Entomologia Experimentalis et Applicata 82: 25–35, 1997. 25c 1997 Kluwer Academic Publishers. Printed in Belgium.

Variation in defences of the plant Barbarea vulgaris and incounteradaptations by the flea beetle Phyllotreta nemorum

Jens Kvist NielsenChemistry Department, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871Frederiksberg C, Denmark

Accepted: July 16, 1996

Key words: Barbarea vulgaris, Cruciferae, Phyllotreta nemorum, Chrysomelidae, Alticinae, flea beetle, plantdefence, resistance, host plant, variation, evolution

Abstract

Several sorts of variation in the interaction between the insect, Phyllotreta nemorum L. (Coleoptera: Chrysomelidae:Alticinae), and the plant, Barbarea vulgaris R.Br. (Brassicaceae), have been discovered: 1) genetic differences inthe levels of defences in the plant, 2) genetic differences in the ability of insects to cope with the plant defences, 3)seasonal variation in levels of defences in the plant, and 4) differences between leaf types in levels of defences.

Two plant accessions were suitable for larval development throughout the season while the remaining nineaccessions were more or less unsuitable for larvae from the ‘susceptible’ T-population at least at certain times ofthe year. All accessions were suitable for the ‘resistant’ E-population throughout the year. There was a seasonalvariation in levels of defences in some accessions which were unsuitable for the T- population during the summerperiod when beetles were present, but not during autumn and spring when the beetles were hibernating. Upper(younger) cauline leaves of these accessions had higher levels of defences than lower (older) cauline leaves. Theresistant E-population used B. vulgaris as a natural host plant while the susceptible T-population did not. The useof B. vulgaris as a natural host plant by the E- population of P. nemorum seems to be an extension of the host plantrange of the species. Variation in plant defences may have facilitated the switch in host plant use by the resistantflea beetle population.

Introduction

The present-day insect plant interactions are the out-come of evolutionary processes. The narrow host plantranges of many insects are formed at least partially byevolutionary adaptations to the defensive chemistry ofplants, especially the secondary compounds (Futuyma& Keese, 1992; Thompson, 1994). Past evolutionaryprocesses leading to the origin of known plant defencesand specialized insect adaptations cannot be unravelledwith any certainty, but several possible scenarios havebeen described (Ehrlich & Raven, 1964; Futuyma &Keese, 1992; Gould, 1988; Jermy, 1984; Thompson,1994). From these scenarios we can develop testablehypotheses on conditions likely to favour evolutionaryinteractions between plants and insects and investig-ate whether such conditions prevail in current eco-

systems. Hypotheses on coevolutionary interactionsbetween plants and phytophagous insects in contem-porary time can only be tested in systems where there issubstantial genetic variation in plant defences as well asin counteradaptations by the insects to these defences.The interaction between the flea beetle, Phyllotretanemorum L. and the crucifer, Barbarea vulgaris R.Br.seems to offer such an opportunity.

B. vulgaris is a biennial or short-lived perenni-al plant (MacDonald & Cavers, 1991). It has beendivided into a number of subspecies in Europe (Hegi,1986; Rich, 1987), but only two subspecies havebeen described from Scandinavia, ssp. arcuata (Opiz.)Simkovics and ssp. vulgaris (Hansen, 1981; Lange,1937; Pedersen, 1958). B. v. ssp. arcuata is by farthe most common Barbarea taxon in Denmark whileB. v. ssp. vulgaris is rare (Hansen, 1981; Peder-

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sen, 1958). B. v. ssp. arcuata has spreading pedicelsand more or less curved siliqua while B. v. ssp.vulgaris has appressed pedicels and straight siliqua(MacDonald & Cavers, 1991). Among the accessionsused in the present investigation, two types of B. v.ssp. arcuata could be recognized by morphologicaland chemical characters. The ‘G-type’ had glabrousleaves and contained the S-isomer of 2-hydroxy-2-phenylethylglucosinolate (glucobarbarin), while the‘P-type’ had pubescent leaves and contained (2R)-glucobarbarin (Huang et al., 1994). The taxonomicaffiliation of these types is currently being investigated(J. Jensen, pers. comm.).

P. nemorum is an oligophagous flea beetle feedingon a restricted number of plants within the family,Cruciferae (Brassicaceae). The larvae are leaf minersand both larvae and adults are crucifer feeders.Eggs arelaid in the soil close to the host plants, and the neonatelarvae have to climb the plant and find a suitable sitefor initiation of a leaf mine (Nielsen, 1977).

A number of crucifers were not utilized as hostplants in a small field experiment (Nielsen, 1977),and in laboratory experiments three major reasons forrejection of plant species have been recognized: 1) lowacceptability of plants to feeding by adult beetles, 2)low acceptability of plants for initiation of leaf minesby larvae, and 3) low survival of larvae after initiationof a leaf mine (Nielsen, 1989a). When the laboratoryexperiments were extended to include the majority ofwild crucifers in Denmark, a high natural variabilityin larval survival in B. vulgaris was observed (Nielsen,unpubl.). The laboratory experiments supported obser-vations from the field that B. vulgaris was an unsuitablehost plant for P. nemorum, but nevertheless at one loc-ation (Ejby) it turned out to be the major host plant forthis flea beetle species (Nielsen, unpubl.).

The present experiments were undertaken in orderto explain the observed variation in laboratory experi-ments with B. vulgaris leaves and the differential use ofthis species as a host plant for flea beetles. Four hypo-theses on causes of variation were tested: 1) genetic dif-ferences in levels of defences among plant populations,2) seasonal variations in levels of plant defences, 3) dif-ferent levels of defences in different types of leaves,and 4) genetic differences among flea beetle popula-tions in counter adaptations to the plant defences.

Materials and methods

Insects. Two flea beetle populations were tested. TheT-population was collected at the agricultural exper-imental Station, Højbakkegaard, Taastrup on radish(Raphanus sativus L.), and the E-population was col-lected at Ejby on the G-type of B. v. ssp. arcuata.Beetles were kept in small groups (about 10 malesand 10 females per group; 4–5 groups per populationper year) in 400 ml plastic vials containing a moistgypsum-charcoal bottom-layer. Both populations werefed with radish leaves. Eggs were laid in the bottom-layer. Egg development lasted 5–6 days, and every 5days the beetles were transferred to new vials. In thisway, the larvae always hatched in a vial with no foodsupply. Inexperienced, neonate larvae were used in theexperiments before they were 24 h old.

Plants. Plant accessions originated from seeds ofB. vulgaris collected at eleven natural growth sites: 1)wood, Nyvang near Bastrup (NEZ), 2) wood, FyrendalSkov (SZ), 3) old grass field, Herlev (NEZ), 4) wetmeadow, Østrup (NEZ), 5) open south slope with afew trees and wet as well as dry habitats, Suserup(SZ), 6) waste area, Gl. Svebølle (NWZ), 7) dry, oldfield, Vindekilde Strand (NWZ), 8) waste area, Ejbynear Glostrup (NEZ), e.g. the site of origin of one ofthe beetle populations, 9) wet meadow at the north endof the lake, Tissø (NWZ), 10), wet meadow, Assentorp(NWZ), and 11) roadside, Vestskoven (NEZ). Descrip-tions of regions (NEZ: North East Zealand; NWZ:North West Zealand etc) follow Enghoff & Nielsen(1977). Voucher specimens of plants are kept at theBotanical Museum in Copenhagen.

Plants were sown in the field in short rows (4 m;0.8 m between rows) in a common garden experiment(Karban, 1992) at the agricultural experimental sta-tion Højbakkegaard, Taastrup. Seeds of all accessions(except no. 8) were collected in 1985 and sown in 1986,1988, and 1989. Seeds from locality no. 8 were not col-lected in 1985 and used in the first experiments sincethe P- and G-type of B. v. ssp. arcuata were growingtogether in Ejby and hybridization could not be ruledout. However, in 1990, the P-type had disappearedfrom the locality, and seeds of the G-type of B. v. ssp.arcuata from this key locality were collected. Theywere sown in 1991 together with accessions no. 3, 4,5, and 11, and used for experiments in 1991 and 1992.

Seeds were sown in April or May, but seed ger-mination was often poor or postponed until late sum-mer, so experiments with young rosette leaves of

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first year plants were not started until August. In1990 germination failed completely, and plants usedin experiments in autumn 1990 and spring 1991 ori-ginated from plants sown in 1989. After flowering, newrosettes were formed from the root neck (MacDonald& Cavers, 1974; Rich, 1984) and these ‘perennial’plants flowered again next spring at the same time asthe biennial plants.

Experiments. Leaves were collected in the field notmore than 24 h before the start of the experiments.Single, intact leaves were placed in plastic vials (12.5,25 or 160 ml depending on the size of the leaf) togetherwith a piece of moist filter paper. One neonate larvawas transferred to each vial. The vials were left indarkness at 22� 2 �C. After three days the leaves wereinvestigated under a preparation microscope, and thesurvival or death of each larva was recorded as well asthe number of leaf mines it had made. Surviving larvaewere about to perform their first moult, while deadlarvae had made 0–5 tiny mines. In a few cases thelarvae had died within the mine, but in most cases thetiny mines were empty and the larvae had died outsidethe mine. The tiny mines were made when the larvae’ate their way’ into the leaf The tiny mines were hardlyextended afterwards, because the larvae left them aftera few hours without much feeding in the mines. Thestimuli which cause termination of mining behaviourare not known (see discussion), but the plant seemsto contain compounds which prevent survival of thelarvae although they accept the plant for mine initiationand make as many as five attempts to colonize the leaf.Survival after three days had previously been foundto be a good estimate of the suitability of plants forP. nemorum (Nielsen, 1989a).

Statistical analysis. The likelihood ratio test (G-test)was made with the GENMOD procedure in SAS (SASInstitute Inc., 1993). A binomial distribution wasassumed, and logits of the probabilities were used in alinear model. G-values were calculated as -2 ln (likeli-hood without the variable/likelihood with the variable)(Hosmer & Lemeshow, 1989) in a type 3 analysis ofvariance. The statistic G is assumed to follow a chi-square distribution and probabilities (P) were calcu-lated based on this assumption (Hosmer & Lemeshow,1989). A significance level of P=0.01 was used (ns:P>0.01).

Figure 1. Seasonal variation in survival rates (S/N) of the T-population of Phyllotreta nemorum on leaves of the G-type of Bar-barea vulgaris ssp. arcuata (means � s.e. for eight accessions).

Results

The majority of larvae initiated leaf mines in all typesof B. vulgaris leaves, but survival rates of larvae fromthe T-population were low in some types of leaves e.g.in young rosette leaves and upper cauline leaves ofB. v. ssp. vulgaris and the G-type of B. v. ssp. arcuata.Survival rates of larvae from the E-population werehigh in all types of B. vulgaris leaves (Figure 1; Tables1, 2 & 4).

Statistical analysis of data obtained with plantsin the rosette stage (Tables 1 & 2) demonstratedhighly significant differences between beetle popu-lations, plant accessions, times of the year, as wellas smaller differences between years (Table 3). Fur-ther subdivision of the data demonstrated clear differ-ences between the two beetle populations (hypothes-is 4). The E-population accepted and survived in allB. vulgaris accessions throughout the year. No sig-nificant differences were found in responses of theE-population to rosette leaves from different acces-sions nor to leaves collected at different times of theyear (Table 3). Responses of the T-population weremore variable, and the most pronounced variation wasfound in survival rates of this population on differentaccessions and on plants collected at different times ofthe year (Tables 1 & 3). The T-population is thereforesensitive to the plant defences, and responses of thispopulation can measure variation in levels of defencesin the plants.

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Table 1. Suitability of rosette leaves of Barbarea vulgaris in thevegetative stage for neonate larvae of the T-population of Phyllo-treta nemorum at different times of the year. N = number of larvaetransferred; I = number of larvae that initiated a leaf mine (% ofN); S/I is the number of larvae that survived for three days dividedby the number that initiated a leaf mine

Accession1 N I (%) S/I (%) N I (%) S/I (%)

August and September2 October

1 65 76.9 24.0 75 93.3 27.1

2 50 74.0 16.2 75 93.3 45.7

3 65 73.8 14.6 115 93.0 24.3

4 50 64.0 15.6 105 91.4 30.2

5 70 67.1 12.8 105 93.3 27.6

6 45 71.1 15.6 75 96.0 54.2

7 40 75.0 10.0 70 92.9 32.3

8 0 – – 35 94.3 48.5

9 50 86.0 86.0 35 91.4 87.5

10 60 91.7 90.9 50 96.0 91.7

11 40 27.5 0.0 56 92.9 17.3

November and December March and April

1 40 97.5 84.6 25 100.0 76.0

2 30 100.0 83.3 10 100.0 90.0

3 50 92.0 78.3 55 90.9 80.0

4 45 93.3 85.7 56 87.5 77.6

5 45 97.8 81.8 55 87.3 75.0

6 40 97.5 82.1 25 80.0 95.0

7 30 96.7 75.9 35 91.4 93.8

8 30 100.0 83.3 60 100.0 76.7

9 40 92.5 94.6 40 92.5 89.2

10 20 90.0 100.0 40 85.0 94.1

11 40 92.5 81.1 65 86.2 64.3

1Accessions no. 1–8: G-type of B. v. ssp. arcuata; no. 9 and 10:P-type of B. v. ssp. arcuata; no. 11: B. v. ssp. vulgaris.2Data for August and September are from 1988 and 1990; in 1991and 1992 a smaller number of accessions were tested and mineinitiation rates as well as survival rates were lower than in 1988and 1990, e.g in 1991, accession no. 3: S = 0%; N = 345 (I notmeasured) and in 1992, accession no. 3: I = 33.3%; S = 0%; N = 45and accession no. 8: I = 33.3%; S = 0%; N = 30.

There were significant differences in mine initi-ation rates (G=10.5; P<0.001) and in survival ratesof the T-population on the two types of B. v. ssp.arcuata (G=270.1; P<0.001). Responses of the T-population to accessions belonging to these two typeswere therefore analyzed separately. It was shown thataccessions belonging to the P-type were acceptablethroughout the year while there were significant dif-ferences between accessions belonging to the G-typeand between leaves of the G-type collected at differ-

Table 2. Suitability of rosette leaves of Barbarea vulgaris inthe vegetative stage for neonate larvae of the E-population ofPhyllotreta nemorum at different times of the year. Symbols asin Table 1

Accession1 N I (%) S/I (%) N I (%) S/I (%)

August and September March and April

1 20 95.0 78.9 0 - -

2 30 86.7 100.0 0 - -

3 25 96.0 100.0 20 95.0 84.2

4 30 96.7 96.6 30 96.7 96.6

5 30 86.7 100.0 20 100.0 85.0

6 30 100.0 96.7 0 - -

7 30 96.7 100.0 0 - -

8 30 100.0 90.0 40 95.0 92.1

9 40 97.5 82.1 30 93.3 100.0

10 40 97.5 92.3 30 93.3 100.0

11 40 97.5 92.3 20 95.0 100.0

1Accessions have been described in Table 1

ent times of the year (Table 3). However, the variationbetween the accessions belonging to the G-type wasmuch smaller (G=20.9) than the variation within thewhole set of accessions (G=270.0) (Table 3).The majorsource of variation within the whole set of accessions istherefore a difference between on the one side the suit-able P-type and on the other side the unsuitable G-typeof B. v. ssp. arcuata and B. v. ssp. vulgaris (Tables 1& 3). These results proved that there were genetic-al differences in levels of defences in different plantpopulations (hypothesis 1), and that these differenceswere correlated with the morphological and chemicalcharacters used to characterize the plant ‘types’.

Differences between plant accessions were mostpronounced in young rosette leaves collected inAugust/September (Table 1). At this time of the year,the rosette leaves of the G-type were the least suitablefor the T-population (Figure 1). Higher survival rateswere obtained in leaves collected in October, and byNovember the G-type had reached the same level ofsuitability as the P-type for larvae of the T-populationand there were no longer any differences betweenaccessions belonging to these two types (Tables 1 & 3).New rosette leaves formed in spring by overwinteredplants of the P- and G-type remained suitable for theT-population (Figure 1; Table 1), but the suitability ofB. v. ssp. vulgaris started on decrease (Table 1) caus-ing a significant difference between accessions alsoin spring (Table 3). Differences between years werevery pronounced in the responses of the T-population

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to young rosette leaves collected in August/September(Table 1), but smaller in older rosette leaves collec-ted in October or later. No differences between yearswere found in responses of the E-population to rosetteleaves. For this reason, data from different years werepooled in Tables 1 and 2, as well as in parts of thestatistical analysis (Table 3). The results demonstratea seasonal variation in levels of defences in rosetteleaves of the G-type of B. v. ssp. arcuata and B. v. ssp.vulgaris (hypothesis 2).

The young rosette leaves of the G-type of B. v. ssp.arcuata and B. v. ssp. vulgaris are the most well defen-ded type of leaves (Figure 1). However, the defencesare effective only against the T-population, while theE-population is able to utilize these leaves (Table 2).There were significant differences in survival rates ofthe two beetle populations on all accessions belongingto these plant ‘types’ (G-test; P<0.001 in all cases) inAugust/September, but the two populations survivedequally well on accessions belonging to the P-type ofB. v. ssp. arcuata (G-test; ns). The major effect of plantdefences in rosette leaves in August was a reductionin survival rates, but a smaller effect was also found inmine initiation rates (Tables 1 & 3).

In the bud stage, there were significant differencesin survival rates between beetle populations (G=140.5;P<0.001), plant accessions (G=156.9; P<0.001), andbetween rosette leaves and cauline leaves (G=26.5;P<0.001). Survival rates of the T-population werelower in the younger cauline leaves than in the olderrosette leaves (Figure 1). This was the first demonstra-tion of a variation between different types of leaves(hypothesis 3). Since the variation between differenttypes of leaves was more pronounced in the floweringstage, the subject will be treated in greater detail here.Variations between beetle populations and plant acces-sions followed the same pattern as described for therosette leaves.

When the plants started flowering, there wereagain significant differences between beetle popula-tions, plants accessions, type of leaves, as well asbetween years (Tables 4 & 5). The effects of plantaccession, leaf type and year on the survival of theE-population were relatively small, but neverthelesssignificant (Table 5). The main reason for differentialresponses of the E-population to plant accessions wasa somewhat reduced survival on B. v. ssp. vulgaris(Table 4). No differences were found in survival ratesof the E-population on accessions belonging to the G-and P-types of B. v. ssp. arcuata (Table 5). Significantdifferences between years were obtained because sur-

vival rates on several accessions were about 15% lowerin 1987 compared to 1990 and later (data not shown).

In the T-population there were no significant dif-ferences between years (Table 5). For this reason, datafrom different years were pooled in Table 4 as wellas in subsequent statistical analyses (Table 5). Sur-vival rates of the T-population were high on lower aswell as upper cauline leaves of the P-type of B. v.ssp. arcuata. As in rosette leaves, the major variationbetween accessions was a difference between on oneside the P-type of B. v. ssp. arcuata and on the oth-er side the G-type of B. v. ssp. arcuata and B. v.ssp. vulgaris (Tables 4 & 5). However, the variationbetween accessions belonging to the G-type was morepronounced (G=26.1) in the flowering stage comparedto young rosette leaves in August/September (G=3.5)(Table 3). There were highly significant differences insurvival rates of the T-population on different types ofleaves, i.e. on upper compared to lower cauline leaves(Tables 4 & 5). Together with the results obtained withrosette leaves, this is a clear demonstration of differ-ences in levels of defences in different types of leaves(hypothesis 3) (Figure 1).

Next to young rosette leaves, upper cauline leaveswere the most defended type of leaves (Figure 1), butagain, the defences were effective only against the T-population except for a small effect also on the E-population in B. v. ssp. vulgaris (Table 4). There weresignificant differences between the two populations insurvival rates on upper cauline leaves of all accessionsbelonging to the G-type of B. v. ssp. arcuata or to B. v.ssp. vulgaris (G-test; P<0.001 in all cases). Theseresults confirmed the presence of genetic differencesbetween the two beetle populations (hypothesis 4).

Discussion

Genetic variation in levels of defences in plants.Genetic differences were found between on one handthe P-type and on the other hand the G-type of B. v.ssp. arcuata and B. v. ssp. vulgaris. Smaller differ-ences were found between different accessions of theG-type of B. v. ssp. arcuata especially in upper cau-line leaves of flowering plants. The single accessionof B. v. ssp. vulgaris was less suitable than the acces-sions of B. v. ssp. arcuata, but more populations ofthis subspecies have to be tested before its status canbe unravelled. Although the inheritance of defences inB. vulgaris have not been resolved, the discontinuousvariation suggests that major genes might be involved

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Table 3. Results of analysis of data from plants in the rosette stage with the likelihood ratiotest (G- test). The test was performed initially on the whole data set, and subsequently ondifferent subsets in order to illustrate the main sources of variation

Subset of Explanatory Initiation of mines Survival

data variable

G P G P df

All data Beetle population 66.2 <0.001 307.5 <0.001 1

Plant accession 36.8 <0.001 249.2 <0.001 10

Time of year 111.2 <0.001 343.5 <0.001 3

Year 13.7 ns 21.2 <0.01 7

E-population Plant accession 9.4 ns 19.9 ns 10

Time of year 0.1 ns 1.1 ns 1

Year 0.7 ns 5.6 ns 1

T-population Plant accession 42.6 <0.001 270.0 <0.001 10

Time of year 112.5 <0.001 368.2 <0.001 3

Year 11.4 ns 27.8 <0.001 7

T-pop. on G-type Plant accession 14.8 ns 20.9 <0.01 7

Time of year 118.6 <0.001 438.1 <0.001 3

T-pop. on P-type Plant accession 0.2 ns 4.0 ns 1

Time of year 1.8 ns 3.3 ns 3

T-population in All accessions 23.8 <0.01 255.1 <0.001 10

August/Sept. G-type 3.5 ns 3.5 ns 6

T-population in All accessions 7.2 ns 110.0 <0.001 10

October G-type 1.6 ns 28.1 <0.001 7

T-population in All accessions 10.5 ns 9.3 ns 10

November/Dec. G-type 8.1 ns 1.8 ns 7

T-population All accessions 25.3 <0.01 32.9 <0.001 10

March/April G-type 22.2 <0.01 10.8 ns 7

(Simms & Rausher, 1992; Via, 1990). Major geneshave often been found to control defences to insects incrop plants, but polygenic inheritance seems to be morecommon in natural ecosystems (Kennedy & Barbour,1992; Simms & Rausher, 1992; Thompson, 1994).

Genetic variation in counteradaptations of insects.In the insect, there were genetic differences in sur-vival rates of the two populations on the most welldefended leaf types, e.g. rosette leaves in August andSeptember. The ability of the E-population to survivein all types of B. vulgaris leaves is controlled by major,dominant genes which seem to be located on auto-

somes as well as on both sex chromosomes (Nielsen,1997). Most previous studies on reciprocal evolution-ary changes in insects and plants have stressed theimportance of polygenes and quantitative inheritance(Via, 1990; Simms & Rausher, 1992). The interactionbetween P. nemorum and B. vulgaris may therefore besuitable for further studies on ecological and evolu-tionary interactions between plants and insects as anexample where major genes have a greater impact.

Effect of season and leaf type on variation in plantdefences. Some plant populations (accessions) weresuitable for both insect populations throughout the

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Table 4. Suitability of leaves from flowering Barbarea vulgaris for neonate larvaeof two populations of Phyllotreta nemorum. Symbols as in Table 1

Plant Beetle Lower cauline leaves Upper cauline leaves

accession population N I (%) S/I (%) N I (%) S/I (%)

1 T 25 88.0 27.3 100 94.0 33.0

E 30 90.0 74.1 85 98.8 83.3

2 T 30 90.0 74.1 90 94.4 28.2

E 25 96.0 91.7 65 100.0 84.6

3 T 45 95.6 67.4 95 91.6 16.1

E 35 100.0 88.6 95 94.7 83.3

4 T 40 90.0 66.7 95 95.8 29.7

E 35 94.3 97.0 85 98.8 83.3

5 T 65 93.8 65.6 120 95.0 50.0

E 50 92.0 93.5 95 98.9 85.1

6 T 30 96.7 69.0 80 96.2 42.9

E 40 92.5 94.6 85 96.5 74.4

7 T 20 95.0 63.2 50 98.0 46.9

E 25 96.0 95.8 55 92.7 86.3

8 T 50 96.0 77.1 60 96.7 34.5

E 40 97.5 100.0 60 98.3 86.4

9 T 40 87.5 94.3 40 95.0 86.8

E 40 80.0 100.0 40 100.0 92.5

10 T 40 90.0 94.4 40 97.5 94.9

E 40 90.0 97.2 40 97.5 97.4

11 T 40 87.5 5.7 50 68.0 5.9

E 50 96.0 83.3 90 95.6 68.6

year. In other plant populations including all the G-type of B. v. ssp. arcuata and ssp. vulgaris there was atemporal variation so that different levels of defencesagainst attack by the susceptible T-population occurredat different growth stages of the same plant. Theseplants were defended during the summer period whenthe risk of insect attack is highest, but undefended dur-ing late autumn and early spring when the beetles arehibernating. These observations support the assump-tion that defences may be costly, and resources fordefences are allocated in direct proportion to the riskof attack by enemies (Zangerl & Bazzaz, 1992). Thetransition from defended to undefended during Octo-

ber occurred without any morphological changes inthe rosette leaves. The seasonal changes in the contentof defensive compounds may be regulated by extern-al factors like temperature or daylength, but it is notyet possible to distinguish between developmental andclimatic influences. There is some indication of a high-er content of defensive compounds in warmer than incolder years, but the correlation between temperat-ure and content of defensive compounds is not simple.Low survival of the T-population in rosette leaves of theG-type of B. v. ssp. arcuata in 1991 and 1992 was tent-atively attributed to temperature effects, since the pre-vailing temperatures were higher in 1991 and 1992 than

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Table 5. Results of analysis of data from flowering plants with the likelihood ratio test (G-test). The test was performed initially on the whole data set, and subsequently on differentsubsets in order to illustrate the main sources of variation

Subset of Explanatory Initiation of mines Survival

data variable G P G P df

All data Beetle population 12.5 <0.001 485.1 <0.001 1

Plant accession 27.1 <0.01 141.0 <0.001 10

Leaf type 3.9 ns 87.7 <0.001 1

Year 10.7 ns 22.8 <0.001 3

E-population Plant accession 11.2 ns 32.9 <0.001 10

Leaf type 9.5 <0.01 31.3 <0.001 1

Year 1.7 ns 36.7 <0.001 3

T-population Plant accession 28.6 <0.01 132.9 <0.001 10

Leaf type 0.1 ns 61.7 <0.001 1

Year 9.6 ns 3.1 ns 3

E-pop. on G-type Plant accession 5.4 ns 8.1 ns 7

Leaf type 4.4 ns 13.6 <0.001 1

E-pop. on P-type Plant accession 0.8 ns 0.2 ns 1

Leaf type 11.9 <0.001 1.5 ns 1

T-pop. on G-type Plant accession 4.8 ns 26.1 <0.001 7

Leaf type 1.2 ns 72.3 <0.001 1

T-pop. on P-type Plant accession 0.4 ns 1.0 ns 1

Leaf type 3.4 ns 0.7 ns 1

in 1988 and 1990, but more studies on these aspectsare needed. The results demonstrate that although theproduction of defensive compounds is under geneticcontrol, there is a strong environmental effect on theactual levels found in the plants (Karban, 1992; Water-man & Mole, 1989).

Mechanisms of plant defences. The mechanisms ofdefences in the plant and counteradaptations in theinsect are still unknown. The defences of B. vulgar-is seem to rely at least partially on the productionof toxic allelochemicals (see below), but the activecompounds have not yet been identified. Glucosino-lates are typical allelochemicals of B. vulgaris andother crucifers. The most abundant glucosinolate inseeds and foliage of unsuitable B. v. ssp. arcuataaccessions (G-type) as well as in B. v. ssp. vulgar-is is (2S)-glucobarbarin while the predominant com-pound in suitable B. v. ssp. arcuata accessions (P-type) is (2R)-glucobarbarin (Kjær & Gmelin, 1957;

Jensen, 1990; Huang et al., 1994; J.A.A. Renwick,pers. comm). The P-type of B. v. ssp. arcuata is erro-neously called B. intermedia by S.K. Jensen (1990),butit is in fact a taxon within B. vulgaris (J. Jensen, pers.comm.). The suggested correlation between glucosino-late content and suitability in B. vulgaris may be fortu-itous, since (2S)-glucobarbarin is also the predominantcompound in B. stricta Andrz. which is suitable forP. nemorum (Nielsen, unpubl.). It is still a possibilitythat different hydrolysis products may be formed from(2S)-glucobarbarin in B. vulgaris and B. stricta. Someglucosinolates stimulate mine initiation in P. nemorumlarvae (Nielsen 1989b), but the compounds present inBarbarea species have not yet been tested. Most cruci-fer specialist insects are able to tolerate high levels ofglucosinolates, and production of other allelochemic-als may be a more effective defensive strategy againstsuch adapted insects (Nielsen, 1978; Sachdev-Gupta etal., 1993a,b). The two isomers of glucobarbarin differin oviposition stimulatory activity against Pieris rapae

33

and P. napi oleracea which both oviposit on B. vul-garis although the plant is toxic for larvae of the latterspecies (Huang et al., 1994).

The defensive compounds in B. vulgaris had onlya minor influence on larval behaviour during initialmine initiation, but they increased the likelihood thatsusceptible larvae from the T-population would leavethe initial leaf mine. A few larvae died in the mines,but most of them died outside the mine. Some larvaemade several mines in unsuitable B. vulgaris leaves,before they finally succumbed.Since the plant defenceshad only a minor influence on initial acceptance ofthe plant, but did influence survival afterwards, it isassumed that the defences rely at least partially onthe production of one or more toxic compounds. Thestimuli which cause the larvae to leave the mine areunknown. It could be a postingestive effect of toxicallelochemicals (Glendinning & Slansky, 1994) con-sumed during the mine initiation process, or it couldbe an effect of noxious allelochemicals acting on thecuticle when the larvae are imbedded in the leaf tissue.Another possibility is that feeding in the mine is con-trolled by other feeding stimulants and deterrents thanfeeding during mine initiation. Unsuitable Barbareaaccessions might then contain a combination of thesecompounds which is satisfactory for mine initiation,but not for continuous feeding in the mine. Further-more, the same allelochemical may well have sever-al effects on the same insect species (Glendinning &Slansky, 1994), and further studies are needed to identi-fy the defensive chemicals in B. vulgaris and unraveltheir effects on resistant and susceptible flea beetle lar-vae. Defences similar to those found in B. vulgaris havepreviously been found in Bunias erucago L., Matthi-ola parviflora (Schousb.) R.Br., and Reseda alba L.(Nielsen, 1989a). Defences in these plant species areeffective against the E- as well as the T-population ofP. nemorum (Nielsen, unpubl.).

Phenological aspects of the interaction between plantand insect. When the beetles appear from thehibernation sites in May, the plants have usually startedflowering. At this time the rosette leaves start wilting,as do the lower cauline leaves later in the floweringperiod. Upper (younger) cauline leaves of typical B. v.ssp. arcuata contain higher levels of defensive com-pounds than lower (older) leaves (Table 4). P. nemorumlarvae initiate mines preferentially in the lower leaves(Nielsen, 1977). As long as rosette leaves and lowercauline leaves are available, larvae of the susceptiblepopulation have a fair chance of survival also in the

G-type of B. v. ssp. arcuata and B. v. ssp. vulgaris, buttheir chances are reduced if they have to climb higherand use some of the more well-defended upper leaves.Moreover, rosettes originating from seeds which havegerminated in spring may be available, and they arehighly defended (Nielsen, unpubl.) in the same wayas the young rosette leaves used in the present experi-ments (Table 1). Therefore, B. vulgaris is a very unpre-dictable resource for P. nemorum larvae. This may beone reason why this abundant plant is rarely used as ahost for P. nemorum in Denmark (Nielsen, unpubl.).

Ecological and evolutionary implications of the inter-action between plant and insect. Until now there isonly circumstantial evidence for ecological and evol-utionary interactions between P. nemorum and B. vul-garis, but some observations from Ejby suggest thatthe presence of ‘resistant’ beetles may have had a pro-nounced effect on the distribution and abundance ofB. vulgaris at this locality. In 1985 when the beetleswere still rare at the locality, both the P- and G-typesof B. v. ssp. arcuata were present. In 1990, the P-typecould no longer be found, and in spring 1992, even theG-type had disappeared from central areas where thebeetles had been very abundant in 1991. However, theplant could still be found in marginal areas, where itwas heavily damaged by the beetles. After 1992, thebeetles have been rare in Ejby and new recruitmentof plants from the seed bank has occurred (Nielsen,unpubl.). When seeds from Ejby were grown in a com-mon garden together with other B. vulgaris accessions,they showed intermediate levels of defences comparedto other accessions of the G-type of B. v. ssp. arcuata(Tables 1 & 3), and there was no indication for a selec-tion towards higher levels of defences in this plantpopulation as a results of flea beetle pressure.

The use of the G-type of B. v. ssp. arcuata as anatural host plant by the E-population seems to repres-ent an extension of the host plant range, since genescontrolling the ability to utilize this plant are rare inDenmark (Nielsen, unpubl.). The host range extensionmay have been facilitated by the temporal variation inthe defences of this plant, since larvae from the sus-ceptible populations may have managed periodicallyto complete development before the defensive com-pounds were produced in sufficient quantities. Thegenes conferring counteradaptations in resistant fleabeetle populations (Nielsen, 1997) may have origin-ated as random mutations in susceptible populationswith a loose association to B. vulgaris owing to theiroccasional ability to survive in this plant. In such pop-

34

ulations there would have been a strong selection pres-sure in favour of genes conferring counteradaptationsto the defences of B. vulgaris leading to fixation ofthe genes in these populations. Host plant shifts ofteninvolve changes in larval performance on the new hostplant as well as in adult behaviour during host plantselection (Thompson, 1994; Via, 1990). Therefore,the host range extension in P. nemorum may have beenfurther facilitated by the dual function of one particu-lar gene which control larval survival as well as adultbehaviour on B. v. ssp. arcuata (Nielsen, 1996).

Acknowledgements

I thank S. Jensen, RVAU, Taastrup for providing thetemperature data, H. Nielsen and J. Jensen, Copenha-gen for help with the identification of plants,F.S. Chew,Boston, J.A.A. Renwick, Ithaca and J.N. Thompson,Pullman for valuable comments on earlier versionsof this manuscript. The work was supported by TheDanish Agricultural and Veterinary Research Council,The Danish Natural Science Research Council, and theCarlsberg Foundation.

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