neatosbtenteaths n h fungalsymbiont. interactions betweentheanthostand employed bythissymbiosisthereforearisesfromcomplex Trachymyces, regardlessoftheanthost.Activitysomeenzymes protease activitywassignificantlyhigherin either fungalspecies.Incontrasttocarbohydratemetabolism, maintain gardenswithsignificantcellulaseactivitywhengrowing *Author (@gmail.com) for correspondence Denmark. Biology, Copenhagen,Universitetsparken 15,DK-2100Copenhagen, University of 75799, USA. 2540 Received 15October 2013;Accepted17April2014 1 al., 2013;McFall-Ngaiet2013).Ofthenumerousexamples of and areabletofunctionasdistinctunits(Douglas,2010;Kültz et understanding howsuchcomplexorganisms maintainhomeostasis ecological consequences.Oneofthelarger mysteriesis has ledtomanykeyevolutionaryinnovationswithfar-reaching Co-operation amongunrelatedorganisms (mutualisticsymbioses) Pectin, Starch,Symbiosis,Xylan KEY WORDS:Attini,Cellulose,Co-,Enzymeactivity, significant metabolizersofcellulose, . Althoughgardensofleaf-cuttingantsarenotknowntobe pectinase activitieswhenT. arizonensis specialization. Incontrast,gardensshowedcomparableamylaseand compared withTrachymyces fungi,consistentwithenzymatic and pectinaseactivitieswhen pectin, xylan,celluloseandcasein.Gardensexhibitedhigheramylase of switchedandsham-switchedfungusgardenstodigeststarch, Trachymyrmex arizonensis switch experimentbetweentheantAttatexana specialization inleaf-cuttingants,weconductedareciprocalfungus- -cutting .To elucidatetheenzymaticbasesofhost– Trachymyrmex grownprimarilybythenon-leaf cutting ‘Trachymyces’), when growingafungusfromthesister-cladeto by exhibitinghigherfungalgrowthratesandenzymaticactivitiesthan unknown. Priorworksuggestedthatderivedleaf-cuttingantsinthe The mechanismsthatmaintainthissymbiontfidelityarecurrently exhibit littleornosymbiontsharing,resultinginhost–fungusfidelity. and thusexhibitdiffuse co-evolutionaryrelationships,whileothers lineages sharefungalsymbiontswithothermembersoftheirlineage fungal symbiontsandothermicrobes.Somefungus-gardeninginsect theirextensiveco-evolutionaryhistorieswithobligate because of Fungus-gardening insectsareamongthemostcomplexorganisms J. N.Seal of fungusgardens speciescombinationsengineerphysiologicalactivity –fungus RESEARCH ARTICLE © 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,2540-2547doi:10.1242/jeb.098483 C0930, Austin, TX78712,USA. INTRODUCTION ABSTRACT Department of Biology, of University of at Tyler,Department 3900 University Blvd, Tyler, TX 2 Integrative Biology, Texas University of at Austin, 1University Station interact synergisticallywithleaf-cutterfungi( 1,2, *, M.Schiøtt ants thatform,correspondingly, thesister-cladeto 3 Centre for Social Evolution, Department of Centre for Social Evolution, Department and reportmeasuredenzymaticactivities 3 A. texana and U.G.Mueller T. arizonensis ants cultivated cultivated eitherfungal Attamyces Attamyces 2 were ableto and theant Attamyces) Attamyces (so-called than in rapid ofstarches,hemicellulosesand proteinsfoundin provisionally as‘Trachymyces’. hencewerefertothem have beentaxonomicallyresolved; 2008; Muelleretal.,1998;Schultz andBrady, 2008).Noneof these cultivate amorediverse assemblage ofcloselyrelated Tschinkel, 2010;Torres etal.,1999). 1994; LealandOliveira,1998;SealTschinkel, 2006;Sealand that maymakethemecologicallyrelevant(BeshersandTraniello, modest ornoworkerpolymorphism,andoccasionalpopulation sizes characterized byrelativelysmallcolonies(hundredsofworkers), Schultz andBrady, 2008).Antsinthegenus of whichmanyinhabitNorthAmerica(Rabelingetal.,2007; Trachymyrmex descended fromacommonancestorsharedwithseveral 2011b; Muelleretal.,1998).Allleaf-cuttingantsarethoughttohave in itsteleomorph(sexual)form(Muelleretal.,2010;Mueller et al., (vegetative, clonal)growthformand called asinglespeciesoffungus, 2003). Leaf-cutterantsgenerallycultivate 2011; Meyeretal.,2011; Seal,2009;Wilson, 1980;Wirth etal., may exertenormousecologicalimpacts(HölldoblerandWilson, proportions (>1millionworkers);asaresult,fungus-growingants pronounced variationinworkersizeandcolonysizesofimmense (genera complex ofwhicharefoundinthecladecontainingleaf-cuttingants occurred inthederivedlineages(the‘higherAttini’),most One ofthemoreprofoundevolutionaryandecologicaltransitions Mueller etal.,2010;1998;SchultzandBrady, 2008). Kellner etal.,2013;Mehdiabadi2012;Mikheyev2010; phylogenetically earlybranchinglineages(Greenetal.,2002; transmission isquiteextensive,especiallyamongthe between cladesofantsandfungiisthenorm,horizontal Although verticaltransmissionofsymbiontsandgeneralizedfidelity macroevolutionary trendswithregardtotheirfarmedsymbionts. Schiøtt etal.,2010). and Biedermann,2012;DeFineLichtetal.,2013;Martin,1987b; (Aanen andEggleton,2005;Aylward etal.,2012b;DeFineLicht enzymes, whichdigestplantfibersexternaltotheinsect’s body integration: theinsecthostfunctionsasadistributoroffungal attributes ofthesesymbiosesisthedegreephysiological Martin, 1987a;Muelleretal.,2005).Oneofthemoststriking 1995; Farrelletal.,2001;DeFineLichtandBiedermann,2012; 2002; Aylward etal.,2012a;Aylward etal.,2012b;Bacci structural carbohydratesofplants(e.g.cellulose)(Aanenetal., tothriveonotherwisenon-digestiblesubstrates,suchas symbionts essentiallyasanexternaldigestiveorgan thatallowsthe macro-symbioses withspecificcladesoffungi,andusefungal such complexity, thefungus-gardening insectshaveevolvedobligate The leaf-cuttingantfungiarethought tobespecializedtowardthe The fungus-gardening(attine)antsexhibitseveral Attamyces bromatificus Atta and species inthe lineages (Mikheyevetal.,2010; Mikheyevetal., ). Theseantsexhibitlarge queens, Trachymyrmex (hereafter, Leucocoprinus gongylophorus Trachymyrmex Attamyces ‘ septentrionalis’ group, Trachymyrmex initsanamorph ) ants typically are

The Journal of Experimental Biology symbiont switchingintwospeciesof were adaptedtotheiranthostssuggestedthatdiseasesconstrain experiments thataddressedwhetherhigher-attine fungallineages 1956) (supplementarymaterialFig. Seal andTschinkel, 2007a;StradlingandPowell,1986;Weber, Mikheyev etal.,2010;Mueller2011b; Muelleretal.,1998; on ecologicalandevolutionaryscales(Mehdiabadietal.,2012; native fungi,andsurveysindicatethatswitchescanoccurnaturally 1:1 co-evolution.Coloniescanbeexperimentallyswitchedtonon- Trachymyces), severalobservationsareinconsistentwithastrict RESEARCH ARTICLE to agarden-deprived replicated experimentswheretheadditionoffresh than non-leaf-cuttingfungi.Additionalsupportderivesfrom non- Attamyces associations(DeFineLichtetal.,2010)showingthat ant–fungus 1986) andinvivo vitro host growingthem).Evidenceforthishypothesisderivesfroman should alwaysshowlowerenzymeactivitiesregardlessoftheant (andconversely, Trachymyces of theantspeciesgrowingthem Attamyces fungal enzymeactivityingardens,andconsequentlypredictsthat identity isoflesserimportanceornotimportantininfluencing testing threehypotheses,describedbelow. investigated thepotentialforconstraintsinsymbiontswitchingby fungus-switched coloniesof prompted ourstudyoftheenzymaticpropertiesgardensin from alowsamplesize(Sánchez-Peña,2005).Thesefindings performance ofleaf-cuttingantcolonies,butthisstudysuffered suggested thatsomeTrachymyces cultivars canlowerthe Mueller, 2014;SealandTschinkel, 2007a).Onepreliminarystudy cultivate fidelity totheirfungallineages(i.e.leaf-cutterantstend Oliveira, 2000;SealandTschinkel, 2007b). caterpillar excrement(HölldoblerandWilson, 2011; Lealand their funguswithvariousdriedplantdebris,flowerpartsand their gardenwithfreshleaves,whereasthenon-leaf-cuttersprovide withtheants;leaf-cutterantsprovide dietary preferencesassociated with et al.,2010).Thesephysiologicaldifferences seemtobelinked Licht etal.,2010;Richard2005;Schiøtt2008; al., 2004;Erthalet2009;DeFineLicht2013; digest cellulose(Baccietal.,2013;D’Ettorre2002;Erthal activities towardthesesubstratesandmayhaveagreaterabilityto , whereasthenon-leaf-cuttingfungihavelowermetabolic srdcdcolonyandgardengrowthwhen as reduced (StradlingandPowell,1986),aswell increased antcolonygrowth ins (i.e. fitness conferhigh specific ant–funguscombinations observedinnature populations. Attamyces cutter antpopulations(Mikheyev etal.,2010),agenerallysuperior Attamyces ants mayavoidtheapparently‘inferior’ Trachymyces. Because onlyexplainswhy cultivated bysympatricleaf-cutterants);it inferior fungalsymbiont(ratherthanswitchto Trachymyrmex problem withthishypothesisisthatbyitselfitdoesnotexplain why forced tocultivateTrachymyces gardens(Sánchez-Peña,2005).A ants cultivating Trachymyces fungi).Underthishypothesis, these The ‘host-independenthypothesis’ postulatesthatantspecies’ Although thetwohigher-attine antlineagesgenerallyexhibit h aatv obnto yohss otltsta nythe The ‘adaptivecombinationhypothesis’ postulates thatonly study (fungusgrownonartificialmedia)(StradlingandPowell, gardens willexhibithigherenzymaticactivitiesregardless Attamyces has indeedhighergrowthratesandenzymaticactivities per ohv wp ylateraltransferthroughleaf- appears tohavesweptby ol aesra likewise through could havespread Atta ants wouldpersistingrowingaphysiologically ants cultivating quantification ofenzymaticactivitynative ; Trachymyrmex urichi Trachymyrmex Atta Attamyces and Trachymyrmex

S1). Theonlyreplicated Trachymyrmex ants tendtocultivate fungus; Atta colony resultedin Attamyces Attamyces Trachymyrmex Trachymyrmex colonies were ants (Sealand ants. We garden strains Atta in reported heremay beinflated. thexylanase were significantlyheteroscedastic. Thus, Furthermore, allxylanaseeffects exceptfungalsymbiont species by antorfungalspecies(Table 3,Table (Fig. regardless offungalspeciesthan in Cellulase activitywassignificantly higherin garden appearedtobeinfluencedbytheantspeciesgrowing it. amylaseand pectinase,cellulaseactivityofthefungus patterns for o nietepteno mls ciiy(Fig. not unlikethepatternofamylaseactivity regardlessoffungalspeciesgrown, comparable pectinaseactivity Fg 2,Table (Fig. combination whereitwas~30%higherthaninallothergroups Pectinase activitywashighestinthe species andalsoexhibitedsignificantsynergistic effects (Table growing. regardless ofthetypefungusthisantspecieswas activity grown byA.texana. amylase activitywassignificantlyreducedwhenTrachymyces was Attamyces ( with theamylaseactivityofthreeothertypesgarden of Trachymyces gardenswhencultivatedby interaction term),whichwascausedbytheloweramylaseactivity to beasynergistic effect offungalandantspecies(significant on thespeciesofanthostcultivatingeitherfungus.Thereappeared higher amylaseactivitythanTrachymyces – butwasnotdependent in Trachymyrmex). Whileenzymaticactivitymaybetypicallyhigher novel combinations(Trachymyces × natural combinationsexhibithigherfungalenzymaticactivitiesthan significantly dependentonfungalspecies– species ofants.Amylase(starchhydrolysis)activitywas toward carbohydratesandproteinswhencultivatedbythetwo The twospeciesoffungiexhibiteddifferent catabolicproperties in otherTrachymyrmex (but seeSánchez-Peña,2005)orinvestigatetheeffects ofswitches examine thereverseswitch(Atta by pathogens(SealandMueller, 2014).Theseexperimentsdidnot (Seal andTschinkel, 2007a),whichwaspossiblyduetoinfections reproductive outputcomparedwithcoloniesgrowingTrachymyces septentrionalis fromfungus-switchexperimentswhere this hypothesisderives combinations wouldresultinloweractivities.Evidencesupporting Enzymatic activity RESULTS influenced byant–fungusinteractions. complex synergism hypothesisbecauseenzymaticactivityis Attamyces Trachymyrmex arizonensis Atta texana symbiont-switch experimentwithcoloniesoftheleaf-cuttingant Trachymyrmex may resultwhen sohigheractivities enzymatic activitiesdonotreflectphylogeny, According tothishypothesis,thecombinationsthatexhibithigher on complexinteractionsbetweenantsandsymbioticfungi. combinationsmaydepend that enzymaticresponsestoant–fungus Attamyces Pectinase activitywassignificantlyinfluencedbyantandfungal etse hs he hypothesesbyperforminga2×2reciprocal We testedthesethree hr osblt,tre h cmlxsnrimhptei’ is ‘complexsynergism hypothesis’, A thirdpossibility, termedthe Attamyces rTahmcs h egto h vdnespot the or Trachymyces. Theweightoftheevidence supports The JournalofExperimentalBiology(2014)doi:10.1242/jeb.098483 cultivated by cultivated by than inTrachymyces, artificialexperimentallyinduced

1). Xylanaseactivitydidnotappear tobeinfluenced

ants aregrowingAttamyces Buckley 1860andthenon-leaf-cuttingant colonies growing 1). Trachymyrmex arizonensis Trachymyrmex arizonensis Atta species. T. arizonensis (Wheeler 1907)thatweregrowingeither A. texana ants aregrowingTrachymyces or

1; supplementarymaterialFig. Attamyces colonies growingTrachymyces) A. texana , oreitherTrachymyces or A. texana Atta Fg ,Tbe1).Thus, 1,Table (Fig. . T. arizonensis did notincreasetheir Attamyces

A. texana 1). Incontrasttothe , orAttamyces had similaramylase growing colonies had × Attamyces Attamyces had much compared P colonies -values 2541

S2).

1). T. ×

The Journal of Experimental Biology were analyzedonlog Starch was doneonlyincases wherethefactorialtestwassignificant toavoidinflatingthesignificance oftheinteraction. dummy variablewas insteadused(seeMaterialsand methods);asaresult,onedegreeof freedomhadtoberemovedfrom the corresponding design (onlyfivecoloniesof differences aredenotedbydifferent letters( time wasasignificantvariable( Because gardensgrewinsizesteadilyduringthe1 E 2542 RESEARCH ARTICLE Interactions with Substrate cultivated by notdependentonfungalspecieswhen collected in2011 was Over thecourseofyear, thegrowthrateofgardenscolonies growth ratesdidnotdiffer amongthecoloniesofeitherantspecies. Despite differences amongenzymaticactivities,fungusgarden combination ofant( Fig. Consequently, theinteractionwasremovedfrommodel. firm conclusionshereintroducetheriskofaType IError. however, becausevarianceswereheteroscedastic,attemptstomake Protein Xylan Cellulose Pectin either Attamyces Table Trachymyces 834(±384)×10 to beindependentofantspecies[ approximately twicethatofTrachymyces gardens andthisappeared Greenhouse–Geisser Trachymyrmex arizonensis rates growth Fungus garden conspecific coloniesgrowingTrachymyces, higher in and fungalspeciesinproteaseactivity(proteasebeing Fig. S3)].Theremighthavebeenaweakinteractionbetweenant μ fungal species(Attamyc es euigsgrprm ml g reducingsugarpermg =0.211 Attamyces –1

.Meanamylaseactivityingardensforeachexperimental 1. Amylase activity (µg per mg ml of fungal extract) Fungal symbiont Ant host 10 15 20 25 30 35 40 45 .Smayo atra NV testsofenzymeactivitiesgardens 1. SummaryoffactorialANOVA 0 5 2.113,12.68 T. arizonensis T. arizonensis P adn xiie rtaeatvt thatwas activity gardens exhibitedprotease >0.25 werepooledandonlymaineffects arelistedhere.Significant tests( , P or Trachymyces fungus Trachymyces 10 Atta texana A. texana <0.0001), buttimedidnotinteractsignificantly transformed data.Errorbarsare±1s.d. F Ant species F F F F a 1,20 1,19 1,19 1,19 1,19 Ε T. arizonensis =0.245, P =1.36, P =23.64, P =9.42, P =3.23, P − =0.145 or Trachymyces). 1 ffnu adnetat Significant of fungusgardenextract. colonies growing (fungus: Attamyces A. texana 3 or Trachymyrmex arizonensis U; Table =0.27 =0.006 =0.09 145,871 b =0.63 =0.0001 Attamyces T. arizonensis F were growing Attamyces , P 1,6 <0.05, Scheffé’s test).Data

1; supplementarymaterial =0.964, b P F Amylase activityisgivenas <0.0001, Huynh–Feldt 10,60 Trachymyces F T. arizonensis =72.25, 1545(±536)×10 Attamyces 1,19 P Attamyces, whereasallothersamplesizeswere 031 i.4A). =0.361; Fig.

b year experiment, =5.95, Fungal species F F F F F 1,20 1,20 1,19 1,19 1,19 P =7.89, P =0.006, P =0.0003, =14.95, P =11.71, P ) and =0.0001, P than in =0.03); 3 =0.01 U, P =0.003 =0.94 <0.001 =0.99 were analyzedonlog hswssaitclyisgiiat( this wasstatisticallyinsignificant Huynh–Feldt P Greenhouse–Geisser differences aredenotedbydifferent letters( E combination ofant( Fig. ( Years werepooledbecauseoflowsamplesize.Time wassignificant interaction. Amylaseandpectinaseactivitieswerehigherwhen arizonensis enzymesingardensof The activityofparticular (F a significantinteractionbetweengardenvolumeandtime the first12 Colonies growingTrachymyces grewslightly larger gardensover patterns regardlessoffungalspecies( with fungalspecies.Coloniesthereforeexhibitedsimilargrowth explaining thevariationinincreaseofgardenvolume. timewasthemostsignificant variable measurements. Thus, to differences betweenthefirst2 examination indicatedthatthesignificantinteractionwaslargely due DISCUSSION Atta texana μ fungal species(Attamyces F euigsgrprm ml g reducingsugarpermg <0.0001, Huynh–FeldtE =0.211 8,80 8,80

–1 .Meanpectinaseactivityingardensforeachexperimental 2. Pectinase activity (µg per mg ml of fungal extract) Fungal symbiont Ant host =3.22, =43.96, 15 20 25 30 35 40 45 50 55 Atta texana 2.113,12.68 α≤0.05) arehighlightedinbold.To accountfortheunbalanced months oftheirlivesthanthosegrowing The JournalofExperimentalBiology(2014)doi:10.1242/jeb.098483 P<0.01, Greenhouse–GeisserΕ=0.388 appears tobeanemergent featureofant–fungal Ant ×fungusinteraction – F F F F 1,20 1,19 1.19 1,19 P , E =0.0001, Greenhouse–GeisserΕ P =1.23, P =5.50, P =8.09, P =6.91, P =0.639 Trachymyces 10 Atta texana and Trachymyrmex arizonensis =0.08). A. texana transformed data.Errorbarsare±1s.d. N a =6) andtomakeupforthe‘missing’ colony, a Ε − =0.31 =0.03 =0.01 =0.02 5.11,51.08 or Trachymyces). 1 =0.145 =0.639 ffnu adnetat Significant of fungusgardenextract. Attamyces A. texana or Trachymyrmex arizonensis , 145,871 b 5.11,51.08 P F T. arizonensis <0.05). However, closer

P Attamyces months andallsuccessive 1,10 , <0.05, Scheffé’s test).Data , a =4.02, P Pectinase activityisgivenas P <0.0001) andtherewas =0.11, Huynh–Feldt F 10,60 Trachymyces T. arizonensis P =3.06, A. texana =0.07; Fig. a ants cultivating 3.1,31.03 Attamyces =0.388 F -tests. This ) and , P P<0.05, =0.003, 3.1,31.03 and , but 4B). A. T. ,

The Journal of Experimental Biology were analyzedonsquare-roottransformeddata.Errorbarsare±1s.d. differences aredenotedbydifferent letters( combination ofant( Fig. RESEARCH ARTICLE population. Thismayalsoexplainwhysome Powell, 1986),ithasnotsweptthroughthe while fungal specieswhengrownby of fungalspeciescultivated.Theequivalencegrowththe two arizonensis role intheevolutionofleafcutting(Figs Trachymyces, andthatthissuperiorityof have intrinsicallyhigherenzymaticactivitiesandgrowthratesthan hypothesis (i.e.thehost-independenthypothesis)that support StradlingandPowell’s (Stradling andPowell,1986) and fungalspecies. activitiesresultfromaninteractionbetweenant symbiosis; rather, enzymaticactivitiesofthe itself norantspeciesbydetermines combinations.Inotherwords,neitherfungalspeciesby ant–fungus tofollowidiosyncraticpatternsasaresultof enzyme appears ofeach the complexsynergism hypothesis,becausetheactivity being cultivated.Theweightoftheobservationsthereforesupports amylase andpectinaseactivitiesregardlessofthefungalspecies T. arizonensis combinations), butthecorrespondingpatternwasnotobservedfor adaptive combinationhypothesis(activitiesarehighestinnatural enzymatic activityregardlessoftheantspeciesgrowingit)and with boththehost-independenthypothesis( texana μ fungal species(Attamyces Attamyces Trachymyces might explainwhy The reducedenzymaticperformanceof hypothesis wassupportedbyobservationsintheA.texana supplementary materialFig. in thefieldgrow aoaoy[niea ale eoton laboratory [unlikeanearlierreport not seemtohavedifficulty ingrowingTrachymyces inthe interactions among themicrobiotaassociated with theantsand/or switched inthelabtoaTrachymyces fungus(Sánchez-Peña,2005). euigsgrprm ml g reducingsugarpermg The weightoftheaccumulatedevidencethereforedoesnot What thenconstrainscultivarswitching? Onepossibilityisthat

–1 .Meancellulaseactivityingardensforeachexperimental 3. Cellulase activity (µg per mg ml of fungal extract) Fungal symbiont Ant host 10 12 Attamyces 0 2 4 6 8 grew , eventhoughourobservations showthat exhibited similarfungusgardengrowthratesregardless Attamyces . Trachymyrmex arizonensis may bemoreproductive Trachymyces Atta texana A. texana a,c than whengrowingTrachymyces, consistent Attamyces − or Trachymyces). 1 ffnu adnetat Significant of fungusgardenextract. Attamyces A. texana

1.However, thehost-independent S1). or Trachymyrmex arizonensis a T. arizonensis A. texana (U.G.M., unpublisheddata; T. arizonensis P taye mexicana Attamyces Attamyces <0.05, Scheffé’s test).Data b Cellulase activityisgivenas gardens exhibitedsimilar Attamyces in vitro is knowntogrowonly T. arizonensis

Attamyces 1–4). A. texana Trachymyces T. arizonensis may explainwhy, b,c Trachymyrmex T. arizonensis A. texana (Stradling and lydakey played has higher Attamyces ) and colonies. growing colonies colonies does untransformed. correspond to±95%confidenceintervals.Datadepictedare heteroscedastic (seeMaterialsandmethods).Inbothcases,errorbars excluded fromtheanalysisbecausevariancesweresignificantly F increase wasnotsignificantlydifferent forthetwofungalspecies(fungus: N Trachymyces of Attatexana variances weresignificantlyheteroscedastic.(B)Growthoffungusgardens P significantly different forthetwofungalspecies(fungus: founding queenscollectedin2011. Fungusgardenvolumeincreasewasnot sizes: Trachymyces colonies overthecourseofoneyear(August2011 toJuly2012).Sample fungal species. Fig. raised whenT. septentrionalis fungi constrainantandfungalcombinations.Thispossibility was ants maynot apply theappropriatetriggers tonovelfungal Rodrigues etal.,2009).Another non-exclusivepossibilityisthat invasions bycompetitorfungi (Haeder etal.,2009;Mueller, 2012; Sen etal.,2009),whichmay ormaynotbeabletowithstand as dootherattinespecies(Ishak etal.,2011; Rodriguesetal.,2011; communities thataredistinctfrom otherspeciesof Trachymyrmex arizonensis garden declinewasnotknownwithcertaintyinthatstudy. weedy fungidestroyinggardens,thoughtheexactmechanism of and Mueller, 2014),whichapparentlywas theresultofinvasionby experienced fitnessreductionsafteradopting 1,10 =0.361). Volumes fromAprilwereexcludedanalysisbecausethe =5 (N Fungus garden volume (cm3)

=4.02, P 4. Fungusgardengrowthrateofeachantspeciesgrowingeither 100 200 300 400 500 600 100 150 200 250 300 350 400 450 50 =1 from2010cohort, 0 0 u.Sp c.Nv e.Jn e.Mr p.MyJn Jul. Jun. May Apr. Mar. Feb. Jan. Dec. Nov. Oct. Sep. Aug. B A u.Jul. Jun. =0.07). Volumes fromAugust,Decemberand Februarywere The JournalofExperimentalBiology(2014)doi:10.1242/jeb.098483 N colonies overthecourseoftheirfirstyearlife.Samplesizes: Attamyces Trachymyces =6 (N (A) Growthoffungusgardens N =3 from2010cohort, taye N=5.Datawerefromthecohortof =3, Attamyces u.Sp c.Nv e.Jn e.Mar. Feb. Jan. Dec. Nov. Oct. Sep. Aug. N =4 from2011 cohort).Fungusgardenvolume likely associatewithdifferent microbial and N =3 from2011 cohort), rcyymxturrifex Trachymyrmex Trachymyrmex arizonensis Attamyces F 1,6 =0.964, Trachymyrmex, fungus (Seal Attamyces Trachymyces Attamyces 2543

The Journal of Experimental Biology 2544 RESEARCH ARTICLE (1000–2000 observation). gardening antswillacceptandfeedtheirfungus;J.N.S.,unpublished shumardii, anaturallyoccurringfungalsubstratethatmanyfungus- at leasttwiceweeklywithoakcatkins(staminateflowersof afterwhichcolonieswerefed cleaned dailyuntilthefirstworkersappeared, Tschinkel, 2007a;SealandTschinkel, 2007b).Queenswere fedandnests libitum andfed 2010 and2011. Coloniesweremaintainedinplaster-lined boxes immediately aftermatingflights(Seal,2009;SealandTschinkel, 2007c)in of bothspecieswereobtainedbyrearingnewlymatedqueenscollected the leaf-cutterant This studyemployedreciprocalsymbiontswitchexperimentsconducted on lignocelluloses andproteinsthat enzymatic machineryrequiredforthemetabolismofcarbohydrates, symbionts thatwouldstimulatethesynthesisofdiverse ubro tde eotsgiiatcluaeatvt for number ofstudiesreportsignificantcellulaseactivity ongoing debateonwhether Wade, 2007). negative inter-genomic epistasis(Heath,2009;Linksvayer, 2007; result inmismatchesatthephysiologicalandgenomiclevel(i.e. colonies growingAttamyces MATERIALS ANDMETHODS gongylophorus species typicalof thevastmajorityofleaf-cutting ants( et al.,2011b; Sanchez-Peña,2010).Attatexana western Lousianaandnortheastern (Muelleretal.,2011a; Mueller 2007). (Rabeling etal., Desert andwesternportionsoftheChihuahuan Desert ellss meritingfurtherstudy. cellulases, symbioses ofT. arizonensis metabolism (Aylward etal.,2012a;Suen2010),thefungal Considering recentinterestintheroleoffungusgardenscellulose abundant ormoreactiveingardensgrownby microbes [e.g.yeasts(Mendesetal.,2012)]areeithermore microbes ingardenbiofilmssuchthatcellulose-metabolizing gardens betweenhostspeciesmaycauseareshuffling ofauxiliary extracted fromsubstrates.A third possibilityisthatswitching fungus togetapointinthegardencyclewhencelluloseis Attamyces substrates (catkins)caninfactmetabolizecellulosebecause suggest thatT. arizonensis resultspresented hereareintriguingbecausethey et al.,2004).The (AbrilandBucher, 2002;Bucher to findsignificantcellulaseactivity Schiøtt etal.,2008;Silva2003),whereasotherstudiesfailed (Bacci etal.,1995;D’Ettorre2002;MartinandWeber, 1969; cultivated fungigrownonavarietyofnaturalandartificialdiets T. arizonensis (Aylward etal.,2013).Forexample,pectinaseactivitywaslowerin and Evaluation ofthishypothesisrequiresknowledgewhether exhausted (DeFineLichtetal.,2010;Moller2011). in refusedepotsoncesimplercompounds(starches,pectins,etc.)are because theantsremoveoldersectionsofgardensandplacethem cutting antsneverallowtheir by genes areactivatedormoreactivelyexpressedthanwhengrown that whenAttamyces onepossibleexplanationofourobservationsis 2013). Therefore, possess atleastthreegenesforcellulosedegradation(Aylward etal., activity than Trachymyrmex arizonensis One ofthemoresurprisingfindingsthisstudysustainsan Atta Trachymyrmex Atta texana in amannersimilartopreviouslypublishedmethods(Sealand ns oeatoshv asdasecondpossibilitythatleaf- ants. Someauthorshaveraiseda m) inmixedoak–juniper–pinyon pine forestsintheSonoran grown byT. arizonensis Attamyces ) (Fisheretal.,1994; Mikheyevetal.,2006;Mueller etal., A. texana colonies growing is foundthroughoutcentral,east andsouthTexas, ants differ inhowmuchtimetheygivetheir is grownbyTrachymyrmex grown byA.texana and thehigher-attine ant is acommonspeciesalongmid-elevations colonies growingAttamyces Attamyces (Fig. Attamyces ants mightbeimportantsourcesof 2). Thusant–fungusswitchesmay Attamyces Attamyces can metabolizecellulose.A exhibited highercellulase gardens todigestcellulose . cultivates asinglefungal Attamyces is knowntopossess T. arizonensis.Colonies than in T. arizonensis . ants, cellulase Leucocoprinus is knownto A. texana on natural Quercus Atta Atta ad - Attamyces queens) (Seal,2009).Attatexana 200 condition andprovidedwith~50 University ofTexas Stengl‘LostPines’ BiologicalStation(30°05.218 collected froma 2007 (30°12.622 2010; Muelleretal.,2011b) (supplementarymaterial Fig. Trachymyces (Mikheyevetal.,2006;Mikheyev2008;Mueller S (31°53.025′ USA colonies werecollectedattheSouthwestResearchStation,nearPortal,AZ, randomly selectedforenzymaticactivityanalysis.All were of whichwerecultivatingTrachymyces); threeofthesecolonies 10 coloniesrearedsimilarlyfromqueenscollectedon25–27July2010(all mortalityamongthese queens,wealsoincludedataon Because ofunequal 2011. Fiveofthesewererearedon 27–28July conducted oneightcoloniesstartedwithqueenscollected 97°10.425′ were suppliedwithfunguscollectedfroman heavy rainofthesummermonsoonseason.Coloniesgrowing substantially lessthanthedifferentiation between Texas andArizona(Muelleretal.,2011b), thisgeneticdifferentiation is Brady, 2008).Despitepopulationdifferentiation between representative ofthetwomajorcladeshigherAttini(Schultzand study arenotsympatric,theseantsandtheircultivatedfungi Fig. the sympatricleaf-cutterant in preparation).Trachymyrmex arizonensis Attamyces (32°18.971′ in2009 reared fromanewlymatedqueencollectednearTucson, AZ,USA, from amature S (30°12′ USA newly matedqueenscollectedafter matingflightsnearHornsbyBend,TX, confounding theresultswithdiet(Kooij etal.,2011). specieswerefedthesame type ofcatkinstoavoid Colonies ofthetwo shumardii) Tschinkel, 2007b).All 11×11×3 uvy,hwvr niaethatsomepopulationsof surveys, however, indicate Leucocoprinus Trachymyrmex cultivates aTrachymyces fungustypicalformost(butnotall) Seal etal.,2012;andMueller, 2014).Incontrast, Attamyces bromatificus reproduces sexually–werefertoitbyitsanamorph(asexual)form, 2011a; Mueller, 2002;Pagnoccaetal.,2001);however, thisspeciesrarely cylindrically shaped,196 containers usedinpreviousstudieson approximately 1 dental plaster(MarjoyEnterprises,SanAntonio,TX,USA). After transferred to7×7 first callowworkersappeared(ca.6 Tschinkel, 2007c).Colonieswerekeptinthesenestingcontainers untilthe were fedandcleaneddailyuntilthefirstworkersappeared(Seal and 9 of fungusintoa4 with cottonsaturatedsterilewater. T all collected inJuly2010attheSouthwestResearchStation.Uponcollection, April 2007( from cohortscollected on18May2010( on 16–18May2010,whereasthe sixcoloniesgrowing Trachymyces, threewerecollected on13May2011 andthreewerecollected

rachymyces weresuppliedwithfungusfroma cm Petridish.Allofthespacebetweenthesetwodisheswasfilled Queens wererandomlyassignedtoeitherthe The experimentalswitcheson The 12Attatexana T. arizonensis g ofgarden(A.texana S1; U.G.M.,inpreparation).Althoughthetwoantspeciesusedthis cm) (SealandMueller, 2014;SealandTschinkel, 2007a;Sealand instead ofTrachymyces (supplementarymaterialFig. W, 141 collected froman N, 110°53.562′ ad libitum N The JournalofExperimentalBiology(2014)doi:10.1242/jeb.098483 =1). Freshlycollected queenswereplacedinsquare plastic T. arizonensis 37.3″ clade thatisthesisterto species thatisplacedinataxonomicallyunresolved ′ N, 97°38.469′

N, 109°12.374′ year ofage,coloniesweremovedtolarger nesting queens weresuppliedwithonemelanizedpupaobtained cm Petridish(gardenchamber)thatwasinsertedinsidea cm plasticboxeswitha5 m elevation). T. septentrionalis N, 97°38′ colonies usedinthisstudyweresimilarly rearedfrom T. arizonensis throughout thedurationofentireexperiment. Kreisel (Muelleretal.,2010;Mueller2011b;

Acromyrmex versicolor queens; owingtothemuchlarger sizeof cm W, 858 colony inthelaboratory, placedwithca.8 A. texana W, 125 3 28.07″ W, 1646 depressions inasquareplasticbox,

T. arizonensis g ofgarden(T. arizonensis Attamyces colonies werefedoakcatkins( nest collectedinApril2010atthe

m elevation).Coloniesgrowing queens weresuppliedwitheither m elevation)orTrachymyces fungus W). Ofthecoloniesgrowing etna utn X S,inMay nest nearAustin,TX,USA,

weeks later),thencolonieswere Trachymyrmex Trachymyrmex arizonensis lvto) 1 m elevation), likely exchangessymbiontswith N

mm-thick bottomofmoistened Acromyrmex versicolor =1), 13May2011 (N and threeonTrachymyces. Attamyces Attamyces used inthisstudywere (supplementary material T. arizonensis T. arizonensis ants (plaster-lined,

day afterthefirst

S1). Attamyces or Trachymyces Attamyces Attamyces clade. Ongoing T. arizonensis T. arizonensis

S1; U.G.M., queens) or Attamyces cultivate Quercus =4) and queens colony colony came from

Atta cm and ′ N, 3

The Journal of Experimental Biology containing 500 by grindingca.120 substrates inthegardens.Enzymeswereextractedfromfungusgardens at least48 for 4 2011), allantcoloniesofbothspecieswerefedastrictdietoakcatkins activities ingardensdependonthesubstratesusedbyants(Kooijetal., (Moller etal.,2011; Suenetal.,2010).Furthermore,becauseenzymatic microbes (e.g.bacterialbiofilms)seemstobegreaterinthelowerportions garden, whereasrelativeactivityofenzymesderivedfromnon-cultivar garden. Mostfungallyderiveddigestionoccursintheupper-most partofthe fungus garden,whichapproximatedasamplefromthetopone-thirdof with pearleaves(Bradfordpear, to the twice aweek. connected toaforagingarenaviaplastictubeandfedcleanedatleast after thefirstworkersemerged. Afterworkeremergence, colonieswere (Fernández-Marín etal.,2004;Seal,2009),theyweregivensubstrateonly were terminatedbyadding50 (Moller etal.,2011; Schiøttetal.,2008;2010).Thereactions the mostcommontypesofplantcarbohydratedigestedbyantfungus Louis, MO,USA).Priorworkhasindicatedthesesubstratestobeamong and xylan(frombeechwood)(allpurchasedfromSigma-Aldrich, St solutions wereused:pectin(fromapple),starch,carboxymethylcellulose solution, andincubatedatroomtemperaturefor60 of waterand50 al., 2003).Accordingly, 10 μloffungusgardenextractwasaddedto40 method, whichassaysreducingsugarconcentration(Miller, 1959;Silvaet estimate foreachcolony. average enzymaticactivitywastakenfromthesefourvaluestoprovidean Each colonywassampledfourtimesoverthecourseof2 activityassays. tube, whichwasusedforboththecarbohydraseandprotease for 15 removal ofvisibleeggs,larvaeandpupae.Extractswerecentrifugedat4°C fungus gardenmaterialwasselectedfromtheuppermost1.5 variation inenzymaticactivity(Molleretal.,2011; Suenetal.,2010), at least1 Enzymatic activityassayswereconductedin2012whenallcolonies Mueller, 2014;SealandTschinkel, 2007a;SealandTschinkel, 2007b). drilled 1 placed ontopofeachnestcover(apiecePlexiglasthatcontainedfivepre- measuredwitharuler The widthandlengthofeachfungusgardenwere the plasternestchamberwascompletelycoveredwithapieceofPlexiglas. (length, widthandheight)ineachnestboxatmonthlyintervals.Thetopof garden volumeswereestimatedbymeasuringthemaximumdimensions lives. Volumes ofgardensweremeasuredmonthlyinbothspecies.Fungus Colonies weremonitoredmonthlyoverthecourseoffirstyeartheir boxes (7×7×3 twice weekly. Because RESEARCH ARTICLE solution andincubatingat99°Cfor15 Fungal enzymatic activityassays measurements growth Fungus garden colorimetrically. A 10 hydrolysis releases theazodye,concentrationof whichcanbeinferred Sigma-Aldrich). Azocaseinisanon-specific proteasesubstrate,whichupon (Semenova etal.,2011). Thismethodusesazocasein(azo-labeledcasein; and Tomarelli, 1947)usedpreviously instudiesonattineproteaseactivity glucose. 540 was addedto150 μlofwaterandthenreadinaspectrophotometerat immediate incubationat99°C.Afterincubation,50 samples weretreatedbyaddingtheDNSandenzymeextractbefore darker thecolor, thehigherconcentrationofreducingsugars).Control dye changescolordependingontheconcentrationofreducingsugars (the Carbohydrase activitywasmeasuredusingthedinitrosalicylicacid Protease activitywasmeasuredusing theAzocaseinmethod(Charney nm. Amountshydrolyzedwereinterpolated usingastandardcurvefor weeks priortoenzymaticassays.Finally, gardenswerealsosampled T. arizonensis min at14,000

mm holes,2 year old.Becausefungusgardensarethoughttoexhibitspatial

h afterthelastfeeding,sothattherewerenofreshlydeposited cm) linedwith5 Atta texana

ml of20 μl of1%(w/v)(=500gsubstrate)carbohydrate

mg offungusgardenmaterialinanEppendorftube colonies, excepttheywerealsoperiodicallysupplied rpm. Thesupernatantwasthentransferredtoafresh μl sampleoffungal extractwasaddedto15 cm apart,fromthecenterofcover)(Sealand Atta colonies werefedsubstratessimilartothose mmol queens donotforageduringthefoundingphase μl of96

mm ofdentalplaster, whichwasmoistened

Pyrus calleryana l − 1 phosphate buffer (pH mmol

min. Athightemperature,theDNS

l − 1 dinitrosalicylic acid(DNS) ).

min. Fourcarbohydrate μl ofeachsample

weeks, andthe 6.9) afterthe

cm ofeach μl of2% μl rehueGisr andHuynh–Feldt-corrected Greenhouse–Geisser- Rohlf, 1995).Ininstanceswheresphericitytestswererejected,wereport variances amongtherepeatedmeasures(Keselmanetal.,2001;Sokaland parametric assumptions,wetestedforsphericity, whichtestsforequalityof at approximately1 effect. Volumes wererepeatedlymeasuredonallcoloniesinthisexperiment time astherepeatedmeasureandwithfungalsymbiontspeciesmain of themissingvaluesgenerated. added to140 μlof1 (TCA). Theresultingsuspensionwascentrifuged(15,000 reaction wasterminatedbyadding120 Data wereeitherlog by subtractingonedegreeoffreedomfromthecorresponding the nullhypothesisfalsely(Type IError),thetestismademoreconservative neither themeannorvariance(Underwood,1997),buttoavoidrejecting belonged (SealandTschinkel, 2007a;Underwood,1997).Thismethodalters calculated fromameanvalueofeachgrouptowhichthemissingdata was solvedusingUnderwood’s techniqueofcreatingadummyvariable arizonensis The problemofunequalsamplesizesresultingfromonlyfiveswitched azocasein solutionandincubatedfor60 by M.S. assays. J.N.S.and U.G.M. wroteandrevisedthemanuscript, withsignificantinput modified theenzymaticassays.J.N.S. executedtheexperimentsandenzymatic J.N.S. andU.G.M.conceiveddesigned theexperiments.M.S.developedand The authorsdeclarenocompetingfinancial interests. Facility suppliedtheplatereader. and theirknowledgeoftheattinesArizona.CecilHarkeyatUT AustinCore Barbara Roth.RebeccaClark,BobJohnsonandDianaWheelersharedcollections and thestaff attheAmericanMuseum’s SouthwestResearchStation,especially We thankKevinAndersonattheHornsbyBendEnvironmentalResearchCenter, Constructive commentsbyreviewersandeditorsgreatlyimprovedthemanuscript. Sequences wereidentifiedusingBLAST atNCBIGenBank. University ofTexas atAustinUniversityDNA SequencingFacilities. et al.,1998;Sen2009;White1990),thensequencing at the PCR amplificationofthe heated tonearboilingfor90 the emerging culturesandplacedinChelexresin(Sigma-Aldrich) Small samples(hyphaeorspores)werecollectedwithsterileforcepsfrom and possiblyitsassociatedbiofilms. effect), itshouldbeinterpretedasaneffect influencedbythefungusgarden a result,whenthestatisticaltestsreportsignificant‘fungal’ effect (or‘ant’ ‘extended phenotype’ ofenzymaticactivityinthesymbiosis they findinthefungusgarden,datapresentedherecorrespondtoan important totheants,becauseantsarepotentiallyconsumingwhatever Leucocoprinus it isunclearwhetherthedistinctionbetweencompoundsproducedby functions inextractingenergy frompectins(Mendesetal.,2012).Although may beimportantcontributorsofcellulaseactivityandperformcrucial (Aylward etal.,2012a;Aylward etal.,2012b).Yeasts inhabitingthegarden biofilms. Themetabolicactivitiesoffungusgardensarelikelyquitediverse between enzymessecretedbythecultivatedfungusorassociatedmicrobial absorbances. activity wascalculatedfromthedifference betweentreatmentandcontrol was addedimmediatelybeforetheapplicationofTCA.Relativeprotease at 25°C.Controlsampleswerepreparedidenticallyexceptenzymeextract assumptions. Statistical analyses Author contributions Competing interests Acknowledgements identification Fungus Garden volumeswereanalyzedwitharepeatedmeasuresANOVA, with h ehd sdt siaeezm ciiydonotdifferentiate The methodsusedtoestimateenzymeactivity colonies (whereasallotherant×fungalcombinationshad The JournalofExperimentalBiology(2014)doi:10.1242/jeb.098483 spp. versusothermicrobesinhabitingthefungusgardensis

month intervals.Inadditiontotestsforstandard mol 10 or square-roottransformedtomeetparametric l ITS − 1 NaOH. Absorbancewasmeasuredat440

min. Purefungalcultureswereidentifiedby gene usingprimersITS4andITS5(Mueller μl of10%trichloroaceticacid

min atroomtemperature.The F -statistics and g) for5minand F sensu lato.As -test foreach P -values. 2545 N

=6) nm T.

The Journal of Experimental Biology sa,H . ilr .L,Sn . od .E,Myr .adMelr U.G. Mueller, and E. Meyer, S.E., Dowd, R., Sen, J.L., Miller, H.D., Ishak, ölolr .adWlo,E.O. Wilson, and B. Hölldobler, ele,K,FrádzMrn . sa,H . e,R,Lnsae,T .and T. A. Linksvayer, R., Sen, H.D., Ishak, H., Fernández-Marín, K., Kellner, 2546 RESEARCH ARTICLE http://jeb.biologists.org/lookup/suppl/doi:10.1242/jeb.098483/-/DC1 Supplementary materialavailableonlineat to M.S. to J.N.S.andU.G.M.,bytheDanishNationalResearchFoundation[DNRF57] Financial supportwasprovidedbytheNationalScienceFoundation[IOS-0920138] ae,D . gltn . oln-eer,C,Glbr-rse,T,Rosendahl, T., Guldberg-Froslev, C., Rouland-Lefevre, P., Eggleton, D.K., Aanen, P. Eggleton, and D.K. Aanen, adr . it,R,Hr,H n ptle,D. Spiteller, and K.D. Heath, H. Herz, R., Wirth, S., Haeder, W. T. Wcislo, and J.K. Zimmerman, and H., J.H. Fernández-Marín, Chung, B.B., Normark, B.C., R.I. O’Meara, Samuels, A.S., and Sequeira, R.M. B.D., Farrell, Cooper, C.P., Silva, Jr, M., Erthal, R.I. Samuels, and C. PeresSilva, Jr, M., Erthal, A.E. Douglas, References Supplementary material Funding ywr,F . unm .E,Sot .J,Se,G,Tig,S . dm,S.M., Adams, S.G., Tringe, G., Suen, J.J., Scott, K.E., Burnum, F. O., Aylward, bi,A .adBce,E.H. Bucher, and A.B. Abril, re,A . ule,U .adAas R.M. Adams, and U.G. Mueller, A.M., Green, ihr .J,Srdig .J n elr D.N. Pegler, and D.J. Stradling, P. J., Fisher, eFn ih,H,Shøt . oosaWzsnk,A,Ngad . Roepstorff, S., Nygaard, A., Rogowska-Wrzesinska, M., Schiøtt, H., De FineLicht, eFn ih,H . cit,M,Melr .G n omm,J. Boomsma, and U.G. Mueller, M., Schiøtt, H.H., De FineLicht, G. Suen, and C. Currie, F., Aylward, hre,J n oael,R.M. Tomarelli, and J. A. Charney, Abril, and V. Marchesini, E.H., Bucher, and A.F. Somera, F. C., Pagnocca, A., Rodrigues, O.C., Bueno, Jr, M., Bacci, F. C. Pagnocca, and M. Anversa, Jr, M., Bacci, J., Moeller, D., Tremmel, C., Teiling, S., Tringe, K., Burnum-Johnson, F., Aylward, eFn ih,H .adBeemn,P. H. Biedermann, and H. De FineLicht, ’tor,P,Mr,P,Dbno,V,Ruad .adErr,C. Errard, and C. Rouland, V., Dibangou, P., Mora, P., D’Ettorre, J.F. A. Traniello, and S.N. Beshers, Trachymyrmex septentrionalis Microbiomes ofantcastesimplicatenew microbialrolesinthefungus-growingant Norton. rhizobia. ule,U.G. Mueller, mutualistic fungalsymbionts. J. Boomsma, and S. rain forest.Curr. Biol. pathogenic fungus 191-195. fungal cultivarsbetweensympatricspeciesoffungus-growingants. (, Formicidae,Attini. and evolutionarysequenceofnestestablishmentinfungus-growing ants Scolytinae andPlatypodinae). B.H. Jordal, enzymes ofleaf-cuttingantfungi. 50, 881-891. distribution inthegutofadultworkersandpartialcharacterization. cutting ants,Acromyrmexsubterraneus ar,K . ioa .D,Peosi .D,Prie .O etal. S.O. Purvine, P. D., ant fungusgardens. Piehowski, C.D., Metagenomic andmetaproteomicinsightsintobacterialcommunitiesinleaf-cutter Nicora, K.W., Barry, cutting antsdoesnotmetabolizecellulose. from alivenestoftheleaf-cuttingantAttacephalotes. 11 between leaf-cuttingantsandfungus-gardensymbionts. J. Boomsma, and P. 2055-2069. Evolutionary transitionsinenzymeactivityofantfungusgardens. activity infungusfarmingambrosiabeetles. of theproteolyticactivityduodenaljuice. nutrient balancebetweenharvestedandrefusematerial. demography intheattineant, herbivory ofleaf-cutterantsonplantstarch. A. Silva, rubropilosa sexdens Leucocoprinus gongylophorus 3778. etal. C. polymers inleaf-cutterantfungusgardens. Nicora, P., Piehowski, gongylophorus K.W., Barry, J.A., Scott, nutritional symbiosesinleaf-cutterants. ants. J.Comp.Physiol.B the symbioticfungusindigestivemetabolismoftwospeciesfungus-growing 0 , 583-587. Evolution (2013). A metabolicpathwayassembledbyenzymeselectionmaysupport (2009). Intergenomicepistasisandcoevolutionary constraintinplantsand (2010). support leaf-cuttingantstoprotecttheirfungusgardenagainstthe produces diverseenzymesforthedegradationofrecalcitrantplant (2013). Co-evolutionary patternsanddiversificationofant–fungus (2001). Theevolutionofagricultureinbeetles(Curculionidae: 64, 1446-1458. . AntonieLeeuwenhoek ISME J. The SymbioticHabit 15, 851-855. (2013). Laccasedetoxificationmediatesthenutritionalalliance (2002). Theevolutionoffungus-growingtermitesandtheir 172 6 Proc. Natl.Acad.Sci.USA , 169-176. Trachymyrmex septentrionalis . ScientificReports , 1688-1701. Evolution . Proc.Natl.Acad.Sci.USA (2005). Fungus-growingtermitesoriginatedinAfrican , thefungusculturedbyleaf-cuttingant (2002). Evidencethatthefungusculturedbyleaf- (1947). A colorimetricmethodforthedetermination (2011). Comp. Biochem.Physiol. Biol. J.Linn.Soc.Lond. 55, 2011-2027. . Princeton,NJ:PrincetonUniversityPress. The LeafcutterAnts (2012b). Theevolutionaryinnovationof J. Biol.Chem. Ecol. Lett.5 Front. Zool. J. InsectPhysiol. 67, 385-386. (1994). Theadaptivenessofworker (2004). Herbivorybyleaf-cuttingants: (2012). Patternsoffunctionalenzyme (Hymenoptera: Formicidae:Attini): Appl. Environ.Microbiol. 3 (1994). , 41-61. 1 (2004). Digestiveenzymesofleaf- , 204. (1995). Cellulosedegradationby (2002). Extensiveexchangeof Mycol. Res. (2009). Candicidin-producing , 325-328. 9 99, 14887-14892. Leucoagaricus basidiomata Biotropica , 13. Proc. Natl.Acad.Sci.USA 171 81, 39-48. 106 152B . Ecology (2004). Ecologicaltraits , 501-505. 59, 525-531. . NewYork, NY: W.W. (2013). , 4742-4746. , 54-59. (2002). Theroleof (2009). Hydrolytic 98, 884-888. J. InsectPhysiol. 36, 327-332. 75, 763-775. Leucoagaricus Mol. Ecol. Evolution 79, 3770- (2012a). (2010). (2011). Atta 64, 11 , ikvyr T. A. Linksvayer, calNa,M,Hdil,M . oc,T . ae,H . oae-oo T., Domazet-Lošo, H.V., Carey, T. C., Bosch, M.G., Hadfield, M., McFall-Ngai, N.A. Weber, and M. Martin, M. M. Martin, M. Martin, P. S. Oliveira, and I.R. Leal, oi,P,Shot . omm,J .add ieLct H. deFineLicht, and J. Boomsma, M., Schiott, P., Kooij, el .R n lvia P. S. Oliveira, and I.R. Leal, Cummings, H.V., Carey, C., Albertson, G.E., Robinson, D.F., Clayton, D., Kültz, edaai .J,Melr .G,Bay .G,Hme,A .adShlz T. R. Schultz, and A.G. Himler, S.G., Brady, U.G., Mueller, N.J., Mehdiabadi, eemn .J,Agn,J n oacu,R.K. Kowalchuk, and J. Algina, H.J., Keselman, eds .D,Rdius . aoOoei . asn .A .adPgoc,F. Pagnocca, and F. A.L. Marson, I., Dayo-Owoyemi, A., Rodrigues, T. D., Mendes, ilr G.L. Miller, Mikheyev P. Abbot, and U.G. Mueller, A.S., Mikheyev, R. Wirth, and M. Tabarelli, I., Leal, S., Meyer, olr .E,D ieLct .H,Hrot . ilt,W .adBosa J. Boomsma, and W. G. Willats, J., Harholt, H.H., DeFineLicht, I.E., Moller, P. Abbot, and U.G. Mueller, A.S., Mikheyev, ule,U.G. Mueller, U.G. Mueller, ule,U . ct,J . sa,H . opr .adRdius A. Rodrigues, and M. Cooper, H.D., Ishak, J.J., Scott, U.G., Mueller, T. R. Schultz, and S.A. Rehner, U.G., Mueller, ule,U . ead,N . ae,D . i,D .adShlz T. R. Schultz, and D.L. Six, D.K., Aanen, N.M., Gerardo, U.G., Mueller, ule,U . ihyv .S,Hn,E,Sn . arn .L,Slmn S.E., Solomon, D.L., Warren, R., Sen, E., Hong, A.S., Mikheyev, U.G., Mueller, ule,U . ihyv .S,Slmn .E n opr M. Cooper, and S.E. Solomon, A.S., Mikheyev, U.G., Mueller, anca .C,Bci . r ugr,M . un,O . eln,M.J., Hebling, O.C., Bueno, M.H., Fungaro, Jr, M., Bacci, F. C., Pagnocca, ihr,F-. oa . rad .adRuad C. Rouland, and C. Errard, P., Mora, F.-J., Richard, orge,A,Cbe .N,Melr .G,Bci . radPgoc,F. C. Pagnocca, and Jr M., Bacci, U.G., Mueller, R.N., Cable, A., Rodrigues, U.G. Mueller, and R.A. Johnson, S. P., Cover, C., Rabeling, brood andworkergenomes. Insectes Soc. savanna: seasonaluseoffungalsubstrateinthecerradovegetationBrazil. fruits, andseedsinCerradovegetationsoutheastBrazil. oga,A . uiir . br,G,Fkm,T,Glet .F etal. S.F. Gilbert, T., Fukami, Sci. USA G., Eberl, N., inabacterialworld,newimperativeforthelifesciences. Dubilier, A.E., Douglas, 1386-1387. cultured bytheAttineant University Press. Enzymes inArthropodBiology culture intheirnests.In in ArthropodBiology (Attini, Formicidae). measures designs:areview. .E,Dwr . dad,S . omn,H . rs,L .etal. L.J. Gross, H.A., Hofmann, S.V., frontiers fororganismalbiology. Edwards, K., Dewar, M. E., Commun. (2012). Symbiontfidelityandtheoriginofspeciesinfungus-growingants. Evol. Biol. cutting antnests.Insects C. coevolution inthefungus-growingantsymbiosis. microclimate. cutting ants:nestsofAttacephalotes Am. Nat. and lepiotaceouscultivarphylogeniesrevealscoevolutionarysynchronydiscord. sugar. Anal.Chem. associations intheasexualfungus-farmingant 10702-10706. ant fungusgardens. (2011). Thedynamicsofplantcell-wallpolysaccharidedecompositioninleaf-cutting mutualists. Pleistocene populationexpansioninafungus-gardeningantanditsmicrobial deconstruction oftheattineant–fungussymbiosis. attine ant–microbesymbiosis. The evolutionofagricultureininsects. Monoculture ofleafcutterantgardens. ants. Science sa,H . opr . ilr .L,Safr .A etal. K.A. Shaffer, northern frontierofatropicalant–fungussymbiosis. J.L., Miller, M., cold-tolerant fungalsymbiontspermitswinterfungiculturebyleafcutterantsat the Cooper, H.D., Ishak, ant–fungus symbiosis. : coevolutionarypatternsatthenorthernrangelimitofleaf-cutter 4053-4056. plant material. leaf-cutting antsandthecontributionof theirfungalcultivartothedegradationof (Hymenoptera: Formicidae). North Americanspeciesofthefungus-gardening antgenus fallax M. Capelari, sterile myceliumofthefunguscultivatedbyleaf-cuttingant and A. Sant’anna, pathogens ofleaf-cutting ants. (2009). Antagonistic interactionsbetweengardenyeasts and microfungalgarden (2012). Generationofnutrientsanddetoxification:possiblerolesyeastsinleaf- . Mycol.Res. .S,V,T n ule,U.G. Mueller, and T. Vo, A.S., , 175 110 3 26, 1353-1362. (1959). Useofdinitrosalicylicacidreagentfordeterminationreducing Mol. Ecol. , 840. (2002). Antversusfungusmutualism:ant–cultivarconflictandthe The JournalofExperimentalBiology(2014)doi:10.1242/jeb.098483 (1987a , E126-E133. (1987b). Thesymbiosesbetweentheattineantsandfungithey , 3229-3236. (2012). Symbiontrecruitmentversusant–symbiontco-evolutioninthe Ecol. Entomol. 47, 376-382. 281 J. Comp.Physiol.B fungus-garden enzymeactivityafterachangeinfungalsubstrate (2007). Antspeciesdifferences determinedbyepistasisbetween , 2034-2038. 105 ). Invertebrate-MicrobialInteractions:IngestedFungalEnzymes 31, 426-428. Insectes Soc. . Ithaca,NewYork, NY: CornellUniversityPress. PLoS ONE 17, 4480-4488. , 173-176. Proc. R.Soc.B 3 , 228-245. (1998). Interactionsbetweenfungus-growingants(Attini), Atta colombicatonsipes (2000). ForagingecologyofattineantsinaNeotropical PLoS ONE Zootaxa 36, 14-24. Br. J.Math.Stat.Psychol. Invertebrate-microbial Interactions:IngestedFungal Curr. Opin.Microbiol. Antonie Leeuwenhoek (1969). Thecellulose-utlizingcapabilityofthefungus BioScience (ed. M.Martin),pp.91-126.Ithaca,NY: Cornell 6 , e17506. 58, 145-151. 175 (2001). RAPDanalysisofthesexualstateand 1664 Annu. Rev. Ecol.Syst. PLoS ONE , 297-303. 278 2 , e994. , 1-53. , 3050-3059. 63, 464-471. drastically alterforeststructureand (2010). Comparativedatingofattineant (2011). Ecosystemengineeringbyleaf- (2006). Crypticsexandmany-to-one (1998). Theevolutionofagriculturein Am. Nat. (2008). Phylogeographyofpost- 5 Proc. Natl.Acad.Sci.USA (2001). Theanalysisofrepeated , e12668. 15, 269-277. Proc. Natl.Acad.Sci.USA . (2005). Digestivecapacitiesof 96, 331-342. Ann. Entomol.Soc.Am. 54, 1-20. Biotropica 6 Suppl.4,S67-S98. 160 36, 563-595. (2007). A reviewofthe (2011). Rapidshiftsin (2011a). Evolutionof Proc. Natl.Acad. (2011b). Frontier 30, 170-178. Trachymyrmex in Panama. (2013). New (2013). (2005). (2010). 103 108 Nat. 62, J. , ,

The Journal of Experimental Biology el .N n shne,W. R. Tschinkel, and J.N. Seal, el .N n shne,W. R. Tschinkel, and J.N. Seal, J. Boomsma, and P. Roepstorff, A., Rogowska-Wrzesinska, M., Schiøtt, el .N n shne,W. R. Tschinkel, and J.N. Seal, el .N n shne,W. R. Tschinkel, and J.N. Seal, S.G. Brady, and T. R. Schultz, J. Boomsma, and L. Lange, H.H., DeFineLicht, M., Schiøtt, el .N n shne,W. R. Tschinkel, and J.N. Seal, U.G. Mueller, and J.N. Seal, RESEARCH ARTICLE orge,A,Melr .G,Ihk .D,Bci . radPgoc,F. C. Pagnocca, S.R. Sánchez-Peña, and Jr M., Bacci, H.D., Ishak, U.G., Mueller, A., Rodrigues, ace-ea S. Sanchez-Peña, el J.N. Seal, Trachymyrmex septentrionalis Trachymyrmex septentrionalis Divers. of phytopathogenicfungi. Leaf-cutting antfungiproducecellwalldegradingpectinasecomplexesreminiscent colony foundinginthefungus-gardeningants degradation ofxylaninthefungusgardensleaf-cuttingants. molecular understandingofsymbiontfunction:identificationafungalgeneforthe 61, 1151-1160. fungus-gardening antsknowwhatmakestheirgardengrow? Funct. Ecol. switching cultivarsdoesnotaltertheperformanceoffungus-gardeningantcolonies. . from northeasternMexico. Formicidae). ant, gardening ants. suggest thatmicrobialinteractionsconstrainhorizontaltransferinhigherfungus- 135-141. Central Texas. (Hymenoptera: Formicidae):ayear-longsurveyofthreespeciesattineantsin (2011). Ecologyofmicrofungalcommunitiesingardensfungus-growingants Texas –Austin. New TheoryontheOriginofAntFungiculture, Ant Symbiosis:Ecology, ExperimentalSymbiontSwitchesandFitnessofAtta,a queens: doesthisexplainwhyleaf-cuttingantsfoundclaustrally? Trachymyrmex septentrionalis 3 , 134-142. (2009). Scalingofbodyweightandfatcontentinfungus-gardeningant Proc. Natl.Acad.Sci.USA 21, 988-997. Ann. Entomol.Soc.Am. FEMS Microbiol.Ecol. Evol. Ecol. (2010). Somefungus-growingants(Hymenoptera:Formicidae) (2005). BMC Biol. Fla. Entomol. 28, 157-176. Essays onOrganismalAspectsoftheFungus-Growing (Hymenoptera: Formicidae). during andafterarecorddrought. (2014). Instabilityofnovelant–fungalassociations (2006). Colonyproductivityofthefungus-gardening (2007b). Complexityinanobligatemutualism:do (2007c). Energeticsofnewlymatedqueensand (2010). Distributionofthefungus-gardeningant, McCook, inaFloridapineforest(Hymenoptera: (2007a). Co-evolutionandthesuperorganism: 8 (2008). Majorevolutionarytransitionsinant , 156. 99, 673-682. 78, 244-255. 105 93, 501-504. , 5435-5440. pp. 156.Austin,TX:Universityof Cyphomyrmex rimosus Physiol. Entomol. Behav. Ecol.Sociobiol. BMC Microbiol. (2008). Towards a Insectes Soc. Insect Conserv. 32, 8-15. (2010). 8 , 40. and 56, iv,A,Bci . r oe eSqer,C,Cre un,O,Pgoc,F. C. Pagnocca, O., CorreaBueno, C., GomesdeSiqueira, Jr, M., Bacci, A., Silva, un . ct,J . ywr,F . dm,S . rne .G,PnoTms A. Pinto-Tomás, S.G., Tringe, S.M., Adams, F. O., Aylward, J.J., Scott, G., Suen, R.J. Powell, and D.J. Stradling, F. J. Rohlf, and R. Sokal, eeoa . uhs . omm,J .adShøt M. Schiøtt, and J. Boomsma, D., Hughes, T., Semenova, e,R,Ihk .D,Etaa . od .E,Hn,E n ule,U.G. Mueller, and E. Hong, S.E., Dowd, D., Estrada, H.D., Ishak, R., Sen, ors .A,Snig,M n agd,M. Salgado, and M. Santiago, J.A., Torres, it,R,Hr,H,Re,R . esha,W n ölolr B. Hölldobler, and W. Beyschlag, R.J., Ryel, H., Herz, R., Wirth, J. E.O. Taylor, Wilson, and S. Lee, T., Bruns, T., White, U.G. Mueller, and J. Gus, J.N., Seal, ae M. Wade, A.J. Underwood, ee,N.A. Weber, 106 Amycolatopsis n prcd eln,M.J. AparecidaHebling, and Generalized antifungalactivityand454-screeningof Panama of Leaf-CuttingAnts:A CaseStudyonAttacolombicaintheTropical Rainforestof etal. K.W. Barry, P. J., Weimer, M., Pauly, microbiomewithhighplantbiomass-degradingcapacity. C.E., Foster, A., 964. fungal strains:afactorinthespeciationoffungusgrowingants. Co. food sources. fungal species:doantscontroltheircrops? patterns ofproteinaseactivityinattineantfungusgardens. ant, e1001129. 143-156. Formicidae: Atta).I.Theoverallpatternin White), pp.315-322.SanDiego,CA:AcademicPress. Guide toMethodsandApplications sequencing offungalribosomalRNA genesforphylogenetics.In septentrionalis Rev.Genet. dry forest.Trop. Ecol. University Press. , 17805-17810. Trachymyrmex jamaicensis . Berlin,Germany:SpringerVerlag. (2007). Theco-evolutionarygeneticsofecologicalcommunities. 8 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.098483 , 185-195. (1980). Casteanddivisionoflaborinleaf-cutterants(Hymenoptera: J. InsectPhysiol. . Ecology innestsoffungus-growingants. (1956). Fungus-growingantsandtheirfungi: (1997). 40, 237-245. 37, 150-161. Experiments inEcology (1995). , onsoilfertilityandseedgerminationinasubtropical 49, 307-313. (2003). Survivalof (1986). Thecloningofmorehighlyproductive Biometry (ed. M.Innis,D.Gelfand,J.SninskyandT. (2012). Fungus-gardeningantsprefernative (1999). Theeffects ofthefungus-growing Behav. Ecol. . NewYork, NY: W. H.Freemanand (1990). Amplificationanddirect Atta sexdens . Cambridge,UK:Cambridge 23, 1250-1256. . Proc. Natl.Acad.Sci.USA Behav. Ecol.Sociobiol. BMC Microbiol. Pseudonocardia (2011). Evolutionary Experientia workers ondifferent PCR Protocols:A (2010). Aninsect (2003). PLoS Genet. Trachymyrmex 11 Herbivory 42, 962- , 15. (2009). 2547 Nat. and 6 7 , ,

The Journal of Experimental Biology