Phenotypic plasticity in a complex world: interactive evects of food and temperature on Wtness components of a seed beetle

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1 Oecologi (2007) 53:9 32 DOI 0.007/s POPULATION ECOLOGY Phenotypic plsticity in complex world: interctive evects of food nd temperture on Wtness components of seed beetle R. Crig Stillwell Willim G. Wllin Lis J. Hitchcock Chrles W. Fox Received: 6 My 2006 / Accepted: 2 April 2007 / Published online: 8 My 2007 Springer-Verlg 2007 Abstrct Most studies of phenotypic plsticity investigte the evects of n individul environmentl fctor on orgnism phenotypes. However, orgnisms exist in n ecologiclly complex world where multiple environmentl fctors cn interct to Vect growth, development nd life histories. Here, using multifctoril experimentl design, we exmine the seprte nd interctive evects of two environmentl fctors, rering host species (Vign rdit, Vign ngulris nd Vign unguicult) nd temperture (20, 25, nd 35 C), on growth nd life history trits in two popultions [Burkin Fso (BF) nd South Indi (SI)] of the seed beetle, Cllosobruchus mcultus. The two study popultions of beetles responded diverently to both rering host nd temperture. We lso found signiwcnt interction between rering host nd temperture for body size, growth rte nd femle lifetime fecundity but not lrvl development time or lrvl survivorship. The interction ws most pprent for growth rte; the vrince in growth rte mong hosts incresed with incresing temperture. However, the detils of host diverences divered between our two study popultions; the degree to which V. unguicult ws better host thn V. ngulris or V. rdit incresed t higher tempertures for BF beetles, wheres the degree to which V. unguicult ws the worst host incresed t higher tempertures for SI beetles. We lso found tht the heritbilities of body mss, growth rte nd fecundity were similr mong rering hosts nd Communicted by Jy Rosenheim. R. C. Stillwell (&) W. G. Wllin L. J. Hitchcock C. W. Fox Deprtment of Entomology, University of Kentucky, S225 Agriculturl Science Center North, Lexington, KY , USA e-mil: rstil2@uky.edu tempertures, nd tht the cross-temperture genetic correltion ws not Vected by rering host, suggesting tht genetic rchitecture is generlly stble cross rering conditions. The most importnt Wnding of our study is tht multiple environmentl fctors cn interct to Vect orgnism growth, but the degree of interction, nd thus the degree of complexity of phenotypic plsticity, vries mong trits nd between popultions. Keywords Plsticity Rection norm Genetic rchitecture Cllosobruchus mcultus Introduction A chnge in n orgnism s phenotype in response to the environment (phenotypic plsticity) is universl chrcteristic of ll orgnisms (West-Eberhrd 2003). While numerous studies hve now investigted phenotypic plsticity, nerly ll of these hve exmined how single environmentl fctor impcts n orgnism s phenotype (Pigliucci 200). Yet, orgnisms exist in ecologiclly complex worlds, simultneously experiencing vrition in mny environmentl fctors tht cn hve interctive evects on growth, development nd life histories (Sultn et l. 998; Sultn 200; Relye 2004; Ris et l. 2004; Relye nd Auld 2005). To predict evolutionry responses to selection in nture it is necessry to understnd how interctions between multiple environmentl fctors Vect rection norm shpe. Two of the most importnt environmentl fctors Vecting the growth nd development of ectotherms, prticulrly insects, re diet nd temperture. Both vribles induce substntil plsticity in number of trits. Animls fed lower qulity diets generlly hve lower survivorship, 23

2 Oecologi (2007) 53:9 32 incresed development time (Nylin nd Gotthrd 998), mture t smller dult body size (Berrign nd Chrnov 994), hve slower growth rtes (Atkinson nd Sibly 997), lower fecundity (Awmck nd Lether 2002) nd produce smller eggs/ovspring (Fox nd Czesk 2000). Animls rered t lower temperture generlly hve higher survivorship (Angillett et l. 2004; Koziowski et l. 2004), longer development time (Atkinson 994), mture t lrger dult size (Atkinson 994; Angillett nd Dunhm 2003), hve reduced growth rte (Atkinson nd Sibly 997), lower fecundity (Ernsting nd Isks 2000; Stillwell nd Fox 2005) nd produce lrger eggs/ovspring (Fox nd Czesk 2000). Though diet nd temperture evects on growth nd life history trits re commonly investigted, the interctions between them re rrely exmined (Kingsolver et l. 2006). Those studies tht hve simultneously exmined the evect of both of these vribles hve generlly found interctive evects on growth nd development (Stmp nd Bowers 990; Gresens 997; Sultn et l. 998; Petersen et l. 2000; Sultn 200; Relye 2004; Ris et l. 2004; Relye nd Auld 2005; Kingsolver et l. 2006), suggesting tht the interctive evect of these two vribles is likely to be importnt. Although dpttion to locl environmentl conditions is ubiquitous in nture, nd popultions frequently evolve diverences in rection norms, few of these studies hve exmined how interctions mong multiple environmentl vribles vry mong popultions. Ptterns of phenotypic plsticity cn lso vry substntilly mong trits. For exmple, temperture rection norms vry considerbly mong morphologicl trits of the cricket, Gryllus Wrmus (Bégin et l. 2004), nd mong life history trits of the seed beetle, Sttor limbtus (Stillwell nd Fox 2005). Likewise, plsticity in response to host species vries mong trits in seed beetles (Fox 993; Fox et l. 994, 996). A more relistic understnding of the evolution of phenotypic plsticity cn thus only be chieved by simultneously exmining the responses of severl trits to multiple environmentl fctors. A complete understnding of the evolution of phenotypic plsticity lso requires knowledge of the genetic rchitecture underlying phenotypiclly plstic trits nd how this genetic rchitecture chnges with environmentl conditions. However, the genetic bsis of plsticity is still poorly understood (Scheiner 993; Promislow 2005; Czesk et l. 2006). Plsticity is often studied by mesuring the heritbility of trits in ech environment, quntifying genotype-by-environment interctions nd mesuring cross-environment genetic correltions (r G ). The cross-environment r G mesures the extent to which trit is correlted mong environments, nd thus how independent trit evolution is cross environments (Scheiner 993; Vi 994). Empiricl studies tht mesure the genetic bsis of plsticity cross multiple environments re lcking due to the diycultly in estimting these genetic prmeters in complex experiments. However, studies tht mesure genetic rchitecture re needed to provide insight into the evolution of plsticity in nture where environmentl complexity is the norm. Here we explore the seprte nd interctive evects of lrvl diet (host species) nd rering temperture on growth nd life history trits in two popultions of the seed beetle Cllosobruchus mcultus. Severl prior studies hve shown tht rering host species (Wssermn nd Futuym 98; Chndrknth nd Mthvn 986; Chndrknth et l. 987; Credlnd 987; Fox 993; Kwecki 995; Timms 998; vn Huis nd de Rooy 998; Boeke et l. 2004; Messin 2004, b; Vmosi 2005) nd temperture (Chndrknth nd Mthvn 986; Chndrknth et l. 987; Gig nd Smith 987; Guntrip et l. 997; Lle nd Vidl 2000, 2003, b; Mbt et l. 2005) hve lrge evects on vriety of trits of C. mcultus, but how these fctors interct to Vect rection norm shpe is poorly understood. Using fctoril experimentl design, we exmine the seprte nd interctive evects of rering host nd temperture on egg-to-dult survivorship, egg-to-dult development time, dult body mss, growth rte nd femle lifetime fecundity of C. mcultus. Also, becuse n understnding of the evolution of plsticity requires knowledge of the underlying genetic rchitecture, we explore how rering host nd temperture inxuence genetic vrinces nd heritbilities of body mss, growth rte nd fecundity, nd how cross-temperture r G s chnge with rering host. Mterils nd methods Nturl history nd study popultions The seed beetle, Cllosobruchus mcultus (Coleopter: Chrysomelide: Bruchine), is generlist seed herbivore of storge crops, but uses primrily species in the genus Vign in nture. Its life cycle revolves round seeds. Femles cement their eggs directly onto the seeds of their host plnt. Eggs htch nd lrve burrow directly underneth the egg into the seed. Lrvl growth nd puption tke plce entirely within single seed. Upon emergence from the seed, dults mte nd femles begin to ly eggs within hours. C. mcultus needs only the resources inside of single seed to complete development nd reproduce; dditionl food nd wter re not necessry (Fox et l. 2004, b). Becuse of its ese of lbortory rering, C. mcultus is widely used model system for life history, behvior nd genetic studies (Bieri nd Kwecki 2003; Fox et l. 2004b; Messin 2004, b; Arnqvist et l. 2005; Vmosi 2005). We exmine the seprte nd interctive evects of rering host nd temperture in two popultions of C. mcultus 23

3 Oecologi (2007) 53: tht re dpted to diverent species of Vign. The South Indi (SI) popultion ws collected in 979 from infested pods of mung ben, Vign rdit (L.) Wilczek, nd the closely relted blck grm, Vign mungo (L.) Hepper, in Tirunelveli, Indi (Mitchell 99). The Burkin Fso (BF) popultion ws collected in 989 from infested pods of cowpe, Vign unguicult (L.) Wlp., in Ougdougou, Burkin Fso (Messin 993). These two popultions diver in lrge number of trits including body size, dult lifespn, lrvl competitiveness, oviposition behvior nd the mount of pternl investment into reproduction (Svlli et l. 2000; Fox et l. 2004, b, c), mny of which hve likely evolved due to diverences in the properties of their host species (Messin nd Krren 2003; Messin 2004b). Both popultions were mintined in lbortory growth chmbers on seeds of V. rdit (SI) or V. unguicult (BF) t >,000 dults per genertion for >00 genertions (BF) or >200 genertions (SI) prior to this experiment. Experimentl design We used completely rndomized design with multifctoril tretment rrngement to exmine the evects of host species nd temperture on egg-to-dult survivorship, eggto-dult development time, dult body mss, growth rte nd femle lifetime fecundity in both popultions of C. mcultus. In short, lrve of full-sib fmilies were rered on three host plnts (mung, V. rdit; zuki, V. ngulris; nd cowpe, V. unguicult) nd t four rering tempertures (20, 25, nd 35 C; ll t 5:9 h, light:drk) yielding 2 tretment combintions for ech popultion. OVspring of ech full-sib fmily were rered on only one host (i.e., no split-brood design) but siblings were divided eqully mong the rering temperture tretments creting spiltbrood design for rering temperture. Cowpe nd mung re the ntive hosts for the BF nd SI popultions, respectively, nd zuki is n lternte host to which neither is dpted. We thus expected tht these popultions would exhibit better responses to rering on their ntive hosts compred to non-ntive hosts. The tempertures we used re within the norml rnge of tempertures t which C. mcultus cn develop nd reproduce (Chndrknth nd Mthvn 986; Chndrknth et l. 987; Mbt et l. 2005). However, becuse the ntive climtes of the BF nd SI popultions re very similr (men temperture diverence between sites is»0.4 C; Ntionl Climtic Dt Center s Globl Surfce Summry of Dy, Asheville, N.C.) nd becuse these popultions hve been mintined in lbortory colonies for more thn 00 genertions under benign nd identicl conditions, we did not expect them to show diverent responses to temperture. To crete fmilies, seeds bering eggs were rndomly selected from our lbortory colonies nd isolted in 35-mm Petri dishes (one seed per dish, one egg per seed). Adults emerging from these seeds were used s prents to generte full-sib fmilies by rndomly piring virgin mles nd virgin femles within ech popultion. Ech pir ws rndomly ssigned to one of three rering hosts (60-mm dishes contining seeds of cowpe, 35-mm dishes contining 40 seeds of mung or 35-mm dishes contining seeds of zuki) nd plced in growth chmber to ly eggs (25 C; 5:9 h, light:drk). Dishes were checked for eggs twice per dy until femles lid eggs on»32 seeds (seeds bering eggs were replced t ech check) fter which dults were discrded. Seeds contining eggs were scrped to one egg per seed (to eliminte lrvl competition) nd plced individully in 35-mm Petri dishes. Egg bering seeds were rndomly ssigned to one of the four rering temperture tretments within 2 h of being lid, such tht ovspring from ech fmily were divided evenly mong the four tretments (split-brood design), with pproximtely eight ovspring per tretment. All ovspring were rered in Petri dishes inside temperture-controlled Percivl rech-in growth chmbers. Developing lrve were rotted dily to control for sptil vrition within growth chmbers. Emerging dult beetles were collected twice dily. Subsmples of six ovspring per fmily per rering temperture were weighed on n electronic blnce (AT26 Delt Rnge; Mettler Toledo, Columbus, Ohio) to the nerest 0. mg. These six ovspring were rndomly selected t the egg stge by mrking their dish. After beetles were weighed, femles were pired with rndomly chosen mle (within ech popultion-by-host-by-temperture tretment combintion) nd plced in 60-mm Petri dishes contining»70 mung seeds. Pirs were llowed to ly eggs until deth t 27.5 C (5:9 h, light:drk) fter which every seed ws exmined nd ll eggs were counted to estimte femle lifetime fecundity. Oviposition host nd temperture will certinly Vect femle egg-lying behvior nd lifetime fecundity (Messin nd Krren 2003; Stillwell nd Fox 2005) but our focus here is on the evect of the lrvl rering environment nd not the dult oviposition environment. We thus use common oviposition environment for ll egg-lying femles. We estimted growth rte s log(mss)/lrvl development time. However, becuse our originl mesure of development time lso includes the durtion of the pupl period, in which growth is not tking plce, we Wrst subtrcted the verge durtion of the pupl period for ech temperture tretment (rering host does not Vect the length of the pupl period; Chndrknth nd Mthvn 986). Averge pupl intervls were obtined from previous study tht used identicl rering tempertures (Chndrknth nd Mthvn 986). Though it is possible tht the length of the pupl period in this study divers from tht 23

4 32 Oecologi (2007) 53:9 32 found in Chndrknth nd Mthvn (986), nlysis of the dt not correcting for the durtion of the pupl period [i.e., log(mss)/egg-to-dult development time] gve qulittively identicl results, indicting tht the correction does not bis our conclusions. Becuse single growth chmber ws used for ech temperture tretment in our study, other sources of environmentl vrition mong growth chmbers re potentilly confounded with temperture. These evects re likely to be smll reltive to the lrge evect of temperture observed for ll trits (see Results), but re nonetheless confounded with temperture. However, our focus here is on the degree to which vrition long diverent environmentl xes interct to inxuence the phenotype of orgnisms, nd the degree to which these interctions vry mong multiple trits. Thus, while vrition mong chmbers Vects the speciwc interprettion of the temperture min evect, this vrition mong chmbers (temperture + unknown environmentl vrition) nonetheless rexects n environmentl xis completely seprte from the other environmentl xis (diet) tht is mnipulted in our study. Thus, chmber evects do not limit our bility to ddress our originl question. In totl, 9,785 dults from 387 full-sib fmilies were rered to dult. 7,658 of these were weighed. Lifetime fecundity ws recorded for 2,85 femles. Sttisticl nlyses All sttisticl nlyses were done with SAS 9. (SAS Institute, Cry, N.C.) using ANOVA (PROC GLM). Norml probbility plots reveled tht ll dt were pproximtely normlly distributed, except egg-to-dult survivorship. However, development time ws log-trnsformed prior to nlysis to stbilize vrinces mong temperture tretments. For our ANOVAs we included popultion, host, temperture nd fmily s min evects. The fmily evect is included becuse of the non-independence of siblings within tretments. Also, becuse fmilies re unique to ech popultion-by-host combintion, the fmily evect ws nested within popultions nd hosts. Consequently, we cn test for fmily-by-rering temperture interction but not fmily-by-host interction. The fmily evect is used s the denomintor men squre for hypothesis tests for ll min evects nd their interctions. For egg-to-dult survivorship we Wrst clculted the proportion of surviving ovspring for ech fmily-by-rering temperture combintion. We then used these mens (rcsine-squre root trnsformed to meet the ssumptions of normlity) for nlysis. Using fmily mens prevented us from including fmily-by-rering temperture interction term in the model. The min focus of this study is on interctions between rering host nd temperture. However, becuse interctions between fctors in n ANOVA mesure chnges in the liner distnce between tretment mens, they re dependent on scle. Becuse of the lrge evect of temperture on mny of the trits we exmined (especilly development time nd growth rte), host-by-temperture interctions, nd other interctions involving temperture, my be detected when no interctions exist. Conversely, signiwcnt interctions cn be msked due to chnges in scle. We thus performed our nlyses s two-step process. First, we exmined min evects. We then creted reltive trit vlues (individul trit vlue/men in ech temperture tretment) to remove the lrge temperture evect. These reltive trit vlues were used for testing for interctions between/mong vribles tht were Vected by temperture (Stnton nd Thiede 2005). All ANOVAs were Wrst performed using the full model with ll possible interction terms present; non-signiwcnt three-wy nd higher order interction terms were dropped from ll Wnl models. Although we initilly included popultion s min evect, we subsequently conducted seprte nlyses for ech popultion due to lrge interctions involving the popultion evect for severl trits. Genetic vrinces (V G ), brod-sense heritbilities (H 2 ) nd cross-temperture genetic correltions(r G ) were estimted using the vrince component procedure in SAS (PROC VARCOMP, REML estimtion; Fry 992; Astles et l. 2006). V G ws clculted s twice the phenotypic vrince (V P ) mong full-sib fmilies (the vrince ws obtined vi the fmily vrince component in PROC VARCOMP) nd H 2 ws clculted s V G /V P (Flconer nd Mcky 996). r G ws clculted for ech pir of tempertures s: σ 2 temperture, 2 /σ temperture σ temperture2, where σ2 temperture, 2 is the fmily vrince component for the mixed model with dt for the two tempertures pooled (the covrince between tempertures), nd σ temperture nd σ temperture2 re squre roots of the fmily vrince component for the two reduced models, one for ech temperture (the vrince within tempertures; Fry 992; Astles et l. 2006). SEs of genetic prmeters were obtined by jckkniwng fmilies (RoV nd Preziosi 994; Windig 997) vi routine creted by the uthors in SAS. We conducted n ANOVA of the pseudovlues of V G nd H 2 tht were generted from the jckkniwng routine to explore the impct of popultion, sex, rering host nd temperture on genetic vrition. We lso used multivrite ANOVA (MANOVA) to exmine the overll evect of popultion, sex nd rering host on r G, nd subsequently used ANOVA to tese prt which speciwc r G s were Vected by the rering hosts. This method ws originlly developed by RoV (2002) for the nlysis of genetic vrince/covrince mtrices. The smpling distributions of the pseudovlues creted by the jckkniwng were ll pproximtely normlly distributed nd were thus not trnsformed prior to nlysis. Genetic prm- 23

5 Oecologi (2007) 53: eters were not clculted for development time due to extremely low H 2 nd lrge mternl evects (Fox 994). Results Popultion, host nd popultion-by-host evects Becuse previous comprisons between the BF nd SI popultions of C. mcultus reveled genetic diverentition between popultions in lrge number of trits (Svlli et l. 2000; Fox et l. 2004, b, c) we predicted tht we would Wnd diverences between the popultions in ll of the trits tht we exmined in this experiment. As expected, BF beetles hd higher egg-to-dult survivorship (F,385 =77, P < 0.000), slightly longer development time (F,38 = 50, P < 0.000), were smller (F,38 =224, P < 0.000), grew slower (F,380 = 9, P < 0.000) nd hd higher fecundity (F,380 = 469, P < 0.00) thn SI beetles (Figs., 2, 3, 4, 5). The evect of rering host on development time, body mss nd growth rte divered between the popultions (highly signiwcnt popultion-by-host interction development time, F 2,38 =34, P < 0.000; body mss, F 2,38 = 35.8, P < 0.000; growth rte, F 2,380 =07, P < 0.000). BF beetles generlly hd the shortest development time (Fig., b; highly signiwcnt rering host evect in Tble ; P < 0.00; see Wgure legends for pir-wise comprisons), were the lrgest (P < 0.00; Fig. 2, b; Tble ) nd grew fstest (P < 0.00; Fig. 3, b; Tble ) when rered on cowpe (except body mss t 20 C). In contrst, SI beetles generlly hd the shortest development time (P < 0.00; Fig., b; Tble ), were the lrgest (P < 0.00; Fig. 2, b; Tble ) nd grew fstest (P < 0.00; Fig. 3, b; Tble ) when rered on zuki nd mung. In ddition, the fecundity of SI femles (but not BF femles; Tble ) ws Vected by rering host; SI femles lid the fewest eggs when rered on cowpe, though the evect ws smll (P <0.0; Fig.4, b; Tble ). There ws lso highly signiwcnt popultion-by-host interction for egg-to-dult survivorship (F 2,385 =5.3, P < 0.000); both popultions hd high survivorship when rered on zuki nd mung, nd the lowest survivorship on cowpe, but the host evect ws much lrger for SI thn BF beetles (P < 0.00; Fig. 5; Tble ). Temperture nd popultion-by-temperture evects Development time decresed substntilly with incresing rering temperture (P < 0.00; Fig., b; Tble ). The temperture evect on development time divered slightly between popultions (F 3,38 = 4.45, P = 0.004; Fig. c, d). As expected, both popultions decresed signiwcntly in Development time (dys) Development time (reltive) c Femles time Temperture Temperture ( o C) b d Temperture ( o C) Mles Burkin Fso South Indi Host species Azuki Cowpe Mung Temperture ( o C) Temperture ( o C) Fig. Egg-to-dult development time of, c femles nd b, d mles of the Burkin Fso (solid lines) nd South Indi (dshed lines) popultions of Cllosobruchus mcultus in response to rering on zuki (squres), cowpe (circles) nd mung (tringles) t diverent tempertures (20, 25,, 35 C). c, d Development time (reltive) re the mens fter removing the lrge temperture evect [individul development time/men development time for ech temperture tretment, following Stnton nd Thiede (2005)]. SEs re included, but re smller thn the symbols for some experimentl tretments. Pir-wise comprisons for Burkin Fso Tukey s test for cowpe versus mung, P < 0.05; cowpe versus zuki, P < 0.05; zuki versus mung, P > 0.05; South Indi cowpe versus mung, P < 0.05; cowpe versus zuki, P < 0.05; zuki versus mung, P >

6 34 Oecologi (2007) 53:9 32 Body mss (mg) Body mss (reltive) c Femles Temperture b d Temperture ( o C) Mles Burkin Fso South Indi Host species Azuki Cowpe Mung Temperture ( o C) Fig. 2 Adult body mss of, c femles nd b, d mles of the Burkin Fso (solid lines) nd South Indi (dshed lines) popultions of C. mcultus in response to rering on zuki (squres), cowpe (circles) nd mung (tringles) t diverent tempertures (20, 25,, 35 C). c, d Body mss (reltive) re the mens fter removing the lrge temperture evect [individul body mss/men body mss for ech temperture tretment, following Stnton nd Thiede (2005)]. SEs re included, but re smller thn the symbols for some experimentl tretments. Pirwise comprisons for Burkin Fso Tukey s test for cowpe versus mung, P > 0.05; cowpe versus zuki, P < 0.05; zuki versus mung, P < 0.05; South Indi cowpe versus mung, P < 0.05; cowpe versus zuki, P < 0.05; zuki versus mung, P > Growth rte (log mg d ) Femles b Burkin Fso South Indi Mles Host species Azuki Cowpe Mung Growth rte (reltive) c 20 Temperture d 20 Temperture ( o C) Temperture ( o C) Fig. 3 Growth rte [log(body mss)/lrvl development time] of, c femles nd b, d mles of the Burkin Fso (solid lines) nd South Indi (dshed lines) popultions of C. mcultus in response to rering on zuki (squres), cowpe (circles) nd mung (tringles) t diverent tempertures (20, 25,, 35 C). c, d Growth rte (reltive) re the mens fter removing the lrge temperture evect [individul growth rte/men growth rte for ech temperture tretment, following Stnton nd Thiede (2005)]. SEs re included, but re smller thn the symbols for some experimentl tretments. Pir-wise comprisons for Burkin Fso Tukey s test for cowpe versus mung, P <0.05; cowpe versus zuki, P < 0.05; zuki versus mung, P < 0.05; South Indi cowpe versus mung, P < 0.05; cowpe versus zuki, P < 0.05; zuki versus mung, P >

7 Oecologi (2007) 53: eggs) Fecundity (no. b Fecundity (reltive) Burkin Fso South Indi Host species Azuki Cowpe Mung Temperture ( o C) Temperture ( o C) Fig. 4 Lifetime fecundity of femles of the Burkin Fso (solid lines) nd South Indi (dshed lines) popultions of C. mcultus in response to rering on zuki (squres), cowpe (circles) nd mung (tringles) t diverent tempertures (20, 25,, 35 C). b Fecundity (reltive) re the mens fter removing the lrge temperture evect [individul fecundity/men fecundity for ech temperture tretment, following Stnton nd Thiede (2005)]. SEs re included, but re smller thn the symbols for some experimentl tretments. Pir-wise comprisons for Burkin Fso Tukey s test for cowpe versus mung, P > 0.05; cowpe versus zuki, P > 0.05; zuki versus mung, P > 0.05; South Indi cowpe versus mung, P < 0.05; cowpe versus zuki, P > 0.05; zuki versus mung, P > Egg-to-dult survivorship (%) Fig. 5 Egg-to-dult survivorship of the Burkin Fso (solid lines) nd South Indi (dshed lines) popultions of C. mcultus in response to rering on zuki (squres), cowpe (circles) nd mung (tringles) t diverent tempertures (20, 25,, 35 C). SEs re included, but re smller thn the symbols for some experimentl tretments. Pir-wise comprisons for BF cowpe versus mung, P < 0.05; cowpe versus zuki, P < 0.05; zuki versus mung, P < 0.05; South Indi cowpe versus mung, P < 0.05; cowpe versus zuki, P > 0.05; zuki versus mung, P >0.05 body mss with incresing rering temperture (P <0.00; Fig. 2, b; Tble ), but SI beetles decresed in mss considerbly more thn did BF beetles (evident s convergence of BF nd SI lines t higher tempertures; signiwcnt popultion-by-temperture interction; F 3,38 =0.5, P < 0.000; Fig. 2c, d). Growth rte incresed substntilly with incresing rering temperture (P <0.00; Fig.3, b; Tble ), though the evect divered between popultions (highly signiwcnt popultions-by-temperture interction; F 3,380 =8.24, P < 0.000) with growth rte of BF beetles incresing with rering temperture fster thn growth rte of SI beetles (evident s convergence of BF nd SI lines t higher tempertures; Fig. 3c, d). The reltionship between femle lifetime fecundity nd temperture ws not monotonic; femles lid the most eggs when rered t the intermedite tempertures (25 nd C) nd lid the fewest when rered t either extreme (20 nd 35 C, P < 0.00; Fig. 4; Tble ). This pttern ws diverent between popultions (signiwcnt popultion-by-temperture interction; F 3,380 = 7.87, P < 0.000); the popultions responded similrly to temperture between 25 nd 35 C, but fecundity of SI femles incresed more drmticlly between 20 nd 25 C thn did fecundity of BF beetles (evident s convergence of BF nd SI lines t 25 C; Fig. 4b). Egg-to-dult survivorship ws highest when beetles were rered t the intermedite tempertures (25 nd C) nd lowest when rered t the extremes, but the evect ws smll (20 nd 35 C; P < 0.0; Fig. 5; Tble ). This evect ws similr in both popultions (non-signiwcnt popultion-bytemperture interction; F 3,385 = 0.94, P = 0.42). Host-by-temperture evects Temperture ( o C) We found signiwcnt interctions between rering host nd temperture for three of the Wve trits we exmined (body mss, growth rte nd fecundity; Figs. 2,3,4; Tble ). This ws most evident for growth rte; the vrince in growth rte mong hosts incresed with incresing temperture (Fig. 3c, d; Tble ). However, this evect divered between popultions (signiwcnt popultion-by-host-by-temperture interction; F 6,380 = 2.33, P = 0.03); the degree to which cowpe ws better host thn zuki or mung incresed t higher temperture for BF beetles, wheres the degree to which cowpe ws the worst host incresed t higher temperture for SI beetles (Fig. 3c, d). Genetic vrinces nd heritbilities Burkin Fso South Indi Host species Azuki Cowpe Mung Overll, the genetic vrince nd heritbility of body mss ws similr for mles (men SEM; V G = ; 23

8 36 Oecologi (2007) 53:9 32 Tble ANOVA (type III sums of squres) for the evects of rering host nd temperture on egg-to-dult development time, dult body mss, growth rte, femle lifetime fecundity nd egg-to-dult survivorship in the Burkin Fso (BF) nd South Indi (SI) popultions of Cllosobruchus mcultus BF SI df F df F Egg-to-dult development time Femles Temperture 3 2,588.02*** 3 8,722.20*** Host *** *** Fmily (host) 99.75*** *** Temperture host Temperture fmily (host) ** Error,860,422 Mles Temperture 3 6,757.64*** 3 8,85.90*** Host *** *** Fmily (host) 99 2.*** 8.73*** Temperture host Temperture fmily (host) Error 2,066,46 Mss Femles Temperture *** *** Host *** *** Fmily (host) *** *** Temperture host * ** Temperture fmily (host) Error, Mles Temperture *** *** Host *** 2 9.5*** Fmily (host) *** *** Temperture host Temperture fmily (host) * Error,462,00 Growth rte Femles Temperture 3 2,5.0*** 3 2,252.26*** Host 2.23*** *** Fmily (host) 99.96*** *** Temperture host * Temperture fmily (host) Error, Mles Temperture 3,260.3*** 3,28.*** Host *** 2 6.2*** Fmily (host) *** *** Temperture host 6 2.9* * Temperture fmily (host) Error,46,099 23

9 Oecologi (2007) 53: Tble continued BF SI df F df F * P < 0.05, ** P < 0.0, *** P <0.00 Interctions from nlyses on reltive trit vlues (individul vlue/men for ech temperture tretment) Fecundity Temperture *** *** Host ** Fmily (host) *** *** Temperture host * Temperture fmily (host) ** Error Egg-to-dult survivorship Temperture ** 3 5.4** Host *** *** Fmily (host) *** *** Temperture host Error H 2 = ) nd femles (V G = ; H 2 = ; F <2.66, P > 0.0). Rering temperture hd lrge evect on V G for mss due to substntil increse in V G t 20 C (35 C, ; C, ; 25 C, ; 20 C, ; F 3,2850 =5.29, P =0.00). However, this ws concordnt with n increse in the phenotypic vrince with temperture such tht there ws no evect of temperture on the H 2 for body mss (35 C, ; C, ; 25 C, ; 20 C, ; F 3,2850 =0.0, P = ). The genetic vrince in body mss of BF femles ws similr to tht of BF mles when they were rered on cowpe (femles, ; mles, ) nd mung (femles, ; mles, ), but lower when they were rered on zuki (femles, ; mles, ; host-bysex interction; F 2,524 =3.39, P = 0.03). However, this pttern ws not observed for heritbilities (host-by-sex interction; F 2,524 =0.2, P = 0.88). There ws no diverence between popultions (F <2.47, P > 0.2) or mong rering hosts (F <0.98, P > 0.37) for either V G or H 2 of body mss, nor were ny of the interction terms signiwcnt (F <.36, P >0.26). The genetic vrince nd heritbility of growth rte ws not signiwcntly diverent between mles (V G = ; H 2 = ) nd femles (V G = ; H 2 = ; F < 0.46, P > 0.5). There ws lrge evect of temperture on V G for growth rte (35 C, ; C, ; 25 C, ; 20 C, ; F 3,2873 =0.82, P < 0.000): this ws concordnt with n increse in the phenotypic vrince with incresing temperture such tht H 2 did not chnge with temperture (35 C, ; C, ; 25 C, ; 20 C, ; F 3,2873 =0.76, P = 0.52). There ws mrginl host-by-temperture interction on the genetic vrince in growth rte (V G, F 6,2873 =2.62, P =0.02; H 2, F 6,2873 =.8, P = 0.09); both V G nd H 2 were highest on zuki t 35 C (V G zuki, ; cowpe, ; mung, ; H 2 zuki, ; cowpe, ; mung, ), but V G ws similr for ll three hosts t 20 C (zuki, ; cowpe, ; mung, ) while H 2 ws highest on mung t 20 C (zuki, ; cowpe, ; mung, ). The genetic vrince nd heritbility for growth rte of SI femles ws pproximtely twice tht of SI mles when they were rered on mung (V G SI femles, ; SI mles, ; H 2 SI femles, ; SI mles, ), but lower thn tht of mles when they were rered on zuki nd cowpe (V G SI femles rered on zuki, ; SI mles rered on zuki, ; H 2 SI femles rered on zuki, ; SI mles rered on zuki, ; V G SI femles rered on cowpe, ; SI mles rered on cowpe, ; H 2 SI femles rered on cowpe, ; SI mles rered on cowpe, ; hostby-sex interction; V G, F 2,338 =3.90, P =0.02; H 2, F 2,338 =2.95, P = 0.05). In contrst, the genetic vrince nd heritbility of growth rte did not chnge with temperture (temperture-by-sex interction; F < 0.59, P >0.62). There ws no evect of popultion (F < 2.3, P > 0.3), rering host (F <.28, P > 0.28) or ny of the other interctions (F <0.94, P > 0.39) on V G or H 2 of growth rte. V G for fecundity of SI femles (3 68) ws double tht of BF femles (63 50; F,33 =4.33, P = 0.04). This ws not due to scle evect; H 2 of fecundity of SI femles ws twice s high s tht of BF femles (SI, ; BF, ; F,33 =9.2, P = 0.003). There ws no evect of rering host (F <0.36, P > 0.7), temperture (F <.48, P > 0.22) or ny of the interctions (F < 3.00, P > 0.05) on V G nd H of fecundity. 23

10 38 Oecologi (2007) 53:9 32 Cross-temperture r G s The overll verge cross-temperture r G for mss (r G = ), growth rte (r G = ) nd fecundity (r G = ) were ner.0. The cross-temperture r G s were not diverent between popultions or mong hosts, nor were there ny signiwcnt popultion-byhost interctions for ny trits (MANOVA, F <.22, P > 0.3; ANOVA of ll pir-wise r G s, F <2.55, P > 0.) with one exception there ws signiwcnt evect of rering host on r G for growth rte (MANOVA, Wilks λ =0.93, F 2,972 =2.79, P = ). However, this ws due minly to lrge host evect on r G t 35 nd 25 C (ANOVA, F 2,49 =5.37, P = 0.005); the correltion ws lowest on cowpe (r G = ) nd highest on mung ( ). Also, there ws signiwcnt sex evect on the cross-temperture r G s for growth rte (MANOVA, Wilks λ =0.95, F 6,486 = 3.9, P = ) due to lrge diverence between the sexes in r G t 35 nd 25 C (ANOVA, F,49 =.6, P = ); the verge cross-temperture r G for mles ( ) ws lrger thn ws the crosstemperture r G for femles ( ). There were no sex-by-popultion or sex-by-host evects on cross-temperture r G s for either body mss or growth rte (MANOVA, F <.22, P > 0.3; ANOVA of ll pir-wise r G s, F <4.52, P >0.03). Discussion In this study we investigted the interctive evects of rering diet (i.e., host species) nd temperture on growth nd life history trits in two popultions of the seed beetle, C. mcultus. We detected signiwcnt host-by-temperture interction for three of the Wve trits we exmined. The host-by-temperture interction for growth rte divered in mgnitude between our two study popultions. This indictes tht the evects of multiple environments on beetle trits cn be either simple (rection norm shpe long one environmentl xis is not Vected by other environmentl xes) or complex (rection norm shpe long one environmentl xis is Vected by other environmentl xes) depending on the trit nd the popultion exmined. In ddition, though estimtes of genetic vrition vried with rering environment, the heritbility estimtes for body mss, growth rte nd fecundity (which re estimtes of the proportion of totl vrince tht is due to genetic vrince, nd thus removes scle evects) were generlly similr mong rering hosts nd tempertures. Cross-temperture r G s were ner.0 for ll trits nd did not diver mong hosts suggesting tht the genetic rchitecture underlying temperture-induced phenotypic plsticity is stble nd not Vected by rering host. Finlly, we found tht these popultions of beetles responded diverently to both rering host nd temperture. Simple versus complex ptterns of phenotypic plsticity Although orgnisms grow nd develop in complex environments where they re exposed simultneously to multiple environmentl fctors, most empiricl ssessments of plsticity hve mnipulted single environmentl fctor. Recent studies hve demonstrted tht environmentl fctors cn hve interctive evects on phenotypes, generting complex rection norms (Stmp nd Bowers 990; Gresens 997; Sultn et l. 998; Petersen et l. 2000; Sultn 200; Relye 2004; Ris et l. 2004; Relye nd Auld 2005; Kingsolver et l. 2006). For instnce, the evects of dietry protein concentrtion on the growth rte of the cterpillr, Mnduc sext, is highly dependent on rering temperture (Petersen et l. 2000). Likewise, the growth rte of the midge, Pseudochironomous richrdsoni, is lwys higher on detritus diet reltive to ditom diet, but this diverence is signiwcntly lrger t high rering tempertures (Gresens 997). In this study, we found tht rering host nd temperture hd interctive evects on body mss, growth rte nd fecundity of C. mcultus. Thus, the shpe of rection norms ws complex. This ws most obvious for growth rte; the vrince in growth rte mong hosts incresed with incresing temperture. However, this evect divered between our two study popultions; the degree to which cowpe ws better host thn zuki or mung incresed t higher tempertures for BF beetles, wheres the degree to which cowpe ws the worst host incresed t higher tempertures for SI beetles. Wheres numerous studies hve previously demonstrted tht rection norm shpe cn vry mong popultions our study shows tht interctions between vribles cn likewise vry mong popultions within species. Consequently, future studies should include severl popultions to fully understnd how these interctions Vect ptterns nd the evolution of phenotypic plsticity. In contrst, we did not Wnd ny signiwcnt interctions between host nd temperture for lrvl survivorship or development time, suggesting tht the evects of host nd temperture re independent for these trits. Are rection norms likely to be simple or more complex in environments where orgnisms re exposed to mny vribles? Recent empiricl investigtions hve found both complex (Stmp nd Bowers 990; Gresens 997; Sultn et l. 998; Petersen et l. 2000; Sultn 200; Relye 2004; Ris et l. 2004; Relye nd Auld 2005; Kingsolver et l. 2006) nd simple (Teplitsky et l. 2004; Hovermn et l. 2005) ptterns of plsticity, but most of these studies hve exmined only one or few trits nd only single popultion within species. It is diycult to generlize becuse few studies hve explored plsticity of multiple trits nd 23

11 Oecologi (2007) 53: popultions under more complex environmentl scenrios. Studying multiple levels of ech of severl environmentl fctors requires complicted experimentl designs nd very lrge smple sizes, mking such studies imprcticl for most orgnisms. Nevertheless, we need to understnd rection norm shpe in more complex environments to hve better understnding of how plsticity evolves, becuse mny environmentl fctors vry simultneously both sptilly nd temporlly in the nturl environments in which orgnisms develop nd experience selection. Genetic rchitecture in complex environments A complete understnding of the evolution of phenotypic plsticity requires knowledge of the genetic rchitecture underlying phenotypiclly plstic trits nd how this genetic rchitecture chnges with environmentl conditions. However, virtully nothing is known bout the genetics of plsticity in environments tht vry long multiple environmentl xes. The evolution of plsticity will be slowed when r G =.0 becuse selection on the trit in one environment will result in similr correlted response in the other environment, though how similr depends on environmentl evects on genetic vrition (Cheverud et l. 985). In this study, we exmined the cross-temperture r G for body mss, growth rte nd fecundity, nd investigted whether rering host inxuenced the cross-temperture r G. The overll verge cross-temperture r G ws not signiwcntly diverent from.0 nd there ws little evidence for genotype-by-temperture (fmily-by-temperture) interction for ny of the mesured trits (Tble ). This suggests tht the evolution of temperture-induced plsticity will be very slow in C. mcultus. Also, the cross-temperture r G ws not generlly Vected by rering host, indicting tht the cross-temperture r G is stble with respect to rering conditions. Interestingly, even when rered in common conditions for >00 genertions some geneticlly distinct popultions of C. mcultus retin distinct genetic rchitectures underlying growth nd life history trits (Bieri nd Kwecki 2003) suggesting tht genetic rchitecture my be evolutionrily stble in ddition to being stble cross environments. Although very high r G will limit the rte of the evolution of plsticity (Vi nd Lnde 985), plsticity cn evolve even with r G =.0 if the heritbility of trit divers between environments, point originlly mde by Cheverud et l. (985) in the context of the between-sex r G. Though the heritbility for fecundity divered between popultions, we hve no evidence tht the heritbility for body size, growth rte nd fecundity vries with rering host or temperture. Thus, the evolution of plsticity in C. mcultus will probbly be hindered by both very high r G s nd similr heritbilities in the diverent environments. However, our estimtes of the genetic prmeters must be interpreted with cution; despite the lrge number of beetles rered in this experiment, the lrge number of tretments led to lrge SEs on individul prmeters. Future studies tht ttempt to investigte genetic rchitecture in complex environments will likewise be problemtic becuse of the number of tretments required to ddress this complexity. Nevertheless, exploring the genetic bsis of plsticity using experiments tht more relisticlly rexect the complex environments found in nture re required to fully understnd the evolution of plsticity. Adpttion of popultions to host nd temperture Adpttion of insect popultions to their ntive host plnts is common in nture. Genetic diverentition in growth nd life history trits between the BF nd SI popultions of C. mcultus is well documented nd is probbly due both to dpttion to their indigenous hosts nd long-term rering on these hosts in culture (Svlli et l. 2000; Fox et l. 2004, b, c), so we expected tht BF beetles would generlly perform better on cowpe nd SI beetles would perform better on mung. We did Wnd tht BF beetles generlly hd shorter development time, were lrger in size (except when rered t the lowest temperture) nd hd fster growth rte when they were rered on their ntive host (cowpe) while SI beetles hd shorter development time, were lrger nd hd fster growth rte when rered on their ntive host (mung) nd the lternte host (zuki), but the evects were smll. In ddition, fecundity of femles from both popultions ws generlly unvected by rering host nd both popultions hd the highest egg-to-dult survivorship on mung nd zuki seeds. Divergence between popultions in therml rection norms for growth trits occur in severl species of insects (Norry et l. 200; Bochdnovits nd De Jong 2003; Stillwell nd Fox 2005). Genetic diverentition in popultion-level therml rection norms is often ttributed to temperturemedited nturl selection when the rection norms mtch diverences in climte between collection loclities of popultions (Stillwell nd Fox 2005). Here, we found tht the BF nd SI popultions responded diverently to rering temperture; the reltive diverence in development time, body size nd growth rte between the two popultions decresed with incresing rering temperture. Likewise, the reltive diverence in fecundity between the BF nd SI popultions decresed from 20 to 25 C. However, this is not likely consequence of dpttion to diverent tempertures becuse the BF nd SI popultions originted from tropicl loctions tht hve very similr climtes (see Mterils nd methods). Moreover, these popultions hve been mintined in lbortory colonies for more thn 00 genertions under benign nd identicl climtes. Consequently, it is unlikely tht the diver- 23

12 320 Oecologi (2007) 53:9 32 ences in responses we observed re cused by dpttion to temperture, but further work is needed to revel why these popultions respond diverently to temperture. Conclusions The most importnt impliction of our study is tht phenotypic plsticity cn be complex for some trits, but simple for others, nd tht popultions cn vry in the mgnitude of this complexity. Understnding the degree of complexity in plsticity nd why trits diver in the degree to which environmentl evects re independent versus interctive requires studies tht mesure multitude of trits in severl popultions of species. Also, though our study found tht genetic rchitecture ws stble cross environments, future studies should investigte how the genetic rchitecture of plsticity is Vected in complex environments so tht more ccurte predictions cn be mde regrding responses to selection in nture. Our dt demonstrte tht studying plstic responses long one environment xis or for only one or few trits, will miss much of the complexity of rection norm shpe tht occurs in nture. Quntifying plsticity long multiple xes will certinly be diycult becuse multifctoril experimentl designs require lrge smple sizes to hve dequte power to distinguish rel from rndom vrition. However, understnding this complexity will yield new nd exciting insights into how plsticity evolves in ecologiclly complex worlds. Acknowledgements We thnk D. Johnson nd O. Njoku for help weighing nd mting beetles during the experiment. We lso thnk K. Hynes for sttisticl dvice, nd thnk Jy Rosenheim nd two nonymous reviewers for providing helpful comments on erlier drfts of the mnuscript. Finncil support ws provided in prt by Ntionl Science Foundtion grnt (NSF DEB ) to C. W. F. Institutionl guidelines were followed for niml cre. References Angillett MJ, Dunhm AE (2003) The temperture size rule in ectotherms: simple evolutionry explntions my not be generl. Am Nt 62: Angillett MJ, Niewirowski PH, Dunhm AE, Leche AD, Porter WP (2004) Bergmnn s clines in ectotherms: illustrting life-history perspective with sceloporine lizrds. Am Nt 64:E68 E83 Arnqvist G, Nilsson T, Ktvl M (2005) Mting rte nd Wtness in femle ben weevils. Behv Ecol 6:23 27 Astles PA, Moore AJ, Preziosi RF (2006) A comprison of methods to estimte cross-environment genetic correltions. J Evol Biol 9:4 22 Atkinson D (994) Temperture nd orgnism size biologicl lw for ectotherms? Adv Ecol Res 25: 58 Atkinson D, Sibly RM (997) Why re orgnisms usully bigger in colder environments? Mking sense of life history puzzle. Trends Ecol Evol 2: Awmck CS, Lether SR (2002) Host plnt qulity nd fecundity in herbivorous insects. Annu Rev Entomol 47: Bégin M, RoV DA, Debt V (2004) The evect of temperture nd wing morphology on quntittive genetic vrition in the cricket Gryllus Wrmus, with n ppendix exmining the sttisticl properties of the Jckknife-MANOVA method of mtrix comprison. J Evol Biol 7: Berrign D, Chrnov EL (994) Rection norms for ge nd size t mturity in response to temperture: puzzle for life historins. Oikos 70: Bieri J, Kwecki TJ (2003) Genetic rchitecture of diverences between popultions of cowpe weevil (Cllosobruchus mcultus) evolved in the sme environment. Evolution 57: Bochdnovits Z, De Jong G (2003) Temperture dependence of Wtness components in geogrphicl popultions of Drosophil melnogster: chnging the ssocition between size nd Wtness. Biol J Linn Soc 80: Boeke SJ, vn Loon JJA, vn Huis A, Dicke M (2004) Host preference of Cllosobruchus mcultus: comprison of life history chrcteristics for three strins of beetles on two vrieties of cowpe. J Appl Entomol 28: Chndrknth J, Mthvn S (986) Chnges in developmentl rtes nd biomss energy in Cllosobruchus mcultus (F.) (Coleopter: Bruchide) rered on diverent foods nd tempertures. J Stored Prod Res 22:7 75 Chndrknth J, Muthukrishnn J, Mthvn S (987) EVect of temperture nd host seed species on the fecundity of Cllosobruchus mcultus (F.). Proc Indin Acd Sci Anim Sci 96: Cheverud JM, Dow MM, Leutenegger W (985) The quntittive ssessment of phylogenetic constrints in comprtive nlyses: sexul dimorphism in body weight mong primtes. Evolution 39: Credlnd PF (987) EVects of host chnge on the fecundity nd development of n unusul strin of Cllosobruchus mcultus (F.) (Coleopter: Bruchide). J Stored Prod Res 23:9 98 Czesk ME, Fox CW, Wolf JB (2006) Experimentl evolution of phenotypic plsticity: how predictive re cross-environment genetic correltions? Am Nt 68: Ernsting G, Isks A (2000) Ectotherms, temperture, nd trde-ovs: size nd number of eggs in crbid beetle. Am Nt 55: Flconer DS, Mcky TFC (996) Introduction to quntittive genetics, 4th edn. Longmn, Essex Fox CW (993) A quntittive genetic nlysis of oviposition preference nd lrvl performnce on two hosts in the bruchid beetle, Cllosobruchus mcultus. Evolution 47:66 75 Fox CW (994) Mternl nd genetic inxuences on egg size nd lrvl performnce in seed beetle (Cllosobruchus mcultus): multigenertionl trnsmission of mternl evect? Heredity 73: Fox CW, Czesk ME (2000) Evolutionry ecology of progeny size in rthropods. Annu Rev Entomol 45: Fox CW, Wddell KJ, Mousseu TA (994) Host ssocited Wtness vrition in seed beetle (Coleopter: Bruchide): evidence for locl dpttion to poor qulity host. Oecologi 99: Fox CW, Hrbin AD, Mousseu TA (996) Suitbility of non-host plo verde for development of Sttor limbtus (Horn) (Coleopter: Bruchide) lrve. Pn Pc Entomol 72:3 36 Fox CW, Bush ML, RoV DA, Wllin WG (2004) Evolutionry genetics of lifespn nd mortlity rtes in two popultions of the seed beetle, Cllosobruchus mcultus. Heredity 92:70 8 Fox CW, Czesk ME, Wllin WG (2004b) Complex genetic rchitecture of popultion diverences in dult lifespn of beetle: nondditive inheritnce, gender diverences, body size nd lrge mternl evect. J Evol Biol 7: Fox CW, Stillwell RC, Amrillo AR, Czesk ME, Messin FJ (2004c) Genetic rchitecture of popultion diverences in oviposition behviour of the seed beetle Cllosobruchus mcultus. J Evol Biol 7:4 5 23