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1 In order to test the hypothesis that the sensitivity of field populations of the rare perennial Arnica montana to slug attack is due to its high palatability, we compared Arnica leaves and seedlings with 20 other plant species in greenhouse food choice experiments.
2 When slugs were given a choice of mature plant leaf discs, Arnica montana was one of the food plants most preferred by all three slug species tested. Arion lusitanicus, Arionsubfuscus and Deroceras agreste differed only slightly in their preference for particular plant species. Greater differences in acceptability were observed when parameters other than area consumed were evaluated. Arnica seedlings displayed similarly high susceptibility to slug grazing.
3 Morphological factors influence mollusc feeding behaviour, with epidermal cell wall thickness and hairiness related to acceptability. Removing the hairs by shaving the leaves increased the acceptability of one species, Centaurea jacea.
4 Leaves of undamaged Arnica montana plants were preferred to leaves of plants that had been predamaged by partial leaf removal, suggesting that there is an inducible component of chemical defence.
5 We discuss our results in comparison with other food choice experiments and attempt to extrapolate them to mollusc/plant interactions under field conditions.
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- Materials and methods
Slugs and snails form an important part of the herbivore fauna of different vegetation types. Although more or less polyphagous, they show distinct food preferences. If a rare plant species shows exceedingly high acceptability to slugs it may become excluded from sites where the presence of other, more abundant food plants supports high mollusc activity. Mollusc herbivory could therefore be a major factor for limiting plant geographical ranges and, in particular, the restriction of montane plant species to higher altitudes may result from higher herbivory pressure in lowlands. We tested this hypothesis for the rare grassland perennial Arnica montana L. (Compositae) in the Harz mountains, Lower Saxony, Germany (Bruelheide & Scheidel 1999), where transplanted Arnica individuals were shown to suffer increasing slug damage with decreasing altitude. Here we present the results of mollusc food choice experiments under greenhouse conditions.
Food selection has been investigated extensively for both slugs (Gain 1891; Pallant 1972; Jennings & Barkham 1975; Dirzo 1980; Lawrey 1983; Rathke 1985; Briner & Frank 1998; Wardle et al. 1998) and snails (Grime et al. 1968, 1970, 1996; Speiser & Rowell-Rahier 1993). However, most studies have involved plant species of high abundance or agricultural importance, and mollusc herbivory of threatened plant species has rarely been investigated. We investigated whether slugs graze on the leaves of a highly endangered species, Arnica montana, in preference to other species from the same montane grassland. Species of the Compositae are generally of high mollusc acceptability, e.g. Leucanthemum vulgare (Cates & Orians 1975; Briner & Frank 1998) and Taraxacum officinale (Dirzo 1980; Cook et al. 1996; Wardle et al. 1998), but although Arnica montana is known to be harmful to cattle, which avoid this plant (Hegi 1987, p. 707), it has not been tested on any of its natural herbivores.
The results of earlier food choice experiments are as diverse as the mollusc species used. For example, the extensively tested Plantago lanceolata was not eaten by Deroceras caruanae (Dirzo 1980), Cepaea nemoralis (Grime et al. 1968) and Arion lusitanicus (Briner & Frank 1998), only slightly by D. reticulatum (Wardle et al. 1998) but greatly by Helix aspersa (Grime et al. 1996). We therefore offered our food plants to three slug species that are normally present in montane meadows to determine whether they show different preference patterns.
Molluscs are known to prefer seedlings to mature plants (Byers & Bierlein 1982). Hanley et al. (1995a) found similar attack rates for 7-day-old seedlings of three plant species, but species-specific changes of palatability with seedling age, possibly due to changing contents of chemical deterrents (Horrill & Richards 1986; Glen et al. 1990) or altered morphological properties (Hanley et al. 1995a). Hulme (1994) highlighted the importance of seedling size as the primary factor determining the risk of mollusc herbivory. For rare or endangered species, seedling survival can be assumed to be of pre-eminent importance for maintaining population sizes and we therefore assessed the extent of slug grazing on Arnica seedlings as well as on leaf discs from mature plants.
It has been suggested that leaf palatability to herbivores has three major components (Grime et al. 1996): chemical defence (as a result of distasteful or toxic substances), physical defence (hardness of plant tissue or presence of epidermal hairs) and substances that promote acceptability (by offering a pleasant taste or lucrative nutrient composition), although neither the effectiveness of any single factor nor the interactions between them is yet understood (Pennings et al. 1998). We examined the potential morphological defence mechanisms of Arnica montana. The effectiveness of epidermal hairs as a barrier against mollusc consumption is controversial. Neither Grime et al. (1968) nor Dirzo (1980) found much evidence to suggest that hairs reduce palatability of leaves, and Cook et al. (1996) stated that the hairiness of leaves does not deter molluscs from eating them. In contrast, Jennings & Barkham (1975) found that several species of Arionidae preferred glabrous, soft-textured plant species, and Westerbergh & Nyberg (1995) detected a preference of hairless ecotypes of Silene dioica in two Arion species. We compared feeding of slugs on normal leaves with those from which the hairs were removed by shaving.
Other physical factors may be important. For example, Dirzo (1980) found a high acceptability of plants with soft epidermis and Grime et al. (1968) suggested that a hard external surface may be an effective barrier against consumption by the snail Cepaea nemoralis. We therefore assessed the thickness of epidermal cell wall of the food plants as a measure of their ‘hardness’.
We also tested the potential for chemical defence. Arnica montana is known to produce Helenalin (Frohne & Jensen 1992, p. 201), a sesquiterpene lactone similar to compounds found to deter snails from consuming plants of the tribe Senecioneae (Compositae) (Speiser et al. 1992; Hägele et al. 1996). Production of such defence chemicals may be constitutive or inducible, and several investigators have found that changes in chemical (Van Dam & Vrieling 1994) or morphological (Pullin & Gilbert 1989) defence mechanisms follow defoliation. We tested whether Arnica montana showed such inducibility by comparing feeding on discs from undamaged plants with those taken from plants that had been damaged in the field 4 weeks previously.
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Rank order differences between slug species which differ significantly in size and habitat demands were remarkably low. Jennings & Barkham (1975) and Rathke (1985) also found a great similarity in the feeding responses to plants when the slug D. reticulatum was compared with several Arionidae species. Extreme differences in palatability of particular plant species can be deduced by comparison of several studies (see compilations in Dirzo 1980 and Briner & Frank 1998) but these are probably due to varying feeding conditions and point to the need to establish a standard method.
As a consequence of this ‘context specificity of preference’ (Peterson & Renaud 1989), we must agree with Rathke (1985) who suggested that ‘fine differences in values should not be overinterpreted’. As with our results (Table 2), Richardson & Whittaker (1982) found altered rank orders when consumption was measured as dry weight rather than leaf area eaten. In addition, these authors highlighted the crucial influence of which plant is selected as a reference material to be offered with each other food plant. Choosing a semi-acceptable plant as reference provides more significant differences between food species than choosing highly or poorly acceptable ones. We analysed our results in this way using species of high (Hieracium), medium (Succisa) or low (Poa) acceptability as ‘reference plant’. For each combination where a particular species occurred together with the reference plant (and any third species) in the eight replicates, we determined whether more of the test or reference species was consumed (+/– results, respectively). We too got different rank orders and different numbers of significant differences between species depending on the reference chosen. In agreement with Richardson & Whittaker (1982), we found the most differences when the semi-acceptable Succisa was used as the reference species (Table 4).
Table 4. Rank orders of the 21 plant species in relation to a ‘reference species’. The values show the numbers of combinations of the eight where both were fed together (experiment 1), when a greater (+) or lesser (–) area of the tested species was consumed than of the ‘reference species’. Combinations where the two species were consumed in equal amounts were excluded. For species codes see Table 1
|Reference species Hieracium laevigatum||+/–||Succisa pratensis|| +/–||Poa chaixii|| +/–|
|To||3/2||Lv||8/0||Hl, To, Am, Lv, Sd, Co, Sp||8/0|
|(Hl)||0/0||Am||7/0||Cj, Ka, Ra, Bo||7/0|
|Sv||2/5||Hl, To||7/1||Pn, Tp, Vc, Hm||6/0|
|Am Lv, Co, Cj, Ka||1/5 1/6||Pn Ra||5/3 4/2||Pl Pb||4/0 3/0|
|Tp, Vc, Ra, Bo, Pl, Pb, Gs, Av, Pc||0/8||(Sp) Bo Sv Tp, Vc Pl, Pb Av Hm, Gs, Pc||0/0 3/5 2/5 2/6 1/7 0/7 0/8|| || |
If each food plant is offered together with reference material the ratio of eaten test species to eaten reference species can be used as an ‘acceptability index’ or ‘palatability index’ (Grime et al. 1968; Dirzo 1980; Richardson & Whittaker 1982). These can be compared statistically by an anova because values of food plant consumption are then independent of each other. However, studies of this kind provide highly artificial results, which are not directly comparable to field conditions. It is more biologically meaningful to offer a range of different test species or ecotypes together, but then many species will remain statistically undifferentiated.
As a consequence, we used combinations of three of our 21 test species at random, in an attempt to approximate field conditions. Rollo (1988) observed the slug D. reticulatum to take two to four meals per night. Speiser & Rowell-Rahier (1993) found an average of one change of food species per day when investigating faeces of the snail Arianta arbustorum, and our preliminary experiments showed that two changes are not unusual.
Unfortunately, the statistical analysis of such experiments is very difficult. As the consumption of one species can be affected by one or both of the other two species in a given trial, the quantities consumed may be interdependent. Therefore, parametric analyses, such as anova, are not valid, although they have been used by several authors, e.g. Westerbergh & Nyberg (1995). Using non-parametric tests, such as the Kruskal–Wallis test, the number of significant differences in single comparisons between plant species is often reduced dramatically, and in this case only the highest ranks remain significantly different from the lowest.
Interestingly, the rank order of preference of D. agreste for seedlings differed only slightly from that obtained with leaf discs. In contrast, Wardle et al. (1998), assessing palatability of 20 herbaceous dicotyledons to D. reticulatum, found no correlation between seedling and leaf disc palatability. We used seedlings that were more than 3 weeks old when subjected to slug grazing and it is possible that younger seedlings would not differ in palatability between the species as Fenner (1987) and Hanley et al. (1995a) found seedling palatability to vary with age. Our results, unlike those of Hulme (1994), do not indicate any correlation between seedling size and mollusc behaviour. Two of the most preferred seedlings, Arnica and Leucanthemum, had the largest and smallest cotyledons, respectively, of the plant species tested.
The main factors influencing mollusc food choice therefore appear to be the same in seedlings and mature leaf discs. There are significant morphological differences between seedling cotyledons and mature foliage leaves, and we therefore suggest that seedlings and older leaves have similar chemical properties and that these are of primary importance for slug food selection. Nevertheless, physical factors may also influence food choice, although they are difficult to detect by comparing different plant species (Wardle et al. 1998). Westerbergh & Nyberg (1995) suggested that it is ‘impossible to evaluate the importance of hairiness on grazing unless the study includes distinct glabrous and hairy morphotypes of a single species’.
We used shaving to change a morphological trait of leaves of a single species. Cytoplasm may leak from the cut surface and this may increase the plant's palatability and attract molluscs, although similar effects are expected when cutting leaf discs and this does not appear to influence feeding. In addition, the method used cannot avoid injuring neighbouring epidermal cells and their walls, which may alter the hardness of the plant material, the glandular hairs might have a chemical effect after shaving, and sharp-edged cell wall residues may have a defensive effect. This approach therefore needs further analysis and improvement.
Similarly, the effect of hardness (experiment 3a) is almost certainly masked by several other plant traits. In Jennings & Barkham's (1975) experiments a behavioural preference for soft textured leaves was least visible in A. ater, the largest slug species studied. The largest of our tested species, A. lusitanicus, also showed the least pronounced preference for leaves with thin epidermal walls.
Although the results suggest that mechanical factors may act as feeding deterrents, the limited correlations indicate the importance of other, mainly chemical, properties.
No differences could be detected in the physical properties of Arnica leaves from predamaged plants that might explain their lower acceptability in comparison to undamaged plants. Chemical defence mechanisms are therefore highly probable, although we did not investigate their basis.
There are, however, also hints of chemical mechanisms that attract slugs to Arnica rather than deter them, perhaps by the same components. For each slug species Arnica ranks lower when number of leaves attacked is measured than when the percentage of eaten leaf area is considered. Once having started to gnaw on Arnica, the slug seems to be compelled to continue eating and, as a result, fewer partly consumed leaf discs remain, possibly because some chemical component causes a kind of ‘addiction’ to Arnica. As with humans eating potato crisps, bags, once opened, rarely remain half empty.
It was a primary intention of this study to extrapolate from the results of greenhouse experiments to slug–plant interactions under field conditions. Field observations of Arnica montana in meadows in the Harz mountains suggest that the risk of herbivory by slugs to whole plants growing in natural surroundings is particularly high adjacent to forest edges and in rainy years (Scheidel & Bruelheide 1999). In field experiments we have been able to show a dramatic increase in damage after releasing slugs into native Arnica populations and that damage by native slugs to transplanted Arnica decreases with increasing elevation in the Harz mountains (Bruelheide & Scheidel 1999). The results indicate that Arnica montana, the species with whose conservation we are concerned, is among the most palatable of those studied, both as leaf discs and seedlings.
In the case of Arnica montana, the results of food choice experiments therefore appear to match the field observations and field experiments. However, the results for some other species illustrate the pit-falls when laboratory results are extrapolated to field conditions. For example, although Hieracium laevigatum displayed the highest acceptability of all in food choice experiments, it was almost never eaten by slugs in the field and suffered almost no damage there. This may be because in the field attached leaves are not on contact with the ground and so are rarely encountered by slugs, which are further discouraged from climbing by the hairy stem. In contrast, in food choice experiments leaves are presented to the slugs in an easily accessible form.
Plants are most accessible at the seedling stage, when slug damage to plant individuals is also most effective. In our experiment, Deroceras individuals were able to kill several seedlings per day. This may be sufficient to prevent seedling establishment of preferred plant species completely, given the slug abundances of 50–60 individuals of Deroceras sp. per m2 reported from grassland (South 1965; Tischler 1980).
Indeed, Hanley et al. (1995b) were able to influence plant community dynamics in artificially created grassland gaps by molluscicide application. These authors suggested the existence of ‘a trade-off between fast growth rate and unacceptability to herbivores in determining competitive ability’ for their test species. Arnica montana, however, faces a particularly disadvantageous position: not only are its seedlings slow growing but they are also highly palatable.