Oviposition tests of ant preference in a myrmecophilous butterfly

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1 Oviposition tests of nt preference in myrmecophilous utterfly A. M. FRASER,* T. TREGENZA,à N. WEDELL,à M. A. ELGARà &N.E.PIERCE* *Deprtment of Orgnismic nd Evolutionry Biology, Hrvrd University, Cmridge, MA, USA Fculty of Environmentl Sciences, Griffith University, Nthn, Queenslnd, Austrli àdeprtment of Zoology, University of Melourne, Victori, Austrli. Present ddress: School of Biology, University of Leeds, Leeds, UK Keywords: diversifiction; Formicide; geogrphicl speciliztion; host selection; host shift; locl dpttion; Lycenide; myrmecophily; oviposition preference. Astrct Butterflies in the fmily Lycenide tht hve oligte ssocitions with nts frequently exhiit nt-dependent egg lying ehviour. In series of field nd lortory choice tests, we ssessed oviposition preference of the Austrlin lycenid Jlmenus evgors in response to different species nd popultions of nts. Femles discriminted etween ttendnt nd nonttendnt nt species, etween ttendnt nt species, nd to some extent, etween popultions of single nt species. When preferences were found, ovipositing utterflies preferred their loclly predominnt ttendnt nt species nd geogrphiclly proximte ttendnt nt popultions. A reciprocl choice test using dults from genertion of utterflies rered in the sence of nts indicted genetic component to oviposition preference. Individul femles were flexile with respect to oviposition site choice, often ovipositing on more thn one tretment during tril. Preferences rose from hierrchicl rnking of nt tretments. These results re discussed in terms of locl dpttion nd its possile significnce in the diversifiction of ntssocited lycenids. Introduction Diversifiction within the utterfly fmily Lycenide is thought to hve een enhnced y the tendency for the mjority of its memers to ssocite with nts (Pierce, 1984). Associtions rnge from mutulistic to prsitic interctions, nd vry in their specificity for, nd dependence on, nt prtners (Cottrell, 1984; Pierce, 1987; Fiedler, 1991, 1997; Pierce et l., 2002). If nt ssocition hs influenced lycenid diversifiction, it should e most evident mong the oligtely myrmecophilous species, which ccount for 15 20% of lycenid species diversity (Fiedler, 1991, 1997). Their ssocitions with nts tend to e species-specific (Cottrell, 1984; Pierce, 1989; Thoms et l., 1989; Fiedler, 1991; Estwood & Frser, 1999) nd dult femles use their nt prtners s cues during oviposition (e.g. Atstt, 1981; Pierce & Elgr, 1985; Seufert & Fiedler, 1996; vn Dyck et l., 2000). Correspondence: Ann Frser, Biology Deprtment, The University of the South, 735 University Ave., Sewnee, TN 37383, USA. Tel.: ; fx: ; e-mil: frser@post.hrvrd.edu The evolution of oviposition preference is seen s driving force in the divergence of phytophgous insect popultions (Futuym, 1986; Diehl & Bush, 1989; Thompson & Pellmyr, 1991). Preference, in this context, refers to oth the order in which femles rnk different hosts nd the specificity or degree to which they prefer one host over nother (Wiklund, 1981; Singer, 1986). Thus, shifts in nt preference y ovipositing lycenid utterflies my contriute to divergence in nt-ssocited lycenid popultions. Ultimtely, this my led to specition events if, for exmple, genetic divergence in host preference hs pleiotropic consequences for reproductive isoltion (Funk, 1998). A phylogenetic study of the lycenid genus Ogyris suggests tht shifts in nt prtners hve een ccompnied y specition events (N.E. Pierce, unpulished dt). Assocition ptterns etween nts nd t lest two other oligtely nt-ssocited lycenid gener, Mculine (Thoms et l., 1989; Elmes et l., 1994) nd Jlmenus (Pierce, 1989; Estwood & Frser, 1999; Bry, 2000) further suggest tht shifts in nt prtners my contriute to popultion divergence nd diversifiction. 861

2 862 A. M. FRASER ET AL. The degree to which ovipositing utterflies discriminte etween different nt species, s well s the extent to which oviposition ehviour hs diverged mong utterfly popultions, hs not een explored. We conducted series of field nd lortory experiments tht ddress these questions in the Austrlin lycenid, Jlmenus evgors. In choice tests, we ssessed oviposition preference t three levels: in response to ttendnt nd nonttendnt nt species, in response to different species of ttendnt nts, nd in response to different popultions of single ttendnt nt species. A reciprocl choice test ws lso conducted to determine whether femles exhiited locl dpttion in oviposition preference, nd whether oviposition preference hd genetic component. Methods nd results Nturl history Jlmenus comprises t lest 10 species, distriuted cross the Austrlin continent (Bry, 2000). All species feed on the plnt genus Acci (some feed on dditionl plnt gener), nd ll hve specilized ssocitions with one or more species of Iridomyrmex or Froggttell nts (sufmily Dolichoderine). There is little overlp in the nt species tht Jlmenus species ssocite with (Estwood & Frser, 1999), nd where Jlmenus species overlp geogrphiclly, they re seprted ecologiclly y their nt prtners (Pierce, 1989; Cost et l., 1996; Bry, 2000). Jlmenus evgors inhits costl nd inlnd res of estern Austrli (Fig. 1). Immture stges re tended y different nt species in different geogrphicl regions (Cost et l., 1996; Pierce & Nsh, 1999), ut re most commonly ssocited with Iridomyrmex species in the nceps nd rufoniger groups (Estwood & Frser, 1999; Pierce & Nsh, 1999). The geogrphicl distriutions of these nts re not fully known, ut in res where severl ttendnt nt species overlp, given ÔcolonyÕ of J. evgors is predominntly ssocited with only single nt species (Cost et l., 1996; Pierce & Nsh, 1999). Adult utterflies tend to remin in their ntl re (Elgr & Pierce, 1988), creting potentil for the evolution of speciliztion in nt preference. Adult femles ly eggs on Acci host plnts in response to the presence of ttendnt nts (Pierce & Elgr, 1985). Typiclly, grvid femles tht lnd on host plnts follow sequence of ehviours leding to oviposition. These include: () drgging, in which femle wlks up nd down the rnches of the plnt drgging the tip of her domen long the sustrte ehviour tht is typicl mong Lepidopter serching for suitle oviposition sites (Renwick & Chew, 1994); () proing, in which drgging femle stops, extrudes her ovipositor nd proes crevice nd (c) oviposition, in which she remins sttionry with the tip of her domen inserted into crck or crevice nd during which she pumps her domen. For the ehviourl nlysis in this study, we grouped drgging, proing nd oviposition into single ctegory of oviposition-relted ehviour. Collection nd identifiction of mteril Butterflies nd nts were collected from vrious loclities in estern Austrli (Fig. 1, Tle 1). Ant identifictions were ssigned in consulttion with Dr Steve Shttuck, Austrlin Ntionl Insect Collection (ANIC), Cnerr nd voucher specimens hve een deposited t the ANIC QUEENSLAND 1 Plum 2 Mt. Neo Nthn 3 Brisne NEW SOUTH WALES Armidle 6,7,8 Eor 4,5 Distriution of Jlmenus evgors AUSTRALIAN CAPITAL TERRITORY 9 Cnerr Sydney VICTORIA ,12 Melourne km 400 Fig. 1 Distriution of Jlmenus evgors nd loclities from which nt species were collected.

3 Ant preference in myrmecophilous utterfly 863 Tle 1 Summry of mteril collected to ssess levels of oviposition discrimintion exhiited y Jlmenus evgors utterflies in response to nts. The outcome of ech experiment is summrized in the fr righthnd column. Geogrphicl distnce refers to the pproximte geogrphicl distnce seprting ech test nt collection loclity from the utterfly popultion used in the experiment. All nt species except I. purpureus nd F. kirii nturlly ttend J. evgors. Experiment Butterfly loclity (site no.)* Tretment offered Ant loclity (site no.)* Geogrphicl distnce (km) Outcome Experiment I: Mt. Neo I. nceps Mt. Neo (2) 0 Butterflies discriminte Choice of (2) I. rufoniger Mt. Neo (2) 1 etween nt species; prefer ttendnt nd I. purpureus Mt. Neo (2) 1 ttendnt I. nceps over nonttendnt F. kirii Plum (1) 1200 I. rufoniger nd nonttendnt nt species nt species (Tle 2) Experiment II: Nthn I. nceps Nthn (3) 0 Butterflies discriminte Choice of locl (3) I. rufoniger Armidle (7) 350 etween tretments; prefer nd foreign (two trils) I. nceps Cnerr (9) 950 locl I. nceps over ttendnt nt No nt I. rufoniger nd foreign species I. nceps (Fig. 2) Experiment III: Nthn I. nceps Nthn (3) 0 Butterflies do not show Choice of (3) I. nceps Mt. Neo (2) 30 cler or consistent locl nd (three trils) I. nceps Eor (4) 350 preference; when foreign No nt preference shown, locl ttendnt nt nt lwys mong popultions Eor I. nceps Eor (4) 0 preferred popultions (4) I. nceps Nthn (3) 350 (Fig. 3) (two trils) I. nceps Mt. Neo (2) 350 No nt Experiment IV: Armidle/Eor I. rufoniger Armidle/Eor (5 8) 0 Nïve utterflies prefer Reciprocl (5 8) I. nceps Melourne (10 13) 1000 locl ttendnt nt species choice of (Tle 4); suggests locl ttendnt nt Melourne I. nceps Melourne (10 13) 0 dpttion in oviposition species (10 13) I. rufoniger Armidle/Eor (5 8) 1000 preference *See Fig. 1 for collection loclities nd corresponding site numers. Ech test nt species tretment ws lso pired with locl, ground-forging I. nceps tretment. nd t the Museum of Comprtive Zoology, Hrvrd University. Species-level txonomy within the nceps nd rufoniger groups of Iridomyrmex is poor. We noted differences in ody colour nd colony structure etween I. nceps popultions from Eor, Nthn nd Mt. Neo compred with those from Cnerr nd Melourne. It is possile tht these two vrints represent seprte species within the nceps groups. Pending systemtic review of the nceps group, however, we dopt conservtive pproch nd refer to ll I. nceps popultions s single species. Choice tests experiments were conducted from Jnury to Mrch over severl yers. Experiment I: Choice of ttendnt nd nonttendnt nt species Methods An ren of four pirs of potted host plnts (Acci irrort) ws rrnged in 5 m 5 m squre in n open field t Mt. Neo, Queenslnd (Fig. 1). Pired plnts were plced within 1 m of ech nother. One plnt in ech pir ws connected to cptive colony of one of four ÔtestÕ nt species (Tle 1). Of these, I. purpureus nd F. kirii do not normlly ttend J. evgors under nturl conditions; I. purpureus tends J. ictinus while F. kirii tends J. pseudictinus nd J. ridus. The second plnt in ech pir served s control, on which the loclly occurring ttendnt nt (I. nceps sp. 25, Pierce et l., 1987) forged. This design permitted us to ssess whether oviposition preferences were influenced y test nt tretment or y tretment position lone. Twelve juveniles of loclly common nt-ssocited memrcid, Sextius virescens (Homopter), were mintined on ech plnt to ttrct nts. Oviposition on plnts y freely flying J. evgors utterflies ws monitored over 15 dys. Plnts were exchnged with fresh trees every 5 dys. Egg msses were counted nd removed t the end of ech dy nd summed cross dys for ech tretment efore nlysis. Femle preference ws ssessed in Chi-squre (v 2 ) test of 2 4 contingency tle. The men numer of nts per plnt ws otined y clculting men for ech of the three trees used in ech tretment over the 15-dy period, nd then finding the men cross these three trees. The numers of nts forging on the different tretments were compred using two-wy repeted mesures nlysis of vrince.

4 864 A. M. FRASER ET AL. Results Egg mss distriution ws not independent of the test nt species (P ¼ 0.006, Tle 2). Femles lid more egg msses on plnts ering I. nceps s the test nt species thn they did on plnts ering other test nt species. The distriution of egg msses on plnts with control I. nceps compred with tht on plnts with test nt species indictes tht preference ws not result of tretment position (Tle 2). I. rufoniger ws s unttrctive to ovipositing utterflies s the two nonttendnt test nt species (Tle 2). Oviposition differences cnnot e ttriuted to the totl numer of nts in ech pired setup (control I. nceps + test species). There were significntly more locl (control) nts thn test nts on plnts (repeted mesures ANOVA, F 1,8 ¼ 9.82; P ¼ 0.014), ut the totl numer of nts (control + test) did not differ mong pirs (F 3,8 ¼ 0.74, P ¼ 0.5) nd the two fctors did not interct (within pirs mong pirs: F 3,8 ¼ 0.17, P > 0.9) (Tle 2). Therefore, nt species nd not the totl numer of nts within pir explins the oserved difference in egg mss distriution. Although it is possile tht only one or two utterflies generted the overll egg mss distriution pttern, this is unlikely given the lrge numers of eggs nd egg msses tht were lid over the course of the experiment (Tle 2). To ddress this uncertinty, however, susequent experiments followed mrked individuls in n enclosed setting. Experiment II: Choice within nd etween ttendnt nt species Methods This experiment ssessed oviposition preference of femles in response to different species of ttendnt nts (Tle 1). When it ws conducted, it ws ssumed tht I. nceps from Nthn nd Cnerr were memers of Tle 2 Numers of egg msses lid y J. evgors utterflies over 15 dys on potted host plnts contining juveniles of the memrcid Sextius virescens tended y workers of different test nt species, or pired control of the predominnt locl ttendnt nt species, I. nceps. The experiment ws conducted in n open field. Host plnts were replced with fresh plnts every 5 dys. Men nts per tree (±1 SE) ws clculted from the three 5-dy verges. Test species I. nceps F. kirii I. rufoniger I. purpureus Egg msses per tretment* Test species Control I. nceps Ants per tree (n ¼ 3) Test species 10.9 ± ± ± ± 2.1 Control I. nceps 16.7 ± ± ± ± 2.9 *v 2 ¼ 12.49, d.f. ¼ 3, P ¼ different species within the nceps group (see ove). Until systemtic review of the nceps group is undertken, however, we will tret them conservtively s single species. Thus, this experiment ssesses oviposition preference oth within nd etween ttendnt nt species. An nt-free control tretment ws lso included (Tle 1). Oviposition trils were conducted in n outdoor screened enclosure ( m) t Griffith University, Nthn, Queenslnd (Fig. 1, Tle 1). Four groups of three potted host plnts (Acci melnoxylon) were rrnged in c. 3m 3 m squre. Within ech group, plnts were in contct with one nother nd one plnt ws connected to cptive nt colony or to n empty nt nest continer (Tle 1). Jlmenus evgors juveniles were plced on plnts to ttrct nts, nd s control in the nt-free tretment. Additionl oviposition sustrte, in the form of rough-rked rnch, ws ttched to the min stem of ech plnt. Butterflies were derived from juveniles collected from the field t Nthn (Tle 1, Fig. 1). Juveniles were tended y their locl nt (I. nceps) until eclosion. Butterflies eclosed nd mted in cges, nd were mrked with numers on the underside of the forewings for identifiction during ehviourl oservtions. An oviposition tril egn y relesing up to 12 mles nd 12 femles into the enclosure. Butterflies tht died over the experimentl period were replced with new individuls of the sme sex. Ants forging on tretments were counted dily nd numers equlized mong tretments y djusting the numer nd or ge clss of J. evgors juveniles on plnts or the numer of nts in nest continer. After 8 dys, egg msses were removed from plnts nd totlled for ech tretment. Egg mss distriution mong tretments ws compred with rndom distriution using the G-test for goodness of fit. Pirwise comprisons were conducted using the G-test, with required significnce vlues djusted for nonindependence using the sequentil Bonferroni procedure (Rice, 1989). Between oviposition trils, plnts were left in plce ut nts nd nest continers were moved to the corner digonlly opposite their previous loction to control for effects of tretment position nd plnt qulity on oviposition. New utterflies were used for ech tril. During the second tril, the ehviour of femle utterflies ws recorded during 5-min scns of the enclosure, conducted dily t hourly intervls etween 07:30 nd 17:30 h. The G-test ws used to nlyse dt on visittion nd ctivity ptterns of individuls t the vrious tretments. Results Femles lid significntly more egg msses on plnts with their locl I. nceps popultion thn on plnts with I. nceps from Cnerr or with I. rufoniger (Fig. 2, P < 0.001, pirwise comprisons P < 0.05). In the first

5 Ant preference in myrmecophilous utterfly 865 Proportion of egg msses lid ' I. nceps (Nthn) Tril 1 (n = 57 egg msses) Tril 2 (n = 94 egg msses) ' I. nceps (Cnerr) I. rufoniger (Armidle) Tretment '' No nt Fig. 2 Oviposition response of J. evgors femles from Nthn on host plnts ering conspecific juveniles tended y their locl ttendnt nt (I. nceps, Nthn), foreign ttendnt nts (I. nceps, Cnerr or I. rufoniger, Armidle) or untended. Totl numers of egg msses lid per tril re given in the figure legend. Oviposition ws nonrndom with respect to tretment (G-test, Tril 1: G ¼ 34.05, P < 0.001; Tril 2: G ¼ 25.57, P < 0.001; d.f. ¼ 3). Brs shring the sme letter do not differ significntly (pirwise comprisons djusted for nonindependence, P > 0.05). tril, femles preferred plnts with the locl I. nceps to nt-free plnts (P < 0.05). Femles did not discriminte mong tretments with foreign nts or nt-free plnts (Fig. 2). Overll, ovipositing femles rnked tretments in the following order: I. nceps Nthn (locl nt) > no nts ¼ I. nceps Cnerr ¼ I. rufoniger Armidle. The nonrndom distriution of egg msses ws relted to differences in femle ehviour on plnts, ut not to visittion ptterns (Tle 3). Visittion to the different tretments ws rndom, oth in terms of the numer of different femles visiting (P ¼ 0.63) nd the totl ' numer of visits received (P ¼ 0.36). However, femles were most likely to engge in oviposition-relted ehviours on plnts contining their locl nt, secondly on nt-free plnts nd lest likely on plnts with foreign ttendnt nt species (P ¼ 0.01). A mjority of femles (8 14) visited nd exhiited oviposition-relted ehviours on more thn one tretment nd five of these femles exhiited oviposition-relted ehviours on t lest three tretments. Egg mss size rnged from 1 to 86 eggs. Men clutch size (±1 SE) per tretment rnged from 17.7 ± 4.0 to 28.2 ± 6.2 eggs; medin clutch size rnge per tretment rnged from 10 to 28 eggs. No significnt differences in egg mss sizes were detected (Kruskl Wllis test: P > 0.05). Experiment III: Choice mong popultions of I. nceps Methods This experiment ssessed oviposition preference of J. evgors femles in response to different popultions of I. nceps (Tle 1, Fig. 1). An nt-free control tretment ws lso included. Experimentl conditions were similr to Experiment II except tht the three host plnts in ech tretment were plced within 50 cm of one nother, ut were not interconnected, nd three nt colonies from ech loclity were used per nt tretment, with ech colony connected to single host plnt. Five oviposition trils were conducted (Tle 1), ech lsting 4 5 dys. Fresh host plnts were used for ech tril to control for ny residul nt or utterfly odours deposited on plnts during the previous tril. All plnts were visully mtched for size nd condition. Ant colonies were moved to different corners of the enclosure etween trils to control for effects of tretment position on oviposition. Butterflies from two loclities were used (Tle 1) nd were derived from field collected, nt-tended juveniles. Egg mss distriution mong tretments ws compred with rndom distriution using the G-test for goodness of fit. Pirwise comprisons were conducted using the Tle 3 Summry of J. evgors femle utterfly visittion nd ehviourl ptterns on host plnts ering conspecific juveniles tended y their locl ttendnt nt (I. nceps, Nthn), foreign ttendnt nts (I. nceps, Cnerr or I. rufoniger, Armidle) or untended. Dt were compiled from hourly scns conducted during tril 2 of Experiment II using utterflies from Nthn. Fourteen femles were used over the course of the tril. Oviposition-relted ehviours (ORB) include drgging, proing nd oviposition. Tretment Nthn utterfly ehviour Locl I. nceps (Nthn) Foreign I. nceps (Cnerr) Foreign I. rufoniger (Armidle) No nts G-test results No. of femle visitors G ¼ 1.74, P ¼ 0.63 Totl no. of visits G ¼ 3.22, P ¼ 0.36 No. (%) of visits 21 (91) 6 (46) 8 (53) 14 (88) G ¼ 10.99, P ¼ 0.01 involving ORB

6 866 A. M. FRASER ET AL. G-test, with required significnce vlues djusted for nonindependence using the sequentil Bonferroni procedure (Rice, 1989). Results Nthn femles discriminted mong nt popultions in the ltter two of three trils, preferring the locl Nthn nd the neighouring Mt. Neo nt popultions over the more distnt Eor popultion (Fig. 3, Tril 2 P < 0.001; Tril 3 P ¼ 0.02; pirwise comprisons P < 0.05). Eor utterflies did not discriminte mong nt popultions, ut preferred nt-tended tretments over the nt-free tretment in Tril 2 (Fig. 3, P < 0.001; pirwise comprisons P < 0.05). Tretment did not influence the numer of eggs per mss in ny of the trils (Kruskl Wllis test: P > 0.05). Egg mss size rnged from 1 to 215 eggs. Men clutch size (±1 SE) per tretment rnged from 16.2 ± 4.2 to 45.0 ± 6.2 eggs; medin clutch size per tretment rnged from 13 to 40 eggs. Experiment IV: Reciprocl choice of two ttendnt nt species using nïve utterflies Methods A reciprocl choice test involving nïve utterflies ws conducted to determine whether femle discrimintion mong I. nceps nd I. rufoniger hd genetic component nd whether femles preferred their locl ttendnt nt species. Jlmenus evgors pupe nd frgment of the ttendnt nt colony were collected from four field sites in ech of two regions (Fig. 1, Tles 1 nd 4) nd rought to the University of Melourne, Victori for rering nd experimenttion. Adults tht eclosed from field-collected pupe were mted in the lortory nd progeny from these dults were rered without nts on A. melnoxylon cuttings, ensuring tht F 1 dults were nïve with respect to nt cues. Oviposition trils using F 1 dults were conducted in the lortory in cylindricl cges, 50 cm high 24 cm dimeter with cler plstic sides nd net roof. Two plstic forging rens were plced in ech cge. One ren contined nts from the sme popultion s the utterfly eing tested, wheres the other contined nts of the foreign ttendnt species. A 40-cm long wooden dowel with numerous pits drilled into the surfce ws mounted verticlly in ech ren nd served s oviposition sustrte for utterflies. Thirty nts were plced on ech dowel t the strt of n oviposition tril. A plstic sleeve contining cotton wool soked in 10% sucrose solution ws plced t the top of ech dowel to ttrct nts onto the dowel. A single, newly eclosed J. evgors femle ws plced in cge with two or three mles s mtes. Cges were checked every morning. When eggs were detected the tril ws terminted nd eggs were counted. Only one femle from ech popultion ws used. Thus, ech popultion represented n independent dt point. Similrly, ech nt popultion ws used only twice: once s the locl ttendnt nt species, nd once s the foreign ttendnt nt species. Oviposition preference ws ssessed using one-tiled inomil proility test. Results Nïve femle utterflies exhiited preference for their locl nt species over the foreign species (Tle 4, P ¼ 0.035, n ¼ 8). This experiment rules out the possiility tht oviposition preference ws ecuse of imprinting on ttendnt nts, s individuls were rered from egg to eclosion in the sence of nts. Moreover, ech femle utterfly ws tested in isoltion from other femles nd over reltively short time period, which ensured tht the presence of conspecific femles or eggs on tretment did not influence ehviour. Discussion Jlmenus evgors femles discriminted etween ttendnt nd nonttendnt nt species, etween ttendnt nt species, nd to some extent, etween popultions of A) Nthn utterflies B) Eor utterflies Proportion of egg msses lid Tril 1 (n = 86 egg msses) Tril 2 (n = 86 egg msses) Tril 3 (n = 81 egg msses) Nthn Mt. Neo Eor No nts Nthn Mt. Neo Eor No nts I. nceps nt popultion Tril 1 (n = 108 egg msses) Tril 2 (n = 115 egg msses) Fig. 3 Oviposition response of J. evgors femles on host plnts ering conspecific juveniles tended y three popultions of I. nceps nts or untended. Butterflies were from () Nthn nd () Eor. Totl egg msses per tril re given in the figure legend. G-tests of oviposition preference: Nthn femles, Tril 1: G ¼ 4.40, P ¼ 0.22; Tril 2: G ¼ 26.80, P < 0.001; Tril 3: G ¼ 9.54, P ¼ 0.02; d.f. ¼ 3; Eor femles, Tril 1: G ¼ 4.64, P ¼ 0.20; Tril 2: G ¼ 26.84, P < 0.001; d.f. ¼ 3. Brs shring the sme letter do not differ significntly (pirwise comprisons djusted for nonindependence, P > 0.05).

7 Ant preference in myrmecophilous utterfly 867 Tle 4 Numer of eggs lid y individul J. evgors femles from Armidle Eor nd Melourne offered simultneous choice of oviposition sustrte ptrolled y their locl ttendnt nt species or y the reciprocl foreign ttendnt nt species (see Tle 1, Fig. 1). Butterfly origin site (No.) Numer of eggs lid on tretment Armidle Eor nts (I. rufoniger) Melourne nts (I. nceps) Armidle Eor Pnton s Gully (5) 9 27 Estwood SF (6) Scry Rod (7) Klind Rod (8) Locl nt preferred* Melourne Violet Town (10) Wlln 1 (11) Wlln 2 (12) LTroe (13) *One-tiled inomil proility, P ¼ 0.035; + locl nt preferred; locl nt not preferred. I. nceps is the predominnt ttendnt nt species t this site (Cost et l., 1996) ut I. rufoniger controls minority of host plnts ering J. evgors. single nt species in oviposition choice tests. Our limited testing lso suggests tht utterflies exhiit locl dpttion in oviposition preference nd tht oviposition preference hs genetic component. A genetic sis for oviposition preference hs previously een reported in Lepidopter nd other insect groups nd is necessry prerequisite for the evolution of oviposition preference (Futuym & Peterson, 1985; Jenike & Holt, 1991; Thompson & Pellmyr, 1991). Oviposition preference cn evolve rpidly in response to the introduction of novel host, the loss of preferred host, or coloniztion of new re where the preferred host is sent or rre (Feder & Bush, 1989; Singer et l., 1993). The degree of evolutionry chnge vries, however, with the orgnism under study, the mount of gene flow mong popultions nd the scle of the nlysis (Thompson & Pellmyr, 1991; Thompson, 1993; Thoms & Singer, 1998). Aspects of the iology of J. evgors nd other oligtely nt-ssocited lycenids tht would likely promote nd mintin locl speciliztion in oviposition preference include the low dispersl rtes of dults from their ntl hitt nd the use of host plnts nd ttendnt nts s rendezvous sites for mting (Atstt, 1981; Pierce & Elgr, 1985; Elgr & Pierce, 1988; Seufert & Fiedler, 1996; Fiedler, 1997). Although this is the first study, to our knowledge, tht ddresses geogrphicl vrition in oviposition preference mong myrmecophilous utterflies, t lest two studies hve exmined vrition with respect to the ttrction of lycenid lrve to nts. Elmes et l. (1994) presented correltive evidence for locl speciliztion in ssocition etween popultions of Mculine lcon nd different Myrmic nt species in Europe. Ant-relted oviposition ehviour ws recently documented in M. lcon (vn Dyck et l., 2000) nd it would e interesting to determine if this contriutes to specificity in ssocition ptterns in this system. In two popultions of Pleejus rgus, in which lrve re ssocited with different Lsius nt species, Jordno & Thoms (1992) found tht lrve were more ttrctive to their nturl host nt species nd tht lrvl differences in ttrctiveness hd genetic sis. We cnnot rule out the possiility tht ehviourl imprinting process, wherey individuls lern dignostic chrcteristics such s smell, shpe nd ehviour of their ntl nt ssocite during development or upon eclosion, influences oviposition ehviour. This my hve influenced femle preference in Experiments I III, ut not in Experiment IV in which n entire genertion of J. evgors ws rered in the sence of nts. Reciprocl choice tests conducted with these dults still reveled n oviposition preference for the locl nt prtner. Our report of locl dpttion in oviposition preference y J. evgors is sed on widely seprted utterfly popultions nd their respective nt ssocites. However, speciliztion within more limited geogrphicl regions my not e evident. Cost et l. (1996) did not find pttern of llozyme vrition relted to nt ssocite in the Armidle Eor re, where J. evgors ssocites with I. rufoniger nd I. nceps, respectively, lthough there ws trend in isoltion y distnce. Cost et l. (1996) suggest tht gene flow mong utterfly supopultions nd frequent extinction-recoloniztion events ccount for the oserved genetic vrition. Our experiments indicte tht nceps-ssocited utterflies lwys prefer I. nceps when discriminting etween I. nceps nd I. rufoniger, regrdless of the geogrphicl distnce tht seprtes utterfly nd nt popultions. Whether rufoniger-ssocited utterflies exhiit similr ehviour in regions where the two nt species overlp is uncler. Interestingly, the one rufoniger-ssocited utterfly tht preferred the foreign I. nceps tretment to the locl I. rufoniger tretment in Experiment IV ws collected from site (Pnton s Gully, Tle 4) where I. nceps is the predominnt ttendnt nt species (Cost et l., 1996).

8 868 A. M. FRASER ET AL. It would e interesting to know if oviposition preference in J. evgors is loclly dptive. Do femles preferentilly ssocite with nt species tht confer the gretest enefits or smllest cost to J. evgors juveniles in given region? Juveniles depend on nts for protection from nturl enemies (Pierce et l., 1987) nd nt species cn differ in the level of protection they provide to their trophoiotic prtners (Addicott, 1979; Buckley & Gulln, 1991; Svignno, 1994; Frser et l., 2001). At Mt. Neo, survivorship of J. evgors juveniles ws higher when I. nceps ws in ttendnce compred with I. rufoniger (Pierce, 1989), suggesting tht nturl selection my fvour discrimintion y ovipositing utterflies nd preference for the most effective ttendnt nt. Adults of J. evgors juveniles tht ssocite with nts incur fitness cost, in the form of reduced ody size (Pierce et l., 1987; Bylis & Pierce, 1992) nd thus my experience lower reproductive success reltive to untended individuls (Elgr & Pierce, 1988) ut I. nceps nd I. rufoniger confer similr costs to developing lrve (Pierce et l., 1987). Therefore, it ppers tht selection cts similrly on juveniles nd dults y promoting preferentil ssocition with nt species tht offer juveniles the most effective protection from nturl enemies. Femles my remin flexile in their choice of oviposition sites, despite selection fvouring ssocition with prticulr nt species. Femles in Experiment I oviposited on host plnts contining ttendnt s well s nonttendnt nt species, indicting tht oviposition ÔmistkesÕ with respect to nt ssocite my occur in nture. Behviourl oservtions from Experiment II further suggest tht individuls re flexile with respect to specificity in oviposition site selection, ut tht overll ptterns of oviposition preference result from hierrchicl rnking of tretments y individuls. This flexile strtegy my e dptive, ecuse femles cn continue to oviposit on plnts with ÔinferiorÕ nt species if the most effective, nd presumly preferred, ttendnt nt species is sent or reltively rre (see lso Wiklund, 1981). On the other hnd, flexile strtegy my e costly if femles deposit eggs on plnts visited y nt species tht do not recognize developing juveniles fvourly. From locl dpttion to specition? A symptric shift in nt ssocite, followed y specition, seems unlikely for J. evgors or other lycenids, owing to the concomitnt shift in chemicl signlling required etween lycenid lrve nd nts (Atstt, 1981; Pierce, 1984; Elmes et l., 1994; Fiedler, 1997). An lloptric model involving coloniztion of new re, in which utterfly s usul ttendnt nt species is sent, together with reduced gene flow with the prentl species, seems more plusile scenrio (see lso Elmes et l., 1994). We speculte tht shift in nt ssocite is proly initited y chnge in dult ehviour rther thn chnge in lrvl chrcteristics ecuse of the greter moility of dults nd the fct tht ovipositing femles ultimtely determine the conditions in which newly htched lrve will emerge. We hve demonstrted flexiility in oviposition site choice y J. evgors nd ssume tht this flexiility would provide opportunities for interctions with novel nt species in nture. For ssocitions to ecome estlished, lrve must e le to ppese their new nt prtner. Oservtions of J. evgors lrve eing tended y I. purpureus nd severl other novel nt prtners in the wild nd under lortory conditions (Pierce, 1989; Estwood & Frser, 1999), indicte tht ppesement of novel nt prtners is possile. Nonetheless, the intensity with which nts prticipte in these novel ssocitions my vry within nd mong nt colonies nd over time (Pierce et l., 1991). As result, mny novel ssocitions my e trnsient. If they persist nd the newly founded lycenid popultion remins geneticlly isolted, selection my ct on trits such s oviposition preference nd lrvl performnce. This my led to sitution where ovipositing utterflies no longer recognize their former ttendnt nt species fvourly when reunited nd lrve re no longer recognized fvourly y their former ttendnt nt species. Specition my e promoted if genes involved in trits relted to oviposition preference nd lrvl performnce hve pleiotropic effects on trits involved in sexul reproduction (Funk, 1998). Acknowledgments We thnk J.D. Frser nd K.L. Nutt for field ssistnce, nd R. Estwood for comments on the mnuscript. R.L. Kitching nd the Fculty of Environmentl Science grciously provided fcilities nd hospitlity t Griffith University. S.O. Shttuck kindly provided identifictions of nts. A.M. Frser ws supported y the Nturl Sciences nd Engineering Reserch Council of Cnd, Hrvrd University Frnk Knox Trveling Fellowship, the Putnm Expedition Fund of the Museum of Comprtive Zoology nd the Explorer s Fund. T. Tregenz ws supported y Royl Society Trveling fellowship. N. Wedell ws supported y the Swedish Nturl Science Reserch Council. References Addicott, J.F A multispecies phid-nt ssocition: density dependence nd species-specific effects. Cn. J. Zool. 57: Atstt, P.R Ant-dependent food plnt selection y the mistletoe utterfly Ogyris mryllis (Lycenide). Oecologi. 48: Atstt, P.R Lycenid utterflies nd nts: selection for enemy-free spce. Am. Nt. 118:

9 Ant preference in myrmecophilous utterfly 869 Bylis, M. & Pierce, N.E Lck of compenstion y finl instr lrve of the myrmecophilous lycenid utterfly, Jlmenus evgors, for the loss of nutrients to nts. Physiol. Entomol. 17: Bry, M.F Butterflies of Austrli: their Identifiction, Biology nd Distriution. CSIRO Pulishing, Collingwood. Buckley, R. & Gulln, P More ggressive nt species (Hymenopter: Formicide) provide etter protection for soft scles nd melyugs (Homopter: Coccide, Pseudococcide). Biotropic 23: Cost, J.T., Mcdonld, J.H. & Pierce, N.E The effect of nt ssocition on the popultion genetics of the Austrlin utterfly Jlmenus evgors (Lepidopter, Lycenide). Biol. J. Linn. Soc. 58: Cottrell, C.B Aphytophgy in utterflies: its reltionship to myrmecophily. Zool. J. Linn. Soc. 79: Diehl, S.R. & Bush, G.L The role of hitt preference in dpttion nd specition. In: Specition nd its Consequences (D. Otte & J. A. Endler, eds), pp Sinuer, Sunderlnd, MA. vn Dyck, H., Oostermeijer, J.G.B., Tlloen, W., Feenstr, V., vnderhidde, A. & Wynhoff, I Does the presence of nt nest mtter for oviposition to specilized myrmecophilous Mculine utterfly? Proc. Roy. Soc. Lond. B 267: Estwood, R. & Frser, A.M Associtions etween lycenid utterflies nd nts in Austrli. Aust. J. Ecol. 24: Elgr, M.A. & Pierce, N.E Mting success nd fecundity in n nt-tended lycenid utterfly. In: Reproductive Success: Studies of Selection nd Adpttion in Contrsting Breeding Systems (T. H. Clutton Brock, ed.), pp Chicgo University Press, Chicgo, IL. Elmes, G.W., Thoms, J.A., Hmmrstedt, O., Munguir, M.L., Mrtin, J. & vndermde, J.G Differences in host-nt specificity etween Spnish, Dutch nd Swedish popultions of the endngered utterfly, Mculine lcon (Denis et Schiff.) (Lepidopter). Memorili Zool. 48: Feder, J.L. & Bush, G.L A field test of differentil host plnt usge etween two siling species Rhgoletis pomonell fruit flies (Dipter: Tephritide) nd its consequences for symptric models of specition. Evolution 43: Fiedler, K Systemtic, evolutionry, nd ecologicl implictions of myrmecophily within the Lycenide (Insect: Lepidopter: Ppilionoide). Bonner Zool. Monogr. 31: Fiedler, K Life-history ptterns of myrmecophilous utterflies nd other insects: their implictions on tropicl species diversity. In: Proceedings of the Interntionl Symposium on Biodiversity nd Systemtics in Tropicl Ecosystems, 1994 (H. Ulrich, ed.), pp Zoologisches Forschungsinstitut und Museum Alexnder Koenig, Bonn. Frser, A.M., Axén, A.H. & Pierce, N.E Assessing the qulity of different nt species s prtners of myrmecophilous utterfly. Oecologi 129: Funk, D.J Isolting role for nturl selection in specition: host dpttion nd sexul isoltion in Neochlmisus eine lef eetles. Evolution 52: Futuym, D.J The role of ehvior in host-ssocited divergence in herivorous insects. In: Evolutionry Genetics of Inverterte Behvior (M. D. Huettel, ed.), pp Plenum Press, New York. Futuym, D.J. & Peterson, S.C Genetic vrition in the use of resources y insects. Annu. Rev. Entomol. 30: Jenike, J. & Holt, R.D Genetic vrition for hitt preference: evidence nd explntions. Am. Nt. 137: S67 S90. Jordno, D. & Thoms, C.D Specificity of n nt lycenid interction. Oecologi 91: Pierce, N.E Amplified species diversity: cse study of n Austrlin lycenid utterfly nd its ttendnt nts. In: The Biology of Butterflies (R. I. Vne-Wright & P. R. Ackery, eds), pp Princeton University Press, Princeton. Pierce, N.E The evolution nd iogeogrphy of ssocitions etween lycenid utterflies nd nts. In: Oxford Surveys in Evolutionry Biology (P. H. Hrvey & L. Prtridge, eds), Vol. 4, pp Oxford University Press, Oxford. Pierce, N.E Butterfly-nt mutulisms. In: Towrd More Exct Ecology (P. J. Gru & J. B. Whittker, eds), pp Blckwell Science Pulishers, Oxford. Pierce, N.E. & Elgr, M.A The influence of nts on host plnt selection y Jlmenus evgors, myrmecophilous lycenid utterfly. Behv. Ecol. Socioiol. 16: Pierce, N.E. & Nsh, D.R The imperil lue, Jlmenus evgors (Lycenide). In: Biology of Austrlin Butterflies (R. L. Kitching, E. Scheermeyer, R. E. Jones & N. E. Pierce, eds), Vol. 6, pp CSIRO Pulishing, Melourne. Pierce, N.E., Kitching, R.L., Buckley, R.C., Tylor, M.F.J. & Benow, K.F The costs nd enefits of coopertion etween the Austrlin lycenid utterfly, Jlmenus evgors, nd its ttendnt nts. Behv. Ecol. Socioiol. 21: Pierce, N.E., Nsh, D.R., Bylis, M. & Crper, E.R Vrition in the ttrctiveness of lycenid utterfly lrve to nts. In: Ant-Plnt Interctions (C. R. Huxley & D. F. Cutler, eds), pp Oxford University Press, Oxford. Pierce, N.E., Bry, M.F., Heth, A., Lohmn, D.J., Mthew, J., Rnd, D.B. & Trvssos, M.A The ecology nd evolution of nt ssocition in the Lycenide (Lepidopter). Annu. Rev. Entomol. 47: Renwick, J.A.A. & Chew, F.S Oviposition ehvior in Lepidopter. Annu. Rev. Entomol. 39: Rice, W Anlyzing tles of sttisticl tests. Evolution 43: Svignno, D.A Benefits to Krner Blue utterfly lrve from ssocition with nts. In: Krner Blue Butterfly: Symol of Vnishing Lndscpe (D. A. Andow, R. J. Bker & C. P. Lne, eds), pp Miscellneous Puliction Minnesot Agriculturl Experiment Sttion, St Pul, MN. Seufert, P. & Fiedler, K The influence of nts on ptterns of coloniztion nd estlishment within set of coexisting lycenid utterflies in south-est Asin tropicl rin forest. Oecologi 106: Singer, M.C The definition nd mesurement of oviposition preference in plnt-feeding insects. In: Insect Plnt Interctions (J. R. Miller & T. A. Miller, eds), pp Springer-Verlg, New York. Singer, M.C., Thoms, C.D. & Prmesn, C Rpid humninduced evolution of insect host ssocitions. Nture. 366: Thoms, C.D. & Singer, M.C Scle-dependent evolution of speciliztion in checkerspot utterfly: from individuls to metpopultions nd ecotypes. In: Genetic Structure nd Locl Adpttion in Nturl Insect Popultions: Effects of Ecology, Life History nd Behvior (S. Mopper & S. Y. Struss, eds), pp Chpmn & Hll, New York.

10 870 A. M. FRASER ET AL. Thoms, J.A., Elmes, G.W., Wrdlw, J.C. & Woyciechowski, M Host specificity mong Mculine utterflies in Myrmic nt nests. Oecologi 79: Thompson, J.N Preference hierrchies nd the origin of geogrphic speciliztion in host use in swllowtil utterflies. Evolution. 47: Thompson, J.N. & Pellmyr, O Evolution of oviposition ehvior nd host preference in Lepidopter. Annu. Rev. Entomol. 36: Wiklund, C Generlist vs. specilist oviposition ehviour in Ppilio mchon (Lepidopter) nd functionl spects on the hierrchy of oviposition preferences. Oikos 36: Received 8 Jnury 2001; revised 18 Mrch 2002; ccepted 21 Mrch 2002

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