Submitted 5 June 2016Accepted 30 September 2016Published 2 November 2016

Corresponding authorRamiro JA Ovejero Aguilarrovejeromendoza-conicetgobar

Academic editorPatricia Gandini

Additional Information andDeclarations can be found onpage 12

DOI 107717peerj2640

Copyright2016 Ovejero Aguilar et al

Distributed underCreative Commons CC-BY 40

OPEN ACCESS

The Ecology of Stress linking life-history traits with physiological controlmechanisms in free-living guanacosRamiro JA Ovejero Aguilar123 Graciela A Jahn4 Mauricio Soto-Gamboa1Andreacutes J Novaro5 and Pablo Carmanchahi3

1 Laboratorio de Ecologiacutea Conductual Instituto de Ciencias Ambientales y Evolutivas Facultad de CienciasUniversidad Austral de Chile Valdivia Chile

2 Laboratorio de Interacciones Ecoloacutegicas Instituto de investigaciones den zonas aacuteridas(CONICET-MENDOZA-LIE-IADIZA) Mendoza Argentina

3Grupo de Investigacioacuten de Eco-fisiologiacutea de Fauna Silvestre (GIEFAS) Asentamiento Universitariode San Martiacuten de los Andes (AUSMA) Universidad Nacional del Comahue(INIBIOMA-CONICET-AUSMA-UNCo) Neuqueacuten Argentina

4 Laboratorio de Reproduccioacuten y Lactancia Universidad de Mendoza (IMBECU-CCT-MENDOZA)Mendoza Argentina

5Programa Estepa-Andino Patagonica-CONICET-PATAGONIA NORTE-INIBIOMA Neuqueacuten Argentina

ABSTRACTBackground Providing the context for the evolution of life-history traits habitat fea-tures constrain successful ecological and physiological strategies In vertebrates a keyresponse to lifersquos challenges is the activation of the Stress (HPA) and Gonadal (HPG)axes Much of the interest in stress ecology is motivated by the desire to understandthe physiological mechanisms in which the environment affects fitness As reported inthe literature several intrinsic and extrinsic factors affect variability in hormone lev-els In both social and non-social animals the frequency and type of interaction withconspecifics as well as the status in social species can affect HPA axis activity result-ing in changes in the reproductive success of animals We predicted that a social envi-ronment can affect both guanaco axes by increasing the secretion of testosterone (T)and Glucocorticoid (GCs) in response to individual social interactions and the ener-getic demands of breeding Assuming that prolonged elevated levels of GCs over timecan be harmful to individuals it is predicted that the HPA axis suppresses the HPGaxis and causes T levels to decrease as GCs increaseMethods All of the data for individuals were collected by non-invasive methods (fe-cal samples) to address hormonal activities This is a novel approach in physiologi-cal ecology because feces are easily obtained through non-invasive sampling in animalpopulationsResults As expected there was a marked adrenal (p-value= 34eminus12) andgonadal (p-value= 0002656) response due to seasonal variation in Lamaguanicoe No significant differences were found in fecal GCs metabolites betweenmalesfemalesseason for the entire study period (p-value= 02839) Despite theseasonal activity variation in the hormonal profiles our results show a positivecorrelation (p-value= 1952eminus11 COR= 050) between the adrenal and gonadalsystem The marked endocrine (r2 = 0806) and gonad (r2 = 07231) response due toseasonal variation in male guanaco individuals highlights the individualrsquos energeticdemands according to life-history strategies This is a remarkable result because no

How to cite this article Ovejero Aguilar et al (2016) The Ecology of Stress linking life-history traits with physiological control mecha-nisms in free-living guanacos PeerJ 4e2640 DOI 107717peerj2640

inhibition was found between the axes as theory suggests Finally the dataset was usedto build a reactive scope model for guanacosDiscussion Guanacos cope with the trade-off between sociability and reproductivebenefits and costs by regulating their GCs and T levels on a seasonal basis suggest-ing an adaptive role of both axes to different habitat pressures The results presentedhere highlight the functional role of stress and gonad axes on a critical phase of a malemammalrsquos lifemdashthe mating periodmdashwhen all of the resources are at the disposal ofthe male and must be used to maximize the chances for reproductive success

Subjects Ecology ZoologyKeywords Stress ecology Reproduction Lama guanicoe Sociality Non-invasive methodsHormonal profiles in wildlife

INTRODUCTIONNatural selection shapes phenotypic traits that permit organisms to respond appropriatelyto intrinsic and extrinsic factors (Darwin 1959 Roff 1992 Stearns 1992 Bozinovic 2002Nespolo Bacigalupe amp Bozinovic 2003 Clutton-Brock et al 2001) Phylogeny and geneticvariation among organisms determine the resource allocation trade-offs among life-history traits (eg age-specific growth reproduction and survival schedules and socia-bility) Environmental ecological and demographic processes (extrinsic factors) createselective pressures that impinge on an organismrsquos lifetime fitness (Stearns 1992 Zeraamp Harshman 2001) Therefore understanding life-history variation among taxa froman evolutionary perspective requires an understanding of the physiological mechanismsthat link genes with phenotype and how extrinsic factors regulate trade-offs betweensurvival and reproduction Furthering our knowledge of ecophysiological mechanismsand particularly endocrine mechanisms is key to understanding how life-history variationand trade-offs arise and are retained (Ketterson amp Nolan Jr 1999 Zera amp Harshman 2001Crespi et al 2013 Becker 2002 Von Holst 1998 Jacobs amp Wingfield 2000)

The stress response is a mechanism by which an individual of a given species copeswith environmental changes (stressors) (Boonstra McColl amp Karels 2001Wingfield 2005Romero Dickens amp Cyr 2009 Ovejero amp Carmanchahi 2012 Ovejero 2013 Boonstra etal 2007) Perception of a stressful situation activates the sympathetic adrenomedullarysystem and the hypothalamic pituitary adrenocortical (HPA) axis (Mostl amp Palme 2002Busch amp Hayward 2009 Sapolsky Romero amp Munck 2000) Activation of the HPA axisstimulates the release of steroid hormones (glucocorticoids GCs eg corticosteronecortisol) GCs in turn activate the mobilization of energy necessary to cope with adverseenvironmental conditions (Dallman et al 2007 Sapolsky Romero amp Munck 2000)

In the last 30 years the term stress has been strongly criticized due to its ambiguityand several authors have researched the allostasis theory and attempted to connectbiomedical and ecological data (McEwen ampWingfield 2003 Romero Dickens amp Cyr2009 Romero Dickens amp Cyr 2009 Allostasis is the daily and seasonal process that allowsorganisms to maintain physiological parameters (homeostasis) within life-sustaining

Ovejero Aguilar et al (2016) PeerJ DOI 107717peerj2640 221

ranges which occur via allostatic mediators (McEwen ampWingfield 2003) The allostaticmodel generated criticism and Romero Dickens amp Cyr (2009) proposed the reactive scopemodel attempting to retain the strengths but avoid the weaknesses of the allostatic modelThe present work contributes to the debate about the applicability of the allostatic modelto nature

Extrinsic factors like anthropogenic disturbance biological invasions or climatechange (in terms of allostasis) can negatively affect behavior reproduction immunefunction and growth through prolonged activation of the HPA axis (Moberg 1991Boonstra amp Singleton 1993 Goymann ampWingfield 2004 Pride 2005 Pereira Duarteamp Negratildeo 2006) Glucocorticoids (GCs) are often considered the allostatic mediatorsof the stress response and they have been the focus of research for decades (SapolskyRomero amp Munck 2000 Ovejero 2013 Cavigelli 1999 Levine 2005Moumlstl Rettenbacheramp Palme 2005 Raouf et al 2006 Romero 2004 Romero amp Remage-Healey 2000 Sheriffet al 2009 Sheriff et al 2011 Smith et al 2012 Soto-Gamboa et al 2009 Tarlow ampBlumstein 2007 Touma amp Palme 2005 Vera Zenuto amp Antenucci 2012Wasser et al2000Wingfield amp Sapolsky 2003) Much less is known however about the role of GCsrsquoin free-living animals (Bonier et al 2009) The role of baseline or stress-induced GC inthe modulation of an individual or populations performance is still essentially unknownUnderstanding social factors and their endocrine correlates (baseline measures of GCsand T) in wild animals has potential implications for wildlife management populationecology reproductive biology and evolution The social environment is therefore oneof the primary sources of information that can induce allostatic responses (BoonstraMcColl amp Karels 2001 Rubenstein amp Shen 2009 Creel et al 2013 Blanchard McKittrickamp Blanchard 2001) Itrsquos expected that in both social and non-social animals the frequencyand type of interaction with conspecifics as well as status in social speciescan affect HPAand HPG axis activity and ultimately the reproductive success of animals

During the breeding season of many mammals males commonly increase testosteronelevels in response to social stimuli such as presence of male competitors or potentialmates (Enstrom Ketterson amp Nolan Jr 1997 Cavigelli amp Pareira 2000 Sinervo et al2000 Ostner amp Kappeler 2002 Soto-Gamboa Villalon amp Bozinovic 2005 Schradin2008 Creel et al 2013) This physiological mechanism (androgen response) modulatedby social environment is likely to be mainly important when considering their roles(testosterone-mediated traits) in the evolution of mating systems and life histories(Wingfield et al 1997 Knapp amp Moore 1997 Faulkes amp Abbott 1997 Hirschenhauseramp Oliveira 2006 Young et al 2006 Rubenstein amp Shen 2009McGlothlin et al 2010Creel 2001 Lofts amp Marshall 1960) In addition testosterone levels can also affect femalepreference (Enstrom Ketterson amp Nolan Jr 1997) thus influencing fitness through bothintra and intersexual selection (Sinervo et al 2000) Although the role of short-termandrogen changes has not been completely described these have long been associatedwith territoriality (McGlothilin et al 2010) The lsquolsquoChallenge Hypothesisrsquorsquo (first describedin bird studies) predicts that androgen levels and agonistic interactions are positivelyrelated and are socially-context dependent (Wingfield et al 1997) Among mammalsthese predictions have recently been tested and a positive correlation was found between

Ovejero Aguilar et al (2016) PeerJ DOI 107717peerj2640 321

social cues and testosterone levels associated with territorial defense group formationsocial dominance and hierarchy formation during the breeding season (Muller ampWrangham 2004 Creel et al 2013) Consequently social stressors provide a contextwhere the individual must respond to a series of repeated stressors over time dependingon the frequency and intensity of the stimulus and this can lead to various physiologicalproblems (see Sapolsky 2002 Cocircte 2000)

Much of the variation in life histories strategies reflects individualsrsquo phenotypicresponses to environmental challenges and perceived risks The adrenal and gonadalsystem strongly influence behavior control the annual cycle of development modulatebehavioral and physiological responses to the environment and establishes importantincompatibilities in life stages It provides a model for studying the connections betweenmammal physiology and life history Guanacos (Lama guanicoe) are the largest socialartiodactyls of South America and these organisms have a polygynous resource-defensemating system This wild and endemic camelid has been successfully used as a studymodel of behavioral interactions De Lamo et al 1998 Franklin 1983Marino amp Baldi2008 Taraborelli et al in press ecological studies (Puig amp Videla 1995 Bank Sarno ampFranklin 2003 Puig et al 2008 Acebes et al 2009 Ovejero et al 2011 Ovejero 2013Marino amp Baldi 2014 Radovani et al 2004 Puig amp Rabinovich 1995 Raedeke 1979Young amp Franklin 2004) and management (Montes et al 2005 Bonacic amp Macdonald2003 Zapata et al 2004) but information about the guanacorsquos physiological ecologyremains scarce More recently guanacos have also been used as a model of stress responsea study was conducted to determine how management including handling shearingand release induce changes in circulating steroid hormones (Carmanchahi et al 2011)however this work was based on an invasive sampling method

To understand how physiology mediates the relationship between life history andthe environment we predict that (A) Social environment positively will affect the HPGand HPA axes by raising the secretion of T and GCs due to individual social interactionsand the energetic demands of the breeding season (B) Male guanacos will show seasonalvariation in the activity of their adrenal and gonadal systems which perform incompatiblefunctions in different life stages (survival vs reproduction) (C) Prolonged elevatedbaseline levels of GCs in male guanacos due to long periods of intense social interactionwill be detrimental costly and decrease fitness and we would expect that in this caseHPA inhibits the activity of the HPG Together these predictions provide a descriptionof the reactive scope model and an understanding of how physiological mechanismsmight constrain patterns of variation in wild South-American camelid life historiesThese predictions might also provide the basis for future mammalian studies targeted atunderstanding phenotype-environment interactions

METHODSEthics statementThe present study did not warrant capturing or handling protected or endangered ani-mals All of the data for individuals were collected by non-invasive methods

Ovejero Aguilar et al (2016) PeerJ DOI 107717peerj2640 421

(fecal samples) to address hormonal activities This is a novel approach in physiologicalecology because feces are easily obtained through non-invasive sampling in animalpopulations This is important because the handling necessary for collection of plasmacan confound estimates of baseline GC measurements The described field studies werecarried out in a protected area with a permit (filesNO 4350-000019112Res 238) from theDRNR (Direccioacuten de Recursos Naturales Renovables Mendoza)

Non-invasive methods to address hormonal activitiesFresh fecal samples were collected from 334 wild adult male guanacos from La Payuniareserve (36000S and 36360S 68340W and 69230W South of Mendoza ProvinceArgentina) The samples were collected during 7 field surveys (15 days each month)over the course of a year Fresh feces samples were collected from focal individuals(one sample per each individual) and were labeled according to the sample locationtime sex age and social structure (family group bachelors group and solitary male)To slow microbial activity and to reduce immunoreactivity problems the samples werefrozen immediately in liquid nitrogen and stored atminus20 C until the time of analysis(Laboratorio de reproduccioacuten y lactancia-LARLAC-IMBECU-CONICET-MENDOZA)Fieldwork was conducted during the breeding and non-breeding months to assessseasonal variation in hormone levels We choose this period (from September to March)because during the beginning of the breeding season guanaco males are exposed tointense social instability caused by agonistic interactions with other males due to territoryestablishment and hierarchies (Ovejero 2013) and at the end males are exposed to femaledefense interactions This suggests that for guanaco males female or territory defensestrategies demand higher energetic costs This is expected if one assumes that defendinga female or territory are the principal objective (and target) of a male that wants tomaximize the probabilities of leaving an offspring We expected that this behaviouralexpression have physiological mechanism that could explain this difference During thenon-breeding season (from April to August) a period with less intense social interactionswe expected lower activity on hormonal mechanism

Furthermore 100 of the sampling surveys were done in the same sites Steroids wereextracted from lyophylized fecal samples according to a protocol developed for fecalsteroid metabolite extraction for free-range ungulates (for more detail see Ovejero 2013Buchanan amp Goldsmith 2004Mateo amp Cavigelli 2005) The commercial125I-cortisoland 125I-testosterone RIA-KITS (BECKMAN Coulter Company IM-1841IM-1119-Immunotech- Prague Czech Republic) were used to quantify the levels of cortisol andtestosterone in the samples (for more detail see Ovejero 2013) The analytical sensitivityof the cortisol assay is 3075 ngmL The antibody used in the immunoassay is highlyspecific for cortisol (100 cross-reaction) The cross-reactivity to other naturally occur-ring steroids is very low for example aldosterone lt01 84 corticosterone cortisone15 11-desoxy-cortisol 18 etc The analytical sensitivity of the testosterone assay is0025 ngmL The antibody used in the immunoassay is highly specific for testosterone(100 cross-reaction) The cross-reactivity to other naturally occurring steroids is

Ovejero Aguilar et al (2016) PeerJ DOI 107717peerj2640 521

very low for example 000014 aldosterone estrone lt03 008 androsterone 5-Dihydrostestosterone 10 α 11β-Hydroxytestosterone 2 corticosterone 84 15cortisone cortisol 11-deoxy-18 etc In addition all samples were assayed in triplicate(extraction efficiency test) and duplicate (binding and parallelism test) and reanalyzedwhenever the resulting coefficient of variation exceeded 20 Intra- and inter-assaycoefficients of variation of GCs-immunoassay were 65 and 112 and for testosteroneimmunoassays were 95 and 134 respectively

Statistical analysesWelch two sample T-tests were used to evaluate the seasonal variation response of steroidhormones Furthermore two-way ANOVAs were used to evaluate the interaction betweensex and season Generalized linear models were developed (n polynomials function=f (x)= anXn

+an1minus1Xnminus1+anminus2Xn

minus2+ ) to describe the hormonal profiles and theirseasonal variation in response to the environment Moreover a Pearsonrsquos correlation wasused to test the activity association between the adrenal and gonadal system throughoutthe year All statistical analyses were conducted using R statistical software version 310(R Development Core Team 2012)

RESULTSEcological process and fecal-hormone profiles in wild guanacosAs expected there was a marked adrenal (t = 74016df = 302174 p-value= 1344eminus121-α (090)= 7661734ndash12057391 mean= 25758641589907 breeding and non-breeding season respectively) and gonadal (t = 32873df = 28948 p-value= 00026561-α(090)= 06213ndash19508 mean= 32098101923700 breeding and non-breedingseason respectively for male groups) GCs and T response due to seasonal variation inLama guanicoe individuals This highlights the individualrsquos energetic demands accordingto life-history strategies No significant differences were found in the amounts of fecalGC metabolites between malesfemalesseason for the entire study period (F = 10757df = 143529 p-value= 02839 male nggr= 22421-α (090)= 16887 std-e= 086n= 251 femalenggr= 20711-α (090)= 26079 n= 76 std-e= 133) This resultwas not expected as it was thought that there would be a difference in the allocation ofresources to cope with the different roles that that males and females have in a populationDespite the seasonal activity variation in the hormonal profiles our results show a positivecorrelation (t = 71524df = 184 p-value= 1952eminus11 CI= 03457400 05719168COR= 050) between the adrenal and gonadal system both peaks of activity concurwith the reproductive season Our last prediction states that prolonged elevated baselinelevels of GCs due to long periods of intense social interaction are detrimental costly anddecrease fitness it is expected that in this context HPA inhibits the activity of the HPGThe marked endocrine (y = 00743x5minus 19677x4+ 19136x3minus 82442x2+ 1474x minus58902r2 = 0806) and gonad (y = 00041x5minus 01102x4+ 10889x3minus 46819x2+7914x minus 07892r2 = 07231) response due to seasonal variation in male guanacoindividuals (Fig 1) highlights the individualrsquos energetic demands according to life-historystrategies This is a remarkable result because no inhibition was found between the axes as

Ovejero Aguilar et al (2016) PeerJ DOI 107717peerj2640 621

Figure 1 HPA ampHPG axis activity patterns throughout the study period Blue line the seasonal base-line fecal testosterone profile (defined by y = 00041x5minus 01102x4+ 10889x3minus 46819x2+ 7914x minus07892r2 = 07231) Pink line the seasonal baseline fecal cortisol profile (defined by y = 00743x5 minus19677x4+ 19136x3minus 82442x2+ 1474x minus 58902r2 = 0806) There was marked adrenal and gonadalactivity during the breeding and non-breeding (CI= 7661734ndash12057391 mean= 25758641589907 re-spectively) season respectively for the male groups the GCs and T response was due to seasonal variationin the guanacos highlighting the individualrsquos energetic demands according to life-history strategies

theory suggests Finally the dataset was used to build a reactive scope model for guanacossee Fig 2 Most studies measure only one physiological mediator at a time here theconcentration of two mediators is placed on the y-axis for a given time point This wasdone because these mediators are correlated in the functional role that each one plays inthe normal reactive scope range In other words both mediators encompass responsesto cope with predictable and unpredictable changes in the environment Our resultshows the ranges of predictivereactive homeostasis and homeostatic overloadfailureThese ranges consist of the seasonal set of point ranges for the physiological mediatorsThe mediators are correlated in terms of the functional role that each one plays in thenormal reactive scope range In other words both mediators encompass responses forcoping with predictable and unpredictable changes in the environment The values ofeach mediator are presumed to exist in four general ranges The minimum (5ndash10 Nggrfor C 1ndash2 Nggr for T) and maximum (20ndash35 Nggr for C 3ndash4 Nggr for T) concentrationof the mediator was presumed to constitute a threshold The results show the ranges ofpredictivereactive homeostasis (PHRH) and homeostatic overloadfailure the stress-gonadal and feedback response indicates the maxndashmin activity patterns for HPA amp HPGaxis during one year These results consist of the seasonal set of point ranges for thephysiological mediators

Ovejero Aguilar et al (2016) PeerJ DOI 107717peerj2640 721

Figure 2 The reactive scope model for guanacosMost studies measure only one physiological mediatorat a time here the concentration of two mediators (cortisol-C testosteroneT) is placed on the y-axis ata given time point The mediators are correlated in terms of the functional role that each one plays in thenormal reactive scope range In other words both mediators encompass responses for coping with pre-dictable and unpredictable changes in the environment The values of each mediator are presumed to ex-ist in four general ranges The minimun (5ndash10 Nggr for C 1ndash2 Nggr for T) and maximun (20ndash35 Nggrfor C 3ndash4 Nggr for T) concentration of the mediator was presumed to constitute a threshold The resultsshow the ranges (double arrows) of predictivereactive homeostasis (PHRH) and homeostatic overload-failure the stress-gonadal and feedback response (gray circles) indicates the max-min activity patternsfor HPA amp HPG axis during one year The C activity patterns in guanacos shows two peak of RH (dottedlines) in the reproductive season between AgO males adopts a territory-defense strategies at the begin-ning of the reprod season during JaF males changes to a female-defense strategies at the ends of the re-prod season These results consist of the seasonal set of point ranges for the physiological mediators

DISCUSSIONOur results confirm the prediction that lsquolsquoSocial environment positively affects the HPGand HPA axes by raising the secretion of T and GCs due to individual social interactionsand the energetic demands of the breeding seasonrsquorsquo Further to this our results suggestthat social environment and breeding season are stressful scenarios for male guanacosThese scenarios are seen to produce associated costs such as increased metabolic rate andenergy expenditure due to territorialfemale defense (male-reproduction strategies) malesocial dominance hierarchies reproduction and increased exposure to predators duringthe breeding season If the data value observed in March is taken as a baseline cortisollevel cortisol is seen to increase by one and a half times during the non-breeding seasonand three times during the breeding season If the March testosterone measurementis taken as the basal testosterone level the same pattern is seen the hormonal profileincreases during the non-breeding and breeding seasons This highlights the importanceof the functional roles of the HPA and HPG axes during the reproductive period as well

Ovejero Aguilar et al (2016) PeerJ DOI 107717peerj2640 821

as the adaptive roles they play in the seasonal pattern observed allowing the species tomeet environmental challenges as well as challenges throughout its life cycle

From the results gathered in this study different inmunoreactive GCs and T secretionpatterns were observed according to the mating system and reproduction strategies (terri-torial defense and female defense) that guanaco males (of a migratory population) adoptduring the breeding season (Ovejero 2013) Our results highlight the functional role ofthe stress and gonad axes during the critical mating period when all of the resources at amalersquos disposal must be used to maximize the chances of reproductive success Generallyit appears that T is immediately involved in aggression associated with reproductionrather than with other forms of aggression (eg anti-predator aggressionMoyer 1968Marler et al 1988) The data here presented support the correlation between T patternsand reproductive strategies (establishment and maintenance of a breeding territory andmate-guarding) proposed byWingfield et al (1990) for male birds However this studyextends the findings of Wingfield et al (1990) to mammals In 1984 the same authorsproposed that males of polygynous species may have higher levels of T for longer periodsduring the breeding season than do males of monogamous species Our results highlightthe functional role that T levels have in determining and maintaining the polygynousmating system in guanacos but we predict that this pattern could be altered in guanacopopulations that coexist with human activities that fragment the guanacorsquos normaldistributional range For example the use of fences by rural settlers has a direct impacton the space used by guanacos and it is possible that this could promote some groups(within a population) to adopt monogamous behavior (Marino amp Baldi 2008)

On the other hand our results highlight the notorious decrease in the activity patternof the stress and gonadal axes during the non-breeding season This result was not ex-pected because individuals must cope with another critical phase of autumnal migrationat this time therefore we expected that all of the resources (in terms of energy) would beavailable to maximize the chances for survival success during the migration trip Theseresults confirm our prediction that lsquolsquoguanacos show a seasonal variation in the activityof their endocrine and gonadal systems and incompatible functions (reproduction anself-maintenance) are established during different life stagesrsquorsquo In winter food intakeby birds and mammals needs to be geared towards preventing starvation while at thesame time maintaining an optimum body mass to permit rapid escape from predatorsGC concentrations may be the critical factor determining the size of the fat reserves andeither environmental severity or predation risk (Wingfield et al 1990) The GC hormonecorticosterone is thought to be important in the physiological orchestration of avianmigration because of the identified elevated level of activity of the avian adrenal glandeither prior to or during migration (Naik amp George 1963 John 1965 Peczely 1976)Preparation for migration and for winter requires a period of hyperphagia and a layingdown of fat reserves (for a review see Holberton 1999) In birdsWingfield amp Silverin(1986) Astheimer Buttemer amp Wingfield (1992) and mammals Dallman et al (1993) GCsplay a vital role in foraging behavior and hyperphagia with low to moderate concentra-tions stimulating feeding behavior As is noted by OrsquoReilly amp Wingfield (1995)migratingbirds show increased baseline concentrations of GCs but a reduced stress response An

Ovejero Aguilar et al (2016) PeerJ DOI 107717peerj2640 921

adaptive explanation for this pattern is the migration modulation hypothesis HolbertonParrish amp Wingfield (1996) and Holberton (1999) It proposes that higher than averageconcentrations of baseline GCs are expressed prior to and during migration to facilitatemigratory fattening However the stress response is lowered when challenged so thatthe negative catabolic effects of high GCs on skeletal muscle do not occur thus criticalskeletal muscles are preserved for flight during migration As such this hypothesis mayexplain the non-breeding season hormonal profiles generated in this study but moredata is needed to test these assumptions Another explanation may suggest that migration(in terrestrial system) could be regulated by other endocrine mechanisms (and otherhormones) involved in generating fat reserves and foraging behavior such as those thatinvolve the thyroid gland Schwabl Wingfield amp Farner (1984)Mattocks (1976) showsthat gonad hormones (at least in some species) have a functional role (fat deposition)before spring migration This may suggest that the increased activities of the gonad axiscompared with the stress axis in guanacos could have an important role in the lsquolsquomigrationto and from the winter rangersquorsquo but to confirm this prediction more experimental and fieldstudies would be needed

The last prediction of this study that lsquolsquoprolonged elevated baseline levels of GCs due tolong periods of intense social interaction will be detrimental costly and decrease fitnessrsquorsquowas not validated by the results gathered Our interest in the relationships between socialor environmental conditions and stress-gonadal hormone concentrations (for bothsocial and non-social species) is motivated by a desire to understand the physiologicalmechanisms by which the environment affects fitness However understanding therelationship between hormonal profiles and fitness is not straightforward therefore thisstudy attempts to obtain proxies that would help us begin to resolve this relationshipWe consider that taken together the interplay between stressgonad hormones acrossseasonal variation could be an interesting proxy to lsquolsquoestablishrsquorsquo causal relationshipsbetween hormonal levels and ecological correlates For example some authors have statedthat prolonged elevated baseline levels of GCs are commonly viewed to be detrimentalcostly and decrease fitness (Bonier et al 2009) However there is scarce evidence forthis negative association Part of the problem with this approach is the variation amongspecies in terms of their hormonesfitness relationships and this variation can beattributed to a variety of demographic social (eg social rank age sex group size)or environmental factors (eg resource availability) Additionally for a variety ofvertebrate taxa seasonal variation in GC levels has been reported to be due to varyingfood availability temperature rainfall and tourist activity (Romero 2002 Tempel ampGutierrez 2004 Kenagy amp Place 2000) however it still remains unclear if this holds truefor mammals The other issue is that many of the studies of HPA-fitness relationshipsreviewed by Bonier et al (2009) were conducted during the breeding season alone Thestudy herein presented provides a full set of data (hormone profiles) across seasonalvariation throughout the course of an entire year As expected guanacos cope with thistrade-off between sociability and reproductive benefits and costs by regulating their GCsand testosterone levels on a seasonal basis like other mammals (Boonstra 2005) and theymay also experience fine adjustments on a daily basis We suggest that elevated levels of

Ovejero Aguilar et al (2016) PeerJ DOI 107717peerj2640 1021

GCs in guanacos due to highly social interactions during the breeding season may playa fundamental role in the regulation of enhanced metabolic needs during reproductionGuanaco males like other iteroparous males exhibit high concentrations of cortisola gonadal axis that is not inhibited by high GC concentrations so the last predictionpresented in this work must be rejected Furthermore during the breeding season ourresults show two GC peaks of activity one at the beginning and another at the end of thebreeding season In the former guanaco males are exposed to intense social instabilitycaused by agonistic interactions with other males due to territory establishment andhierarchies In the latter guanaco males are exposed to female defense interactionsThis suggests that for guanaco males female defense strategies demand higher energeticcosts when compared to territory defense strategies This is expected if one assumes thatdefending a female (rather than a territory) is the principal objective (and target) of amale that wants to maximize the probabilities of leaving an offspring Moreover birth ratedata for the population was collected from 2007ndash2008 (hormonal measures)-2009 so ifone expects a negative relationship between the hormonal profiles one would also expectto see a negative value for the proportion of offspring per adult The birth rate values forthose years were 014 028 and 029 respectively thus with these results one can expectthat there is no detrimental effect on individual-population fitness

The lesser HPA axis activity measured during the non-breeding season confirms theprediction lsquolsquothat reproduction and sociability may be long-term and predictablersquorsquo Thisis likely the case because this period is associated with fewer social interactions Theindividual hierarchies are more relaxed because the animals are preparing to migrate (ANovaro pers comm 2015) and the species strategy at this time is to survive and to notreproduce

Finally from the data collected here a reactive scope model for guanacos has beengenerated The results show the ranges of predictivereactive homeostasis and homeostaticoverloadfailure ranges these consist of the seasonal set of point ranges for the physio-logical mediators For guanacos the normal reactive scope is described with two GCs andT set point ranges The model was made to reflect the natural progression of life-historystages such as breeding Furthermore the minimum concentration of the mediator waspresumed to constitute a threshold Subsequently a GC and T threshold was proposedand below this limit it is seen that the individualpopulation enters into homeostaticfailure This threshold is predicted to indicate the minimum concentration of GCs andT mediator necessary to sustain guanacos seasonal life changes (in natural conditions)However more studies are needed to test this prediction On the other hand abovethe upper end of the normal reactive scope when a physiological mediator exceeds thereactive homeostatic range the individualpopulation enters into homeostatic overload(pathological states) This threshold is predicted to indicate the maximum concentrationof GCs and T mediator necessary to sustain seasonal life changes in guanacos (in naturalconditions) However more studies are needed to test this prediction especially consid-ering that pharmacological stimulation may allow individuals to reach a higher thresholdthough this would not be considered within the scope of natural changes

Ovejero Aguilar et al (2016) PeerJ DOI 107717peerj2640 1121

CONCLUSIONSKnowledge of physiological and behavioral mechanisms is key to understanding how life-history variation and trade-offs might arise and help organisms cope with environmentalvariation Mechanistically trade-offs result from the need to differentially allocate limitedresources to traits like reproduction versus self-maintenance with selection favoring theevolution of optimal allocation mechanisms Guanacos cope with the trade-off betweensociability and reproductive benefits and costs by regulating their GCs and T levels on aseasonal basis This seasonal regulation suggests the adaptive and functional role of bothaxes in regulating energy allocation for predictable life-history events (like reproduction)During breeding and event with highly social interactions elevated baseline levels of GCsand T may actually be predictive of high reproductive success From this we proposethat guanacos anticipate stressors with acute impacts and the duration is moderated byphysiological consequences (ie reproduction) During the non-breeding season a periodwith less intense social interactions lower baseline levels of GCs and T may actually bepredictive of a high probability of survival Our study provides a model for further studyof the links between physiology and life history

Studying both the HPA and the HPG axes was an informative approach to understandthe functional mechanisms that animals use to cope with stress Studying these two axessimultaneously is important because the key circulating steroid hormones of the stressaxis GCs influence the expression of approximately 10 of the genome Some of thetargets of GCs include genes controlling metabolism growth repair reproduction andmanagement of resource allocation (Le et al 2005) However both axes play fundamentalroles not just when animals are experiencing stress but also during normal periods ofsurvival and growth Therefore these axes mediate the adaptation of the organism to itsenvironment At the individual level the stress axis plays a key role in allowing animals torespond to the changes and challenges of both environmental certainty and uncertaintyAt the species level the stress axis plays a central role in evolutionary adaptation toparticular ecological pressures

ACKNOWLEDGEMENTSWe appreciate the comments and suggestions made by Dr Francisco Bozinovic DonaldR Strong and Tim Coulson on earlier versions of this manuscript We thank Nelly Horakand Emily Giles for help with the English language The park rangers and other staff of LaPayunia Provincial Park helped us throughout different stages of this work

ADDITIONAL INFORMATION AND DECLARATIONS

FundingThis study has been funded by the Rufford Small Grant Foundation (RSGF 120608)The Scientific Research SocietySigma-Xi and FONDECYT-CONICYT-PROGRAM(No 3140237) We thank for partial support by FONDECYT 11060132 (MSG)rsquorsquo The

Ovejero Aguilar et al (2016) PeerJ DOI 107717peerj2640 1221

funders had no role in study design data collection and analysis decision to publish orpreparation of the manuscript

Grant DisclosuresThe following grant information was disclosed by the authorsRufford Small Grant Foundation RSGF 120608Scientific Research SocietySigma-XiFONDECYT-CONICYT-PROGRAM 3140237FONDECYT 11060132

Competing InterestsThe authors declare there are no competing interests

Author Contributionsbull Ramiro JA Ovejero Aguilar conceived and designed the experiments performed theexperiments analyzed the data contributed reagentsmaterialsanalysis tools wrote thepaper prepared figures andor tables reviewed drafts of the paperbull Graciela A Jahn conceived and designed the experiments contributed reagentsmateri-alsanalysis tools reviewed drafts of the paperbull Mauricio Soto-Gamboa and Andreacutes J Novaro contributed reagentsmaterialsanalysistools reviewed drafts of the paperbull Pablo Carmanchahi conceived and designed the experiments reviewed drafts of thepaper

Animal EthicsThe following information was supplied relating to ethical approvals (ie approving bodyand any reference numbers)

The present study did not warrant capturing or handling protected or endangeredanimals All of the data for individuals were collected by non-invasive methods (fecalsamples) to address hormonal activities

Field Study PermissionsThe following information was supplied relating to field study approvals (ie approvingbody and any reference numbers)

The described field studies were carried out in a protected area with a permit (filesNO4350-000019112Res 238) from the DRNR (Direccioacuten de Recursos Naturales RenovablesMendoza)

Data AvailabilityThe following information was supplied regarding data availability

The raw data has been supplied as a Data S1

Supplemental InformationSupplemental information for this article can be found online at httpdxdoiorg107717peerj2640supplemental-information

Ovejero Aguilar et al (2016) PeerJ DOI 107717peerj2640 1321

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