in testosterone more closely related toacquisition of mates (via rank acquisition) than to mating itself.

“When the animal is excited all the naked parts become much morevividly tinted” (Darwin, 1871, p 293, referring to the mandrill)

Male mandrills (Mandrillus sphinx) possess a suite of secondarysexual traits, including brightly colored sexual skin on the face, rumpand genitalia (Hill, 1970). Studies of a semi-free-ranging colony ofmandrills at the Centre International de Recherches Médicales,Franceville (CIRMF), Gabon, have found that facial red coloration ispositively correlated with serum testosterone, and is most fullyexpressed in alphamales, while subordinates show reduced colorationand lower serum testosterone (Setchell and Dixson, 2001c; Wickingsand Dixson, 1992b). Males that gain alpha rank increase in bothtestosterone levels and red coloration, although the loss of alpha rankdoes not necessarily result in reduced testosterone or coloration (n=4males in each case, Setchell and Dixson, 2001a). Males appear to usecolor as a “badge of status” to assess individual differences in fightingability (Setchell and Wickings, 2005). These results suggest that colormaymirror changes inmale physiology, in a similar fashion to bill colorinmale blackbirds (Faivre et al., 2003), and convey current informationconcerning male testosterone levels to rival males, as well as topotential mates (Setchell and Dixson, 2001a,c). Thus, it seems thatDarwin may be correct, and that male mandrills do become morevividly colored when “excited”, i.e. when testosterone levels are high.However, measurements of serum testosterone in these earlier studiesrepresent point samples, taken during captures once or twice per year.Serum hormone levels are likely to be influenced by the stress ofcapture and response to anesthesia, and the measures obtained areunlikely to be representative of male testosterone levels (Sapolsky,1991). Most importantly, the serumvalues ignore potential differencesbetweenmales in their endocrine response to capture (Sapolsky,1991).

In this study, we used fecal measures of testosterone over a13 month period for 17 adult male mandrills living in two groups toinvestigate relationships among testosterone, ornamentation, andbehavior in greater detail. Fecal sampling presents advantages incomparison to the previous serum measures, both by avoiding theinfluence of capture stress on hormone levels, and by allowing thecollection of multiple samples over time (Hodges and Heistermann,2003; Whitten et al., 1998). Our study had three objectives:

1. To examine how dominance rank, the presence of receptivefemales, and social stability influence fecal levels of immunoreac-tive testosterone (fecal iT) levels in male mandrills.

2. To test the hypothesis that differences in fecal iT levels in individualmales (differences in “excitement”, Darwin, 1871) are accuratelyreflected in male ornamentation.

3. To examine the temporal relationships between fecal iT, rank andcoloration.

Methods

Study population

Mandrills are found in the dense rainforests of Gabon, Congo, mainland EquatorialGuinea and southern Cameroon to the south of the Sanaga river (Grubb, 1973). It hasproven impossible to habituate and observe known individuals in the wild (Abernethyet al., 2002; Harrison, 1988). However, the semi-free-ranging mandrill colony at theCIRMF provides an opportunity to study reproduction and behavior under relativelynatural conditions, with the advantages of regular observations and occasionalcaptures. The CIRMF mandrill colony was established in 1983/4, when 15 animals (7males, 8 females) were released into a 6.5 ha forest enclosure (E1). Between 1984 and2004 no subsequent additions were made to the colony, other than by breeding,although animals were removed occasionally. A second semi-free-ranging group wasestablished in 1994 in a smaller enclosure (E2, 3.5 ha) by transferring 17 mandrills(including 6 adult females and 4 adult males) from the first enclosure. In 2002 a third,small enclosure (E3, 2 ha) was added, to accommodate animals that had previouslybeen housed in outdoor group cages. The mandrills foraged freely and received dailysupplements of monkey chow, fruit and vegetables. Water was always available from astream, which runs through all three enclosures.

This study was conducted between February 2004 and March 2005. Subjectsincluded all adult males (aged 9+ yr, Setchell et al., 2006b) living in E1 (n=8), E2 (n=7)and E3 (n=2) during the study period. One adult male spontaneously transferred fromE1 to E2 in December 2004, and thus contributed to both groups, giving a total of 16males (rather than 17). The size and age–sex composition of the study groups during thestudy period is summarized in Table 1 and corresponded to the smaller end of groupsizes observed in the wild (Rogers et al., 1996). During the study, 19 infants were borninto E1, 4 were born into E2 and none were born into E3. Two adult males, two adultfemales and four juveniles were added to E2 in June 2004. One infant and one adultmale died in E1. Two adolescent males died or escaped from E2 (one in June 2004, onein December 2004).

Dominance rank

Observations of male behavior weremade ad libitum during twice daily observationperiods (approx. 10h00–11h30 and 15h30–17h30). Dominance rank was calculatedusing dyadic interaction matrices for each month of the study, based on all interactionswhere one male avoided or fled when another male approached. Males ranked 1 out-ranked all other males (alpha male), males ranked 2 out-ranked all but the alpha male,etc. Changes in alpha male in the colony are abrupt and unambiguous. Two changes inalpha male occurred in E1, and one in E2, during the study. In E1, the dominancehierarchy was stable from five months before the beginning of the study until 31 July2004, when the alpha male died of injuries likely sustained when falling out of a tree.Rank instability occurred between August and October 2004, with the second-rankingmale taking-over as alpha male for 40 days, before being challenged, injured anddeposed by a third male on September 20th 2004. This unstable period occurred duringthe mating season. The hierarchy then remained stable until the end of the study(March 05). At the beginning of the study, the alpha male in E2 had been dominant for8 yr. He was deposed in December 2005 by the male who jumped into E2 from E1, whothen remained alpha male until the end of the study period. This take-over occurredoutside the mating season.

In order to investigate the influence of rank stability on fecal iT and male coloration,we defined months as “stable”, when no changes in alpha male occurred, and“unstable” where a change in alpha male occurred, or had occurred in the previousmonth. We also calculated the net rank change across the study period for eachindividual male (initial rank compared with final rank) for use in some analyses.

Table 1Size and age–sex composition of the study groups in February 2004

Enclosure Total Males Females Number of femalesavailable to cyclea

Number ofadult malesb

E1 75 30 45 30 8E2 72 25 34 4 7E3 25 11 14 0 2

a Females of reproductive age,without a contraceptive implant (melengestrol acetate,MGA, provided by Contraception Advisory Group of the American Zoological Associa-tion). Females with implants showed no sexual swellings during the study period.

b Males aged N9 yr.

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Presence of receptive females

The reproductive state of females was noted daily as one of the following:

• Cycling: females in any stage of the menstrual cycle, during which females showconspicuous perineal swellings (Dixson, 1998). Females show conspicuous swellingsfor 11±1 (mean±SEM, n=45 cycles) days per cycle (Setchell and Wickings, 2004a)

• Pregnant: assigned post-hoc from the birth of an infant, beginning with the finaldetumescence of the perineal skin, mean 175 days (Setchell et al., 2002)

• Lactating: the period following the birth of an infant to the resumption of cycling(mean post-partum amenorrhoea following birth of a live infant is 208 days, Setchellet al. (2006a))

• Other: not pregnant, lactating or cycling.

Days of the study were split into “mating” and “non-mating”, depending on thepresence (“mating”) or absence (“non-mating”) of females with maximal sexualswellings in the group (termed “receptive” females). Receptive females were presentduring seven of the 13 months of the study in E1 (June–December 2004), and threemonths in E2 (June, September and November 2004). No females cycled in E3. Animalshoused in E1/E2 and E2/E3 were in visual contact with one another, meaning thatreceptive females in one enclosure might have influenced males in the neighboringenclosure. However, we assumed that visual contact would be less influential thancontact with females in a male's own enclosure, and any interactions between en-closures would serve only to reduce the likelihood of finding a significant relationshipbetween the presence of receptive females and fecal iT or red coloration.

Male coloration

The color of the red skin on the nose is closely related to the degree of expression ofother secondary sexual traits in male mandrills (Setchell and Dixson, 2001a,c), and wasused as ameasure of secondary sexual development. We quantified facial color monthlyfor non-anesthetized males using digital images captured using a Nikon Coolpix 5700digital camera and saved as fine quality jpegs. Images required calibration, to accountfor exposure and light drift (Gerald et al., 2001). Colony conditions meant that it wasimpossible either to obtain images of animals in the same frame as a photographicwhite and black standard, or to place a standard in the same position as the animal andcapture a second image immediately following that of the subject. We, therefore, usedonly images takenwhen males were in either an open grassy area or in an open feedingpen, and where color ranged the full spectrum from white to black, and used the“Autolevels” command in Adobe Photoshop 6.0 (Image Mode set to RGB) to define thelightest and darkest pixels in each color channel as white and black. This correctionwasintended to reduce the influence of variation in ambient lighting conditions on colorvalues as far as possible under colony conditions. Once calibrated, the midnasal stripwas outlined in a standard fashion using the polygonal lasso tool in Adobe Photoshop6.0. The mean luminosity and the mean red intensity value (mean number of pixels ateach intensity level) of the highlighted area were measured using the “Image N

histogram” command. Red intensity divided by luminosity of the image best describedthe red color of an image (as ranked by independent observers), and was highly andsignificantly positively correlated with previous measures made using quantified colorcharts (Setchell and Dixson, 2001c).

Fecal sampling

A total of 317 fecal samples were collected from study subjects between January2004 and March 2005. We attempted to collect three samples per month for each male,achieving a mean of 1.6±1.3 samples per male per month, and a total of 20±3 samplesper male. Samples were collected immediately after defecation during either morning(10h00–11h30) or afternoon (15h30–17h30) observation periods. The identity of theindividual, date, time (am or pm) and consistency of the sample were noted, the feceswere homogenized, and a portion (mean±SEM=7.12±1.44 g) stored at −20C in 40 ml of90% ethanol until extraction. Diarrheic samples were not used.

Steroid extraction

Fecal samples were homogenized in their storage ethanol, vortexed for 1 min, andshaken for 3 h. Theywere then centrifuged for 15 min at 2000 rpm. A 2ml aliquot of thesupernatant was dried under N2 at 40 °C, then resuspended in 1 ml EIA Phosphatebuffered saline (0.1 M phosphate buffered saline, pH 7.0, with 0.1% bovine serumalbumin). The rest of the supernatant was stored at −20 °C. The fecal pellet was dried toconstant mass at 60 °C to determine the dry mass of the sample. Extraction efficiencywas determined by measuring recovery of 3H-testosterone (3000 counts per min)added to ten samples prior to extraction. Mean extraction efficiency was 77.6%(SEM 2.4%). Hormone concentrations were corrected for this extraction loss andexpressed as ng of hormone per mg of dry feces. Mean dry fecal mass was 1.23±1.13 g.We allowed for a 24 h time lag to peak steroid excretion when comparing testosteronelevels with other variables (Bahr et al., 2000).

Assay methodology

Fecal extracts were measured for immunoreactive testosterone by microtitreplateenzyme immunoassay (EIA), using an antiserum and labeled testosterone conjugate

(horseradish peroxidase: HRP) provided by Coralie Munro (University of California,Davis). This assay measures testosterone immunoreactivity, comprising multipleimmunoreactive elements, of which native testosterone is most likely a very minorcomponent, we therefore refer to the results as fecal immunoreactive testosterone(fecal iT). We validated the assay by demonstrating parallelism, accuracy, andsensitivity. Serial dilutions of pooled fecal extracts gave displacement curves parallelto that obtained for the testosterone standard for 20–90% binding, indicating that theamount of testosterone measured varied directly with the volume of extract for thisportion of the curve. Accuracy was determined by spiking a low and high quality controlpool with standard preparations in the range 500–31.25 pg. The pool comprisedsamples from several adult and several sub-adult males. The recovery of the standardpreparations was 115.3±7.2% (r2=0.980, n=3 plates). Assay sensitivity was 1.95 pg/well.

We validated the assay biologically in two ways. First, a comparison of fecal iTvalueswith the concentration of free testosterone in serum samples collected during bi-annual captures for a subset of males (n=36 samples from 13 males) showed that thetwo were significantly related (using a general linear mixed model (GLMM) withidentity as a random factor to take repeated samples from the same individual intoaccount: F1,26.6=7.461, p=0.011). Second, fecal iT values for males aged 6–7 yr (n=55samples, mean±SEM 5508±475 ng/g), when the testes are still small, and serumtestosterone values are low (Setchell and Dixson, 2002; Wickings and Dixson, 1992a),were significantly lower than those of prime adult males (n=30 samples for males aged11–12 yr, mean±SEM 8973±6658 ng/g, t83=3.14, p=0.002). Together, these resultssuggest that the assay may reasonably be regarded as indicative of testicular androgensecretion in these mandrills.

Fecal extracts were diluted 1:6 in assay buffer (0.1 M phosphate buffered saline,pH 7.0, with 0.1% bovine serum albumin) and 50 µl aliquots assayed along with 50 µlaliquots of reference standard in doubling dilutions (range 1.95–1000 pg/well). Sampleswere organized by date, and were re-run if duplicates had coefficients of variationgreater than 5%. Samples binding N90% or b20% were re-diluted from the originalextraction as necessary and re-assayed. The intra-assay coefficient of variation for asubset of 30 samples was 2.75±1.71%. Inter-assay variation for high (HQC) and low(LQC) pooled fecal quality controls were high (HQC 35.3%, LQC 34.3%, n=24), indicatingassay drift. However, HQC and LQC concentrations were correlated highly across allplates (rN0.9, n=24), the range of the QCs was similar across all plates, and values forthe same samples on different plates also yielded highly correlated results (rN0.9,n=33).We therefore normalized all data to themean of the quality controls on the sameplate as follows: we calculated the mean of the high and low QCs for each plate, andtook the mean of these values across all plates. We then adjusted the assay results foreach plate by the difference between the mean for the individual plate and theoverall mean for all plates. This procedure reduced inter-assay variation to 8.0% (HQC)and 2.8% (LQC).

Statistical analyses

Fecal iT levels were normalized via log transformation. We used a general linearmixed model (GLMM, in SPSS 15) to assess the effects of predictor variables on bothfecal iT levels and coloration. We included male identity as a random factor, to accountfor the fact that we sampled the same individuals repeatedly, and tested formain effectsand two-way interaction terms. Predictor variables were as follows:

• dominance rank (see above for details)• age (covariate)• presence of receptive females (categorical variable: mating vs. non-mating, see abovefor definitions)

• dominance rank stability (categorical variable: stable vs. unstable, see above fordefinitions)

• the enclosure that a male lived (categorical variable: E1, E2, E3) to detect anydifferences in fecal iT and red coloration that were due to group membership.

Hormone concentrations may change over time when fecal samples are stored inethanol, although these patterns are dampened by storage at sub-zero temperatures(Khan et al., 2002). We, therefore, also included the number of months for whichsamples were stored prior to extraction as a variable in the model. Finally, we alsoincluded whether samples were collected during am or pm observation periods in theanalysis (categorical variable), to detect any influence of circadian rhythms (Beattie andBullock, 1978; Perachio et al., 1977; Plant, 1981).

To examine the relationship between fecal iT and red coloration, we also used aGLMM, with red coloration as the dependent variable, fecal iT as a covariate, andindividual identity as a random factor. Next, we investigated the significant relation-ships revealed in the above analyses at the level of individual males. We exploredrelationships among fecal iT, red coloration, age and rank using correlation analyses(using non-parametric Spearman correlations for analyses involving dominance rank).We investigated differences in fecal iT and red coloration in mating and non-matingperiods, and in stable and unstable periods using paired t-tests.

We used a method developed by Beehner et al. (2006) to investigate temporalrelationships between current and future fecal iT, dominance rank and red coloration.This involves comparing the mean value of one variable for each male in any givenmonth with mean values of a second variable for that month and for each subsequentmonth. We did this pair-wise for the three combinations: fecal iT/dominance rank, redcoloration/dominance rank and fecal iT/red coloration. Because this method involvesmultiple tests, we used Qvalue (Storey, 2002) to calculate the q-value for each test. The

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q-value measures the minimum false discovery rate that is incurred when calling thattest significant, and represents a more balanced approach to the risk of Type 1 and TypeII statistical errors than the Bonferroni correction, particularly when statistical power islow (Storey and Tibshirani, 2003).

All statistical analyses included adult males in E1, E2 and E3 (n=16). Omitting thetwo males in E3 (where females were all contracepted) did not change the significanceof the results.

Results

Results of GLMMs

Neither the number of months for which samples were stored priorto extraction, nor whether they were collected during am or pmobservation periods, significantly influenced fecal iT (Table 2). Using allavailable data, and including male ID as a random factor, we found thatdominance rank and the presence of receptive females influenced bothfecal iT levels and red coloration significantly (Table 2). Male rankstability also influenced fecal iT levels significantly, but did not influencered coloration significantly (Table 2). Male age and the enclosure that amale lived in did not significantly influence either fecal iT or redcoloration (Table 2). There were no significant interaction terms in themodels and we therefore present the results of main effects only.

Again using all available data, and including male ID as a randomfactor, we found that red coloration co-varied significantly with fecaliT (GLMM: F1, 261.4=16.416, pb0.001).

Age

Mean values for red coloration and fecal iT for individual malesacross the study period were significantly positively related (r=0.518,p=0.040, n=16). Fecal iT was not significantly related to age (fecal iT:r=0.246, p=0.341, n=17), although red coloration did increase sig-nificantly with age (r=0.604, p=0.013, n=13).

Dominance rank

When mean values for individual males across the study periodwere compared, the overall relationship between fecal iT and domi-nance rank was positive and significant (rs=0.587, p=0.017, n=16).However, top-ranking males did not necessarily have the highest fecaliT levels in their group (Fig. 1A), and the relationship between fecal iT

and dominance rank was non-significant if groups were examinedseparately (E1: rs=−0.429, p=0.289, n=8; E2: rs=−0.500, p=0.253,n=7; E3 included only two adult males). Male rank and age were notsignificantly correlated (rs=−0.049, p=0.858, n=16).

Of 17 males, 6 showed a net increase in rank across the studyperiod (gaining 1–7 rank positions), 5 suffered a net decrease in rank(falling 1–3 positions), while 5 showed no net change in rank duringthe study. Therewas no significant relationship betweenmean fecal iTand net rank change for individual males (r=−0.087, p=0.748, n=16).

Mean dominance rank and mean red coloration were not signifi-cantly correlated (rs=0.396, p=0.129, n=16, Fig. 1B). This was not dueto younger males being less red, because the partial correlation ofred coloration and rank, controlling for age, was also non-significant(r=−0.409, p=0.130, n=13). In E1 and E3, the top-ranking males werealso the reddest males. However, in E2, the top-ranking male was oneof the least colorful males, while a male with a mean rank of 4.1was most colorful. Here, the top-ranking male was newly dominant(and still developing his color), while the male ranked 4.1 was post-dominant, and had not entirely lost his coloration.

Temporal relationships among dominance rank, fecal iTand red coloration

Whenwe compared a male's mean fecal iT for a given month withhis mean value for dominance rank for that month and for eachsubsequent month, we found that fecal iT was significantly correlated

Table 2Results of GLMMwith male identity as a random factor assessing the effects of differentpredictor variables on fT levels and red coloration

Sourcea Numerator df Denominator df F Sig.

Dependent variable: log fTIntercept 1 13.482 364.450 b0.001Time to extraction 1 264.381 0.007 0.933am vs. pm sample 2 288.062 0.653 0.521Enclosure (E1, E2, E3) 2 17.751 1.552 0.239Receptive females(present/absent)

1 293.675 29.783 b0.001

Dominance rank 1 83.349 9.683 0.003Age 1 8.939 0.241 0.636Rank stability(stable vs. unstable)

1 289.708 4.856 0.028

Dependent variable: red colorationIntercept 1 17.506 47.423 b0.001Enclosure (E1, E2, E3) 2 22.695 0.581 0.568Receptive females(present/absent)

1 253.999 24.095 b0.001

Dominance rank 1 232.411 65.845 b0.001Age 1 16.630 2.446 0.137Rank stability(stable vs. unstable)

1 250.935 1.286 0.258

a Results are presented for main effects only; no two-way interaction termssignificantly influenced either dependent variable.

Fig. 1. Mean log fecal iT (A) and red coloration (B) vs. mean dominance rank across thestudy period for males in the three enclosures. The relationship between fecal iT anddominance rank was significant (Spearman correlation: rs=0.550, p=0.022, n=17). Therelationship between red coloration and dominance rankwas non-significant (rs=0.396,p=0.129, n=16).

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with dominance rank for the current month and for two subsequentmonths (correcting for multiple testing using Qvalue, Fig. 2). A verysimilar pattern was found with the reverse comparison: dominancerank was significantly correlated with fecal iT for the current monthand two subsequent months, although here the correlation was alsosignificant in months 3 and 5 (Fig. 2A).

Current red colorationwas significantly correlatedwith dominancerank for anygivenmonth, and for two subsequentmonths (Fig. 2B). Thereverse relationship was similar: current dominance rank was signifi-cantly correlatedwith red coloration for the currentmonth and for twosubsequent months (Fig. 2B). However, the correlation coefficient be-tween current rank and future red coloration was more negative than

the correlation coefficient between current coloration and future rankfrom month 2 onwards, suggesting that current rank was a betterpredictor of future coloration than vice versa.

Finally, amale's mean fecal iT for any givenmonthwas significantlycorrelated with his red coloration for that month only, but not for anysubsequent months (Fig. 2C). By contrast, however, red colorationwassignificantly correlated with fecal iT for the current month and for 2subsequent months (Fig. 2C), suggesting that red coloration was abetter predictor of future fecal iT than vice versa.

Presence of receptive females

Thepresence of receptive femaleswas linked to a significant increasein fecal iT, with 11 of 14 males showing an increase in fecal iT whenreceptive femaleswere present (paired t13=2.913, p=0.012, Fig. 3A). Thethree males that experienced a decrease in fecal iT during matingperiods ranked 4 or lower. The presence of receptive females was alsolinked to a significant increase in red coloration: 12 of 14 males showedan increase in red coloration when receptive females were present(t13=3.807, p=0.002, Fig. 3B). Of two males who showed a decrease inred coloration, one had recently been deposed as alpha male, and theother was low-ranking (rank 6).

Rank stability

There was a significant increase in fecal iT from stable to unstableperiods (t12=3.305, p=0.006, Fig. 3C). Of 13 males for whom datawere

Fig. 2. Correlation coefficients between males' fecal iT and dominance rank (A), redcoloration and dominance rank (B) and fecal iT and red coloration for current andsubsequent months. Points below the horizontal line represent significant correlationsat pb0.05, after post-hoc correction for multiple tests (see Methods). Sample sizesnecessarily decrease from131 (currentmonth) to 65 (+12months) (A), from111 (currentmonth) to 58 (+12 months) (B), and from 113 (current month) to 56 (+12 months) (C).

Fig. 3. Comparison of (A) fecal iT levels and (B) red coloration in individual males duringperiods when receptive females were absent (no mating) and present (mating). Bothcomparisons are significant: A: paired t13=2.913, p=0.012, B: paired t13=3.807, p=0.002.

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available during both stable and unstable periods,10 increased in fecaliT, while three decreased.

Discussion

In this studywe demonstrated the feasibility and biological validityof a novel tool for assessing fecal iT levels in mandrills. Previousmeasures of serum testosterone levels have yielded important infor-mation, but have been severely limited by the restricted possibilitiesfor sampling, and the influences of capture and anesthesia on serumhormone levels. The non-invasivemethod developed here allows us toaddress new questions and hypotheses in the CIRMF colony, and willalso be useful for studies of wild mandrills, as fecal samples can becollected even when habituation is impossible. Our findings that fecaliT levels, dominance rank and red colorationwere significantly relatedto one another corroborate the results of previous studies based onserum testosterone levels (Setchell and Dixson, 2001a,c), and suggestthat differences in androgen levels in individual males are indeedaccurately reflected in male ornamentation. Here we extend ourprevious findings with more detailed analyses of the influence ofdominance rank, the presence of receptive females, and social stabilityon androgens and coloration for a larger sample of males, and use afiner-grained measure of androgen levels to investigate the temporalrelationships between androgens, rank and coloration.

Dominance rank

A previous study of the CIRMF mandrills found that that alphamales had the highest serum testosterone levels, based on a singlepoint sample taken during the mating season, and the brightest facialcoloration (Setchell and Dixson, 2001c). Serum testosterone was alsosignificantly correlated with facial coloration (Setchell and Dixson,2001c). In the current study, based on a far more comprehensiveinvestigation of androgen levels, together with a more detailed inves-tigation of behavior, we found that fecal iT levels were significantlyrelated to dominance rank, independent of rank stability and themating period. However, in contrast to the results for serum testos-terone, the dominant male did not have the highest androgen levelsin either of the two main study groups. Alpha males were also notnecessarily the reddest male in their social group. These findingsimply that other factors, in addition to dominance rank, may alsoinfluence androgens and coloration. For example, 9 yr old males wereless red than older adult males, newly alpha males were less red, andpreviously alpha males retained some red coloration. This last findingis interesting, as it suggests that post-dominantmalesmay continue tosignal the traits that enabled acquisition of high rank to females (whileexposing themselves to increased aggression from other males).

Positive correlations between androgen levels and dominance rankhave also been reported for wild chimpanzees (Muehlenbein et al.,2004; Muller and Wrangham, 2004), but not for chacma baboons(Beehner et al., 2006). Although alpha males did not necessarily havethe highest androgen levels in this study, the overall associationbetween rank and androgen status suggests that male mandrills maylive in a permanently aggressive context in which they must activelymaintain their dominance status. In such a situation, the benefits ofhigh levels of androgens, in terms of rank-related reproductivesuccess, may always outweigh the physiological costs, in terms ofimpaired immune function (Folstad and Karter, 1992), and increasedmetabolic expenditure (e.g. Marler and Moore, 1988). While thealmost daily presence of receptive females during themating period isan obvious context for aggressive challenge, competition for high rankmay also occur during non-mating periods, for several reasons. First,although mandrills show a seasonal peak in mating between July andSeptember, there is always the possibility that a female will cycle outof season: females cycle and conceive in 11 of 12 months of the year inthe CIRMF colony (Setchell et al., 2002; Setchell andWickings, 2004b).

Second, males may aim to remain dominant until the next matingpeak, as it is extremely unusual for a male to regain high rank once hehas lost it (Setchell et al., 2006b). Third, a male may strive to maintainhis top-ranking position in order to protect previous offspring frominfanticide, which is known to occur in the colony (Setchell et al.,2006b). Indeed, Ostner et al. (2008) have suggested that the threat ofinfanticide leads to the increases in testosterone that they observedduring the birth season in male red-fronted lemurs.

Fecal iT levelswere not related to net rank change in this study. Thisis in contrast to results for chacma baboons, where fecal testosterone ismore strongly associated with changes in rank (rank trajectory) thanwith dominance rank itself (Beehner et al., 2006). This may be due tothe influence of the semi-captive situation and/or to fewer rankchanges in themandrills studied here. Fivemalemandrills in this studyshowednonet rank change across our study,while thiswas the case foronly one male in the baboon study (Beehner et al., 2006). The malebaboons also underwent more dramatic changes in rank than themandrills. Finally, while the baboon study included two immigrantmales, only one male mandrill approximated this situation (the malethat jumped from E1 into E2). Immigrant males may enter thedominance hierarchy at a low level, but have high androgen levels, behighly aggressive, and quickly rise in rank. However, the relationshipbetween net rank change and fecal testosterone was still evident inbaboons when the two immigrant high achievers were removed fromthe analysis (Beehner et al., 2006).

Current fecal iT levels were predictive of both current and futurerank for two months in these mandrills and the relationship betweencurrent rank and future fecal iT was very similar. This suggests that thebehavioral effects of androgens, likely in terms of aggressive behavior,are relatively fast-acting in these mandrills. This is in line with therapid changes in testosterone concentrations that occur following awin or a loss in a dominance interaction (Bernstein et al., 1974; Mazurand Booth,1998), and suggests that the relationship between rank andandrogen status is a relatively tight one. These results for mandrillsagain contrast with those reported for wild chacma baboons, wherecurrent fecal testosterone was more predictive of future rank than ofcurrent rank, and continued to predict future rank over the entire11 month study, while dominance rank was significantly related tofecal testosterone levels for only the current month and one sub-sequent month (Beehner et al., 2006). It seems unlikely that thesedifferences are linked to higher rank stability in themandrills, becauseif current rank correlates significantly with current androgen status inan entirely stable situation, then both relationships between currentrank and future androgens and the reverse (current androgen levels–future rank) should also be significant.

The predictive relationship between dominance rank and redcoloration was also quite short-term, significant for only 0–2 months,and relationships between current fecal iT and future red coloration,and vice versa, were also of relatively short duration. Currentdominance rank predicted future red coloration slightly better thancurrent red coloration predicted future dominance rank. This fits withour observations that the acquisition of top rank precedes develop-ment of full red coloration (this study and unpublished observations).Similarly, loss of dominance rank precedes loss of coloration, whileprevious alpha males retain some red coloration (this study, Setchelland Dixson, 2001a). This time interval may be associated with thetime required for hormone receptor populations to increase (in thecase of rank gain) or decrease (in the case of rank loss) in the skin.

The latter findings have important implications for signalinghypotheses: while red reliably signals both androgen status and rank,possession of red coloration does not lead to high dominance rank.Instead, males that become top-ranking then develop more coloration.Like fleshy ornaments in birds (Faivre et al., 2003; Negro et al., 2006;Velando et al., 2007), red skin coloration appears to be a dynamic traitthat is able to mirror changes in male physiology relatively rapidly. Thisis in contrast to bird plumage, for example,which is producedduring the

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annual moult, and cannot convey up-to-date information concerningmale testosterone levels (Hill, 2006). Thus, male mandrills that basetheir interactions on relative red coloration (Setchell and Wickings,2005), are using an honest signal of their opponent's current androgenstatus, competitive ability, and willingness to engage in a fight. At thesame time, red coloration should allow direct assessment of currentcondition by females (Velando et al., 2007). Females that showpreferences for redder males as mates (Setchell, 2005), are choosingmales that signal the possession of high rank, and possibly also thepossession of high rank for the time taken to develop full coloration.

Presence of receptive females and social instability

Both fecal iT and red coloration increased when receptive femaleswere present. This contrasts with a previous study of the same colony,in which changes in male ornamentation did not appear to relate tothe female reproductive cycle, although serum testosterone didincrease during the mating period (Setchell and Dixson, 2001b).However, the earlier study was based on a small sample size (sevenmales in two groups) and did not take rank changes into account. Herewe show that male rank stability also influenced androgen levelssignificantly, although it did not influence red coloration signifi-cantly. Why this is the case is unclear, although it may be due tothe time delay between changes in testosterone and changes in color(2–3months), which is the approximate length of the unstable periodsin this study.

The observed increases in fecal iT levels when receptive femaleswere present, and during periods of social instability, support thechallenge hypothesis, as these are both likely to be “critical” periods formales (Wingfield et al., 1990). Males compete directly for access toreceptive females during themating season: mate-guarding dominantmales receive frequent challenges from subordinates, while subordi-nates attempting to mate risk aggression from dominant males, andinjuries are more likely to occur in the mating period (Setchell et al.,2006b). During periods of instability, males compete actively to im-prove their dominance rank. However, increases in fecal iT duringthe mating season may also result from the presence of receptivefemales themselves. Testosterone secretion in male mammals, includ-ing primates, increases as a result of exposure to receptive females(review in Dixson, 1998), and a study of ring-tailed lemurs hasdemonstrated an increase in fecal testosterone in response to sexualactivity, but not to male–male aggression (Lynch et al., 2002).

Conclusions

The faecal immunoreactive testosterone assay validated in thisstudy allowed us to make a detailed comparison of fecal androgens,ornamentation and behavior in these mandrills. We found that redcolor in male mandrills is closely linked to fecal androgen levels: fecaliT levels and color are both higher in dominant males, and bothincrease receptive females are present. Fecal iT also increases duringunstable periods. These findings suggest that male mandrills live in apermanently aggressive context, and support the challenge hypoth-esis. Our results also confirm Darwin's observation that when malemandrills are “excited”, they increase in coloration. However, thesecolor changes occur across a longer time-scale than Darwin envisaged,occurring over months rather than immediately.

Acknowledgments

We thank CIRMF for making this study possible. We are grateful tothe staff of the Primate Centre for their help during the study. We aregrateful to Pierre Rouquet, Oliver Bourry, Bettina Salle, Paul Bamba,Philippe Engandja and ChabannelMbou, and to other staff and interns atthe CIRMF Primate Centre for their help during the study. We thankBenoît Goossens and Aurelie Gauthier for the help with fecal sampling,

Lyndsey Skyner and Rob Coleman for their guidance in the laboratory,Alison Forhead and John Waterhouse for their assistance in assayvalidation, and Robin Bernstein for the advice and analyses of freetestosterone. The CIRMF is financed by the Gabonese government, TotalGabon and the Ministère Français des Affaires Etrangères. This studyforms part of a Leverhulme Trust project grant (award no. F/01576/B).

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