Are associational refuges species-specific?

Transcription

1 Functional Ecology 2003 Are associational refuges species-specific? Blackwell Science, Ltd P. A. HAMBÄCK*, J. PETTERSSON and L. ERICSON *Department of Ecology and Crop Production Science, Section for Landscape Ecology and Department of Entomology, Swedish University of Agricultural Sciences, PO Box 7044, SE Uppsala, Sweden, and Department of Ecology and Environmental Sciences, Umeå University, SE Umeå, Sweden Summary 1. Associational refuges reduced herbivory on plants in presence of other plant species may be caused by general and species-specific plant characteristics. However, the species specificity of associational refuges has rarely been evaluated. 2. This study examined the species specificity of one known example of associational refuges, the forb Lythrum salicaria and the monophagous insect herbivores Galerucella calmariensis, G. pusilla and Nanophyes marmoratus. The underlying mechanism was examined in order to evaluate connections between mechanisms and species specificity. 3. Laboratory studies showed that N. marmoratus but not Galerucella individuals were attracted by odour from undamaged host plants, and that neither species was distracted by odour from Myrica gale. 4. Field experiments showed that three non-host plant neighbours with an appearance similar to M. gale, and artificial shrubs, reduced the abundance and egg-laying of Galerucella species by 70 90%. The abundance of N. marmoratus was increased 18-fold on plants in thickets compared with outside. 5. The different responses by N. marmoratus and the Galerucella species to plant neighbours appear to be because N. marmoratus, but not Galerucella, uses olfactory information in the initial host-finding process. Key-words: Associational refuges, Galerucella calmariensis, Galerucella pusilla, host-finding interference, Lythrum salicaria, Nanophyes marmoratus, olfactometer Functional Ecology (2003) Ecological Society Introduction Plant productivity increases with plant diversity in many plant communities (Hector et al. 1999; Tilman, Wedin & Knops 1996), and one mechanism that may account for this positive correlation is reduced herbivore damage on plants in species-rich habitats (Andow 1991; Koricheva et al. 2000; Mulder et al. 1999; Tonhasca & Byrne 1994). However, it is less well understood whether these effects are caused by biodiversity per se or by the presence of specific plant types (Bengtsson 1998), but the degree of specificity probably depends on the mechanism involved. For instance, the reduced herbivory hypothesis, also called associational refuges (Pfister & Hay 1988) or associational resistance (Tahvanainen & Root 1972), may be caused by general plant characteristics such leaf shape or colour (Brown & Lawton 1991), or by speciesspecific characteristics such as unique plant odour compounds. The importance of different mechanisms has rarely been evaluated, but associational refuges appear to arise usually because of non-host plant Author to whom correspondence should be addressed. interference with the herbivore s host-finding process (Andow 1991; Finch & Collier 2000; Risch, Andow & Altieri 1983). In this paper we examine the species specificity of one particular associational refuge, and the underlying mechanisms. We examine the potential of olfactory repellence (a species-specific mechanism) and visual masking (a general mechanism) in a system with the forb Lythrum salicaria and three monophagous herbivore species. The background is an earlier study showing that L. salicaria experiences reduced herbivory, and an increased seed set by over a magnitude, when associated with the aromatic shrub Myrica gale (Hambäck, Ågren & Ericson 2000). That study also showed that reduced herbivory was probably caused by host-finding interference, where the presence of Myrica affected the host-finding process by the monophagous leaf-feeding beetle Galerucella calmariensis but not by the flower-feeding weevil Nanophyes marmoratus. In that study we were unable to differentiate between an olfactory and a visual interference mechanism. Here we tested the potential of olfactory interference in laboratory trials with olfactometers, and the specificity of the associational refuge through a set of field experiments. In the laboratory, 87

2 88 P. A. Hambäck, J. Pettersson & L. Ericson we examined (1) if olfactory cues are involved in host-finding by the three main insect herbivores (G. calmariensis, G. pusilla and N. marmoratus) attacking L. salicaria; and (2) if the odour produced by M. gale either repels or masks the presence of L. salicaria. In the field, we tested if the reduced herbivore abundances and reduced herbivory on L. salicaria also occurred when plants were placed in thickets of other shrub species or in artificial thickets. In these experiments we selected shrub species, and constructed artificial thickets, that were structurally similar to Myrica thickets. Hence any differences in the herbivore response to these treatments are more likely to be a consequence of olfactory rather than visual interference of the host-finding process. Materials and methods STUDY SITES The field work was performed in two regions. Two experiments were performed in areas close to Umeå in northern Sweden, Brännölandet (63 42 N, E) and Vitskärsudden (63 39 N, E), and two experiments were performed at the coast of Uppland in southern Sweden, Jungfruholm (60 26 N, E) and Själgrundet (60 38 N, E). All areas are similar in structure, with shallow and rocky shorelines, but have different mixtures of grasses, shrubs and forbs. The areas are also characterized by land uplift, and by ice-scouring in winter. In all areas, L. salicaria occurs from the lower part of the shore, close to the average water table, to the Alnus fringe (A. incana in the north and A. glutinosa in the south) in front of the Spruce/Pine forest. All four shrub species included in the study (A. glutinosa, Hippophaë rhamnoides, M. gale and Salix myrsinifolia-phylicifolia agg.) occur on the upper part of the shore. However, different areas are dominated by different shrub species: S. myrsinifoliaphylicifolia agg. and M. gale grow at Brännölandet and Vitskärsudden, while A. glutinosa and H. rhamnoides grow at Jungfruholm and Själgrundet. STUDY SPECIES Purple Loosestrife, Lythrum salicaria L. (Lythraceae), is a perennial, insect-pollinated forb that is native to Eurasia and has been introduced to North America (Hultén & Fries 1986). In northern and central Sweden it is mainly found on the shores of the Gulf of Bothnia. Reproducing plants are 50 cm tall on average, and produce several flowering shoots. Flower buds develop in leaf nodes in the upper part of the flowering shoot. In Fennoscandia, L. salicaria flowers for 6 8 weeks in July August and seeds mature 6 8 weeks after flowering. Plant establishment is predominantly from seed. Galerucella calmariensis L. and G. pusilla (Coleoptera: Chrysomelidae) are the main leaf-feeding herbivores on Purple Loosestrife in study areas, and both species Fig. 1. Two-armed olfactometer used to investigate insect responses to plant odours. The arena for the beetle consists of areas labelled A C, and plants were placed in a chamber at one or both ends (labelled empty vs plant). To create a unidirectional odour flow, the centre of the olfactometer was connected to an air-suction apparatus. At the start of a trial, beetles were released in the arena centre (B) and positions within the arena were then recorded every 3 min. To prevent beetles reaching plants, a fine net covered the tube connecting the behavioural arena with plant chambers. are monophagous herbivores feeding on leaves and flower buds. In the southern area (Uppland) both species occur, although G. pusilla is the more abundant species, while G. calmariensis is the only species occurring in the northern area. The two species have a similar life history, the only apparent difference being that G. calmariensis adults are slightly larger. Their life cycle is univoltine and the adults overwinter in the litter (Hight et al. 1995). In southern Sweden adults appear in mid-may; in northern Sweden they appear in mid-june. The adults feed on leaves and lay their eggs in batches on the stem and on the lower leaf surface. The larvae hatch 7 10 days later, feed on leaves and flower buds for 2 3 weeks, then pupate in the ground. Feeding can cause extensive damage to the host plant (Blossey 1992; Hambäck et al. 2000), and both species are presently used as biocontrol agents for Purple Loosestrife in North America (Hight et al. 1995). There is no distinct dispersal period for either species, but dispersal could occur throughout the growing season. Nanophyes marmoratus Goeze (Coleoptera: Curculionidae) is the main flower predator on Purple Loosestrife in the study area. It feeds solely on L. salicaria flowers and has a univoltine life cycle (Blossey & Schroeder 1995). Adults appear by the end of June, and the newly emerged adults feed on young leaves and flower buds. The females oviposit on young flower buds. The emerging larva consumes the reproductive parts of the flower and pupates at the bottom of the bud (Blossey & Schroeder 1995). While more than one egg may be deposited in each flower, it is unusual for more than one larva to reach pupation. OLFACTOMETER STUDIES The abilities of G. calmariensis, G. pusilla and N. marmoratus to locate host plants through olfactory cues, and their respective responses to M. gale, were examined using two-armed olfactometers (Fig. 1; for a detailed description of the methodology see Petterson et al. 1998). The olfactometers were made of perspex

3 89 Associational refuges and Galerucella and consisted of three layers with an arena cut out in the middle sheet. This arena had a four-sided central zone ( cm) and two tapered arm zones, each 3 8 cm long and ending with a 0 6 cm broad space where odour sources were connected with Teflon tubing via 4 mm holes through the periphery. An air stream over the arena was created by attaching a water pump to the centre of the arena, and the air flow was regulated to approximately 3 ml s 1. This was sufficient to give an even cover of the arm zone with the applied odour. Each trial was run as follows. At the start of the trial, one beetle was placed in each olfactometer and was allowed to move for 5 min before measurements started. Following this acclimatization period, we recorded the position of the beetle every third minute for 60 min (20 recordings). At each recording we scored one of three positions, where the beetle was in either arm (Fig. 1, areas A and C) or in the centre (area B). If an individual was immobile (moved less than 5 mm) for more than three consecutive periods, this individual was excluded and we tested a new individual. For each stimulus we tested 36 individuals of G. pusilla (18 males and 18 females) and 18 individuals of G. calmariensis and N. marmoratus (females only), and the same individual was used only once for each stimulus. We found no sex differences, and therefore present only pooled data. To test beetle responses we used a Wilcoxon sign-rank test. The replicate in this test was the individual beetle, and the test was performed on the number of intervals that each individual was recorded in either arm. All recordings of an individual in the central part were excluded from the analysis. Olfactometers were cleaned between runs, first with water and mild detergent, then with 70% ethanol. To test the hypothesis that olfactory interference was involved in reduced herbivory on L. salicaria when plants grew in Myrica thickets, we used the following combinations of stimuli: (i) Lythrum control; (ii) Myrica control; (iii) Lythrum/Myrica control; (iv) Lythrum/Myrica Lythrum. We also included a zero treatment, control control, to examine the possibility of unexpected responses caused by other aspects of the laboratory environment, and a humidity control, wet vs dry filter paper, to examine the possibility of attraction to water vapour emitted by plants. The possible outcomes of the olfactory experiment are as follows. If olfactory cues are involved in the host-finding process, we would observe attraction to the Lythrum side in experiment (i). If Myrica is repellent (active avoidance), we would observe avoidance of the Myrica side in experiments (ii) and (iv). If Myrica is masking the presence of Lythrum (confusion), we would observe zero response in experiment (ii), attraction to the Lythrum side in experiment (iv), and reduced attraction in experiment (iii) compared with (i). As these hypotheses are nested, we performed a trial only if previous trials indicated that this was necessary to distinguish hypotheses. FIELD EXPERIMENTS We examined whether reduced herbivory on Purple Loosestrife occurred only in thickets of M. gale, or if the same response occurred when plants grew in thickets of other shrub species that were unrelated to M. gale but had a similar physical structure. We placed paired transplants of L. salicaria in and outside similarly sized thickets (0 5 5 m 2 ) of Salix (Vitskärsudden in the northern area, initiated 25 June 2001, N = 29); A. glutinosa (Själgrundet in the southern area, initiated 4 June 2001, N = 20); and H. rhamnoides (Jungfruholm in the southern area, initiated 2 June 2001, N = 28). We also placed paired transplants in and outside artificial thickets (Jungfruholm, initiated 2 June 2001, N = 28; Brännölandet, initiated 25 June 2001, N = 28). Artificial thickets (0 5 m 2 ) were constructed from a coarse plastic net with tied-on green cotton stripes (2 cm wide) up to a height similar to that of natural shrubs (30 50 cm). If differences in herbivore abundance between potted L. salicaria in and outside thickets are equal for thickets of all four shrub species and for artificial thickets, this indicates that the associational refuge on L. salicaria is general. If olfactory interference of the host-finding process is the mechanism underlying reduced herbivory on L. salicaria in Myrica thickets, we would expect this difference to disappear when using artificial thickets. The experimental plants originated either from a population 15 km north of Brännölandet (for experiments in the northern region), or from a population 20 km north-east of Jungfruholm (for experiments in the southern region). Plants had been grown from seeds in experimental gardens at either Umeå or Uppsala, and were 2 4 years old at the time of the experiment. To prevent possible negative effects due to nutrient competition from the shrub, or positive effects due to nitrogen release from N-fixing nodules on the roots of Myrica, Hippophaë or Alnus, plants were transplanted in pots. The potted plants were paired based on size at the time of transplantation to the field site. One plant in each pair was placed in a natural or artificial thicket, while the second plant was placed 2 m away and outside the thicket. All potted plants were watered regularly throughout the experiment. On all potted plants we counted the number of G. calmariensis and G. pusilla (adult beetles and eggs) once, twice or three times, all within 3 weeks of transplantation. We did not differentiate between the two Galerucella species, as this is difficult for adults and eggs in the field. In two areas (Jungfruholm and Brännölandet) we also counted the number of adult N. marmoratus. Finally, at Jungfruholm we counted the number of Nanophyes larvae and pupae and the number of flowers. Larvae and pupae of Nanophyes were counted by collecting and dissecting all flowers on each plant. This procedure is laborious and has to be performed on fresh flowers. It was therefore performed only in one area.

4 90 P. A. Hambäck, J. Pettersson & L. Ericson after 5 days (Galerucella adult); after about 2 weeks (Galerucella eggs); after about 4 weeks (Nanophyes adults); and after 2 months (Nanophyes larvae/pupae). These periods depended on the species life histories. Results OLFACTOMETER STUDIES Fig. 2. Responses of Galerucella pusilla (Gp), G. calmariensis (Gc) and Nanophyes marmoratus (Nm) to the odour of the host plant Lythrum salicaria and to the non-host Myrica gale (***, P < 0 001; *, P < 0 05). Trials were performed in a twoarmed olfactometer (Fig. 1). The differences in the number of Galerucella and Nanophyes between potted L. salicaria in and outside thickets was tested by the Wilcoxon sign-rank test. Prior to testing we excluded double-zeros where numbers were zero in both treatment and control within a pair. The respective tests were performed on numbers The zero treatment indicated no significant difference between the two arms of the olfactometer (z = 0 04, N = 18, P > 0 1): no artefact was inadvertently included in the olfactometer experiment, and there was no response to humidity for either Galerucella species (Gp: z = 0 28, Gc: z = 0 45, N = 17, P > 0 1 for both species). However, we found a negative response to humidity for N. marmoratus (z = 2 0, N = 19, P < 0 05). In the olfactometer treatments with plants, the two Galerucella species showed similar responses (Fig. 2). Neither species responded to the odour of their host plant L. salicaria (Gp: z = 0 87, N = 33, P > 0 1; Gc: z = 0 08, N = 18, P > 0 1), and both species were attracted to the odour of M. gale (Gp: z = 3 54, N = 38, P < 0 001; Gc: z = 2 20, N = 18, P < 0 05). This indicates that olfactory responses were less important in the initial host-finding process of G. calmariensis and G. pusilla, and therefore that olfactory interference by M. gale is less likely. As a consequence, we did not perform experiments (iii) and (iv), which were aimed only at separating two olfactory interference mechanisms, masking and repellence. The response of N. marmoratus was different from that of Galerucella. The weevil responded positively to odour from Lythrum (z = 2 28, N = 36, P < 0 05) and not to that of Myrica (z = 0 20, N = 18, P > 0 1). FIELD EXPERIMENTS Fig. 3. The number of Galerucella calmariensis/g. pusilla [adults (a); eggs (b), mean ± SE; ***, P < 0 001; **, P < 0 01; *, P < 0 05] on the host plant Lythrum salicaria when host plants were placed in either an open control area (open bar) or inside Ecological a thicket Society, [solid bar; thickets include Myrica gale, Alnus glutinosa, Hippophaë rhamnoides, Salix myrsinifolia-phylicifolia agg., and artificial thickets at Brännölandet (1) 17, and Jungfruholm (2)]. Data for the M. gale study are from Hambäck et al. (2000). The response by Galerucella was similar for all treatments (Fig. 3), although tests were hard to perform for adult numbers because of many double zeroes. For adults, numbers were reduced in artificial thickets in the northern area (Brännölandet, z = 2 91, N = 21, P < 0 01), but a similar trend was not significant for artificial thickets (z = 1 06, N = 8, P > 0 1), and was marginally significant for Hippophaë thickets (z = 1 63, N = 9, P = 0 1) in the southern area (Jungfruholm). At other sites, numbers were too few for a meaningful test. For eggs, numbers were reduced in Hippophaë thickets (z = 3 33, N = 18, P < 0 001); Alnus thickets (z = 2 20, N = 7, P < 0 05); Salix thickets (z = 4 27, N = 25, P < 0 001); and artificial thickets in both the northern (Brännölandet, z = 3 20, N = 26, P < 0 001) and southern area (Jungfruholm, z = 2 41, N = 20, P < 0 05). The response by N. marmoratus was different from the Galerucella response (Fig. 4), but again tests were hard to perform for adult numbers. For adults, numbers were higher in both Hippophaë (z = 2 10, N = 6, P < 0 05) and artificial thickets

5 91 Associational refuges and Galerucella (z = 2 10, N = 6, P < 0 05) in the southern area, while no difference was observed between treatments in the artificial thicket experiment in the northern area (Brännölandet, z = 0 10, N = 6, P > 0 1). The experiment at Brännölandet ended prematurely due to destruction by waves, and adults were therefore scored prior to flowering. Larval and pupal numbers of Nanophyes showed a pattern similar to adult numbers, where numbers were higher in Hippophaë thickets (z = 3 78, N = 19, P < 0 001) and in artificial thickets (z = 1 99, N = 14, P < 0 05) at Jungfruholm. Finally, flower numbers were higher in both Hippophaë thickets (z = 2 62, N = 21, P < 0 01) and artificial thickets (z = 2 50, N = 18, P < 0 05) at Jungfruholm, and the effect size was similar to that for Nanophyes larvae/pupae. Discussion This study shows that the associational refuge for L. salicaria is not specific to thickets of M. gale, but a similar refuge is provided by three other shrub species. The reduced herbivore abundance of the two leaffeeding beetles G. calmariensis and G. pusilla was Fig. 4. Number of Nanophyes marmoratus [adults (a); larvae/pupae (b), mean ± SE, ***, P < 0 001; **, P < 0 01; *, P < 0 05] on the host plant Lythrum salicaria, and flower 2003 number British (b) when host plants were placed in an open control area (open bar) or inside Ecological a thicket Society, [solid bar; thickets include Myrica gale, Hippophaë rhamnoides and artificial Functional thickets Ecology, at Brännölandet (1) and Jungfruholm (2)]. Data for the M. gale study are 17, from 87 93Hambäck et al. (2000). quantitatively similar for thickets with M. gale, A. glutinosa, H. rhamnoides, S. myrsinifolia-phylicifolia agg., and artificial thickets (Fig. 3). It is therefore also likely, even though this was measured only in three of six experiments, that the reduced herbivory translates into an increased reproductive output by L. salicaria. The probable reason for this generalistic associational refuge is that the two Galerucella species do not appear to use olfactory cues for locating undamaged Lythrum individuals, and are not repelled by odours emitted from M. gale (Fig. 2). It appears that the associational refuge for L. salicaria inside shrubby thickets is due to visual interference, an interference mechanism that is equally well provided by all shrub species investigated and by the artificial shrubs. The fact that olfactory cues are not used for primary host plant selection by G. calmariensis and G. pusilla suggests that undamaged hosts are located by Galerucella individuals mainly by a more-or-less random movement (Grevstad & Herzig 1997). Once a host plant has been located, and verified through gustatory stimuli, egg-laying commences. The presence of nonhost plants increases the movement rates and therefore also increases the probability of moving away from the host plant (similar to the reasoning of Kareiva & Odell 1987; Finch & Collier 2000). The resulting asymmetrical distribution of Galerucella individuals in and outside shrubs may then be reinforced through aggregation, occurring as a consequence of beetle attraction to damaged Lythrum individuals (P.A.H., J.P. and S. Al Abassi, unpublished results; see also Grevstad & Herzig 1997; Peng & Weiss 1992). The response by Galerucella individuals to non-host plant neighbours contrasts with that of N. marmoratus. Nanophyes showed dramatic increases in density inside shrubs (Fig. 4), and appeared to use olfactory cues to a greater extent than Galerucella individuals (Fig. 2). It is probable that the presence of non-host plant neighbours therefore has no effect on host-finding by Nanophyes, because they are better able than Galerucella to differentiate between undamaged host and non-host plants during the host-finding process. This lack of response would explain a zero difference on plants in and outside shrubby thickets, while the higher Nanophyes density inside thickets demands an additional explanation. The most likely mechanism is that the higher flowering frequency of L. salicaria in the thickets (Fig. 4), an effect caused by the reduced abundance of and herbivory by Galerucella adults and larvae (Hambäck et al. 2000), is a strong attractant to Nanophyes individuals. These mechanisms suggest that the development of a general theory on associational refuges which would enable us to predict when plant neighbours reduce herbivore numbers should take account of the herbivore host-finding process. This study suggests that reduced herbivory in the presence of non-host plant neighbours is mainly achieved for herbivore species that rely on visual or gustatory cues in the host-finding process.

6 92 P. A. Hambäck, J. Pettersson & L. Ericson However, the generality of this hypothesis is difficult to evaluate, as few previous studies have combined field measurements of herbivory rates in the presence and absence of non-host plant neighbours with observations of underlying behavioural mechanisms. An exception is Finch & Collier (2000), who found reduced herbivory by cabbage root flies (Delia radicum) in mixed croppings. By observing the egg-laying behaviour of individual root flies, they argued that egglaying was initiated only following a repeated number of contacts with host plants, and that the presence of non-host plants disturbed this process leading to rejection of an egg-laying site. Hence, a larger non-host plant density near cabbage plants would reduce egglaying by root flies. This response contrasts with that of diamondback moth (Plutella xylostella), which approaches host plants differently, and in this case intercropping is a less effective means of reducing attack rates on cabbage. It would be of interest to examine the host-finding ability and the mechanisms involved for a larger set of herbivore species, and to relate these to successes and failures in intercropping experiments. It is probable that this knowledge would aid in understanding the large variability observed in these studies (Andow 1991) and assist in the development of more effective intercropping systems. In a wider context, if generalistic associational refuges are more common than species-specific associational refuges, as suggested in this study, there will be consequences for the interpretation of recent studies relating plant diversity and productivity. Generalistic associational refuges through physical interference would cause herbivory to level off quickly with increasing plant diversity. Once a sufficiently large non-host plant neighbour density is established, it is unlikely that additional plant neighbours cause further reductions in herbivory. Hence, a productivity increase through reduced herbivory is also likely to level off at moderate plant diversities. At present few data are available to support this conclusion, but future studies that mix herbivore exclusions with plant diversity manipulations may provide important insights. It would be helpful if future studies on plant diversity effects on herbivore abundance and herbivory were accompanied by mechanistic studies. 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