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1 ( ( ( ( ( ( ( ( SocialBehaviorandGutMicrobiotainWild Red7belliedLemurs(Eulemur(rubriventer) (In SearchoftheRoleofImmunityintheEvolutionof Sociality( ProGradu AuraRaulo DepartmentofBiosciences DivisionofEcologyandEvolutionaryBiology UniversityofHelsinki May2015 1

2 Table&of&Contents& 1. INTRODUCTION GUTMICROBIOTAANDTHEHOLOBIOMEAPPROACH SOCIALBEHAVIORANDMICROBETRANSMISSIONINASOCIALGROUP 5 SOCIALBEHAVIORANDDIVERSITYOFMICROBIOTA 5 SOCIALBEHAVIORANDSIMILARITYOFMICROBIOTA 6 SOCIALBEHAVIORANDPRESENCEOFCERTAINBENEFICIALMICROBES STRESSANDMICROBIOTA AIMS STUDYSPECIESANDSTUDYSITE MATERIALSANDMETHODS COLLECTIONOFBEHAVIORALDATA 12 CONSTRUCTINGSOCIALITYINDICESANDINTERACTIONMATRICES FECALSAMPLECOLLECTIONANDANALYSISFORCORTISOLMETABOLITES FECALSAMPLECOLLECTIONANDGUTMICROBIALDNAANALYSIS STATISTICALANALYSES 16 DESCRIBINGMICROBIALDATA 16 CLUSTERINGOFMICROBIALDATA 17 ANALYSESUSINGDISSIMILARITYINDICES 18 ANALYSESUSINGDIVERSITYANDRICHNESS 18 WHICHBACTERIALTAXAAREBEHINDTHETREND? RESULTS ENVIRONMENTAL,DEMOGRAPHICANDGENETICFACTORSAFFECTINGMICROBIOTA SEASONALCHANGEINGUTMICROBIOTA GUTMICROBIALCOMPOSITIONANDINTERACTIONMATRICES GUTMICROBIALCOMPOSITIONANDSOCIALITYINDICES GUTMICROBIALCOMPOSITIONANDSTRESSHORMONES DISCUSSION ENVIRONMENTALANDDEMOGRAPHICVARIATIONINGUTMICROBIOTA GROUPASPECIFICMICROBIOTA MICROBIALTRANSMISSIONINASOCIALNETWORK GUTMICROBIOTA,HPAAAXISANDINDIVIDUALDIFFERENCESINSOCIALBEHAVIOR SEASONALCHANGEINGUTMICROBIOTA FUTUREDIRECTIONSANDAFRAMEWORKFORSOCIALTRANSMISSIONOFBENEFICIAL GUTMICROBIOTA CONCLUSIONS ACKNOWLEDGEMENTS 36 2

3 1. Introduction& 1.1. Gut&microbiota&and&the&holobiome&approach&& Microbialcommunitieslivinginthegut,skinandglandsofvertebratesareofgrowing scientificinterest,duetotheiremergingroleasafunctionallinkbetweentheindividual anditssurroundingecosystem.recentdevelopmentsinmolecularmethodshave inspiredagreatamountofresearchonmutualisticmicrobiotainalltaxa.infields rangingfromendocrinologyandphysiologytobehavioralandevolutionaryecology,this new holobiomeapproach (Rosenbergetal.2007)focusesonthereciprocaland dynamicrelationshipsofmutualisticmicrobiotaandtheirhost. Mostofourcurrentknowledgeongutmicrobiotacomesfromhumanmedical researchfocusingonpathogens,whilethemutualisticmicrobiotaofwildvertebrate specieshasreceivedmuchlessattention.furthermore,whiletheeffectsofmicrobiota onhealthareincreasinglyacknowledged,currentresearchisforthemostpartlacking evolutionaryperspectiveonthistopic(butseestillingetal.2014).itisknown, however,thatvertebrategutmicrobiotahaveanimportantinfluenceonindividual immunity(reviewedbyhooperetal.2012)andisneededforprovidingenzymesfor digestion(mackie2000).forexampleruminantsareknownfortheirbacteria7rich rumenthatslowsdownthepassageoffoodmaterialtoenableefficientfermentationof cellulosebymutualisticmicrobes(mackie2000).newevidencealsosuggeststhat consistentwiththe"fermentationhypothesis"(alboneetal.1974,albone1990), microbiota,especiallythatlivinginmammalianscentglands,affectssocialrecognition bysynthesizingpheromones(sinetal.2012,theisetal.2012,2013,douglas&dobson 2013).Inadditiontothispotentialtoalterbehaviorindirectly,anorganism s microbiotaisknowntohavedirecteffectsonitsmoodandbehavior(bravoetal.2011, Ezenwaetal.2012).Thedirecteffectsofgutmicrobesonbehaviorlikelyhappen throughmicrobe7gut7brain(mgb)7axis,aphysiologicalpathwayinvolvingthevagus7 nerve(bravo2011,montiel7castroetal.2013). Mutualisticmicrobesplayanotableroleinananimal sphenotype,butthe determinantsofindividual smicrobiotaarescantlyknown.studiesofwildspecies, 3

4 laboratoryrodentsandhumansyieldinconsistentresultsregardingtherelative importanceofgeneticbackground,maternaltransmission,andsubsequent environmentalcontactsindetermininggutmicrobialcompositionsinceallthesefactors canhaveamajorimpactonthemicrobiota(pendersetal.2006,lanyonetal.2007, Khachatryanetal.2008,Turnbaughetal.2009a,b,Bensonetal.2010,Degnanetal. 2012,Ubedaetal.2012,Stevensonetal.2014,Leclaireetal.2014).Firstly,itisknown thatthecompositionofmicrobiotacanhaveageneticbasis.forexample,immunity7 relatedgenesareresponsibleforselectivelyenablingtheestablishmentofcertain microbesinthebody,andthusformthephysiologicalbasisformicrobiotainmice (Lanyonetal.2007,Zomeretal.2009,Bensonetal.2010).Ontheotherhand, relatednesswithinhumanfamiliesdoesnotpredictsimilarityofgutmicrobiota (Turnbaughetal.2009b).Secondly,themicrobesthatcolonizeananimalbodycome primarilyfromthemother.manygutmicrobescannotliveoutsidethehostbody,and mustrelyentirelyonmaternaltransmission(reviewedbyfunkhouser&bordenstein 2013).Thus,aswithmanyphenotypictraits,earlylifeisimportantindetermining microbiota.forexamplebirth(mandaretal.1996)andearlypostnatalexposureby nursing(jinetal.2011,fernandezetal.2013)orevenbyeatingsoilorfeces(troyer 1984b,Soave&Brand1991)havebeenconsideredthemostimportantperiodsof transmissiondeterminingindividualmicrobiota.inaddition,recentevidencesuggests thatpartofthemicrobiotacanbeacquiredpriortobirth(funkhouser&bordenstein 2013).Lastly,gutmicrobialcompositionisneverthelessknowntofluctuatewith changingdiet,hormonalprofiles,seasonandenvironment,hencehighlightingthe environmentalimpactonmicrobiota.whilesomeoftheseeffectsareduetoachanging dietandtheabioticenvironment,socialenvironmentislikelyanimportantfactoras well.thusamixtureofvertical(genetic,maternal)andhorizontal(social, environmental)transmissionmostlikelydetermineindividualmicrobiota.forexample, bothspottedhyenas(crocuta(crocuta)andchimpanzees(pan(troglodytes)havebeen foundtohavemoresimilarmicrobiotawithingroupsduetosharedenvironmentand socialcontact,butimmigrantindividualsstillretainedaslightlydifferentmicrobiota afteryearsofco7living(degnanetal.2012,theisetal.2012) 4

5 1.2. Social&behavior&and&microbe&transmission&in&a&social&group& Microbialtransmissionhasbeenmainlystudiedinthecontextofpathogensand parasites.itisknownthatoneimportantfactoraffectingpathogenorparasite transmissionissocialcontactandthisisconsideredoneofthemaincostsofsociallife (Alexanderetal.1974,Mölleretal.1993,Altizeretal.2003).Forexamplevulnerability toinfectionsincreaseswithincreasinggroupsizeininsects(turnbulletal.2011). However,itislessoftensuggestedthatinadditiontoentailingimmunologicalchallenge, socialitymightactuallyalsoenhanceimmunitythroughtransmissionofbeneficial microbes(troyeretal.1984a,lombardo2008,archie&theis2011,gilbert2015).this isanimportantandunderstudiedpointofviewwhenbalancingtheprosandconsof sociallifestyletounderstandtheevolutionofsociality.tosketchtheroleofgut microbiotaintheevolutionandsocialdynamicsofsocialspecies,oneneedstopay attentiontothreeimportantaspectsofthemicrobiotainasocialcontext:diversity (affectsimmunity),similaritybetweenindividuals(affectsrecognitionandimmunity) andthepresenceofcertainbacteriawithknownbehavioralimpacts. Social'behavior'and'diversity'of'microbiota' Manysocialbehaviorsincludephysicalcontactandcanfunctionaspotentialpathways formicrobialtransmission.forexample,kulkarni&heeb(2007)experimentally infectedzebrafinches(taeniopygia(guttata)withbacillus(licheniformis(bacteria(and followedhowtheinfectionspreadintheirsocialgroup;preeningandsexualbehaviors transmittedthebacteriatoallgroupmembersinlessthanaday.similarly,frequent intimatekissingenhancedmutualtransmissionofmouthmicrobiotainhumans(kortet al.2014).behaviorcanaffectmicrobialtransmissionandrecentfindingssuggestthat microbiotacanaffecthostbehavioraswell(ezenwaetal.2012,montiel7castroetal. 2014).Forexamplegerm7freemicewerefoundtobemoreactiveandlessanxiousthan micewithnormalmicrobiota(heijtzaetal.2011).similarly,autism7relatedlackofpro7 socialityisassociatedwithalteredmicrobiota(mingetal2012,caoetal.2013) Manyauthorshavesuggestedthatpro7socialandaffiliativebehaviors,suchas grooming,licking,preening,kissingorsocio7sexualbehaviors,mighthavepartly evolvedtoservebeneficialmicrobialtransmission(troyer1984,lombardoetal.2008, Ezenwaetal.2012,Montiel7Castroetal.2013).Insocialgroups,exchangingmutualistic 5

6 microbesbetweenmultipleindividualsallowsmorediversemicrobiotatobepresent. Diversemicrobiotainturnhasbeenlongsuggestedtobearequisiteforaresilient immunity(hooperetal.2012,lozuponeetal.2012),inthesamewaybiodiversity makesecosystemsmoreresilienttochange(levineetal.1999,gunderson2000).for example,sociallytransmittedmicrobiotawasfoundtoprotectbumblebees(bombus( terrestris)fromacommonparasite(koch&schmid7hempel2011).whilesociality bringsimmunechallengesintheformofincreasedexposure(infectionfromothers)and evensusceptibility(immunosuppressivesocialstress)tosimilarlytransmittable parasites(alexander1974,mölleretal.1993,altizeretal.2003,turnbulletal.2011), transmissionofbeneficialbacteriamightcompensatethis.thisisanimportantissuefor thetheoriesoftheevolutionofsociality.becausemutualisticmicrobescanaffect behaviorandsocialbehaviorservesasatransmissionrouteforthesemicrobes,a positivefeedbackloopofincreasedsocialityandmicrobialtransmissionisapossible mechanismintheevolutionofsociality.thiscouldhelpexplaintheassociationbetween socialityandhealthfoundinhumansandotheranimals(houseetal.1988,cohenetal. 2003,Sapolskyetal.2004,Holt7Lunstadetal.2010,Archieetal.2014) Social'behavior'and'similarity'of'microbiota' Manygroup7livinganimalsusefriendlyandaffiliativebehaviorstoenhancesocial bondsorgroupcohesionandtoavoidorreconcileconflictsbetweengroup7members (Vasey1995,Aurelietal.2002,Wittigetal.2008,Hohmannetal.2009,Radford2011). Whenmicrobesaretransmittedthroughsocialbehaviorinasocialnetwork,individuals mostcloselysociallylinkedcanbeexpectedtosharethesamemicrobes.mutualistic microbiotainsocialsystemshavereceivedrelativelylittleattention,whereas transmissionpatternsofparasitesinsocialnetworksarewellstudied(dreweetal. 2010,Godfreyetal.2010,Griffin&Nunn2011,Fenneretal.2011,Bulletal.2012, MacIntoshetal2012,Rimbachetal.2015).Forexample,MacIntoshetal.(2012)found thatfemalejapanesemacaques(macaca(fuscata(yakui)withmorecentralpositionina socialnetwork,andhencemoregroomingpartners,hadmoreendoparasites.consistent withthis,malesocialrolepredictedgutmicrobialcompositioninmeerkats(suricata( suricatta,leclaireetal.2014).whensocialrelationshipsaresymmetric,transmission leadstomoresimilarmicrobiotabetweensocialpartners.forexamplehumanfamily memberslivingtogethersharemoresimilargutmicrobiotawitheachotherthanwith 6

7 peopleoutsidethefamily,regardlessofrelatedness(turnbaughetal.2009b,songetal. 2013).Actually,evendogssharemoresimilargutmicrobiotawiththeirhumanowners thanotherdogsoutsidethefamily(songetal.2013).suchgroup7specificmicrobiota hasbeenfoundinyellowbaboons(tungetal.2015),gorillas(gomezetal.2015), hyenas(theisetal.2012),meerkats(leclaireetal.2014)andchimpanzees(degnanet al.2012).similarmicrobialcompositionsinsocialgroupshavebeensuggestedtoplaya roleinchemicalcommunication,sincemutualisticbacteriaareresponsiblefor synthesizingmostoftheodorsandpheromonesusedinrecognitionbyavarietyof species.forexampleinspottedhyenas,groupmemberssharea scentmarksignature, asimilarscentmarkchemicalcompositioncreatedbyasimilarscentglandmicrobial community(theisetal.2012). Thefactthatindividualsdifferintheirsocialpersonality(Englishetal.2010,Koski 2011,Seyfarthetal.2012,Neumann2013,Carteretal.2014)andpossiblyunderlying physiology,speaksforafluctuatingbalancebetweentheimmunologicalbenefitsand challengesofsociallife.thisbalancedeterminesforexampletherelativevaluesof microbialdiversityandsimilarityunderselection.eventhoughgainingadiverse microbialcommunitythroughsocialcontactmightbebeneficial,contactwith individualswithtoodissimilarmicrobiotamightbeharmful,becauseindividuals becomeco7adaptedtotheirmicrobesandunfamiliarbacteriamightbepathogenicina non7adaptedindividual(fengetal.2011,stillingetal.2014).inlinewiththis, Barribeauetal.(2012)concludedthattadpolesmostlikelypreferthecompanyof individualswithsimilarmhc7genotypeaswellassimilarmicrobiota.furthermore,such avoidanceofopportunisticpathogensmightbethereasonbehindmatechoicedrivenby similarmicrobiota.forexample,drosophila(melanogastermaleswereobservedtomate morereadilywithfemaleswhohadhadthesamedietandsimilargutmicrobiota (Sharonetal.2010). Social'behavior'and'presence'of'certain'beneficial'microbes' Gutmicrobiotaconsistsofavarietyofbacteria,ofwhichsomearemorerelevantfor socialbehaviorandtransmissionthanothers.microbesthatvarylittleinabundance andaresharedmoreorlessbyallindividualsofagivenspeciesarethoughttoformthe CoreMicrobiota (Turnbaughetal.2007,butseealsoLozuponeetal.2012).Moreover, coremicrobiotacanbethoughttoconsistofthematernallytransmittedpartof 7

8 microbiota.inaddition,somemicrobesaremoreeasilysociallytransmittableand highlyvariableinabundance(heretermedas PeripheralMicrobiota ).Forexampleina recentstudy,tungetal.(2015)foundthataspecificsetof sociallystructuredbacteria weretransmittedthroughsocialinteraction,leadingmoresimilarmicrobiotabetween socialpartners.oneimportantgenusamongthesesociallystructuredbacteriawas Bifidobacterium,whichisassociatedwithmanyhealthbenefitsinhumans(Turronietal. 2008).Anotherbacteriallineagewithemergingimportanceinsocialbehavioris Lactobacillus.RecentstudieshaverevealedthatLactobacillusbacteriaareassociated withdecreasedanxietyinmice(bravoetal.2011),possiblyduetotheircapabilityto promoteoxytocinsecretion(poutahidisetal.2013) Stress&and&microbiota& Socialbehaviorandmicrobiotaseemtobemoreintimatelylinkedthanpreviously thought.onefactorentwinedwiththisrelationshipisstressphysiologysincestress affectsbothsocialbehaviorandmicrobiota.stressisknowntoaffectphysiological homeostasisandtoresultinmoreorlesssocialbehaviordependingonthecontext. Specifically,theeffectsofsocialstresscandifferfromtheeffectsofnon7socialstress. Theevolutionofsociallifehascreatedawholenewnicheforthehormonalstress response,mediatedbythehypothalamic7pituitary7adrenalaxis(hpa).whereas environmentalstresscanreducesocialproximity(barbosa&dasilva7mota2009, Overdorff&Tecot2006,Tecot2013),socialstresscaninfactenhanceaffiliationaimed atreducingstressandavoidingconflicts(devriesetal.1996,carter1998,enghetal. 2006,Stöweetal.2008).Itisclearthatstresscanaffectmicrobialtransmissionthrough itseffectsonsocialbehavior,butglucocorticoidstresshormonesalsohavedirecteffects onmicrobiota.hormonalprofilesareassociatedwithmicrobiotaingeneral,whichis evidentinmicrobiotadifferencesbetweenthesexes(alexyetal.2003,saagetal.2011, Markleetal.2013,Leclaireetal.2014),butglucocorticoidstresshormoneshave especiallyfastandprofoundeffectsongutmicrobes.inmiceandprimates,stressis knowntoreducegutmicrobialdiversity(bailey&coe1999,baileyetal.2010),reduce therelativeamountoflactobacilli(bailey&coe1999,galleyetal.2014,galley&bailey 2014)andchangerelativeabundancesofresidentbacteriaingeneral(Parketal.2003). Inhumans,stressfultimeperiodswithhighercortisollevelsareassociatedwith changesingutmicrobiota,mostimportantlyreducedlactobacilli(lizkoetal.1987, 8

9 Knowlesetal.2008).However,stresseffectsonmicribiotaarenotunidirectional; throughthevagusnerveofthegut7brain7axis(bravoetal.2011,montiel7castroetal. 2013)microbiotaisknowntoaffectHPA7axisfunctionaswell.Forexample,lackof functionalmicrobiotasensitizesmicetostressorsandincreasedhpa7axisreactivityand anxiety7likebehavior(sudoetal.2004,crumeyrolle7ariasetal.2014). Figure 1 9

10 Figure1depictstherelationshipsbetweenmicrobiota,immunity,socialbehavior andstressphysiology.thisholobiomicmaplinksanindividualtoitssurrounding ecosystem,blurringtheboundariesofindividualsasfunctionalunitsofnature.some interactions,suchasthosebetweenimmunityandmicrobiotaorstressphysiologyand socialbehavior,areextensivelystudied,butotherinteractions,suchasthosebetween socialbehaviororstressphysiologyandmicrobiotaorstressphysiologyand microbiota,callformoreresearchbeforewecandrawanextensivemapofthese interactions Aims& Toshedlightontheinteractionsofgutmicrobiota,socialbehaviorandHPA7axis,I collecteddataaboutsocialbehavior,taxonomiccompositionofgutmicrobiotaandfecal cortisolmetabolitesfromawildpopulationofred7belliedlemurs(eulemur(rubriventer)( intheranomafananationalpark,easternmadagascar.iaskwhetherpatternsofsocial contact(group7membership,groupsize,socialnetwork)areassociatedwithpatternsof gutmicrobialcomposition(similarity)andwhetherindividualdifferencesinsociality arerelatedtospecificaspectsofgutmicrobiota(diversity,(presence(of(certain(microbes).( Furthermore,Iaddressthequestionofgeneticvs.environmentaldeterminantsof microbiotabycomparinggutmicrobialcompositionoffamilymemberswithdifferent relatedness.lastly,iexaminecorrelationsbetweenfecalcortisollevelsandgut microbialcompositiontotakethefirststeptowardsrelatinghpa7activityandgut microbialcompositioninthisspeciesandtoraisefurtherquestionsabouttheoriginof possiblecovariance. Red7belliedlemursarecooperativeandhighlysocialprimates.Individualsvaryin howmuchtheycontributetosocialactionsandwithwhom.forexampleindividual significantlydifferinhowmuchallomaternalcaretheyprovidetoinfants(tecot& Baden,unpublisheddata).Byusingsocialnetworkanalysisandbyconstructing individualsocialityindicesfromtheamountofsocialbehaviorsanindividualexhibits,i examinetheintra7groupdifferencesinsocialbehavior.myoverallhypothesisisthatthe amountanddirectionofclosesocialinteractionispositivelycorrelatedwithgut microbialdiversityandsimilarity,respectively.thispatternshouldbevisiblewhen comparingi)individualswithdifferentlevelsofsocialinteractionsinthesamegroup,ii) 10

11 individualsbetweengroups(nointeraction),andiii)individualsfromgroupsof differentsizes.inaddition,theassociationbetweengutmicrobiotaandsocialbehavior couldpartlybemediatedbyinteractionwiththestresshormonesofthehpa7axis.to testthishypothesis,severalspecificpredictionscanbemade: 1. Gutmicrobialcompositionshouldbemoresimilarbetweengroup7membersthan betweenindividualsfromdifferentgroups 2. Gutmicrobialdiversityshouldbegreaterinlargergroups 3. Moresocialindividualsshouldhavemorediversegutmicrobiotathanlesssocial individuals. 4. Pairswithshortdistanceinasocialnetwork(frequentmutualinteraction) shouldhavemoresimilargutmicrobialcompositionthanpairswithagreater distance(rarelyinteractwitheachother) 5. Gutmicrobialcompositionandfecalcortisolmetabolitesshouldcovary. Specifically,highfecalcortisollevelsshouldbeassociatedwithlowgutmicrobial diversity Inadditiontosociallife,Iinvestigatewhethergeneticrelatednessandearly environmentwithingrouparedeterminantsofgutmicrobiotainthisspecies.totest thishypothesisipredictthefollowing:ifgenesaremoreimportantthanthesocial environmentindetermininggutmicrobiota,adultsshouldhavemoresimilargut microbialcompositionwiththeiroffspringthanwitheachother Study&species&and&study&site& IcarriedoutthebehavioralobservationsandfecalsamplecollectionintheRanomafana NationalPark,EasternMadagascar,fromAugust2013toFebruary2014.Theareaisa rainforestwithtopographicvariation,montanecloudforestgrowingonridgesand lowlandrainforestinvalleys(wright1999).myspecificstudysite,vatoharanana,isa highaltituderegionlocated5kmsouthofthecentrevalbioresearchstation,bythe edgeoftheforest.thereareatleast12lemurspecieslivinginthearea. Withinthisstudyarea,thefocalstudysubjectswereeightfamilygroupsofred7bellied lemurs(eulemur(rubriventer).additionalfecalsampleswerecollectedfromother groupswhenencountered(total36individuals).red7belliedlemursarefrugivorous strepssirrhines,livinginsmall,relativelystable,monogamousfamilyunitsof276 11

12 individualswithonebreedingpairandtheiroffspringofdifferentages(overdorff 1996b,OverdorffandTecot2006,Tecot2008).Theyanegalitarianspeciesthatexhibit allomaternalcareandhaveaseasonalbreedingcycle(tecot2008,2010).somefathers andjuvenileshelptoraiseinfantsandallfamilymembersparticipateinsocial interactions,suchasgrooming,huddlingorplayingwitheachother.groupsofred7 belliedlemurshavefixedterritoriesthatvaryintheirfruitavailabilityduringtheyear (Overdorff1996a,Tecot2008).Mydatawerecollectedduringthetimeoftheyearwhen infantswerebornandfruitavailabilitywasgenerallylow(tecot2008),thoughwithin thisseasonthedietoftheanimalschangedmanytimesdependingonwhichparticular treeswerefruiting.furthermore,becauseofthegeneralfruitingpatternsofthefood plants,allstudyindividualshadarelativelysimilardietatanygiventime. 2. Materials&and&Methods& 2.1. Collection&of&Behavioral&Data&& Therewerefourpeoplecollectingbehavioraldatainourfieldteam.Twoofuswere localfieldtechnicianswithextensiveexpertiseinlemurbehavioralandhormonal research.wefollowedtheeightfamilygroupsofred7belliedlemursinthe Vatoharananaareaopportunistically:Wheneveragroupwasfound,itwasfollowedfor therestoftheday.ikepttrackofgroupsizeandcompositionandofanychanges therein.continuousbehavioraldatawerecollectedfrom27focalindividualsineight groups,byfollowingeachgrouponewholedayopportunisticallyeverytimetheywere found(approximatelysevenhoursperday,everyonortwoweeks).behavioraldata wereobtainedbyusingscansamplingmethodwithfive7minuteintervals;everyfive minutes,eachgroupmemberwasclassifiedtoonebehavioralcategoryofthered7 Bellied7LemurAllomaternalCareProjectethogram(Table1;Tecot&Baden, unpublishedfieldmanual).forsocialbehaviors(mutualgrooming,huddling,playing),i recordedwithwhomthebehaviorwasexhibited.becausefourdifferentpeople participatedinthebehavioralclassification,inter7observer7reliabilitywastested repeatedly(gwet2001).weaccumulated157150hoursofbehavioraldatafromeach 12

13 group(table2).onlythegroupswithmorethantwoindividualsandmorethan50 hoursofbehavioraldatawereusedtoinvestigatequestionsrelatedtosocialbehavior. Table 1. Ethogram, Tecot & Baden, unpublished field manual Mark Behavior Description M( Move Movinginsideasingletree(lessthan3meters) T( Travel Directedmovementmorethan3meters R( Rest Animalismotionless(eyesopenorclosed) HUD( Huddle Restinginphysicalcontactwithothers,tailswrapped aroundeachother PPLY( PartnerPlay Disorientednon7continuousplayfulbehaviorbetween morethanoneindividuals SPLY( Self7play Disorientednon7continuousplayfulbehaviornot directedtootherindividuals SGR( Self7groom Groomingone sownfur AGR( Allogroom Groominganotherindividual RGR( ReceivingGroom Receivinggroomfromanotherindividual MGR( MutualGroom Twoindividualsgroomingeachothersimultaneously FD( Feed Handling,sniffingandeatingfooditems AGG( Aggression Fastandloudchasingorcuffingotherindividual AGM( Scentmarking Ano7genitalscentmarkingbehavior ' Table 2. Amount of behavioral data per group and the criteria used to choose groups to study social behavior. All groups were used in other analyses described in the methods Group GroupSize Hoursofbehavioraldata Usedtostudybehavior X X X X

14 Constructing'sociality'indices'and'interaction'matrices' Individualsocialityindiceswereconstructedbasedonthebehavioralscansampledata. Theywerecalculatedastheproportionsoftimeeachsubjectspentinparticularsocial behaviors.specifically,iconstructedtwoindicesfortwodifferentsocialbehaviors, respectively: 1. Huddleindex:proportionoftimespenthuddlingwithothersoutofthetotal amountofresttime. 2. Socialgroomindex:proportionoftimespentsociallygrooming(mutualgroom, allogroom,receivinggroom)outofthetotalamountoftimeobserved. Toconstructinteractionmatrices,Iusedastandardmethodforsocialnetworkanalyses (Whitehead2008).Pairwisecomparisonsofmutualinteractionsyieldedsocialdistance valuesforeachpairofindividuals.distancevalueswerecalculatedforapairof individualsastheproportionoftimespentinmutualinteractionoutoftotaltimespent insocialinteractionofthetwoindividualsineachpair.interactionmatriceswere constructedfortwosocialbehaviors: 1. Huddlematrix:adistancematrixofhowmucheachpairspenthuddling together 2. Groommatrix:adistancematrixofhowmucheachpairspentgroomingeach other Socialityindicesandinteractionmatriceswereconstructedseparatelyfortwotime periods:1)beforeinfantswereborn(september7octoberin2013),and2)andafter infantswereborn(december2013untiljanuary2014). 2.2&& Fecal&Sample&Collection&and&Analysis&for&Cortisol&Metabolites& Icollectedfecalsamplesforhormonalassaysbywrappingtheminfoilanddryingthem byfire(tecot2001,2008,2013).sampleswerekeptdrybystoringtheminabagfullof silicapearls.hormonalsampleswerecollectedfromindividualsofthefocalgroups wheneverpossibletodetectchangesinhormonalstatus.sampleswerekeptdryand broughttothelaboratoryfortheevolutionaryendocrinologyofprimates(leep)in UniversityofArizona.Fecalsampleswereground,siftedand0.1gwasextractedfrom eachsampleintoasolutionofhalfethanolhalfwater.sampleswerethenmixed 14

15 (vortexed),centrifugedandthesupernatantwaspouredoff.onemlofeachsamplewas takenintonewtubesand4mlofethylacetatewasaddedtofreeconjugantsteroids. Samplesweremixedandcentrifugedagainandthetoplayerwasaspiratedintoculture tubes,evaporatedinwaterbathafterwhich1mlof30%methanolwasadded.samples wereconditionedwith1mlofmethanoland1mlofwater(distilled).1mlofsample wasaddedandwashedwith5%methanol.steroidswereelutedfromthecolumnwith 2mlofmethanol,whichwasthenevaporatedandreplacedwith1mlofethanol. Cortisolassaysweredoneusinganenzymeimmunoassay(EIA)protocolby CoralieMunro:Enzyme7labeledantigen(horseradishperoxidasecortisol)wasdiluted ineia7buffer(1:100000)andmixed.0.5mlofsampleswerefirstevaporatedand0.3 mloftheantigendilution(t:hrp7eia)wasaddedtoallsamplesandstandards.samples andstandardswerethenmixedandtransferredtoassaytubes.forcontrol,0.3mlpure EIA7bufferwasaddedtoNSB7tubesand0.3mlT:HRP7EIAwasaddedtoB07assaytubes. Platescoatedwithsteroidconjugatewerewashedand0.1mlofeachsampleand standardmixturewereplatedinduplicate,shakenandincubatedfor2hoursina humiditychambertoenablebinding.plateswerethenwashedtoseparateboundand freecortisol.1mlsubstratewasadded,theplatewasmixedforfiveminutesand absorbancewascalculatedwithabiotekplatereader.theslopeofseriallydilutedfecal extractswasparalleltothatofthecortisolstandard,f=0.12(11,12),p=0.730.fecal cortisolassaysweredeterminedtobeaccurate(96.68±1.79%standarderrorofthe mean) 2.3& Fecal&sample&collection&and&gut&microbial&DNA&analysis&& IcollectedfecalsamplesformicrobialDNAanalysisinEppendorftubesfilledwith RNAlater.Minimumofonesamplewascollectedfromallfocalindividuals.Second samplesforseasonalcomparisonwerecollectedfrom15individualsinfivegroups.to increasesamplesize,icollectedadditionalsamplesopportunisticallywhenever encounteringindividualsfromnon7focalgroups.eventhoughididnothavematching behavioraldataforthesesamples,theywereusedintheanalysisofdemographic correlates,suchaswithage,sexorgroupsize.altogether110microbialfecalsamples werecollected. 15

16 Eppendorftubeswerekeptinafreezer(780C)for6monthsbeforesendingthem totheknightlaboratoryintheuniversityofcoloradotobeanalyzed.dna7extraction wasdoneusingmobiopowersoil kits.pcrwasrunwithprimerstargetedatv17v3 regionofthe16srrnagene.theresultingampliconsweresequencedusingillumina MiSeqplatform.IusedQIIMEsoftwaretomatchbarcodeswithsamples,toeliminate badqualitydnaandtoflipreversesequences(caporasoetal.2010).here,quality filteringwasdonewithdefaultsettingsofthe split_libraries_fastq.py command(q>20, 1%riskmarginalinbases).Sequenceswereclusteredandmatchedwithknown taxonomicdatabase(greengeens)usingopenreferenceotupicking(pick_openm reference_otus.py)whileenablingreversestrandmatching.thiswaythelistofthe knownotus(operationaltaxonomicunit,comparableto species )thatmatchedwith thedatabasecouldbeenrichedwiththedenovoclusteredunknownotus.thisis important,becauseasignificantproportionofgutmicrobiota,especiallythatoflemurs, belongstotheseunknowntaxa(katherineamato,personalcommunication).herefor example,openreferenceotupickingyielded1,131,454reads(sequences)whereas closedreferenceotupicking(withoutdenovoclustering)yieldedonly304,163reads. Theamountofsamplesthatweresuccessfullysequencedandhadaminimumof3,000 readswas92.thedatawasrarefiedto3,000readspersample Statistical&analyses& Describingmicrobialdata IperformedstatisticalanalysesusingR(Rversion3.1.2, PumpkinHelmet,RCore Team2014).Themicrobialdatawerefirststandardizedasrelativeabundances(the frequencyofcountsofeachoturelativetothetotalcount)andconvertedinto dissimilarityindices.theuseddissimilarityindicesdescribetherelativedifferencesof microbialspeciescommunitiesbetweenindividualsbasedonpresence absence information(sørensendissimilarityindex)orrelativeabundance(bray Curtis dissimilarityindex)(legendre&legendre2012).thebray Curtisdissimilarityindexis anindexusedtocalculatethedissimilarityofspeciescompositionbetweentwosites (here,betweensamples).itiscalculatedasfollows: " " = 1 ", 16

17 where " isthesumofthelessercountsofeachotupresentinbothsamples"#, and "# arethetotalnumberofotuspresentinbothsamples.thesørensen dissimilarityindexisasimilarindex,butunlikebray Curtis,itonlyappliesfor presence absencedata.itiscalculatedasfollows: " =, wherecistheabsolutenumberofotussharedbythetwosamples,andaandbarethe totalnumberofotusineachsamples,respectively.iusedthesetwodifferentindicesto getanoverviewofgutmicrobialdissimilaritybasedonboth(a)havingdifferentotus presentorabsent(sørensen),and(b)havingdifferentrelativeabundancesofotus (Bray Curtis).SørensenindiceswerealsousedtoreinforceBray7Curtismetricsof dissimilarity,becausedifferencesinthepresence absenceofcertainbacteriaarea morereliablemeasureofdissimilarityofmicrobialcommunitiesthandifferencesin relativeabundances.inadditiontodissimilarityindices,iusedshannondiversityindex andrichnesstodescribethegutmicrobialdiversity. Clusteringofmicrobialdata Iuseddissimilarityindicestoconstructdissimilaritymatricesofallindividuals,and thesewereusedtoclusterdatatorevealthedistributionofgutmicrobiotaacrossthe studypopulation.clusteringfromthedissimilaritymatriceswasdoneusingthek7 meansmethod,whichisanalgorithmthatassignsdatapointstoclustersbyminimizing thesumofsquareddistancesofpointstothecenterofeachcluster(legendre& Legendre2012).Eightclusterswerefoundusingthismethod,whichcoincideswiththe numberofstudygroups. Sincethemonthlychangesinthegutmicrobiotaweresmall(controlledfor individualandpregnancy,r 2 =0.02,p=0.012),Ipooledthesamplesfromtwomonths beforeandafterinfantswereborn(september OctoberandDecember January)to formtwocomparabletimeperiods(seasons).clusteringwasthendoneonasubsetof thedatacontainingonlyonesampleperindividual,alltakenduringthefirstseason (Sep Oct).Here,clusteringwasdonewithadefinednumberofeightclusters,derived fromtheobservedclusteringpatternofthewholedata.thiswasfurthervisualized usingprincipalcoordinateanalysis(pcoa),whichprovidesagoodoverviewofthedata 17

18 distributionbeforeanyfurtheranalyses.pcoaaimsatfindingtheaxisalongthe multidimensionaldistributionthatexhibitsthemostvariation(legendre&legendre 2012);thisaxisiscalledthefirstprincipalcoordinate. Analysesusingdissimilarityindices Totesttheeffectoftime(monthandseason)andpregnancyonthemicrobiota composition,iusedpermutationalmultivariateanalysisofvarianceusingdissimilarity indicesasdistancematrices.thisanalysiswasperformedwiththeadonisfunctionas implementedintheveganpackageinr(oksanenetal.2014).permutational multivariateanalysiswaschosenbecausetheelementsinadissimilaritymatrixarenot independent.thistestisbasedoncomparingtheobservedassociationbetween dissimilarityindicesandothervariableswiththosearisingwhentheindexvaluesare repeatedlyrandomlypermuted.thisallowsreliableestimationofstatistical significance.thesametestwasusedforthesmallersubsetofdatawithonesampleper individualcollectedinthefirstseasontotestfortheeffectsofage,sex,groupandgroup size.similarly,icomparedsamplesofasetoffemalesandtheirmatesbeforeandafter pregnancytotestforthepossibleeffectsofpregnancy,andusedprincipalcoordinate analysistovisualizethis. Analysesusingdiversityandrichness Inadditiontothese,Iusedgeneralizedlinearmodelstotestfortheeffectofseasonand socialityindicesongutmicrobialdiversityandrichness.thiswasdonebyfittingaline bylinearregressionthedata(minimizingthesumofsquaredresiduals)andcalculating whetherthisregressionexplainssignificantlymoreoftheobservedvariationthat wouldbeexpectedbychance,assumingadistributionofexponentialfamily(grafen& Hails2002).Furthermore,totestcorrelationsbetweeninteractionmatrices(converted fromsocialproximitytosocialdistancematrices)andmicrobialdissimilaritymatrices,i usedthemanteltestofmatrixcorrelation(legendre&legendre2012).themanteltest isapermutationaltestofcorrelation,wherestatisticalsignificanceistestedby repeatedlyrandomlypermutingtherowsandcolumnsofoneofthematrices,andthe correlationcoefficientsarecomparedwiththatobservedfortheoriginalmatrices.p7 valueiscalculatedasaproportionofpermutationsthatleadtohigherthanthe observedcorrelationcoefficient.fortestsinvolvingsocialityindicesandinteraction 18

19 matricesiuseddataonlyfromgroups,forwhichtherewassufficientbehavioraldatato constructtheseindicesandmatricesforbothseasons(beforeandafterinfantswere born).thissubsetofdataconsistedof26samplesencompassing13individualsfrom fourgroups. Whichbacterialtaxaarebehindthetrend? Tofindoutwhichbacterialtaxaareresponsibleforagiventrend,IusedDufrene LegendreindicatoranalysistocalculateindicatorvaluesforeachOTUinthegroupsof samplesundercomparison(dufrene&legendre1997).thedufrene Legendre indicatorvalueiscalculatedasfollows: "#$%& " = " " 100,where " isthenumberofotuiinclassj(suchasthefamilygroup)dividedbythe totalnumberofotui,and " isthenumberofsamples(individuals)inclassjthat containotuidividedbytotalamountofsamplesinclassj.thestatisticalsignificanceof theresultingindicatorvaluesarethentestedbyrandompermutation.here,p7valueof 0.01(except0.05whenanalyzingtheeffectofpregnancywithasmalldataset)wasused asathresholdforsignificance,andofeachclass,tenotuswiththehighestindicator valueswerechosen. 3. Results& 3.1. Environmental,&demographic&and&genetic&factors&affecting&microbiota&& Thegutmicrobiotaofred7belliedlemurswasdominatedbythephylaBacterioidetes, ProteobacteriaandFirmicutes(Fig.5).Withinoneseason,gutmicrobiotaclustered stronglyaccordingtothefamilygroupandthegroupsformedthreebiggerclusters partiallyparallelingthelocationoftheirterritories(fig.2and3).thatis,thegroupsin thetwosmallerclustershaveneighboringoroverlappingterritories,butnotallgroups inthebiggestclusterseemtobesocloselylocated.individualdifferencesingut microbialcomposition(bybothdissimilarityindices)werebestexplainedbygroup identity(seetable3).inaddition,within7groupdifferencesweresignificantlyexplained 19

20 bypregnancy(seetable3).additionalanalysesrevealedthatmicrobiotaduringand afterpregnancychangedsignificantlyinadultfemales(r 2 =0.16,p=0.02withboth dissimilarityindices;seefig.4),butnotinacontrolgroupofthecorrespondingmales (p=0.5).thisdifferencewasnotinthegutmicrobialdiversity,butratherindifferences inthespecies(otus)ofthebacterialcommunity. Indicatoranalysisrevealedthatdifferencesbetweenthemicrobiotaofthesame femalesduringandafterpregnancyweredrivenbyphylum7levelchanges.specifically, pregnantfemales gutmicrobiotawascharacterizedbybacteriabelongingtophyla ProteobacteriaandVerrucomicrobia,whereaspost7pregnantfemales gutmicrobiota wascharacterizedbyabundanceofbacteriabelongingtophylumfirmicutes.moreover, thebestindicatorspeciesforgroup7specificdifferencesingutmicrobialcomposition werebacteriabelongingtotheorderclostridiales(inphylumfirmicutes).figure5 summarizesgroupdifferencesingutmicrobialcompositionatthephylum7level.group size,age,sexandage7sexinteractioncontrollingforpregnancyhadnosignificanteffect ongutmicrobialcomposition(seetable3). Table 3. Correlations of demographic factors (age, sex, group, group size) with the gut microbial composition (Sørensen dissimilarity index). All data are from the same season. Df SS MS F7value R 2 p( Pregnancy <0.01 Age Sex Group <0.01 GroupSize Age:Sex Residuals Total

21 Figure 2. PCA-ordination of the filtered data. Gut microbes cluster as family groups (numbered colored areas) and these groups form three bigger clusters, partially paralleling their territory location (See Fig. 3). Figure 3. Geographical territories of the study groups. Group colors are the same as in Fig

22 pca[,2] DURING AFTER pca[,1] Figure 4. Breeding females gut microbial compositions in relation to each other (lines) and during and after pregnancy (colors) visualized with principal component analysis Bacteroidetes Proteobacteria Verrucomicrobia Firmicutes Euryarchaeota Acidobacteria Planctomycetes Figure 5 Gut microbial composition of the eight study groups, allocated by phyla. White area covers unknown species 22

23 3.2. Seasonal&Change&in&Gut&Microbiota& Seasonalchangeinthegutmicrobialcompositionwasinvestigatedwitha datasetwherebreedingfemaleshadbeenremovedtoeliminatethestrongeffect ofpregnancy.therelativeabundancesofgutmicrobes(bray7curtisdissimilarity index)variedbetweenthetwoseasonssignificantly,thoughtheeffectwasquite small(table4).analysisusingpresence7absencedataformicrobialcomposition (Sørensendissimilarityindex)showednosignificanteffectoftheseason(Table 5).Gutmicrobialdiversity(Shannon)andrichnessdecreasedsignificantlyfrom beforetoafterinfantswereborn(p=0.02forrichness,p=0.04fordiversity,fig. 5).Asimilartrendindiversitywasnotdetectedinbreedingfemaleswhenthis wasanalyzedseparately.indicatoranalysisrevealedthatthetrendofdecreasing gutmicrobialdiversitywasassociatedwithincreasingabundanceofbacteria belongingtothephylumbacteroidetes,mostimportantlygenusprevotella. Table 4. The effect of season on gut microbial composition using relative abundances (Bray- Curtis dissimilarity index). Because data were unevenly distributed in time (Different groups had different amount of data in different months), Month, Group and their interaction were added to the analysis as control variables before Season. Df SS MS F7value R 2 p Group Month <0.01 Season <0.01 Group:Month Residuals Total Table 5. The effect of season on gut microbial composition using relative abundances (Sørensen dissimilarity index). Because data was unevenly distributed in time (Different groups had different amount of data in different months), Month, Group and their interaction were added to the analysis as control variables before Season. Df SS MS F7value& R 2 p Group Month <0.01 Season Group:Month Residuals Total

24 A (Before) B (After) A (Before) B (After) Figure 6 Gut microbial diversity (left) and richness (right) are higher before than after infants are born 3.3. Gut&Microbial&Composition&and&Interaction&Matrices& Therewasnosignificantcorrelationbetweengutmicrobialdissimilarity(by Bray7CurtisorSørensendissimilarityindices)andsocialconnectednesswithin groupwithanyoftheinteractionmatrices(huddlematrixandgroommatrix beforeandafterinfantswereborn) Gut&Microbial&Composition&and&Sociality&Indices& Individualsvariedintheamountofrestingtimetheyspenthuddlingtogether andtheamountoftimetheyspentgroomingothers(fig.7).groomindiceswere significantlynegativelycorrelatedwithgutmicrobialdiversitybetween individualsthroughtime(p=0.01).afteraddingtimetotheanalysis,negative correlationswereapparentinbothindices(table6),butonlybeforeinfants wereborn(fig.7.a,b).richnessfollowedasimilartrend(fig.7.c,d),thoughit wasnotsignificantlycorrelatedwiththehuddleindexatanytime.overall, huddleindexdecreased(p<0.01)andgroomindexdidnotchangefrombeforeto afterinfantswereborn. 24

25 Table 6. Results of generalized linear model testing for the effects of both sociality indices on gut microbial diversity, after the effect of time (Pre/Post infant) is taken into account. Estimate( Std.Error( tvalue( p (Intercept) <0.001 Pre/PostInfant GroomIndex Estimate( Std.Error( tvalue( p (Intercept) <0.001 Pre/PostInfant HuddleIndex Figure7Gutmicrobialdiversityandrichnessinrelationtosocialityindices.Beforeinfantsare born(inred),diversityisnegativelycorrelatedwithgroomindex(a)andhuddleindex(b),but correlationisnotapparentafterinfantsareborn(inblack).richnessfollowssimilartrends(c,d), butcorrelationwithhuddleindex(d)isnotsignificant 25

26 3.5. Gut&microbial&composition&and&stress&hormones& TocontrolfortheeffectsofpregnancyonHPA7axisactivity,breedingfemales wereexcludedfromtheanalysisofcorrelationsbetweencortisolprofilesandgut microbialcomposition.fecalcortisollevelswerepositivelycorrelatedwithgut microbialcomposition(r 2 =0.14,p=0.05withSørensendissimilarityindex,R 2 = 0.14,p=0.04withBray7Curtisdissimilarityindex).Dissimilarityindices correlatedwitheachother(p<0.01),indicatingthattheyspeakforthesame thing.afteraddinggroupandsex7ageclassintotheanalysisascontrolvariables, theresultwasevenmoresignificant(table7).theeffectofcortisolongut microbiotawasnotrelatedtodiversityorrichness,butrathertodifferencesin thespeciesofthebacterialcommunity,specificallyspeciesbelongingtophylum Firmicutes. Table 7. Correlation of Cortisol levels (CORT) with gut microbial composition using relative abundances (Bray-Curtis dissimilarity index). Group and sex-age class are control variables. Df SS MS F7value R 2 p Group <0.01 Sex7AgeClass CORT Residuals Total Discussion& 4.1. Environmental&and&Demographic&Variation&in&Gut&Microbiota& Groupidentity,age,andseasonalchangewerethemainfactorsexplaining variationinthegutmicrobiotaofred7belliedlemurs,whichwasverysimilar betweenallindividualswithinonegroup.noeffectofrelatedness(differencesin microbiotabetweenparent7offspring,parent7parentandoffspring7offspring pairs)wasdetected.themainfactorexplainingwithin7groupdifferencesingut microbiotawaspregnancy.inparticularfemalesgutmicrobiotadifferedfrom theothergroupmember smicrobiotaandbreedingfemalesalsoexperienceda changeinthegutmicrobialcompositionatthetimeofgivingbirth.similarlyto 26

27 theseresults,themicrobiotaofpregnantwomen(colladoetal.2008,korenetal. 2012,Romeroetal.2014)aswellaswellbreedingfemaleEuropeanbadgers (Meles(meles)werefoundtodifferfromnon7breedingfemales'microbiota. Moreover,parallelingthetrendfoundbyKorenetal.(2012)inpregnantwomen, ourresultsshowthatpregnantfemalesgutisabundantinbacteriabelongingto thephylumproteobacteria,whilepost7pregnantfemales gutsareabundantin bacteriabelongingtothephylumfirmicutes.sincebirthandearlyenvironment areimportantinthedevelopmentofahealthymicrobiota,pregnancy7related modificationofgutmicrobiotamightbeanadaptivewayofreadyingthe microbialenvironmentfortheinfant(korenetal.2012).inadditionto pregnancy,noothersexoragedifferenceswerefound.thisisquitesurprising, giventhenumberofstudiesindicatingthatmicrobiotacandifferbetweensexes (Alexyetal.2003,Saagetal.2011,Markleetal.2013,Leclaireetal.2014)and tendstochangeduringdevelopmentuntilindividualsreachmaturity(palmeret al.2007,mariatetal.2009,sinetal.2012,leclaireetal.2014). Thedissimilarityofgutmicrobiotabetweenfamilygroupsmightbe partiallyduetodifferencesinthefoodsourcesandmicrohabitatsofdifferent territories.geographicalrangehasbeenpreviouslylinkedwithdifferencesingut microbiotainprimates(degnanetal.2012,moelleretal.2013,gomezetal. 2015)However,sincetheterritoriesofourstudygroupshaveoverlap, environmentaldifferencesseemaninadequateexplanationfortheobserved dissimilarityofmicrobiota.furthermore,red7belliedlemursarefrugivoresand allindividualswithinthesameareaexperiencemoreorlesssynchronized changesindietbecauseofthefruitingpatternsoftheirfoodplants.despitethis, theseasonalchangeingutmicrobiotawasnotnearlyasrobustasdifferences betweengroups,suggestingthatspatialvariationindietorenvironmental conditionscannotaloneexplainthedifferencesbetweengroups. However,group7specificmicrobiotasformedthreebiggerclustersthat mightbeexplainedbythelocationofgroupterritories(fig3).indeed,thegroups inthetwosmallerclusters(8+11and5+10)haveneighboringoroverlapping territories,butnotallgroupsinthebiggestcluster( )seemtobeso closelylocated.thisterritoryinformationisnotbasedongps7coordinates,buta trail7locatingsystemandmightbeslightlymisleading.morespecificinformation 27

28 ofterritoriesfromvariousdistancesofeachotherisneededbeforetheeffectof territorydistanceongutmicrobialcompositioncanbedescribed GroupKspecific&microbiota& Group7specificgutmicrobiotareflectsthesociallifeofthisspecies,withhigh within7groupcohesionandextremelylowinteractionbetweenmembersof differentgroups.theseresultsaddtotheincreasingevidenceofgroup7specific microbiotainhighlysocialspecies(turnbaughetal.2009b,songetal.2013, Degnanetal.2012,Theisetal.2012,Leclaireetal.2014,Tungetal.2015,Gomez etal.2015).red7belliedlemurs,justlikecooperativelybreedingmeerkatsand hyenas,provideallomaternalcareandrelyonin7groupcooperationincaringfor theyoung.inthiscontext,sharedmicrobiotamightbecrucialforgroup7specific scentmarks,recognitionandbonding.moreimportantly,however,shared microbiotainasmallgroupcanbeseenasamechanismofimmunity synchronization;individualsbecomeaccustomedtotheirgutmicrobesand indeedthesamegutbacteriamightbemutualisticinanaccustomedindividual butpathogenicasanovelinfection(barribeauetal.2011,fengetal.2011, Stillingetal.2014).Sharingmicrobiotamakesitcertainthateverybodyis accustomedtothesamebacterialallies,andwillnotinfecttheirgroupmembers withpotentialpathogens.insupportofthishypothesis,cooperativebreeders andatleastonespecieswithsharedinfantcarearegenerallyknowntopossess synchronizedphysiologicalprocesseswithinthegroup.forexample,inmany cooperativelybreedingprimates,othergroupmemberscanreflectthehormonal statusofthepregnantfemale(storeyetal.,2000,nunesetal.,2001,ziegleretal., 2004,Tecot2008)possiblyreadyingthemtotheirtaskinprovidingallomaternal care(ziegler2000).furthermore,cooperativelybreedingmarmosets(callithrix( jacchus)werefoundtohavemoresimilarpersonalities,andthusmostlikely moresimilarhormonalprofiles(koolhaasetal.,1999,carereetal.2010, Koolhaasetal.,2010Sih2011)thanchimpanzeesrelyingondyadicratherthan group7widecooperation(koski&burkart2015).toexplorethisfurther, synchronizedhormonalpatternsincooperativegroupsneedstobestudied more. 28

29 4.3. Microbial&transmission&in&a&social&network& Ifoundnocorrelationbetweensocialcontactandsimilarityofmicrobiotawithin groups,butthisislikelybecausefamilygroupsofthisspeciesaresmall(276 individuals)andsocialconnectednesswithingroupisrelativelyhighbetweenall individuals.however,group7specificgutmicrobiotaspeakfortheimportanceof socialcontactindeterminingthemicrobiotainthisspecies.furthermore,even thebreedingpairsharedsimilargutmicrobiota,whichismostlikelyformedonly throughsocialorsexualcontact,sincetheyarenotrelatedanddonotshareearly environment(i.e.theyderivefromdifferentnatalgroups).thisisinteresting, becauseifsocialcontactcanfunctionasatransmissionrouteforgutmicrobes (Kulkarni&Heeb2007,Kortetal.2014,Tungetal.2015),thesemicrobescan carryinformationofsocialcontactaswell.inwildanimalresearch,thereisa strongbiasinthestudyofsocialbehaviortowardsdiurnalandhighlysocial species.gutmicrobiotacouldbeusedtodrawmapsofsocialnetworksalsoin asocial,nocturnalorotherwisecrypticspecies,whose(socialorsexual)behavior ishardtostudyinwild.parallellingthisidea,zohdyetal.(2012)managedto capturethepatternofsocialcontactsinwildnocturnalmouselemurs (Microcebus(rufus)bymarkingandrecapturingtheirparasites(lice).Whereas socialmicrobetransmissioninasocialspecieswouldmainlymeansexualor agonisticcontact,insocialspeciessimilarityofmicrobiotamightbeagoodproxy forthestrengthofasocialbond(montiel7castroetal.2013). Inadditiontosimilaritywithingroups,sociallytransmittedgutmicrobes mightenhancegutmicrobialdiversityasgroupsgetbigger.however,ifoundno correlationbetweengroupsizeandthegutmicrobialdiversity.thisislikelyto beduethesmallvariationingroupsize(276).inaddition,montiel7castroetal. (2013)pointedoutthatitisnotgroupsizeperse,butrathermodulargroup organizationthatisassociatedwiththebenefitsofsociallytransmittedmicrobes. Unlikeourpair7living,cooperativestudyspecies,biggerprimategroupstend havehighmodularity,leadingtopartialsocialisolationofdifferentsocial moduleswithinonegroup.thediversityofmicrobiotamighthavebeenone factordrivingtheevolutionoflargegroupsize,butselective(modular)bonding 29

30 patternsmayhaverisenwithaneedtobalancethetransmissionofbeneficial microbesandpathogens(montiel7castroetal.2013).indeed,socialmodularity isknowntoreduceparasitetransmission(griffin&nunn2011). Sincebondingbehaviorinprimatesservesasagoodwayformicrobial transmission,oneneedstodecidecarefullywithwhomtoaffiliate.affiliating withasmallsetofclosekinorfriendsisknowntoreducestress(wittigetal. 2008).Inadditiontothis,itmightalsoreducetheriskofinfection.Thus,asocial bondortheassociatedbondingbehaviorinhighlysocialspeciescanbe additionallyseenasamanifestationofimmunologicaltrust.inspecieswith allomaternalcare,likered7belliedlemursallindividualsaretightlybondedand seemtosharethemicrobiota,hencenomodularityisneeded Gut&Microbiota,&HPAKaxis&and&Individual&Differences&in&Social& Behavior& Contrarytomyprediction,bothsocialityindices(HuddleindexandGroom index)werenegativelycorrelatedwithgutmicrobialdiversitybetween individuals.thenegativecorrelationbetweensocialityandgutmicrobial diversitywassignificantandstrongbeforeinfantswereborn,notaftertheywere born.whereasgutmicrobialdiversitydecreasedfrombeforetoafterinfants wereborn,individualhuddleindicesdecreasedbutgroomindicesstayedthe same.thefactthatthenegativetrenddisappearsafterinfantsareborncouldbe explainedbydecreasingphysiologicaldissimilaritybetweenindividualsatthe timewheninfantsareborn.familymembersmostlikelyhavemoredifferent physiologicalstateswhilefemalesarepregnant.inaddition,individualscould havemoresimilarphysiologicalstatesafterinfantsarebornwhenallindividuals mightexperiencestressorothersynchronizedhormonalchangesrelatedto allomaternalcare(ziegler2000). Beforeinfantswereborn,individualsthatparticipatedmoreingrooming, groomedothersmoreandsleptinclosecontactwithothers,hadthelowestgut microbialdiversity.thisisoppositetowhatwaspredicted,assumingthat groomingfunctionsasawayofgutmicrobialtransmission.increasedmicrobial transmission,however,mightnotalwaysleadtohighermicrobialdiversity. 30