Received 7November 2002 Accepted 20November 2002 Publishedonline 12March 2003

Proximateand ultimate control of sexratios in Myrmicabrevisp inosa colonies J. M. Bono1* and J.M.Herbers 2 1Departmentof Biology, Colorado State University, FortCollins, CO 80523,USA 2Collegeof Biological Sciences, OhioState University, 484West 12th Avenue, Columbus, OH 43210,USA The literature onsexratio evolutionin coloniesis dominatedby inclusivefitness arguments. In general, genetictheory makes goodpredictions about sexual investmentin ant populations,but understanding colony-level variance in sexinvestment ratios has proven more difficult.Recently, however, more studies have addressedecological factorsthat influencecolony-level sexinvestment ratios. Foodavailability, in particular, has beenmanipulated becauselarval nutrition influencesfemale castedetermination, thus implying that resourceavailability shouldbe of critical importance for colonysex investment ratios. How- ever,results from foodsupplementation experiments are equivocal, andit is clear that antcolony response tofood supplementation is dependenton the ecological background ofthepopulation. Wepresentedfield coloniesof the ant Myrmicabrevispinosa with twofood types (proteins and carbohydrates), andassessed their relative impact oncolony-level sexinvestment ratios. Weshow that coloniesreceiving carbohydrate enhancementinvested in more female sexualsand produced more female-biasedsex allocation ratios than protein-fedor controlcolonies. Thus, our study is the first, to our knowledge, to demonstrate that sex ratios in social insectcolonies might besensitive to resource quality. Male investmentwas not influenced by foodtreatment, but was positively correlated with colonysize. Therefore, the shift in sexratios in our studymust have beenmediated through nutritional influenceson female castedetermination rather than male broodelimination. Wealso usedour data toevaluate evidencefor sexratio compensationby queen- right coloniesin responseto male productionby workersfrom orphanedcolonies. Keywords: foodsupplementation; sex investment; sex ratio compensation;kin selection;

1. INTRODUCTION occursvia male cannibalism in Solenopsisinvicta , Linepi- themahumile and Formicaexsecta (Aron et al. 1995; Pas- Hamilton’s (1964) theory ofkin selectionsuccessfully sera& Aron1996; Sundstrom et al. 1996) butby selective explains theevolution of social behaviour in Hymenop- feeding in Leptothoraxacervorum (Hammond et al. 2002). tera, andhas also servedas a framework tostudy popu- The ability tobias sexratios hasproduced a rich literature lation-level sexinvestment ratios (Trivers &Hare 1976). onevolutionary strategies tobeemployed by queensver- In general, genetictheory makesgood predictions about susworkers (Crozier &Pamilo 1996; Queller & sexinvestment ratios producedby populations (Bourke& Strassmann1998; Chapuisat &Keller 1999; Reuter& Franks 1995; Griffin &West2002). However,there is Keller 2001). considerablevariation in thesex investment ratios among The literature onsexratio evolutionin social Hymenop- colonieswithin apopulation that canreflect relatedness tera is dominatedby inclusivefitness arguments. Only structure,queen– worker conflictand worker repro- recently,however, have studiesaddressed ecological fac- duction, inter alia (Crozier &Pamilo 1996). Yetthe proxi- torsthat might influencecolony-level sexinvestment ratios mate mechanismsused by coloniesto adjust sex (Backus& Herbers1992; Deslippe &Savolainen 1995; investmentratios are poorly understood. Herbers& Banschbach1998, 1999; Morales& Heithaus In hymenopteran societies,queens can control the pri- 1998; Aron et al. 2001). Foodavailability, in particular, mary sexratio by varying theproportion offertilized and has beenstudied because it is easyto manipulate andits unfertilizedeggs laid; workerscan influence the secondary link tofemale castedevelopment implies that it shouldbe sexinvestment ratio either by cannibalizing male larvae or ofcritical importance totheproximate controlof sex ratios by directing female larvae todevelop into gynes(virgin (Ho¨lldobler &Wilson1990; Wheeler 1986). Thus,we queens)rather than intoworkers. Female castedetermi- expectcolonies with more foodresources to producemore nationis influencedby several factorsincluding larval female-biasedsex investment ratios relative tofood- nutrition,with larvae receiving more fooddeveloping into stressedcolonies. Similarly, coloniesexperiencing food gynesand starved larvae becoming workers(Wheeler stressshould invest in males andworkers that are typically 1986; Ho¨lldobler &Wilson1990). It is generally believed smaller andless costly to produce than gynes(Nonacs that thefate of female larvae is notfixed genetically, 1986; Rosenheim et al. 1996). though recentevidence suggests that ageneticcomponent Predictionsfrom this resource-basedmodel are straight- is likely, at least in somespecies ( Julian et al. 2002; forward,but experimental manipulations offood avail- Volny &Gordon2002). Worker manipulation ofsex ratios ability have producedcontradictory results.Morales & Heithaus(1998) foundthat providing elaiosomes(a lipid- rich foodgained from anant– plant mutualism) to Aphaen- *Authorfor correspondence ([email protected]). ogasterrudis coloniesincreased gyne investmentbut not

Proc.R. Soc.Lond. B (2003) 270, 811–817 811 Ó 2003 TheRoyal Society DOI10.1098/ rspb.2002.2287 812J. M.Bonoand J. M.Herbers Sexratios in Myrmicabrevispinosa colonies male orworker investment.Deslippe &Savolainen (1995) Herbers& Banschbach1998, 1999). Our interest,then, also observeda shift towardgyne investmentfollowing wasto separate outthe effects of different food types for foodsupplementation, but they didnot report data on sexinvestment. productionof males or workers.Aron et al. (2001) In this study,we manipulated both foodquantity and reportedan increase in theproduction of males andgynes foodquality for theant, M.brevispinosa. We relied on following foodsupplementation, but they argued that extensiveinformation on Myrmica biology in theliterature. theseshifts were best explained by lowerrates of fracticide Manyspecies are polygynous,with theaverage queen in well-fedcolonies, and not simply by nutritional influences numberper nestranging from lessthan 1togreater than onfemale castedetermination. Herbers & Banschbach 15 (Elmes1991). Queensproduce male andfemale eggs (1998) observeda shifttoward gyne productionin astudy throughout theactive period,but female sexualsrequire of Leptothoraxlongispinosus ,butthis contrastedwith ashift twoseasons to develop. Large larvae that overwinter main- towardmale productionin aprevious,identical experiment tain thecapacity todevelop into workersor gynes, onthe same population. Furthermore, manipulations of dependingon environmental rearing conditionsin early foodavailability in twodifferent populations of Myrmica spring (Elmes1991). Workers are continually produced punctiventris producedstrong shifts toward male production throughout spring andsummer, but late seasonlarvae may anddecreased nest-mate relatedness in onepopulation, but enterwinter diapause. Thus, we designed an experiment producedno changes in theother population (DeHeer et totake advantage ofthis information andto separate out al. 2001; Herbers& Banschbach1999). This welterof foodquality from quantity. By presentingdifferent food resultsdoes not allow ustogeneralize about howant col- types(proteins and carbohydrates) tocolonies, and oniesrespond to resource enhancement. assessingtheir relative impact oncolony-level sexinvest- Clearly, ecological contextis a key determinantof ant mentratios, weshow that differentcomponents of sex colonyresponse to food supplementation. The ecological investmentare sensitiveto these two nutrient types. background ofa population canvary from year toyear, andmay differeven for speciesin thesame community (DeHeer et al. 2001; Herbers& Banschbach1999). More- 2. MATERIALAND METHODS over,sexual investmentstrategies among colonieswithin apopulation canvary in responseto other factorssuch as Wefound coloniesof M.brevispinosa nesting underrocks in queenturnover andworker reproduction.We recognize aspruce/firforest located26 kmnorthwest of Fort Collins,CO, theimportance ofecological context,but we suggestthat USA.The study site(elevation 2000 m) has steep rockyslopes theconfusion of results from previous investigations where M.brevispinosa coloniesexcavate shallow nests under reflectsanother factor:investigators have confoundedfood rocks.These arescavengers, and we observedforagers quality with foodquantity. returningto nests with insectprey. Although nest chambers Differential nutrientavailability couldhave strong underrocks are often extensiveand extendbeyond the rock, consequencesfor sexinvestment in ant colonies,parti- coloniesdo not appear to occupymultiple nesting sites. cularly if certain resourcesfavour theproduction of one sexover theother (Grafen1986; Boomsma 1993). In ant colonies,proteins are generally fedto larvae andare essen- (a) Foodsupplementation experiment tial for theproduction of broodin someant (Brian Ninety-six colonieswere randomly assigned to four food treat- 1956, 1973; Markin 1970; Porter 1989; Sorensen& mentgroups (24per treatment): protein supplementation, Vinson1981). Therefore,we expectprotein supplementa- carbohydrate supplementation,protein and carbohydrate sup- tionto influence total sexual investment.By contrast, plementation,and no supplementation.Supplemented nests investmentratios wouldbe changed only if proteinshave received4.5 mg of tuna and/oraccessto a20%sucrose feeder differing effectson male andfemale production.Protein twiceweekly (Tim Judd, personal communication). We pos- supplementation,potentially, couldlead toincreasedgyne itionedthe feedingstations veryclose to the target nest ’s investment,mediated through influenceson female caste entranceto ensurethat its foragers wouldfind the resourceand determination.However, this couldbe balanced by that other colonieswould not. Indeed,foragers quicklyfound reducedlevels ofmale cannibalism in protein-sup- and recruitednest-mates to both types of food source.Colonies plementedcolonies leading toincreased male investment. receivingboth proteinand carbohydrate supplementationwere Carbohydrates are themain sourceof energy for workers givenaccess to onlyone food type perfeeding session to prevent in mostant colonies (Brian 1956, 1973; Porter 1989), and selectiveforaging for onefood type. Thus, coloniesin this treat- also serveas a critical sourcefor fat synthesisin gynes mentgroup receivedhalf of the amount of proteinand carbo- during theperiod betweeneclosion and nuptial flights hydrates givento coloniesin the puretreatment regimes.We (Passera &Keller 1990; Passera et al. 1990). Thus,we checkedfeeders periodically to ensurethat onlytarget colonies expectcarbohydrate supplementationto increase adult wereusing them. gyne weight. Boomsma (1993) suggestedthat gynesare Wecarried out food supplementationover the courseof two mosteasily producedwhen ample proteinsare available activeseasons runningfrom 29 May –12November2000 and 6 early in larval developmentand carbohydrates are avail- May 2001until nests werecollected at the endof July2001. By able after gyneshave eclosed.Therefore, we might expect the endof the first fieldseason adisproportionate numberof thesenutrient types to act synergistically with higher gyne coloniesassigned to the proteintreatment group appeared to investmentin coloniesgiven both foodtypes. Most food have moved,as they nolonger visited food sources.Therefore, supplementationstudies have usedproteins (Aron et al. to boost samplesize, we found sevennew coloniesand assigned 2001) or acombination ofproteins and carbohydrates them to the proteintreatment group priorto the secondfield (Backus& Herbers1992; Deslippe &Savolainen 1995; season.

Proc.R. Soc.Lond. B (2003) Sexratios in Myrmicabrevispinosa colonies J.M.Bonoand J. M.Herbers 813

(b) Colony collection anddemographic analysis producefemales, seven invested in males,presumably by Adultalates began to emergein late July, and we collected worker reproduction.These data suggestthat ahigh pro- coloniesover a 3-day period(29 –31July 2001). We used hand portion ofcolonies lose their queensevery year, andsome trowels to shovelants and soilinto large plastic bags. Nest cham- ofthem persistfor upto 2 years ascongregations ofwork- berswere often extensiveso carewas taken to ensurethat colon- ersthat lay haploid eggs.Food type had noeffect on the ieswere collected fully. We continued digging to adepth of distribution ofqueens among nests( G-test, p . 0.05) or severalcentimetres below whereants weresighted and we thenumber of queens per nest(regression, p = 0.276 for checkedsoil under nearby rocksfor any sign of ants orbrood. protein; p = 0.960 for carbohydrates). Moreover,food Wefeel confident that we collectedall of the ants residingin supplementation(regardless offood type) didnot affect onenest area. thelikelihood ofa colonycontaining aqueen( G-test, Wetransported coloniesto the laboratory wherethey were p . 0.05). Foodtype had noeffect on the number of countedand sortedby caste.We dried up to 10ants of each workersper colony(regression p = 0.617 for protein; caste(except worker pupae) inan ovenat 80 °Cfor at least p = 0.852 for carbohydrates), andthere was no difference 3days. These werethen weighed individuallyto the nearest in worker numberbetween fed and unfed colonies (one- 0.01mg. Dry weight data wereused to calculatethe energetic way ANOVA, p = 0.932). Furthermore, therewere no dif- cost ratio (ECR;Boomsma 1989), which was then usedto com- ferencesin colonysize among queenright colonies,colon- pute sex-allocationratios ( P m) = [(numberof males)/(number iesrecently orphaned and colonies orphaned for longer of males 1 ECR ´ numberfemales)]. These ratios ranged from periodsof time. 0(allfemale broods) to 1(allmale broods), and werearcsine- Meansand confidence intervals for weightsare presented transformedfor statisticalanalysis. in table 1. Workers weresmaller in coloniesreceiving pro- teinsupplementation (to be fully explored below)but we (c) Dataanalysis foundno evidence that weightsof reproductive pupae or Weused regression analysis to assess the effectsof ourfood adultswere affected by foodtreatment. However, our fail- treatments on demographicand reproductiveparameters. Data ureto detect differences among treatmentsmight bedue werefirst analysedusing afullmodel including the two food toalack ofpowerfrom small sample sizes,as implied by types (carbohydrate and protein)with theirthree levels (none, thewide confidence intervals (table 1). half and full),and acarbohydrate –proteininteraction term. We Sexual investmentcomparisons were complicated by includedworker numberas acovariatein the analysis of repro- thefact that sexual developmenttimes were not consistent ductiveparameters. All variables were log-transformed with the acrosscolonies, meaning that somecolonies contained exceptionof Pm (arcsine)and worker number(square root). In adult sexuals,while otherscontained sexual pupae.Male allcases the interactionterm was not significantand was there- pupaewere heavier than male adults( t-test, p = 0.041), so foreeliminated from the model.Qualitative treatment effects comparisonsacross nests would be problematic. Moreover, wereindicated when the effectof oneor both food types was only asmall numberof colonies contained both sexual significantin the model.Protein and carbohydrate supplementa- pupaeand adults, so it wasnot possible to reliably predict tionwere not simultaneouslysignificant in any of ourmodels, adult weightswith pupal weights.However, since average which allowedus to further examineour data for quantitative sexual adult weightsdid not differ by foodtreatment, we effects;we useda simpleregression across treatments,which calculated sexual investmentas the product of thenumber codedas none,half (forcolonies fed alternately with carbo- ofmale orfemale sexualsreared by acolonyand the popu- hydrate and protein)and fullsupplementations (for colonies fed lation-wideaverage weight ofadults in that caste. onlyone type of nutrient). Wealso tested for ageneraleffect of food supplementation (b) Analysis ofreproductive parameters (poolingall supplemented colonies) with one-way ANOVAs.We Outof the 56 coloniesused in this study,41 invested used the G-test ofindependenceto determinewhether the likeli- in sexual reproductives,13 reared only workersand two hood of investingin males or gynes was affectedby ourfood coloniesdid not produce brood of any kind.Most of the treatments orby the presenceof aqueen.There were no differ- coloniesthat reared sexualsspecialized in male production encesfor any parameterbetween colonies receiving protein (29 total), while five coloniesspecialized in gyne pro- treatment for oneseason and those receivingthe treatment for ductionand seven colonies produced mixed sexual two seasons (ANOVAs, p . 0.05for alltests), so allcolonies broods.Because queen presence/ absencedid not influence receivingprotein were combined for subsequent analyses. thelikelihood ofcoloniesinvesting in gynesor males ( G- tests, p . 0.05), weconsidered colonies capable ofpro- ducingboth gynesand males if they reared any diploid 3. RESULTS (gyne or worker) broodat all; coloniesnot fitting that (a) Demographicanalysis descriptionwere considered orphans. Seven colonies in Coloniescontained 445 ± 50 (mean ± s.e.; n = 56) thepopulation wereorphaned but still produceda sub- workers,and only onewas polygynous. The majority stantial numberof males (268, or25% ofthe population (78%), though,were queenless and presumably orphaned. total), prompting usto analyse ourdata for evidenceof To determinehow long queenlesscolonies had been sexratio compensation(Taylor 1981; Crozier &Pamilo orphaned,we looked for female (diploid) pupae.The 1996). Sex ratio compensationis expectedin populations presenceof worker and/or gyne pupaein aqueenlesscol- whenorphaned colonies produce a preponderanceof onyimplied arecentorphaning. Only 9ofthe 44 queen- males,such that queenright coloniesbenefit by overprod- lesscolonies had nofemale pupae,indicating that most ucing females.Consequently, queenright coloniesproduce orphanedcolonies were still able toproduce male and amore female-biasedsex ratio than otherwiseexpected female broods.Out of the nine colonies that failed to andthe populational sexratio is more male-biased than

Proc.R. Soc.Lond. B (2003) 814J. M.Bonoand J. M.Herbers Sexratios in Myrmicabrevispinosa colonies

Table1. Back-transformed meansand confidence intervals (given in brackets)for dry weights(mg) of individuals reared by M.brevispinosa colonies. (Colonies received different food treatments over thecourse of two active seasons.)

carbohydrate protein mixed control

workers 0.53 [0.45, 0.62] 0.43 [0.38, 0.49] 0.48 [0.42, 0.54] 0.51 [0.45, 0.58] male pupae 0.53 [0.36, 0.77] 0.50 [0.39, 0.66] 0.48 [0.36, 0.66] 0.57 [0.43, 0.75] male adults 0.42 [0.33, 0.54] 0.42 [0.33, 0.53] 0.46 [0.35, 0.63] 0.43 [0.30, 0.62] gyne pupae 3.24 [0.95, 11.01] —a 2.17 [1.07, 4.40] 2.52 [0.74, 8.57] gyne adults 2.88 [1.77, 4.69] 2.69 [1.84, 4.10] 3.23 [1.98, 5.26] 4.10 [2.05, 8.19] a Missing data.

Table2. Valuesfor Pm (sex allocation ratio) predicted bya sexratio compensation model (Crozier &Pamilo 1996) compared withmean values and pooled population values obtained from a M.brevispinosa population. (Meansare back-transformed.)

actual mean values

mean predicted under mean predicted under [95% confidence pooled Pm for queen control worker control interval] population

mean Pm for queenright colonies 0.46 0.14 0.88 [0.75, 0.97] 0.49

mean Pm for entire population 0.53 0.28 0.92 [0.82, 0.98] 0.57

Table3. Back-transformed meansand confidence intervals (given in brackets)for reproductive parameters in M.brevispinosa colonies receiving food supplementation over thecourse of two active seasons.

carbohydrate protein mixed control all fed colonies

withorphans: total 17.76 [4.69, 60.89] 8.42 [3.26, 19.87] 15.09 [5.82, 36.96] 6.12 [2.01, 15.76] 12.15 [6.86, 20.99] male investement 6.26 [1.64, 18.93] 6.06 [2.60, 12.83] 4.49 [1.65, 10.35] 3.41 [1.13, 8.12] 5.47 [3.21, 8.94] gyne investment 4.88 [0.38, 24.10] 0.86 [ 20.29, 3.89] 3.41 [0.55, 11.53] 0.78 [ 20.37, 4.06] 2.21 [0.71, 5.05]

Pm 0.85 [0.46, 1.00] 0.97 [0.82, 0.99] 0.80 [0.51, 0.97] 0.96 [0.79, 0.99] 0.89 [0.77, 0.98] without orphans: gyne investment 40.16 [3.71, 358.63] 0.97 [ 20.27, 4.35] 4.00 [0.67, 13.95] 0.94 [ 20.39, 5.18] 2.83 [0.84, 6.98]

Pm 0.38 [20.44, 0.95] 0.96 [0.78, 0.99] 0.77 [0.43, 0.96] 0.95 [0.73, 0.99] 0.86 [0.69, 0.98]

wouldbe expected without orphaned colonies (Crozier & producing either male or female reproductives( G-tests, Pamilo 1996, p.151). p . 0.05 for all tests).Moreover, we found no evidence For queencontrol and 25% ofall males producedby that foodsupplementation (regardless offood type) had orphanedcolonies, the mean male allocation ratio in aneffect on total sexual investment,gyne production, queenright coloniesshould be 0.462, andthat over the male productionor sex-allocation ratio ( Pm ) (one-way entirepopulation (including orphans) shouldbe 0.533. ANCOVA, p . 0.05 for all tests).However, we noteagain For worker control,the expectation for queenright colon- that lowpower, as evidenced by thewide confidence inter- iesis 0.139, andfor theentire population is 0.276 (table vals, may have limited ourability todetect differences 2). In ourpopulation, themean male allocation ratio for among treatments(table 3). queenright colonieswas 0.884 andthe average value for Regressionswere performed with fourresponse vari- theentire population was0.920, both more male-biased ables:total sexual investment,male investment,gyne than predictedunder queen or worker control.We note investmentand Pm (sexratio). Sinceorphaned colonies thelarge discrepancyfor queenright coloniesbetween werenot capable ofproducing female sexuals,we also meanmale allocation ratio (0.884) andpooled male allo- analysed gyne investmentand Pm with orphanedcolonies cation ratio (0.493), which indicatesthat mostqueenright excluded.When orphaned colonies were included in the coloniesproduced only afewmales each,but some pro- analysis wedidnot detect an effect of either foodtype on duceda large numberof gynes. total sexual investment,male investmentor Pm . The data Meansand confidence intervals for reproductivepara- in table 3strongly suggestthat coloniesreceiving carbo- metersare presentedin table 3. Foodtype andfood hydrate enhancementhad increasedtotal sexual invest- supplementation(regardless offood type) didnot affect mentand gyne investment,but again thewide confidence thelikelihood ofcolonies investing in reproduction,or of intervals imply lowpower for thesecomparisons.

Proc.R. Soc.Lond. B (2003) Sexratios in Myrmicabrevispinosa colonies J.M.Bonoand J. M.Herbers 815

a) 30 2.0

) 25 1 +

t

n 1.5 s e e 20 i m n t s o l e o v c n 15

i 1.0 f

o e

l . a o n m 10

g 0.5 o l 5

0 0.05 0.25 0.450.65 0.85 b) 2.5 sex allocation ratio )

1 Figure 2. Sexallocation ratios for M.brevispinosa colonies + 2.0 t receiving three levels of carbohydrate supplementation. n

e When orphans were excluded from theanalysis, colonies m t

s 1.5 receiving carbohydrates hadmore female-biasedsex e

v allocation ratios (regression, p = 0.009). Seven colonies with n i

l asexallocation ratio = 1were orphans (three from full a 1.0 u treatment (black), one from halftreatment (grey) and three x e

s from no carbohydrate treatment (white)).

g 0.5 o l canteffect of protein supplementationwas to produce 0 5 10 15 20 25 30 35 40 smaller workers,an effect that wasalso additive (table 4). square root worker number) That is,we found quantitative effectsof carbohydrates on sexual reproduction,but of proteins on worker weight. Figure 1. (a)Maleproduction asa function of colony size in colonies of theant M.brevispinosa .On average, large colonies invested more biomassin malesthan smallcolonies 4. DISCUSSION (regression, p = 0.0005 for worker number). ( b)Total sexual investment asa function of colony size in colonies of theant Other studieshave shownthat foodsupplementation M.brevispinosa .There wasa trend for larger colonies to canaffect sex investment ratios producedby ant colonies invest more biomassin sexual individuals thansmall colonies (Backus& Herbers1992; Deslippe &Savolainen 1995; (regression, p = 0.103 for worker number). Herbers& Banschbach1998, 1999; Morales& Heithaus 1998), butour study is the first, to our knowledge, to demonstratethat colonysex investment ratios are sensitive Oneclear resultwas that whenwe pooled our data tothe type offood used for supplementation.When we acrosstreatments, we foundthat colonysize was positively examined theresponses of colonies that weresup- correlated with male investment( p = 0.0005; figure 1 a), plementedwith carbohydrates and/orproteinsversus con- an effectthat drovea weaker,but similar, relationship trols,only negative resultswere obtained. By contrast, betweencolony size and total investmentin sexuals whenwe separated out the effects of carbohydrate and (p = 0.103; figure 1 b)Thus,larger coloniesput more protein supplementation,important patternsemerged; energy intomale reproduction,thereby increasing total overall, theprotein supplementationhad noimpact onsex sexual reproduction. allocation in ourant colonies,but carbohydrate supple- Whenorphaned colonies were excluded from theanaly- mentationproduced a positive linear responsein gyne pro- sis,carbohydrate supplementationclearly influencedgyne duction.Thus, it is important todistinguish foodquantity investment,as carbohydrate-supplemented colonies pro- from foodquality in order tounderstand proximate mech- ducedmore gynes(regression, p = 0.009 for carbohydrates; anismsunderlying conflictover sexallocation. table 4). Moreover,the quantitative effectof this nutrient Moreover,our approach provides an insight intothe wasadditive, asthe level ofgyne investmentfor themixed mechanismsused by M.brevispinosa coloniesto adjust sex treatment group wasintermediate tothe other treatments investmentratios. Sincegyne productionbut not male (table 4). Coloniesthat receivedcarbohydrate enhance- productionwas influenced by foodtype, manipulation of mentalso producedlower sex-allocation ratios, andthe sexinvestment ratios musthave occurredby differential quantitative effectof nutrient enhancement was additive as determination offemale larvae rather than elimination of well (regression, p = 0.020 for carbohydrates; table 4). Fig- male brood.Our resultstherefore corroborate Hammond ure2 showssex-allocation ratios for all coloniesin the et al. (2002), whoshowed that this mechanism is population, sortedby carbohydrate treatment.Out of the important for theant Leptothoraxacervorum .The resource- 29 colonieswith asex-allocation ratio of1, sevenwere basedmodel (Rosenheim et al. 1996) predictsthat orphanedand therefore unable to produce female sexuals increasedgyne investmentshould lead toa decreasein (threefrom full treatment,one from half treatment and worker production.Unfortunately, we were unable to threefrom nocarbohydrate treatment).The only signifi- collectreliable data onthe number of new workers pro-

Proc.R. Soc.Lond. B (2003) 816J. M.Bonoand J. M.Herbers Sexratios in Myrmicabrevispinosa colonies

Table4. Back-transformed meansand confidence intervals (given in brackets)for theresponse variablesthat were significantly influenced byfood type. (Datafor log worker weightand gyne investment are given in milligrams. p values were obtained byregression analysis.)

significant factor full half none p value

log worker weightprotein 0.43 [0.38, 0.49] 0.48 [0.42, 0.54] 0.52 [0.47, 0.57] 0.024 gyne investment carbohydrate 40.14 [3.90, 344.48] 4.00 [0.71, 13.66] 0.96 [ 20.07, 3.13] 0.009

Pm carbohydrate 0.38 [20.43, 0.94] 0.77 [0.44, 0.96] 0.96 [0.84, 1.00] 0.020

duced by M.brevispinosa colonies,so we do not know other Myrmica species(Evans 1998; Herbers& Mouser whethermore gyneswere produced at theexpense of new 1998). Giventhese complications, wecannotpredict with workers,as predicted by theresource-based model precisionthe optimal ratio for queenright coloniesin (Rosenheim et al. 1996). Yet,we saw no corroborating ourpopulation. evidenceof that effectwhen we compared colonysize: Acomparison ofmean sex allocation ratios tothe worker numberdid not vary betweentreatments, implying pooledpopulation sexallocation ratio does,however, that differentials in gyne productionthat weobserveddid imply sexratio compensationfrom somequeenright col- notoccur at theexpense of new workers, but were in onies.Despite the ambiguities ofrelatedness and worker addition toworker production.Thus, it is possiblethat laying, anunbiased allocation ratio over theentire popu- theincreased gyne investmentcould reflect a higher lation coupledwith ahighly male-biased meanmale allo- diploid egg-laying rate by queensin carbohydrate- cation ratio meansthat coloniesinvesting in gynes supplementedcolonies. produceda large numberof them,just as predicted by the Wedid not expect to see an effect of carbohydrate sexratio compensationmodel. This effectwas particularly enhancementon reproductive parameters, sincecarbo- evidentfor coloniesreceiving carbohydrate supplementa- hydrates are generally retainedby ant workers(Markin tion(figure 2), implying that theavailability ofthis nutri- 1970; Sorensen& Vinson1981) andare accumulatedby entmight constraina queenright colony ’sability toadjust sexualsafter pupal emergence(Passera &Keller 1990; its sexratio. With high queenmortality andmost colonies Passera et al. 1990). Yetour data showedno differences producing only males,colonies with thenecessary in alate sizebetween carbohydrate treatments.Rather, our resources(carbohydrates for example) gain more fitness resultsimply that gyne –worker developmentis influenced by producing alarge numberof gynes.Thus, support for directly by carbohydrates, aneffect that mustoccur during thesex ratio compensationhypothesis is mixed, andthe larval development.Alternatively, carbohydrate enhance- possibility deservesfurther study. mentmay have increasedfemale investmentindirectly: Colonysize strongly influencedthe production of males sincecarbohydrates serveas fuel for workers(Brian 1956, (figure 1) butnot gynes. A positive relationship between 1973; Porter 1989), ourtreatment may have released worker numberand male productionis inconsistentwith workersfrom foodstress, allowing them toprovide more aresource-basedmodel if small colonieshave lowforaging prey itemsto developing larvae. Our studyspecies is a successrelative tolarger colonies.Under those conditions, generalist scavenger,for which carbohydrates may be weexpect small coloniesto specialize in producing less more limiting than protein.Certainly laboratory colonies costlymales. It ispossible that thepositive relationship of Leptothoraxcurvispinosus and Solenopsisinvicta , fed sugar reflectsenhanced worker reproductionin larger colonies water andinsects, grew at fasterrates than coloniesfed butthe effect would have tobe much stronger than here- only (Evans & Pierce 1995; Porter 1989). Repeat- toforeobserved for any antcolony (Crozier &Pamilo ing ourexperiments with aspeciesthat tendshomopterans 1996). The relationship couldalso beexplained by the andthus are notcarbohydrate limited wouldallow usto effectof local resourcecompetition (Bourke& Franks testthis alternative. 1995; Crozier &Pamilo 1996), butthe extent to which Wewere also able touse our data toexamine ultimate femalescompete for resourcesin this population is controlover sexratios in this species.Specifically, thehigh unknown.Rather, wesuspect that larger andtherefore rate oforphaning in this population allowed usto test for older colonieshave experiencedmore queenturnover. If sexratio compensationby queenright colonies(Crozier & true,then larger colonieshave lower levels ofwithin-col- Pamilo 1996; Taylor 1981). Our meanmale allocation onyrelatedness than smaller colonies,leading toincreased ratios for queenright coloniesand for thepopulation are male investmentas predicted by geneticmodels of sexual muchmore male biasedthan theoptimal valuesderived investmentin ants(Trivers &Hare 1976). Clearly, from amodelof sex ratio compensation.However, this detailedgenetic work to uncover family structureis modelassumes mongynous colony structure and no neededto differentiate these possibilities. worker reproductionin queenright colonies,assumptions Our studyprovides important insight notonly intofac- that may have beenviolated for this population. While torsthat influenceratios in M.brevispinosa colonies,but monogyny seemsto predominate in ourstudy system, also intothe mechanisms used by coloniesto adjust them. queensupercedure may beimportant; if unrelatedqueens Oursis thefirst study,to our knowledge, to demonstrate take over orphanedcolonies then queenright colonies that foodquality is at least asimportant asfood quantity. comprise amix offamily groups (Evans1996). Similarly, Colonynutritional statustherefore emerges as an wedo not know whether workers of this speciesproduce important additional variable for interpreting sexallo- males in queenright colonies,as has beenreported for cation data.

Proc.R. Soc.Lond. B (2003) Sexratios in Myrmicabrevispinosa colonies J.M.Bonoand J. M.Herbers 817

Elmes(1991) suggestedthat theproduction of gynes and Hamilton, W.D.1964 Genetical evolution of social behaviour males in Myrmica coloniesare controlledthrough separate I & II. J.Theor.Biol. 7, 1–52. mechanisms.Our data lendsupport to his hypothesis:gyne Hammond, R.L., Bruford, M.W.&Bourke, A.F.G.2002 productionwas sensitive to nutritional statusbut male pro- Ant workers selfishlybias sex ratios bymanipulating female 269 ductionresponded to colony size. Investigations ofsex allo- development. Proc.R. Soc. Lond. B , 173–178. (DOI 10.1098/rspb.2001.1860.) cationin social insectsmust therefore disentangle factors Herbers, J.M.&Banschbach, V.S.1998 Food supplyand affecting investmentin gynesversus males. Furthermore, reproductive allocation in forest ants: repeated experiments futurework on proximate mechanismsof control within give different results. Oikos 83, 145–151. thosecolonies must be careful toensure that foodquality Herbers, J.M.&Banschbach, V.S.1999 Plasticity of social is notconfounded with foodquantity. organization in aforest ant species. Behav. Ecol. Sociobiol. 45, 451–465. We thankRick and VickieJordan for granting accessto our Herbers, J.M.&Mouser, R.L.1998 Microsatellite DNAmar- field site, and Chris DeHeer and Susanne Foitzik for helpwith kers reveal details of social structure in forest ants. Mol. Ecol. theexperimental design. We also thankRumsais Blatrix, 7, 299–306. Emmanuel Chiche, Craig Hill and TimJudd for help withlab- Ho¨lldobler, B.&Wilson, E.O.1990 The ants.Cambridge, oratory and fieldwork and for stimulating discussions that MA:Belknap Press of Harvard University. improved thequality of our work. We also thankJoe Fontaine and Andrea Cutone for helpful comments on early versions of Julian, G.E.,Fewell, J.H., Gadau, J., Johnson, R.A.&Lar- themanuscript. 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