Entomologia Experimentalis et Applicata 104: 207–215, 2002.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

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Host plant use of Phyllotreta nemorum: do coadapted gene complexes playa role?

Peter W. de Jong1 & Jens Kvist Nielsen2

1Laboratory of Entomology, Wageningen University, PO Box 8031, 6700 EH Wageningen, The Netherlands(Phone: ++31 317482244; Fax: ++31 317484821; E-mail Peter.dejong@ento.dpw.wau.nl); 2Royal Veterinaryand Agricultural University, Chemistry Department, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen,Denmark

Accepted: May 14, 2002

Key words: Barbarea vulgaris, Chrysomelidae, coadapted gene complex, Coleoptera, Cruciferae, epistasis, fleabeetle, local adaptation, Phyllotreta nemorum, resistance

Abstract

The view of (insect) populations as assemblages of local subpopulations connected by gene flow is gaining ground.In such structured populations, local adaptation may occur. In phytophagous insects, one way in which localadaptation has been demonstrated is by performing reciprocal transplant experiments where performance of insectson native and novel host plants are compared. Trade-offs are assumed to be responsible for a negative correlationin performance on alternative host plants. Due to mixed results of these experiments, the importance of trade-offs in host plant use of phytophagous insects has been under discussion. Here we propose that another geneticmechanism, the evolution of coadapted gene complexes, might also be associated with local adaptation. In thiscase, however, transplant experiments might not reveal any local adaptation until hybridization takes place. Wereview the results we have obtained in our work on the host plant use of the flea beetle Phyllotreta nemorumL. (Coleoptera: Chrysomelidae: Alticinae), and propose a hypothesis involving coadapted genes to explain thedistribution of genes that render P. nemorum resistant to defences of one of its host plants, Barbarea vulgaris R.Br. (Cruciferae).

Introduction

At the very heart of the study of biodiversity lies adap-tive evolution. Adaptive evolution can only be fullyunderstood when the genetic basis of adaptations isunravelled. Despite the crucial need for knowledge ofthe genetics of natural adaptations, this knowledge isstill lacking to a large extent (e.g., Orr & Irving, 1997;Jones, 1998; Endler, 2000). One type of system thatholds promise that such knowledge might be obtainedin the short term, is that of host plant use by phy-tophagous insects. To explain the host plant range ofphytophagous insects, genetic variation in preferenceand performance of the insects on different host plantshave been studied (e.g., Futuyma & Peterson, 1985;Thompson, 1988; Via, 1990; Jones, 1998; Sezer &Butlin, 1998). In some cases, quantitative inheritance

of host plant adaptation has been demonstrated (e.g.,Via, 1990), but other studies have found evidence forthe influence of a limited number of genetic factorson host plant adaptation (e.g., Jones, 1998; Sezer &Butlin, 1998). Our own work on the genetics of theuse of an atypical host plant, Barbarea vulgaris ssp.arcuata (Opiz.) Simkovics (Cruciferae) (G-type) bythe flea beetle Phyllotreta nemorum L. (Coleoptera:Chrysomelidae: Alticinae) has shown evidence for theinvolvement of a limited number of genes with ma-jor phenotypic effect (Nielsen, 1997b; de Jong et al.,2000). However, results on the geographic distributionof the resistance of P. nemorum to Barbarea-defenceshave suggested that the genetic basis of this adaptationmay not simply involve major genes, but that modi-fiers may also affect this trait (de Jong et al., 2001,and see below). These modifiers would then interact

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epistatically with the resistance genes. The aim of thispaper is to explore under which circumstances theseepistatic interactions between genes of major pheno-typic effect and modifiers are likely to evolve. To thisend, we review our work on the genetics of host plantuse in P. nemorum which has lead us to the hypothesisthat epistasis plays a role in the evolution of flea beetleresistance to Barbarea-defence.

The case for local adaptation. The past decade hasshown a flurry of interest in the role of populationstructure and local processes in evolutionary trajec-tories. Examples include metapopulation modelling(Hanski, 1999), the geographic mosaic theory of co-evolution (Thompson, 1994, 1999; Nuismer et al.,2000; Gomulkiewicz et al., 2000), and the study of lo-cal adaptation (e.g., Mopper & Strauss, 1998). This in-terest may well have been fuelled, among other things,by new possibilities opened up by the development ofmolecular techniques, but actual thinking about theeffects of local processes on evolution dates all theway back to Darwin (1859). The discussion about theimportance of local processes in evolution began inearnest with the Fisher-Wright controversy (for a re-view see Wade & Goodnight, 1998, and also Coyneet al., 1997, 2000). Whereas Fisher essentially consid-ered populations to be large with panmixis, Wright putmore emphasis on processes of evolution in structuredpopulations (Wade & Goodnight, 1998). Obviously,many organisms have discontinuous distributions, andindeed examples have accumulated where populationsclearly show genetic structure, and processes of lo-cal selection, genetic drift, and migration play animportant role in evolution.

One process within the context of structured popu-lations which has recently received particularly muchattention is local adaptation (e.g., Mopper & Strauss,1998). Local selection may favour particular geno-types that are best adapted to the local environment.One type of system in which local adaptation has beenparticularly well studied is the adaptation of insects totheir host plants. The local environment may in thatcase be an individual plant, but also a patch with a cer-tain host plant species. Typically, experiments demon-strating local adaptation in insect-host plant systemsinvolve reciprocal transplant experiments, in whichperformance of insects on original and novel hostplants are compared. If performance on the originalhost plant is better than on the novel one, the insect issaid to be locally adapted to its native host. This localadaptation may be governed by negative pleiotropic

gene action; genes that lead to better performance onone host are unlikely to simultaneously improve per-formance on an alternative host (cf. Joshi & Thomp-son, 1995; Mopper, 1996; Strauss & Karban, 1998).The same mechanism is presumably involved in hostplant specialization of phytophagous insects (Rausher,1984; Mackenzie, 1996), and where different hostplants have a patchy distribution, insects might lo-cally adapt to different host plant species. However,evidence for genetic trade-offs with host plant use isrelatively scarce (e.g., Fry, 1996; Joshi & Thompson,1995; Thompson, 1996; Via & Hawthorne, 2002).

What might be the role of epistasis? In the mecha-nism of local adaptation (or host plant specialization)described above, selection is exerted by the (physi-cal and biological) environment of the phytophagousinsect. If we adopt a slightly different perspective,and focus on selection at a particular locus, we canextend the environment with the genetic environmentof that locus. In this view, epistasis between lociis important, and selection may lead to coadaptedgenes, or genomic integration (Wallace, 1991). Herewe come to the essence of the Wright-Fisher con-troversy: whereas Fisher thought epistasis to be ofminor importance in adaptive evolution, it is a centralconcept in Wright’s shifting balance theory of evolu-tion (Fenster et al., 1997; Brodie III, 2000; Johnson,2000). Since the introduction of the term ‘coadap-tation’ (Dobzhansky, 1955) it has been a challengeto assess its importance in natural systems (Wallace,1991). Timofeeff-Ressovsky (1940) and Dobzhansky(1941) provided early evidence for the existence ofcoadapted (locally adaptive) gene complexes (see alsoEndler, 1977). Coadapted gene complexes are thoughtto evolve more readily in structured populations (Fen-ster et al., 1997), and may be a manifestation of localadaptation or may be independent of the local envi-ronment (‘intrinsic coadaptation’; Edmands, 1999).What is crucial, is that coadapted gene complexeswhich have evolved for example in insects using aparticular host plant species may not lead to a sim-ple prediction of performance on an alternative hostplant. Fitness of individuals may be only reduced af-ter the coadapted gene complexes are broken up byhybridization between, for example, sub-populationsusing different host plant species (‘outbreeding de-pression’; e.g., Lynch, 1991; Edmands, 1999; Waseret al., 2000; see Figure 1). Outbreeding depression,or hybrid breakdown, is perhaps the most prominentmethod to demonstrate the presence of epistasis or

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Figure 1. Hypothetical change in fitness of an insect adapted to plant A when: (a) the insect migrates to plant B, and trade-offs across hostplants reduce fitness; (b) the insect’s adaptation to plant A involves coadapted genes, which remain associated with each other as long as thereis no outbreeding. Therefore, fitness on plant B may only be reduced when hybridization occurs between insects adapted to plant A and B,respectively (c).

coadaptation (Fenster et al., 1997). There is ampleempirical support for hybrid breakdown (Ford, 1971;Wallace, 1981; Edmands, 1999; Waser et al., 2000),both at the inter- and intraspecific level (Fenster &Galloway, 2000). The speed with which coadaptedgene complexes break up during hybridization is in-fluenced by the degree of linkage between the lociinvolved in the complex. With rather tight linkage, itmay require several generations of outbreeding beforeany effects on fitness are measurable. Therefore thefailure to find evidence for trade-offs in phytophagousinsects across different host plants by means of, forexample, reciprocal transplant experiments, does notnecessarily mean that these insects are not adapted totheir native host plant. This argument applies at anyscale of population structuring, including, e.g., localadaptation to individual host plants. Crucial to an un-derstanding of the role of coadaptation and epistasisin evolution is the simultaneous study of the geneticbasis of (local) adaptation and of population structure(cf. Fenster & Galloway, 2000).

Coadaptation does not necessarily involve largecomplexes of genes; Endler (1977) describes models

of coadaptation between a gene of major phenotypiceffect and one or a few modifiers. Examples of empir-ical studies involving such a relatively simple geneticbasis, but with evidence for coadaptation, include anexperimental cline for the Bar gene in Drosophila(Endler, 1977), mimicry in Papilio butterflies (Clarke& Sheppard, 1963), and insecticide resistance in thesheep blowfly (McKenzie et al., 1982, but see Clarkeet al., 2000). In the remainder of this paper we willreview the work we have carried out on the adapta-tion of a phytophagous insect, Phyllotreta nemorum,to its host plants. We will summarize the evidencethat has lead us to believe that coadaptation of genesplays a role in the evolution of host plant use by thisphytophagous insect (see Table 1).

Variation in suitability of the host plant B. vulgarisand in flea beetle ability to use this plant. Phyllotretanemorum is an oligophagous flea beetle. It attacksa limited number of cruciferous host plants (includ-ing commercial crops: Nielsen, 1977; Alford, 1999),where the adults feed on the leaves and the larvae areleaf-miners. Work on host plant use in this species

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Table 1. Evidence for the involvement of coadapted gene complexes in the adaptation of Phyllotreta nemorum to a novel host plant, Barbarea

Observations Implications

A single copy of an R-gene can transfer a susceptible genotype Major dominant genes (R-genes) are important in the adaptation

(rr; no survival on Barbarea) into a resistant genotype (Rr) which of P. nemorum to the novel host plant Barbarea

survives equally well on Barbarea and "old" host plants (Nielsen,

1997b, 1999; de Jong et al., 2000)

R-genes are specific for defences in Barbarea and have no effects R-genes have evolved as adaptations to defences in Barbarea

on flea beetle fitness on other host plants (Nielsen, 1999; de Jong,

Meijer & Nielsen, unpublished)

Autosomal R-genes predominate in one population (Kværkeby) There are local differences in genetics of adaptation to Barbarea

whereas autosomal as well as sex-linked genes are found in another

flea beetle population (Ejby) living on Barbarea (Nielsen, 1997b;

de Jong et. al., 2000)

Flea beetles living on Barbarea are homozygous or contain several Homozygous (RR) beetles collected in the field on Barbarea seem

copies of R-genes (e.g., autosomal as well as sex linked genes) not to have lower viability than heterozygous and non-resistant beetles

(Nielsen, 1997b; de Jong et. al., 2000)

Homozygous beetles (RR) produced in the laboratory by There are important genetic differences between field collected

crossing heterozygous beetles amongst each other (Rr x Rr) and laboratory-produced homozygous beetles (RR)

are less viable than heterozygous (Rr) and non-resistant beetles

(de Jong & Nielsen, 2000)

Low frequencies of R-genes are found in non-adapted flea beetle There are limitations to the spread of R-genes from adapted to non-

populations living on other plants than Barbarea. Only adapted populations. In the absence of trade-offs across host plants

heterozygous beetles are found in these populations selection against homozygous beetles (RR) in non-adapted populations

(de Jong & Nielsen, 1999) may be involved in this limitation

was started in Denmark in the 1970s (Nielsen, 1977,1978a, b) with an emphasis on the study of host plantchemistry influencing the suitability of the plants forP. nemorum. One plant genus, Barbarea, contains sev-eral species that are suitable host plants for P. nemo-rum (Nielsen, 1996). However, one species, Barbareavulgaris R. Br., initially showed very variable suitabil-ity for larval development of P. nemorum in laboratorybioassays (Nielsen, 1992). It was therefore consid-ered an unlikely host plant of this flea beetle, until apopulation was discovered in Denmark (Ejby) wherelarge numbers of P. nemorum used B. vulgaris as hostplant (Nielsen, 1992). It was subsequently discoveredthat there was both variation in suitability of B. vul-garis for P. nemorum, and variation among individualP. nemorum to use B. vulgaris as host plant.

In Denmark, two subspecies of B. vulgaris are dis-tinguished: B. vulgaris ssp. arcuata (Opiz.) Simkovicsand ssp. vulgaris, of which the latter is rare. Further-more, in B. vulgaris ssp. arcuata two ‘types’ havebeen distinguished on the basis of morphological andchemical differences: the ‘G-type’ and the ‘P-type’

(Nielsen, 1997a), of which the former, like B. vulgarisssp. vulgaris, is unsuitable for the majority of P. nemo-rum during summer (but not from autumn to spring),whereas the latter is not. Beetles that were collectedin Ejby on B. vulgaris ssp. arcuata (G-type) producedlarvae that could complete development on this hostplant, whereas beetles collected on radish (Raphanussativus L.) in a different locality (Taastrup) could onlyuse leaves of B. vulgaris ssp. arcuata (G-type) duringautumn – spring, but B. vulgaris became unsuitableto them during summer. Both the variation in suit-ability of B. vulgaris, and the ability of P. nemorumto use this plant, were found to have a genetic basis(Nielsen, 1992, 1997a, b). In B. vulgaris the majorsource of variation in suitability was correlated withthe morphological and chemical traits characterizingthe P- and G-type (Nielsen, 1997a), and the geneticbasis of the ability of P. nemorum to use the G-type ofB. vulgaris ssp. arcuata was demonstrated by studyingperformance of larval offspring from particular crosseson this plant (see below).

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Genetic basis of the ability of P. nemorum to useB. vulgaris as host plant. Detailed studies have beencarried out to investigate the genetic basis of the abil-ity of P. nemorum to use B. vulgaris ssp. arcuata(G-type; the Barbarea-type we focused on in ourstudies, and henceforth referred to as ‘Barbarea’) ashost plant throughout the breeding season (in sum-mer). Initially, crosses (F1, F2, and backcrosses) weremade between beetles collected on Barbarea in Ejby,and beetles collected on radish in Taastrup (Nielsen,1997b). Neonate larvae were put on Barbarea leavesin laboratory bioassays. Whereas most larvae initiatedmines in the leaves, some did not show any growth,and died within three days, while others were ableto complete development on the leaves. Mendeliansegregation ratios of surviving (resistant) versus non-surviving (susceptible) larvae showed that survival onBarbarea was determined by major, dominant genes.Briefly summarized, the larval survival after cross-ing beetles collected in Ejby with those collected inTaastrup (F1) was higher than 90%. Larval survival inthe F2 was slightly below 75%, which is expected ifone locus is involved, where a dominant allele con-fers the ability to survive on Barbarea. As expected,backcrosses of the F1 with Taastrup-beetles yielded50% larval survival. Determination of the sex ratiosof the surviving individuals (in flea beetles, males arethe heterogametic sex; Segarra & Petitpierre, 1990)showed X- as well as Y-linked inheritance of the genes(Nielsen, 1997b), although at present it is not cer-tain whether these genes were actually located on thesex-chromosomes themselves (J.K. Nielsen, unpubl.).

Another series of detailed experiments was carriedout with beetles that were collected on Barbarea ina second locality in Denmark (Kværkeby). Crossesand backcrosses were performed between beetles col-lected in the field and a susceptible line from Taastrup(see below). Crosses between field-collected and sus-ceptible beetles yielded a uniformly high larval sur-vival (close to 90%). Crossing resistant F1 offspringamongst each other and genotyping the F2 (reared onradish) showed a 1:2:1 ratio of homozygous resistant,heterozygous, and susceptible beetles. No evidencewas found for biased sex ratios in any of the crosses inthis series of experiments. Hence, Mendelian segrega-tion ratios revealed that all the beetles (16 individuals)collected on Barbarea in Kværkeby were homozygousresistant at one autosomal locus (the same locus for allindividuals), resistance being dominant (de Jong et al.,2000).

Fitness-effects of resistance genes. From the Taas-trup population (collected on radish) a line was devel-oped in the laboratory, which was fully susceptible.This line was subsequently used to produce a series ofnear-isogenic lines of resistant beetles. This was doneby backcrossing resistant beetles that were collected indifferent localities in Denmark and/or which seemedto show different modes of inheritance of the resis-tance (R-) genes with the susceptible line for a numberof generations, and rearing the offspring on Barbarea.One line, carrying a Y-linked R-gene originating fromEjby, was studied in detail (Nielsen, 1996, 1999).Results showed that the R-gene not only influencedlarval performance on Barbarea, but also determinedadult acceptance of this plant for feeding in non-choicetests (Nielsen, 1996). Secondly, the results showedno evidence for any influence of the Y-linked R-geneon larval survival on other host plants than Barbarea,suggesting that the R-gene is highly specific for per-formance on Barbarea. Finally, the Y-linked R-genedid not influence development time and body weightof beetles on other host plants than Barbarea, althoughdevelopment time was longer on Barbarea than on theother host plants (Nielsen, 1999).

A similar lack of evidence for trade-offs associatedwith the R-genes was found for the Kværkeby popu-lation. Beetles were backcrossed once with the sus-ceptible line, and F1-offspring were crossed amongsteach other. The resulting F2 larvae were reared onradish and the F2 was genotyped. The three resistance-genotypes emerged in the expected Mendelian ratio,showing that there was no differential survival of thesegenotypes on radish (de Jong et al., 2000). A secondexperiment with beetles collected at this locality, inwhich various life history traits (body weight, devel-opment time, larval and pupal mortality, and starvationresistance) were measured, showed no evidence fortrade-offs associated with resistance genes across hostplants either (P.W. de Jong, P. Meijer & J.K. Nielsen,unpubl.).

Geographic distribution of R-genes. The R-genes al-low P. nemorum to extend its host plant range, includ-ing Barbarea in its diet. In the absence of trade-offsacross host plants, it is expected that the genes en-abling the beetles to use this host plant will spreadrapidly (depending, among other things, on abundanceof the various host plants, host plant phenology, etc.).However, a study to examine the occurrence of re-sistance to Barbarea defences at various localities inDenmark did not support this prediction. Beetles were

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Figure 2. Proportion of resistant P. nemorum that were collected on different host plants. Samples collected from geographically distinctpopulations of the same plant species were pooled.

sampled during summer on different host plants (in-cluding Barbarea) in twelve locations distributed overEast Denmark (de Jong & Nielsen, 1999). The distrib-ution of the different host plants is patchy, and samples(9–25 individuals per locality) were also taken fromdifferent host plant patches within one locality. Bee-tles from each sample were crossed and backcrossedwith the laboratory susceptible line, yielding informa-tion about the proportion of resistant beetles in eachsample, and also giving some information about themode of inheritance and the genotype of the beetles.Two samples collected on Barbarea contained 100%resistant beetles (N=18 and 20, respectively) and manyof these were apparently homozygous (Kværkeby andEjby) or contained autosomal as well as sex-linkedgenes (Ejby; larval survival on Barbarea in crosseswith susceptible beetles was close to 100%). Thisis hardly surprising, since beetles without R-genesare unable to use Barbarea (G-type) as a host plantand such beetles would be produced continuously bymatings between heterozygous beetles. What is moresurprising is that beetle populations sampled on otherhost plants invariably contained lower frequencies ofbeetles with R-genes (Figure 2), although some ofthese populations are only a few kilometers away froma Barbarea patch with 100% resistant beetles (de Jong& Nielsen, 1999 and unpublished results). Moreover,for none of the beetles on other plants than Barbareafor which information was obtained about their geno-type, evidence was found that they were homozygousresistant, since larval survival on Barbarea in crosseswith susceptible beetles was at most close to 50%.These observations can be explained in various ways,

but, assuming that we are looking at an equilibriumsituation, they suggest that something is limiting thespread of resistance to Barbarea defences. Since notrade-offs across host plants associated with resistanceto Barbarea defences have been found so far (seeabove), another explanation was sought.

Population structure. It has already been mentionedthat the host plants of P. nemorum are patchily dis-tributed. Since the beetles presumably feed and mateon or near the plants where eggs are later deposited,there is a possibility that flea beetle populations arestructured. Migration might either be limited betweengeographically disjunct plant patches, or between dif-ferent host plant species (the latter would lead tohost plant races). Limited migration may influencethe spread of genes conferring resistance to Barbarea.To study possible population structure, beetles weresampled on different host plant patches, at differentgeographic distances from each other and consistingof different host plant species. Supposedly neutral al-lozyme loci were used to investigate the extent andsource of population structure (de Jong et al., 2001).The results of this study showed that there was noevidence of an influence of host plant species on pop-ulation structure (de Jong et al., 2001). However, therewas a significant effect of the distance between theplant patches on which samples were collected onthe genetic differentiation between the beetle-samples.Albeit significant, the extent of differentiation wasvery small: Fst-values were below 0.08 when sam-ples were compared that were separated by 45 km.This suggests that, although there is an effect of ge-

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ographic distance on genetic similarity of the beetlepopulations, the extent of migration on the geographicscale that we studied is high. It is, therefore, unlikelythat a mere restriction of migration limits the spread ofresistance genes from Barbarea plant patches to otherplants. So what factor then could be responsible for therelatively low proportion of resistant beetles on otherplants than Barbarea?

Attempt to produce a homozygous resistant line inthe laboratory. A clue to the possible answer wasobtained by an unexpected result we got when weattempted to produce a pure breeding line from oneof our near-isogenic (backcrossed) lines (de Jong &Nielsen, 2000). After 4–5 generations of backcross-ing a resistant line carrying an autosomal resistancegene (originating from Ejby) with the laboratory-keptsusceptible line, we attempted to produce a line ofbeetles which would be homozygous for this resis-tance gene (RR), by simply crossing heterozygousbeetles amongst each other (Rr × Rr). Instead of theexpected proportion of homozygous individuals (onefourth when reared on radish if all genotypes sur-vive, one third when reared on Barbarea where onlyresistant genotypes survive), we found only a fewhomozygous individuals whereas the heterozygote re-sistant and susceptible beetles emerged in the expectedMendelian proportion when reared on radish, andnearly all survivors were heterozygous resistant whenreared on Barbarea. No evidence was found that thesurvival of heterozygous resistant beetles was reducedin these crosses (de Jong & Nielsen, 2000). Interest-ingly, we had evidence that the original, field-collectedfounder of the line was in fact homozygous resistant,just like the beetles we had collected elsewhere onBarbarea (see above), since backcrosses with suscep-tible beetles produced a larval survival on Barbareawhich was higher than 50% (J.K. Nielsen, unpubl.).The discrepancy between the results of this exper-iment and the common occurrence of homozygousresistant beetles in the field led us to the conclusionthat the backcrossing in maintaining our near isogeniclines must have been responsible for the apparentmortality of homozygous resistant individuals in ourexperiment.

Coadapted gene complexes involving modifiers may‘rescue’ homozygous resistant flea beetles. Withoutclaiming that this is the only (or complete) possi-ble answer, we will now integrate all of our findings(summarized in Table 1) into a plausible, testable

explanation. Genes of major phenotypic effect usu-ally are assumed to confer large negative pleiotropiceffects (e.g., Lande, 1983; Orr & Coyne, 1992). Ex-amples are known where the negative effect on fitnessof major genes is associated with being homozygous atthat locus (e.g., Orr & Coyne, 1992; Cavalli-Sforza &Bodmer, 1971; Watt, 1977). Our results are consistentwith such a genotype-dependent negative pleiotropiceffect of an autosomal resistance gene in flea beetles.Due to Mendelian segregation, homozygous resistantbeetles are continuously produced in the field wherethe gene frequency for resistance is high (i.e., onBarbarea). This sets the stage for selection favouringmodifiers to counteract the negative fitness effects ofbeing homozygous resistant (see, e.g., Sezer & But-lin, 1998). These modifiers coadapt with the resistancegenes, and they might get linked, or strong selec-tion or drift might fix the modifiers. The presence ofmodifiers would explain the common occurrence ofhomozygous resistant flea beetles on Barbarea in thefield. Following this possibility, what has happened inour backcrossed lines is that the association betweenthe resistance genes and the modifiers was disrupted.Therefore, the negative fitness effects of the resistancegenes in homozygotes were no longer compensated,explaining the high mortality of homozygous resistantbeetles in our experiment.

If this were true and if it occurred generally, in-dependent of the origin of the beetles (the modifier-hypothesis is presently based on results from back-crossed lines carrying an autosomal R-gene derivedfrom one population; the same or different sets ofmodifiers might be involved in different populationsand R-loci), a similar break up of the associationbetween resistance genes and modifiers may occurafter resistant migrants, originating from Barbarea-patches, interbreed with beetles living on alternativehost plants, where there is no selection favouring re-sistance genes and modifiers. This would explain ourobservation that, although there is a large amount ofmigration between local flea beetle populations (judg-ing from neutral allozyme loci), resistant beetles onalternative host plants are less common, and appar-ently always heterozygous. This hypothesis wouldrequire a level of population structure that is suffi-cient to maintain coadapted gene complexes in patcheswhere these are selected, while it allows for theirbreaking up where selection is relaxed. In fact, we didfind significant spatial differentiation between localflea beetle populations.

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This hypothesis implies that local flea beetle pop-ulations are in fact locally adapted through the evolu-tion of coadapted gene complexes involving resistancegenes and modifiers. However, transplant-experimentswould not show any evidence of local adaptationuntil hybridization would take place. The strongestarguments favouring the coadapted gene complex hy-pothesis in the flea beetle system (Table 1) are: (a) thedifference in viability of naturally occurring, andlaboratory-produced homozygous resistant beetles atan autosomal locus, and (b) the low frequencies of(especially homozygous) resistant beetles in popula-tions living on other plants than Barbarea. A crucialand simple way to test the hypothesis would be tocollect resistant flea beetles on other host plants thanBarbarea, and cross them. Assuming that these beetleshave been outcrossed with beetles that are not adaptedto Barbarea in the field, this experiment should show areduction in survival of homozygous resistant beetlesbecause of the break-up of coadapted gene complexesduring the outcrossing. Obviously, our assumption thatthe distribution of resistance in flea beetles reflectsan equilibrium situation also needs to be tested bymonitoring the frequency of resistance in our study-populations. Although we can not claim at present thatthe coadapted gene complex hypothesis reflects themechanisms involved in the evolution of host plant usein our flea beetle system, we feel that the possibilityof the evolution of coadapted gene complexes and therole of epistasis in the evolution of insect-plant interac-tions deserves more attention, and might explain somecases where expected negative trade-offs across hostplants appeared to be absent.

Acknowledgements

We thank Bas Zwaan, Caroline Mueller, Kathleen Vic-toir and two anonymous referees for helpful commentson a previous draft of the manuscript. Martin Brittijnproduced the figures. The work was supported by theDanish Agricultural and Veterinary Research Council.

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