/. Moll. Stud. (1990), 56, 37-45. © The Malacological Society of London 1990

THE RELATIVE IMPORTANCE OF MOLLUSCS AND AS SELECTIVE GRAZERS OF ACYANOGENIC WHITE CLOVER (TRIFOLIUM REPENS)

D. RAFFAELLI1 and A. J. MORDUE2 1 Culterty Field Station, University of Aberdeen, Newburgh, Ellon, Aberdeenshire, AB4 OAA 2 Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB9 2TN Scotland. (Received 16 December 1988, accepted 22 February 1989)

ABSTRACT populations, cyanogenic morphs may be selec- Downloaded from ted because of their better growth rates and Selective grazing of acyanogenic and cyanogenic white increased competitive ability (Ennos, 1981). clover was investigated by a field experiment using biocides to maintain (a) mollusc only, (b) only, White clover supports a wide variety of her- (c) both mollusc and insect, and (d) neither mollusc bivores, including molluscs, insects, birds and mammals. There is now much evidence from nor insect, grazing regimes. Molluscs fed more on http://mollus.oxfordjournals.org/ acyanogenic clover throughout the year, whereas field and laboratory studies for preferential graz- insects damaged more acyanogenic plants in late sum- ing by molluscan herbivores on the acyanogenic mer, but more cyanogenic plants in spring. When both morphs of T. repens (Crawford-Sidebotham, molluscs and insects were present, the net effect was 1972; Angseesing & Angseesing, 1973; Ang- greater damage to acyanogenic plants. However, in seesing, 1974; Dirzo & Harper, 1982a, b; Horril situations where insects are more abundant than mol- & Richards, 1986; Burgess & Ennos, 1987). The luscs there might be a net selection against cyanogenic clover. field studies to date, although demonstrating the grazing capacity of herbivore groups other than molluscs have not strictly controlled for their presence. This is important when considering

INTRODUCTION the maintenance of cyanogenic and acyanogenic at New York University on July 15, 2015 morphs of T. repens, because differential graz- In common with many other plants, white clover ing by different herbivore taxa may act in oppos- (Trifolium repens) possesses cyanogenic glu- ing directions. Therefore, a field trial was set cosides, thought to confer some protection up using taxon-specific biocides so the impact against herbivores. Free cyanide is produced of molluscan and insect herbivores could be through the reaction between these cyanogenic assessed when one or the other or both were glucosides and a hydrolysing enzyme. In T. present. Vertebrate herbivores were completely repens this trait is polymorphic and governed excluded. by two unlinked loci, AC/ac and Li/li, which determine the presence of the glucosides (lina- marin and lotaustralin) and enzyme (linama- rase) respectively (Corkhill, 1943). In plants METHODS possessing the dominant alleles to both loci Four genotypes of 'Blanca' strain T. repens, hence- (AcLi) damage to the leaf brings the enzyme forth called BL5, BL11, BL66 and BL67 were cloned into contact with the glucosides with the sub- to produce 20 plants of each. BL5 and BL11 were sequent release of cyanide. homozygous positive (cyanogenic) having both the The expression of this polymorphism is con- enzyme and glucoside, and BL66 and BL67 were trolled by a complex array of environmental homozygous recessives (acyanogenic), containing factors. On a geographical scale, the frequency neither the enzyme nor glucoside (P. Maher and D. Innes, unpublished). Eighty plants, each with about of the different morphs of T. repens has been 60 expanded leaflets, were dispersed between four related to latitude (Daday, 1965), altitude (de vegetation-bare plots 1.5 x 1.5 m square, such that Auraujo, 1976) and drought (Foulds & Grime, each plot contained five individuals of each genotype 1972), the cyanogenic morph decreasing with located randomly 30 cm apart within a grid. The pot low temperatures and drier conditions. Within rims were level with the soil surface. The plots were 38 D. RAFFAELLI & A.J. MORDUE located adjacent to rough grazing land where T. repens 300 leaflets for the 5 plants of each genotype in each supported large populations of insect and molluscan treatment and 4800 leaflets per sampling occasion. herbivores at Pitmedden, Aberdeenshire, N.E. Scot- The damage scores for each genotype were obtained land (OS ref. 905 294). by summing the score for the five individuals of that A plot was randomly allocated one of the four genotype. The damage categories of different geno- following treatments. types were compared using the log-likelihood ratio for a 2 (genotype) x 3 (damage category) contingency Plot 1: molluscs excluded. This was achieved by liberal table (Sokal & Rohlf, 1969). application of commercially available metaldehyde- It was originally intended to compare each acy- based slug pellets, 'Murphey slugits' and 'pbi mini- anogenic (BL66 and BL67) with each cyanogenic (BL5 pellets' containing 3% and 6% w/v metaldehyde and BL11) genotype. This comparison is only possible respectively. Pellets were added to the plot every 5 or if the genotypes are similar morphologically, par- 6 days to maintain a high pellet density. The treatment ticularly with regard to leaflet size. Despite the initial appeared effective under all weather conditions in similarity of the four genotypes selected, between- that the pellets remained visible and no live slugs were genotype differences in leaflet size became apparent as recorded in this treatment. There were no obvious the plants grew and these differences were maintained effects on the insect population density. throughout the experimental period. Since BL11 and B167 had similar leaflet areas throughout the exper- Downloaded from Plot 2: insects excluded. Insects were excluded from imental period these were chosen for the comparisons this plot by regular spraying (ideally every 5 or 6 days) of leaf damage shown here. However, both BL5 and with a commercial malathion insecticide ('Murphey BL66 showed similar trends to BL11 and BL67 Liquid Malathion' 50% w/v a.i.) at the rate of 8 ml respectively. solution per plant (equivalent to .01 ml of 50% a.i. per Observations and collections of molluscs were made

plant). Spraying frequency was weather dependent, over the experimental period. Molluscs, principally http://mollus.oxfordjournals.org/ rain, snow cover and high wind speeds sometimes slugs, were observed only in plots 1 and 4, which did preventing application. The frequency achieved was not have the molluscicide. Thorough collections of sufficient to exclude insects for most of the exper- insects were made in September 1982 when the plants imental period, but was probably insufficient on two were taken up in their pots, shaken over paper and occasions (see below). Malathion was inactive against returned to the soil, and again on the termination of slugs and snails at these concentrations (also, A. the experiment in July 1983 when the plants and their Culpon, Murphey, pers. comm.), but had a phytotoxic roots were searched for both foliage and soil insects. effect on the plants, seen as a paling of the leaves and slight reduction in plant growth rate. These effects were similar for all genotypes within a plot, and, since only within-treatment (i.e. between-genotype) RESULTS differences were of interest, the effect was not con- at New York University on July 15, 2015 sidered a problem. The main molluscan species found in the study plots was Deroceras reticulatum (Miiller). Arion Plot 3: both slugs and insects excluded. This treatment subfuscus (Draparnaud) and Arion ater (Lin- was a combination of the two described above. All naeus) occurred less frequently. Limax mar- herbivores were theoretically excluded and any ginatus (Muller) was also recorded frequently in damage to plants in this plot would indicate inef- the study area. This species causes damage to fectiveness of one or both of the biocides. ornamental plants (Godan 1983), but is not usually a foliage feeder, preferring algae and Plot 4: neither slugs nor insects excluded. Neither of fungi (Kerney & Cameron 1979). This was con- the treatments described above were applied to this firmed in feeding trials, where L. jnarginatus plot, thereby allowing slugs and insects free access to did not ingest T. repens leaflets even when no the plants. When spraying plots 2 and 3 with malathion, plots other food was available. Snails are generally 1 and 4 were covered with large polythene sheets to uncommon in the study area. Cepaea spp. were prevent contamination by drifting spray. Plants in the largest snails found, but these were only plots 1 and 4 were sprayed with water at the same recorded infrequently. frequency and intensity as those sprayed with Adult weevils (mainly Sitona lepidus) were by malathion in plots 2 and 3 to avoid between-plot far the most common insect herbivores found, differences in plant water relations. Rabbits, hares, whilst weevil larvae were the dominant root deer and birds were excluded by a 2.5 cm mesh totally feeders (Table 1). surrounding all four plots. Leaf damage was scored on nine occasions between Leaf damage in BL11 and BL67 is plotted to August 1982 and July 1983 on a 5-point scale of no show % leaves undamaged, % leaves nibbled damage, or up to 25, 50, 75 and 100% of leaflet (less than 25% removed) and % leaves heavily area removed. In subsequent analyses the last three damaged (more than 25% removed) throughout categories were pooled as >25% damage. Sixty the year (Figs. 1-4). Heavy damage occurred leaflets were scored for each plant, making a total of immediately after the introduction of the plants MOLLUSCS AND INSECTS GRAZING ON CLOVER 39 Table 1. Insects recorded from T. repens in Plots 1 to 4 (September 1982 and June 1983). All adults unless otherwise stated.

Unsprayed Sprayed (1 &4) (2&3)

COLEOPTERA Curculionidae: Sitona lepidus + Sitona sp. + + Otiorhynchus singularis + Apionidae: Apion assimile + Apion dichroum + Apion virens + + Weevil larvae + Weevil pupae + Chrysomelidae: Chrysolina varians + Chrysolina sp. + Downloaded from Longitarsus sp. + : Cateretidae: Cateretes pedicularis + Coccinellidae: Coccinella decempunctata + Hydrophiloidea sp. + Scolytidae: Hylaster sp. + Scarabidae: Scarabid sp. +

Cantharidae: Cantharis fusca + http://mollus.oxfordjournals.org/ Staphylinidae: Ocypus olens +

HYMENOPTERA : Tenthredo arcuata-schaepperi larva + myosotidis larva , + larva + Xiphydria camelus + Ichneumonidae: Phygadenontinae sp. + Gelis sp. . . . + Chalcidae: Aphelinus abdominalis paras.toids + Braconidae: Praon sp. (cocoon in aphid) + at New York University on July 15, 2015

LEPIDOPTERA Noctuidae: Laconobia oleracea larva + Autographa gamma larva + Noctuid larva sp. +

DIPTERA Tipulidae: Tipula sp. larva + + Tipula sp. pupa + Cecidomyidae: + Agromyzidae: Agromyza nana +

COLLEMBOLA Sminthuridae Sminthurus viridis +

THYSANOPTERA Thripidae Chirothrips sp. ' + Thrip sp. + DERMAPTERA Forficulidae: Forficula auricularia +

HEMIPTERA Aphididae: Aphididae sp. + Berytinidae: Berytinus minor + Cercopidae: Philaenus spumarius nymph + Aphrophora salicis larva + 40 D. RAFFAELLI & A.J. MORDUE

n.s. » • *

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82 83 Fig. 1. Seasonal variation in leaf damage of Trifolium repens in Plot 2, from which insects were excluded. Open circles = BL67 (acyanogenic), closed circles = BL11 (cyanogenic). *** p< .001, ** p< .01, * p < .05 (chi square). MOLLUSCS AND INSECTS GRAZING ON CLOVER 41

n.s. • n.s. « . . n.s. n.s. 501 Insect damage (Plot 1) Downloaded from http://mollus.oxfordjournals.org/

100 at New York University on July 15, 2015

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Fig. 2. Seasonal variation in leaf damage of Trifolium repens in Plot 1, from which slugs were excluded. Key as for Fig. 1. 42 D. RAFFAELLI & A.J. MORDUE

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Fig. 3. Seasonal variation in leaf damage of Trifolium repens in Plot 4, from which neither slugs nor insects were excluded. Key as for Fig. 1. MOLLUSCS AND INSECTS GRAZING ON CLOVER 43

•> n.s n.s. n.s. * n.s. n.s. CO CD T> * No slugs or insects (Plot 3)

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82 83 Fig. 4. Seasonal variation in leaf damage of Trifolium repens in Plot 3, from which both slugs and insects were excluded. Key as for Fig. 1. 44 D. RAFFAELLI & A.J. MORDUE to the field and during spring and early summer did not consistently select particular genotypes. of the following year (Figs. 1-4). Slug damage In Plot 1, acyanogenic plants were preferred in (Plot 2) occurred throughout the whole year late summer and early autumn, whereas in early with significantly more acyanogenic leaves being spring more cyanogenic leaflets were eaten (but grazed than cyanogenic on all but two occasions see below). The high levels of damage in both (in September 1982 and March 1983) (Fig. 1). autumn and spring were due mainly to the Insect grazing (Plot 1) produced a different pat- removal of small semicircular pieces from the tern of leaf damage with a very definite seasonal edges of the leaflets, damage characteristic of variation (Fig. 2). Grazing was low during the the adult weevil Sitona. This might imply a winter months and damage peaks occurred dur- switch in feeding preference by this species, but ing the autumn and spring. The relative amounts such a high diversity of insects fed on clover of insect damage to the different morphs throughout the year that it is not possible to changed throughout the year, with more damage attribute damage to any one. It is likely that all to acyanogenic plants during September and species of phytophagous insect do not discri- October 1982, whereas cyanogenic plants were minate between cyanogenic and acyanogenic damaged more from February to April 1983 clover in a similar way, if at all. For instance, Downloaded from (Fig. 2). When both insects and slugs were in laboratory choice experiments the weevils present (Plot 4) a compounding effect of the two Sitona lineatus, S. hispidulus and Apion sp. did herbivore types was seen with the acyanogenic not discriminate between the two morphs, morph significantly more heavily damaged than whereas the grasshoppers Omocestus viridulis the cyanogenic morph from August to October and Melanophus sp., the locust Schistocerca gre-

1982 and in May and July 1983, suggesting an garia and the aphid Myzus persicae did. Also, http://mollus.oxfordjournals.org/ over-riding effect of slugs (Fig. 3). ovipositing females of the leaf mining dipteran The data for Plot 3, where both insects and Agromyza nana did not discriminate between slugs were excluded, showed that exclusion was the two morphs, but larvae were significantly successful, except for two occasions (September smaller in cyanogenic leaves (Mordue, unpub- 1982 and March 1983) when leaves were nibbled lished). by herbivores (Fig. 4). This damage was prob- Dirzo & Harper (1982b) also suggested that ably due to insects resulting from a too insects as a group were inconsistent in their infrequent application of malathion. From Feb- selection of cyanogenic or acyanogenic morphs. ruary to March 1983 snow and high winds pre- They used the patterns of leaf damage found in vented regular spraying whilst in September the field to assign grazing to either molluscs 1982 wet conditions probably made spraying or insects. We presented a range of potential at New York University on July 15, 2015 ineffective. These two peaks of insect grazing herbivores with T. repens leaves and observed are of importance when considering the damage the pattern of damage by each species. Whilst due to molluscs (Fig. 1). The only time mollusc it is true that some herbivores do leave charac- grazing does not show a significant selection for teristic feeding marks, e.g. the weevil Sitona, it acyanogenic morphs is during these two periods, is not possible to confidently assign damage to when presumably insects were not adequately particular species, or even to groups of species. excluded from this plot. Apart from the reduced For instance, slugs rasp away leaf tissue often effectiveness of malathion in September and causing a large, irregular wound with a frayed March, field observations on herbivore activity edge, but the damage pattern can vary from confirm that the biocides performed well. small holes in the leaflet to complete removal of all three leaflets. Slugs may also gnaw through the base of the petiole. Feeding trials with the. leatherjacket larva of the craneflay Tipula palu- DISCUSSION dosa, as well as various lepidopteran and sawfly larvae, produced, in a number of cases, damage The experiment has demonstrated conclusively similar to that caused by slugs. Heavy damage that slugs feed preferentially on acyanogenic by the adult weevil Sitona sp. and light damage clover throughout the whole year. Levels of by slugs can also be remarkably similar. We damage appear to vary seasonally being lower conclude that it is not possible to separately in winter than in spring and summer, although estimate the extent of herbivory on T. repens by Deroceras reticulatum is reported active at molluscs and insects, using the pattern of leaf 0.8°C, can withstand -8°C for several hours damage. (Godan, 1983), and could cause considerable Our use of group-specific biocides has allowed damage in mild winters. Unlike slugs, insects for the first time a clear distinction between MOLLUSCS AND INSECTS GRAZING ON CLOVER 45 mollusc and insect mediated damage under field morphism in Trifolium repens. Heredity, 31, 276- conditions. This distinction is important if the 282. role of invertebrate herbivores in maintaining ANGSEESING, J. P. A., 1974. Selective eating of the the cyanogenic polymorphism is to be properly acyanogenic form of Trifolium repens. Heredity, 32, assessed. Like Dirzo & Harper (1982b), we have 73-83. shown preferences by some insects for cyano- AURAUJO, A. M. de, 1976. The relationship between altitude and cyanogenesis in white clover. Heredity, genic plants. In the present study the effects of 37, 291-293. slugs overwhelmed this opposing effect of BURGESS, R. S. L. & ENNOS, R. A., 1987. Selective insects in Plot 4 in summer (Fig. 3), but on the grazing of acyanogenic white clover: variation in two occasions when insects fed in the herbivore- behaviour among populations of the slug Deroceras free, and presumably in the slug only plots (Figs. reticulatum. Oecologia, 73, 432-435. 1 and 4), there was evidence of selection against CORKHILL, L., 1943. Cyanogenesis in white clover cyanogenic morphs. At the same time, however, (Trifolium repens L.). V. The inheritence of cyan- insects (in the absence of slugs) fed selectively ogenesis. New Zealand Journal of Science and Tech- on acyanogenic clover (Fig. 2). This paradox nology, 23B, 178-193. CRAWFORD-SIDEBOTHAM, T. J., 1972. The role of merely underlines the complexity of insect graz- Downloaded from slugs and snails in the maintenance of the cyan- ing on clover and the need to assess the affects ogenesis polymorphisms in Lotus corniculatus and of insects on a species by species basis. Never- Trifolium repens. Heredity, 28, 405-411. theless, it is conceivable that in situations where DADAY, H., 1965. Gene frequencies in wild popu- slugs are scarce there could well be overall selec- lations of Trifolium repens. IV. Mechanism of nat- tion against the cyanogenic morph by inver- ural selection. Heredity, 20, 355-365.

tebrate herbivores. It is important therefore to DIRZO, R. & HARPER, J. L., 1982a. Experimental http://mollus.oxfordjournals.org/ consider the separate effects of these two groups studies on slug-plant interactions. III. Differences when trying to understand the factors main- in the acceptibility of individual plants of Trifolium taining the cyanogenic polymorphism of T. repens to slugs and snails. Journal of Ecology, 70, repens. 101-117. DIRZO, R. & HARPER, J. L., 1982b. Experimental studies on slug-plant interactions. IV. The per- formance of cyanogenic and acyanogenic morphs of Trifolium repens in the field. Journal of Ecology, ACKNOWLEDGEMENTS 70, 119-138. ENNOS, R. A., 1981. Manifold effects of the cyano- We are very grateful to Dr. P. Maher, Open genic loci in white clover. Heredity, 46, 127-132. University, Edinburgh for the provision of clo- FOULDS. W. & GRIME, J. P., 1972. The influence of at New York University on July 15, 2015 soil moisture on the frequency of cyanogenic plants vers and to Dr. V. K. Brown, Imperial College, in populations of Trifolium repens and Lotus cor- London for useful advice at the start of the niculatus. Heredity, 28, 143-146. project. We would like to thank R. R. Askew, GODAN, D., 1983. Pest Slugs and Snails. Springer- M. M. Blight, B. Laing, D. Marriott, H. G. Verlag, Berlin. Morris, M. R. Shaw and M. R. Young for help HORRILL, J. C. & RICHARDS, A. J., 1986. Differential in the identification of molluscs and insects. grazing by the mollusc Arion horlensis Fer. on cyanogenic and acyanogenic seedlings of the white clover Trifolium repens L. Heredity, 56, 277-281. KERNEY, M. P. & CAMERON, R. A. D., 1979. A REFERENCES Field Guide to the Snails of Britain and North-Wesl Europe. Collins, London. ANGSEESING, J. P. A. & ANGSEESING, W. J., 1973. SOKAL, R. R. & ROHLF, F. J., 1969. Biometry. W. H. Field observations on the cyanogenesis poly- Freeman & Co., San Francisco.