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University of Joensuu, PhD Dissertations in Biology No:10 Leaf beetle feeding patterns on and variable plant quality in Betulaceous and Salicaceous hosts by Arsi Ikonen Joensuu 2001 Ikonen, Arsi Leaf beetle feeding patterns on and variable plant quality in Betulaceous and Salicaceous hosts. - University of Joensuu, 2001, 154 pp. University of Joensuu, PhD Dissertations in Biology, n:o 10. ISSN 1457-2486. ISBN 952-458-074-8 Keywords: Alnus, Betula, Populus, Salix, Agelastica, Chrysomela, Galerucella, Lochmaea, Phratora, Phytodecta, Plagiodera, chemical defence, chlorogenic acid, condensed tannins, feeding cue, herbivory, host race, nitrogen, performance trade-off, phenolic glucosides. Many European leaf beetle species have been recorded both on Betulaceous and Salicaceous host plants. Accordingly, Agelastica alni L. and Galerucella lineola F. were frequently found on hosts of both of these plant families, in eastern Finland. However, the host utilization strategies of these two leaf beetles are very different. For A. alni, alders (Alnus spp.) are the natural primary hosts, and certain willows (Salix spp.) are used as secondary hosts, especially in high population densities. A. alni individuals found on different plant families appear to be equally adapted to individual plant species. In contrast, G. lineola populations associated with Betulaceous and Salicaceous host plants differ in their behaviour and physiology. In fact, Betulaceae- and Salicaceae-associated G. lineola beetles may represent distinct host races. Since the hosts of alternative plant families seem to favour different physiological adaptations in beetles, further genetic differentation of the host races is likely. Another studied five leaf beetle species were mainly found on Salicaceous hosts, but in some cases they can obviously have analogous host races, as demonstrated with G. lineola. Host utilization patterns of studied leaf beetles are governed by secondary leaf phenolics, due to the high deterrent activity of many of these substances. Even an association with the phenolics of a certain structural group does not necessarily pre-adapt leaf beetles to all individual compounds in a particular group. Thus, willow phenolic glucosides strongly restrict the utilization of Salicaceous plants by A. alni, even though the leaves of its primary hosts, alders, also contain certain specific phenolic glucosides. A. alni feeds only on Salicaceous plants with a low level of phenolic glucosides in their leaves. Similarly, most other studied leaf beetles also appear to be sensitive to high amounts of willow glucosides. Moreover, leaves of certain phenolic glucoside-rich willows also contain high amounts of another deterrent phenolic, chlorogenic acid. The effects of these potent repellents, however, may depend on the background composition of other plant biochemicals. This indicates that the distribution of stimulants can also affect the food selection of leaf beetles. A lack of specific stimulants probably explains why A. alni do not even accept the deterrent-poor willows as well as alders. On the other hand, it is uncertain what those leaf traits are to which G. lineola beetles associated with the alternative host plant families respond differently to. However, the phenolic glucosides specific to plant families are probable candidates for this due to the generally high bioactivity of phenolic glucosides. At the intraspecific level of plants, among conspecific clones and within individual plants, leaf nutritive traits, such as nitrogen content, may have a decisive role as determinants of leaf quality for leaf beetles. In addition, the variation in abiotic environmental conditions may sometimes outweigh the effects of variable food quality on host utilization patterns of leaf beetles. Consequently, leaf beetles may, in some cases, even aggregate on low-quality host plants. Arsi Ikonen, Department of Biology, University of Joensuu, P.O. Box 111, FIN-80101 Joensuu, Finland CONTENTS LIST OF ORIGINAL PUBLICATIONS 1. INTRODUCTION 7 2. THE SYSTEM 9 2.1. Plants 9 2.2. Insect herbivores 9 3. GENERAL METHODOLOGY 10 4. PATTERNS IN LEAF CHEMISTRY 11 5. PATTERNS IN LEAF BEETLE HERBIVORY 13 5.1. Utilization of the host plants of two families by leaf beetles 13 5.2. Leaf chemicals as determinants of leaf quality 15 5.3. Plant hybridization and leaf beetle herbivory 17 5.4. The importance of variable food quality in relation to other factors 18 6. CONCLUDING REMARKS 19 ACKNOWLEDGEMENTS 20 REFERENCES 20 LIST OF ORIGINAL PUBLICATIONS This thesis is based on the following articles, referred to in the text by Roman numerals. I Ikonen, A., Sipura, M., Miettinen, S. and Tahvanainen, J.: Preference and performance patterns of alder- and willow-associated Galerucella lineola beetles. Submitted (Entomologia Experimentalis et Applicata). II Ikonen, A., Tahvanainen, J. and Roininen, H.: Phenolic secondary compounds as determinants of the host plant preferences of the leaf beetle, Agelastica alni. Submitted (Chemoecology). III Ikonen, A., Tahvanainen, J. and Roininen, H. 2001: Chlorogenic acid as an antiherbivore defence of willows against leaf beetles. Entomologia Experimentalis et Applicata 99: 47-54. IV Ikonen, A.: Preferences of six leaf beetle species among qualitatively different leaf age classes of three Salicaceous host species. Accepted (Chemoecology). V Sipura, M., Ikonen, A., Tahvanainen, J. and Roininen, H.: Why does the leaf beetle Galerucella lineola F. attack wetland willows? Submitted (Ecology). VI Hallgren, P., Ikonen, A., Hjältén, J. and Roininen, H.: Inheritance patterns of phenolics in F1, F2 and back-cross hybrids of willows: implications for herbivore responses to hybrid plants. Submitted (Journal of Chemical Ecology). Raupp & Sadof 1989; Baur et al. 1991; Seldal et al. 1994; Mutikainen et al. 1996). Damaged plant individuals can induce this kind of change even in their undamaged neighbours (Dolch & Tscharntke 2000). Thus, increasing population densities of insect herbivores are likely to lead to inhibitory feedback-loops through decreased resource quality. For herbivorous insects, the quality of plant tissues for food depends mainly on the concentrations of essential nutrients and defensive secondary compounds (Tahvanainen et al. 1985; Bryant et al. 1987; Lindroth et al. 1988; Kelly & Curry 1991; Clancy 1992; Rank 1992; Matsuki & MacLean 1994; Hemming & Lindroth 1995; Van Dam et al. 1995; Stamp & Yang 1996; Orians et al. 1997; Kause et al. 1999). Nitrogen, in particularly, seems to be generally scarce and a limiting nutrient for herbivorous insects, since plant tissues contain remarkably less nitrogen than animal tissues (Feeny 1970; McNeill & Southwood 1978; Mattson 1980). In concordance, an increase in diet nitrogen content has been frequently found to result in improved insect herbivore performance (Tabashnik 1982; Bryant et al. 1987; Lindroth et al. 1997; Ritchie 2000). Consequently, insect herbivores are expected to evolve behavioural mechanisms to find and utilize plant tissues with optimal nitrogen content (McNeill & Southwood 1978). For example, herbivorous insects can use amino acids as feeding stimulants (Hsiao & Fraenkel 1968). In contrast, secondary plant compounds often inhibit growth and the development of insects and can even cause mortality (Todd et al. 1971; Isman & Duffey 1982; Manuwoto & Scriber 1986; Bryant et al. 1987; Lindroth et al. 1988; Kelly & Curry 1991; Matsuki & MacLean 1994; Hemming & Lindroth 1995; Ayres et 1. INTRODUCTION Outbreaks of herbivorous insects (e.g. Barbosa & Schultz 1987; Mattson & Haack 1987; Tikkanen et al. 1998) indicate that the greenness of nature is not a self-evident fact. Due to their high reproductive potential and diversity, herbivorous insects could probably exterminate green plants in the absence of any limiting factors. Normally, insect herbivore populations, however, seem to be strongly limited by the resource-based bottom-up forces (Hunter & Price 1992; Roininen et al. 1996; Ritchie 2000). Apparently insect herbivores can only properly adapt to a restricted range of qualitative variation in plants, since an increase in adaptation to a certain type of food entails a decreased adaptation to alternative resources (Gould 1979; Fry 1990; Jaenike 1990; Via 1991; Joshi & Thompson 1995). Thus, herbivorous insects tend to be more or less specific in their host utilization (Smiley 1978; Bernays & Graham 1988; Roininen & Tahvanainen 1989; Jaenike 1990; Kolehmainen et al. 1994; Baur & Rank 1996; Roininen et al. 1999). Even a relatively small inter- and intra-specific qualitative variation in plants usually has a great effect on the behaviour and physiology of insect herbivores (Larsson et al. 1986; Matsuda & Senbo 1986; Bryant et al. 1987; Meyer & Montgomery 1987; Kelly & Curry 1991; Bingaman & Hart 1993; Matsuki & MacLean 1994; Hemming & Lindroth 1995; Van Dam et al. 1995; Orians et al. 1997). Consequently, herbivorous insects have qualitatively adequate food available only in limited amounts and for limited times. Furthermore, insect feeding on the basically high-quality host plants may cause the qualitative detorioration of these plants (Raupp & Denno 1984; Haukioja & Hanhimäki 1985; Neuvonen et al. 1987; 7 reduce the relative disadvantage gained from feeding on poor-quality hosts (see also Feder 1995; Feder et al. 1995; Rossi et al. 1999). If herbivorous insects obtain an ’enemy-free space’ on low-quality hosts, these hosts may even be the most heavily attacked ones (Damman 1987; Thompson & Pellmyr 1991). On the other hand, because of the physiological constraints of insects the variation in abiotic environment, in microclimatic conditions for example, can also have a pronounced effect on the host utilization patterns of herbivorous insects in the field (Willmer et al. 1996; Larsson et al. 1997). In some cases, variation in abiotic factors may outweigh the effect of food quality and lead to an even higher insect herbivore load on lowquality hosts (Sipura 2000; Sipura & Tahvanainen 2000). Overall, top-down forces by natural enemies and abiotic factors are likely to interact with the resource-based bottom-up forces in the regulation of natural insect herbivore populations (Williams 1983; Hunter & Price 1992; Ritchie 2000). This thesis mainly focuses on the consequences of variable food quality on the feeding patterns of several Scandinavian leaf beetle species (Coleoptera: Chrysomelidae). Natural host plants of the studied leaf beetles belong to the Betulaceae and Salicaceae families. Firstly, it is asked if plants of these two families require and favour different behavioural and metabolic adaptations in leaf beetles, and if an increase in adaptation to one host plant family entails a reduction in adaptation to another host family (I, II). Results obtained in laboratory experiments are discussed in relation to natural host utilization patterns of the studied species of leaf beetles. Secondly, it is examined which traits determine leaf quality for the leaf beetles (II-V). To answer this, both al. 1997). Secondary plant substances also frequently act at the behavioural level of insects as deterrents and feeding inhibitors (Kraft & Denno 1982; Matsuda & Senbo 1986; Kelly & Curry 1991; Mori et al. 1992; Gross & Hilker 1994/1995; Van Dam et al. 1995). Overall, many insect herbivores may continuosly be faced with an optimization problem of how to make a compromise with the intake of essential nutrients and harmful secondary compounds (see Rhoades & Cates 1976; Cates 1980; Meyer & Montgomery 1987; Van Dam et al. 1995). However, there are also well-adapted and highly specialized insect herbivores for which the effects of specific secondary compounds of their natural main hosts are either neutral or positive (Kraft & Denno 1982; RowellRahier & Pasteels 1986; Denno et al. 1990; Rank 1992; Kolehmainen et al. 1994; Matsuki & MacLean 1994; Rank 1994; Soetens & Pasteels 1994; Gross & Hilker 1994/1995; Van Dam et al. 1995; Van der Meijden 1996; Orians et al. 1997; Roininen et al. 1999). Apart from plant quality, a herbivorous insect also encounters abiotic conditions and natural enemies, parasites and predators, on its host plant (Hunter & Price 1992). It is known that natural enemies can remarkably decrease survival and population densities of herbivorous insects and, consequently, herbivoryinduced damages on the host plants (Häggström & Larsson 1995; Sipura 1999, 2001a). When herbivorous insects aggregate on high-quality hosts and when their natural enemies act in a densitydependent manner, enemy-induced mortality of a herbivore may be higher on the high-quality hosts (Sipura 2001b). This means that feedback by natural enemies can equalize insect herbivore densities on the qualitatively different host plants and 8 especially economically important source of raw material for plywood and paper industry (Lines 1984; Oksanen 1987). As fast-growing plants, alders and many Salicaceous species can be grown in shortrotation forestry as a renewable source of energy (Salmi 1993; Kendall et al. 1996). preference and performance experiments with chemically analysed plant material and preference studies with pure secondary compounds were conducted in laboratory. A study with parental willow species and their four hybrid categories tries to reveal the inheritance patterns of main leaf secondary compounds of these plants (VI). Patterns in leaf chemistry are compared to plant susceptibility for a natural community of several leaf beetle species. In addition, one of the articles explores the importance of variable plant quality in relation to topdown and abiotic factors as determinants of the field distribution pattern of Galerucella lineola F. (V). 2.2. Insect herbivores Betulaceous and Salicaceous plants in northern Europe harbour a diverse community of leaf-eating insects (Kreslavsky et al. 1981; Shaw 1984; Dodge et al. 1990; Soetens et al. 1991; Koch 1992; Hanhimäki et al. 1994; Chinery 1997; Rousi et al. 1997; Tikkanen et al. 1998; Kause et al. 1999; Nyman 2000; Sipura 2000). One of the most prominent groups among them are leaf beetles. Many European leaf beetle species have been found to feed both on Betulaceous and Salicaceous plants (Tischler 1977; Kreslavsky et al. 1981; Dodge et al. 1990; Koch 1992; Palokangas & Neuvonen 1992; Seldal et al. 1994; Gross & Hilker 1994/1995; Baur & Rank 1996). In some of these species conspecific beetles associated with different host plant families seem to be separate host races (Kreslavsky et al. 1981; see also Gross & Hilker 1994/1995). However, there are also species of leaf beetles that only utilize the hosts of one of these two plant families (Koch 1992). All these leaf beetles are univoltine insects which overwinter as adults (see Baur & Rank 1996; Kendall et al. 1996; Häggström 1997; Kendall & Wiltshire 1998; Sipura 2000). The overwintered adults colonize their host plants in the spring at budbreak, after which they mate and feed on host plant leaves for a few weeks before oviposition. The eggs laid on the leaves hatch within a few weeks. Larvae also feed on the leaves of the same 2. THE SYSTEM 2.1. Plants In northern Europe, Betulaceous plants, alders (Alnus spp.) and birches (Betula spp.), and Salicaceous plants, aspen (Populus tremula L.) and willows (Salix spp.), often naturally grow together, especially in early successional habitats. All these plants are deciduous trees or shrubs. The taxonomy of these plants is complicated by the fact that in both plant families con-generic pure species may form inter-specific hybrids (e.g. Hanhimäki et al. 1994; Hjältén 1997). In both families, plant leaves contain phenolic secondary substances such as cinnamic acid derivatives, condensed tannins, flavonoids and phenolic glucosides, but only few individual compounds are recorded both in Betulaceous and Salicaceous plants (Matsuda & Matsuo 1985; Julkunen-Tiitto 1986, 1989; Daniere et al. 1991; Mori et al. 1992; Suomela et al. 1995; Ossipov et al. 1996; Keinänen & Julkunen-Tiitto 1998; Rank et al. 1998; Kause et al. 1999). Among Betulaceous plants, birches are an 9 young G. lineola adults from separate laboratory cultures and naturally overwintered adults of several populations both on Betulaceous and Salicaceous species, it is examined to see if behavioural differences of conspecific beetles result from differences in genetics, earlier host experience or both (I). The performance of naive larvae of four G. lineola populations both on Betulaceous and Salicaceous hosts was studied in order to find out if conspecific beetles associated with the different host plant families are differently physiologically adapted. Laboratory experiments were also conducted to study what leaf traits are crucial in determining plant quality for leaf beetles (II-V). In order to see if plant secondary phenolics govern the host utilization of A. alni, the effects of five pure phenolic secondary compounds on its behaviour were tested in 2-choice bioassays with naturally overwintered adults (II). For the bioassays, all A. alni adults were collected from A. incana, and the beetles were also kept on their original hosts in laboratory prior to experiments. Test compounds and the used concentrations were selected on the basis of secondary chemical analyses of the potential host plants (see below). Test solutions were applied onto natural substrates, host plant leaves. Leaves treated with the solvent were used as controls. The effects of chlorogenic acid were also tested for G. lineola, L. capreae, Ph. polaris and P. versicolora (III). In addition, the preference tests among the chemically screened plant species (II, III), conspecific leaf age classes (IV) and willow clones (V) were done with naturally overwintered adult beetles of several species. In article V, the larval performance of G. lineola on chemically analysed Salix phylicifolia L. clones was also measured. The plant material for all plants. As they are rather poor dispersers, adult host choice largely determines larval food plant. After the larval period, fullygrown larvae move to the soil beneath the host plant in order to pupate, but some species may pupate on the shoots or stems of the host (see Tischler 1977; Baur & Rank 1996; Häggström 1997; Sipura 2000). Adults of the new generation emerge in early autumn after a short pupal stage. Under certain circumstances, leaf beetles can cause severe defoliation that entails stunted plant growth and the death of young shoots (Kendall et al. 1996). 3. GENERAL METHODOLOGY This thesis is based both on experimental and observational data (I-VI). Since the main interest was the effect of variable food quality on leaf beetles, the majority of the experiments were done in a laboratory where it is possible to standardize all other factors that may affect leaf beetle behaviour and performance. In the laboratory experiments, adults and larvae belonging to seven species of leaf beetles were used: Agelastica alni L., Chrysomela populi L., Galerucella lineola F., Lochmaea capreae L., Phratora polaris Schneid., Phytodecta rufipes Geer and Plagiodera versicolora Laich. Plant species used in the laboratory experiments were: Alnus incana (L.) Moench, A. glutinosa (L.) Gaertner, B. pendula Ehrh,, P. tremula L., Salix caprea L., S. cinerea L., S. lapponum L., S. myrsinifolia Salisb., S. pentandra L. and S. phylicifolia L. As A. alni and G. lineola were rather frequently found both on Betulaceous and Salicaceous host plants even in sympatry, it was examined if adults of conspecific populations naturally associated with different host plant families behave differently in standardized host selection experiments (I, II). By rearing 10 comparing their retention times and UV-vis spectra to those of authentic compounds. Polymeric phenolics, condensed tannins, were analysed with a colourimetric Butanol-HCl assay (II-VI). The total nitrogen concentrations of leaves were measured from subsamples of powdered leaves with an automatic CHN-analyzer (IV, V). Water content (V) was calculated based on the weight difference between fresh and dried leaves. laboratory experiments was collected from naturally growing plants. Because of the possibility of intra-specific hybridization (e.g. Hanhimäki et al. 1994; Hjältén 1997), only characteristic individuals of each plant species were used. In article VI, the seeds of two parental willow species, S. caprea and S. repens L., as well as their four hybrid categories were produced by handpollination. Subsequently, potted clones were exposed to the feeding of the natural communities of several leaf beetle species and other leaf-eating insects in the experimental field. After the experiment, it was possible to separately determine leaf damages caused by the leaf beetles and other herbivores. As leaf samples of all the 193 study plants were individually screened, it was possible to relate the patterns in herbivore feeding to the patterns in the leaf chemistry. In order to examine how well plant quality measured in laboratory predicts insect distribution in the field, field surveys were conducted in articles II and V. Field observations and experiments were also used to evaluate the importance of abiotic factors and natural enemies, along with variable food quality, as determinants of the field distribution of G. lineola (V). Most studies were done in eastern Finland in the city of Joensuu or nearby (I-V), but the field experiment of a hybrid study was conducted in Umeå in north-eastern Sweden (VI). The leaf samples used in the chemical analyses were firstly dried and ground to a fine powder in a plant mill (IIVI). Extraction of phenolics from the powdered leaf samples was done as described below (III). High performance liquid chromatography (HPLC) was used to analyze low-molecular weight phenolics in leaf extracts (II-VI). Individual compounds in chromatograms were identified by 4. PATTERNS IN LEAF CHEMISTRY Both Betulaceous A. incana and several Salicaceous plant species studied contained low-molecular weight phenolics such as cinnamic acid derivatives, flavonoids, phenolic glucosides and phenolic polymers such as condensed tannins in their leaves (II-VI). In relation to the generally rather restricted distribution of phenolic glucosides in the plant kingdom, it is remarkable that both Betulaceous and Salicaceous plants contain phenolic glucosides in their leaves. Earlier studies corroborate this observation (JulkunenTiitto 1986, 1989; Kelly & Curry 1991; Mori et al. 1992; Bingaman & Hart 1993; Keinänen & Julkunen-Tiitto 1998; Rank et al. 1998; Mutikainen et al. 2000). Apart from A. incana, phenolic glucosides can also be found in the leaves of the following Scandinavian Betulaceous plant species: A. glutinosa, B. pendula and Betula pubescens Ehrh. (Keinänen & Julkunen-Tiitto 1998; Mutikainen et al. 2000; Ikonen unpubl. data). Structurally, phenolic glucosides can be divided into two main categories (e.g. Julkunen-Tiitto 1989). Salicylates are derivatives of salicin in which a variable number of structural groups is linked to the salicin core of molecules. In contrast, nonsalicylic phenolic glucosides are a structurally more heterogenous group. The 11 and condensed tannins are produced in a shikimate pathway, but in different branches of the pathway (e.g. Lavola 1998; Keinänen et al. 1999). The common precursor of condensed tannins and phenolic glucosides is cinnamate. Thus, if a plant allocates a lot of cinnamate for the production of phenolic glucosides, only a small amount of this precursor is available for production of condensed tannins in another branch of the pathway and vice versa. The trade-off between the content of condensed tannins and phenolic glucosides is also obvious among the different leaf age classes of those Salicaceous plants that produce glucosides in detectable amounts (IV). The highest phenolic glucoside content and the lowest level of condensed tannins are found in young leaves. Young leaves also contain more nitrogen. Leaves of a tea-leafed willow, S. phylicifolia, are characterized by the presence of a low-molecular weight flavoinoid, ampelopsin, in very high amounts, whereas other simple phenolics can be found only in small amounts (II, V). In addition, leaves of S. phylicifolia contain high-molecular weight condensed tannins. In article V, S. phylicifolia clones grown in three habitats along a soil moisture gradient (wetland, flood zone and dry zone) differed significantly in their leaf chemistry. Namely, leaves of wetland willows contained less nitrogen and water but more secondary phenolics than leaves of clones growing in drier habitats. In general, clonespecific variation in leaf chemistry may be due to variable plant genetics, environmental conditions, or both (Larsson et al. 1986; Bryant et al. 1987; Rousi et al. 1997; Hakulinen 1998; Mutikainen et al. 2000). As the studied S. phylicifolia clones (V) were naturally growing plants, it is impossible to surely say whether their variable susceptibility results from some of common character for all of them is that a molecule contains one phenolic ring which is, in various ways, linked to the glucose moiety. Both types of glucosides can be found in leaves of Salicaceous plants, but Betulaceous plants appear to contain only certain non-salicylic phenolic glucosides (Julkunen-Tiitto 1986, 1989; Mori et al. 1992; Keinänen & Julkunen-Tiitto 1998; Mutikainen et al. 2000; II; Ikonen unpubl. data). Most individual glucosides seem to be restricted either to the leaves of Betulaceous or then Salicaceous plants. Data from leaf chemistry of different willow hybrid categories and parental species indicate that the levels of the main secondary compounds, condensed tannins and salicylic phenolic glucosides, are additively inherited (VI). The F1 and F2 hybrid forms were intermediate to pure parental species and back-crosses were intermediate to the respective parental species and F1 and F2 hybrid forms. Earlier, Orians et al. (2000) have reported that in another complex of two pure willow species and their F1 hybrids condensed tannin content of hybrid leaves is also intermediate to parental species. However, phenolic glucosides were in this case below the midpoint of parental species in hybrid leaves indicating their dominant inheritance. Moreover, our data from hybrid willows show that leaf concentrations of phenolic glucosides and condensed tannins correlate negatively among the plant individuals of all categories in which a considerable fraction of individual plants contain detectable amounts of glucosides in their leaves (VI). In concordance, Julkunen-Tiitto (1989) and Orians et al. (2000) have earlier reported negative correlations of leaf condensed tannin and phenolic glucoside contents in willows. This kind of trade-off may result from the fact that both phenolic glucosides 12 Koch 1992; Palokangas & Neuvonen 1992; Seldal et al. 1994; Gross & Hilker 1994/1995; Baur & Rank 1996). In concordance, A. alni and G. lineola were found on hosts of both of these plant families in the main study area in eastern Finland (I; II). The most frequently used hosts for both beetles appear to be A. incana, A. glutinosa and S. phylicifolia, but birches (Betula spp.) are also occassionally utilized by them. Host utilization strategies of A. alni and G. lineola in a BetulaceaeSalicaceae host plant complex seem to be, however, totally different. In standardized laboratory feeding trials, both Betulaceaeand Salicaceae-associated sympatric A. alni beetles preferred grey alder, A. incana, over tea-leafed willow, S. phylicifolia (II). Thus, in A. alni no indication of intra-specific host-associative differentation or conditioning on alternative hosts was found. For A. alni, birches and willows appear to be secondary hosts which are especially used in times of high population densities (II; see also Tischler 1977; Baur & Rank 1996). This may be linked to the fact that A. alni is sensitive to herbivoryinduced detorioration of alder leaves (Baur et al. 1991; Baur & Rank 1996; Dolch & Tscharntke 2000; see also Seldal et al. 1994). If a host race of a herbivorous insect species is defined as a population that is partially reproductively isolated from other conspecific populations because of its hostspecific adaptations (see Jaenike 1981; Diehl & Bush 1984; Prokopy et al. 1988; Craig et al. 1993), Betulaceae- and Salicaceae-associated G. lineola beetles seem to represent separate host races (I). Firstly, Salicaceae-associated G. lineola populations accept only willows as their hosts in standardized laboratory feeding trials, whereas conspecific Betulaceaeassociated populations also accept the above factors or from all of them. However, since S. phylicifolia as a terrestrial plant species has no clear adaptations to grow subemerged, it is likely that differences between the clones of three moisture zones are largely due to the floodstress of willows in wet habitats. Moreover, as both ampelopsin and condensed tannins were at the highest level in the leaves of the same clones (V), in S. phylicifolia there did not appear to be a similar trade-off between the production of low- and high-molecular weight phenolics as found in other, phenolic glucoside-producing willow species (VI). Accordingly, data from different leaf age classes suggest that young leaves of S. phylicifolia contain both higher concentrations of ampelopsin (unpubl. data) and condensed tannins (IV). Young S. phylicifolia leaves also contain more nitrogen than conspecific older leaves suggesting that decreasing concentrations of nitrogen with increasing leaf age is a rather universal pattern in higher plants (Feeny 1970; Raupp & Denno 1983; Langenheim et al. 1986; Julkunen-Tiitto 1989; Denno et al. 1990; Van Dam et al. 1995; IV). 5. PATTERNS IN LEAF BEETLE HERBIVORY 5.1. Utilization of the host plants of two families by leaf beetles Herbivorous insects generally tend to be more or less host-specific (Smiley 1978; Bernays & Graham 1988; Roininen & Tahvanainen 1989; Jaenike 1990; Kolehmainen et al. 1994; Baur & Rank 1996; Roininen et al. 1999). Nevertheless, many European species of leaf beetles feeding on deciduous woody plants utilize both Betulaceous and Salicaceous hosts (Tischler 1977; Kreslavsky et al. 1981; 13 but successfully demonstrated in only a few cases (Gould 1979; Hare & Kennedy 1986; Fry 1990; Jaenike 1990; Via 1991; Fox 1993; Joshi & Thompson 1995; Lazarevic et al. 1998). Thus, the evidence for performance trade-offs found in G. lineola (I) is remarkable. In general, performance trade-offs would be a potentially powerful mechanistic explanation for the specialization of insect herbivores, since in order to maximize their performance insects should specialize. However, Salicaceous plants appear to be superior hosts for even the most Betulaceae-adapted G. lineola beetles (I). This result is in line with the increasing evidence that in many cases modifications in insect host plant utilization may take place primarily at the behavioral level, whereas specific metabolic adaptations of insects are consequences rather than causes of the host shifts (Smiley 1978; Futuyama 1984; Prokopy et al. 1988; Roininen & Tahvanainen 1989). Consequently, even shifts onto poor-quality host plants appear to be possible if there exists some factor that balances the negative effect of lowquality food on an insect’s overall fitness (see Feder 1995; Feder et al. 1995; Rossi et al. 1999). In G. lineola, the fundamental factor driving host-race formation may be the patchy distribution of the original main host, S. phylicifolia. Since G. lineola is, because of its phylogenetic constraints (see below), restricted to the microclimate of shoreline habitats, it has to utilize those plants it can find in its preferred habitat. If S. phylicifolia is locally rare or absent, it may be possible for G. lineola to gain the highest overall fitness by readily accepting alternative host species irrespective of their poor quality. Accordingly, Betulaceaeassociated G. lineola populations seem to be most frequently found at sites where willows are rare or absent (I). Betulaceous plants or prefer them (I). Even the sympatric G. lineola populations associated with the hosts of different plant families differ significantly in their feeding behaviour. Moreover, these behavioural differences of G. lineola populations seem to have an underlying genetic basis. In the field, behavioural differences of conspecific insects are likely to lead to reproductive isolation even in sympatry, since matings in nature are most probable between insect individuals sharing equal host preferences (Craig et al. 1993; see also Kreslavsky et al. 1981). However, in G. lineola this behaviour-based pre-zygotic reproductive isolation appears to be incomplete since Betulaceae-associated populations usually also accept Salicaceous host plants (I). Moreover, G. lineola beetles can be conditioned to ’wrong’ hosts if forced to feed on them. This is in line with other findings which indicate that the association of an individual insect with a certain plant species may increase its probability to be attacked in future encounters (e.g. Prokopy et al. 1982). On the other hand, G. lineola beetles were found to be physiologically better adapted to their respective hosts than their conspecifics from other host species, and increasing adaptation to a certain host appears to entail a decreased adaptation to other hosts (I). This means that Betulaceous and Salicaceous host plants seem to favour and require different adaptations in G. lineola. Thus, natural selection may largely eliminate those G. lineola genotypes from a certain host species which are successful and abundant on alternative hosts possibly producing a post-zygotic reproductively isolating mechanism. Performance trade-offs, the negative genetic correlations of insect performance on the alternative host species, have been recently frequently sought out 14 and S. pentandra, with high leaf phenolic glucoside content. Clearly, association with non-salicylic alder phenolic glucosides have not entailed in A. alni a general behavioural tolerance for willow phenolic glucosides. These results support earlier findings that willows with high phenolic glucoside content are usually avoided by the insect herbivores which are not well adapted to willows (Rowell-Rahier 1984; Orians et al. 1997). Moreover, the fact that three other leaf beetle species, L. capreae, Ph. polaris and P. versicolora, avoided high-glucoside S. myrsinifolia (III) indicates that they are also sensitive to phenolic glucosides in high amounts (see also Tahvanainen et al. 1985; Palokangas & Neuvonen 1992; Kendall et al. 1996). On the other hand, some willow species with a high leaf phenolic glucoside level also contain chlorogenic acid in high amounts in their leaves (Rank et al. 1998; II; III). In bioassays, chlorogenic acid particularly deterred the feeding of A. alni and L. capreae (II; III). Earlier, Matsuda & Senbo (1986) have demonstrated a deterrent effect of chlorogenic acid on several Japanese leaf beetles. Overall, the high resistance of S. myrsinifolia and S. pentandra against certain leaf beetles seems not to result solely from their high leaf salicylate content (II; III). Apart from their inhibitory effects on behaviour, both chlorogenic acid and phenolic glucosides may negatively affect the performance of non-adapted insects (Todd et al. 1971; Isman & Duffey 1982; Lindroth et al. 1988; Kelly & Curry 1991; Hemming & Lindroth 1995; Stamp & Yang 1996). In contrast, condensed tannins may generally be rather ineffective at the behavioural level of leaf beetles, since even A. alni primarily associated with low-tannin hosts, alders, was not deterred by low-molecular weight basic structural unit of condensed tannis, (+)-catechin (II). In my study area, Ch. populi, L. capreae and Ph. polaris seem to be restricted to Salicaceous plants, even though they have been recorded elsewhere in Europe to live also on Betulaceous host plants (Kreslavsky et al. 1981; Koch 1992; Palokangas & Neuvonen 1992). It is likely, that these three leaf beetle species also have Betulaceae-associated races (Kreslavsky et al. 1981; see also Gross & Hilker 1994/1995) as found here in G. lineola (I). However, the conditions favouring the formation of Betulaceae-associated races in Ch. populi, L. capreae and Ph. polaris may be rather specific and rarely occur, since the majority of their populations obviously lives on Salicaceous plants (see Matsuda & Senbo 1986; Dodge et al. 1990; Augustin et al. 1993; Köpf et al. 1996; Chinery 1997; III; IV). Among the leaf beetles studied in this thesis, only Ph. rufipes and P. versicolora seem to be completely restricted to Salicaceous plants (see Raupp 1985; Jones & Coleman 1988; Raupp & Sadof 1989; Dodge et al. 1990; Soetens et al. 1991; Koch, 1992; Orians et al. 1997; III; IV). 5.2. Leaf chemicals as determinants of leaf quality In concordance with earlier studies (Tahvanainen et al. 1985; Kelly & Curry 1991), present results suggest that leaf beetle herbivory on Salicaceous plants is strongly limited by their characteristic lowmolecular weight secondary compounds, phenolic glucosides. Even though leaves of the natural primary hosts of A. alni, alders, contain their specific non-salicylic phenolic glucosides, both salicylic and non-salicylic willow phenolic glucosides deterred this leaf beetle from feeding (II). In feeding preference experiments, A. alni also avoided two willow species, S. myrsinifolia 15 living on salicylate-rich Salicaceous plants (Rank 1992; Soetens & Pasteels 1994; Kolehmainen et al. 1995; see also Smiley et al. 1985). Consequently, the preference of Ch. populi and Ph. rufipes for young leaves of their host plants (IV) may be partially due to the higher content of salicylates in younger rather than in conspecific old leaves. Experiments with chlorogenic acid and A. alni (II) lend support to earlier studies which suggest that effects of individual secondary substances on insect herbivores may depend on the background variation of other plant chemicals (Renvick & Radke 1987; Soetens & Pasteels 1994). Namely, for A. alni chlorogenic acid was a deterrent only on the leaves of the secondary host, S. phylicifolia, while the effect on the leaves of the primary host, A. incana, was neglible (II). Moreover, current data show that A. alni prefers grey alder, A. incana, even over the willow species that contain little feeding deterrents in their leaves (II). Overall, leaves of A. incana seem to contain some very specific and strong stimulants for A. alni. These stimulants may obviously outweigh even the effects of potent deterrents. However, these stimulants have not yet been identified. It is not known which leaf traits Betulaceae- and Salicaceae-associated G. lineola beetles respond differently to (I). For another leaf beetle, Chrysomela lapponica L., a salicylic phenolic glucoside, salicin, is a deterrent for beetles associated with Betulaceous plants, whereas for Salicaceae-associated conspecifics salicin acts as a stimulant (Gross & Hilker 1994/1995). Salicin is present only in the host plants of the Salicaceae-associated Ch. lapponica beetles. Analogously, it is possible that G. lineola populations associated with Whether or not condensed tannins have effects on performance of the studied leaf beetles remains an unsolved question. In general, condensed tannins are known as potential inhibitors of insect herbivore performance (Manuwoto & Scriber 1986; Bryant et al. 1987; Ayres et al. 1997). Extremely abundant flavonoid ampelopsin in S. phylicifolia leaves probably has no strong antiherbivore activity even against A. alni, since S. phylicifolia is rather palatable for A. alni (II) and is also suitable for complete larval development (Baur & Rank 1996). A. alni also prefers young S. phylicifolia with higher ampelopsin content than in conspecific older leaves (IV). Among the experimentally studied leaf beetle species (I-V), Ch. populi and Ph. rufipes are species which live on Salicaceous plants with high contents of salicylate-type phenolic glucosides (IV). In general, many leaf beetle species living on salicylate-rich willows and poplars are known to use host plant salicylates as precursors to their own defensive larval secretion (Rowell-Rahier & Pasteels 1982; Pasteels et al. 1983; Smiley et al. 1985; Rank 1994). In contrast, non-salicylic phenolic glucosides are inappropriate precursors for the production of salicylaldehyde-containing defensive larval secretion. The secretion of Ch. populi larvae also contains salicylaldehyde (Pasteels et al. 1983), but larval defenses of Ph. rufipes appear to be unknown. In any case, in salicylate-using leaf beetles the amount of larval secretion and, therefore, the effectiviness of larval defense positively correlates with the amount of its precursors in the food (Rowell-Rahier & Pasteels 1982; Pasteels et al. 1983; Smiley et al. 1985; Denno et al. 1990; Rank 1994). Following evolutionary logic, it should not be surprising that salicylates are found to stimulate the feeding of many leaf beetles 16 Moreover, experiments with differently aged leaves of three Salicaceous hosts indicate that young leaves with high nitrogen content and low toughness tend to be generally preferred over less nitrogenous and tougher older leaves (IV). The preference for young leaves is true irrespective of whether these young leaves contain more or less condensed tannins than conspecific older leaves. Moreover, both salicylate-using leaf beetle species and those generally sensitive to high amounts of phenolic glucosides preferred young leaves of glucoside-rich S. pentandra, even though young leaves contain more glucosides than older leaves. This result contradicts the observations made of several other leafeating insects. Many herbivorous insects minimize the intake of harmful lowmolecular weight secondary compounds rich in young leaves by selecting the older rather than younger leaves of their hosts (Rhoades & Cates 1976; Cates 1980; Meyer & Montgomery 1987; Van Dam et al. 1995). Obviously, even some of the studied leaf beetle species living on glucoside-poor willows (IV) have, however, to some degree adapted to these typical secondary substances of willow leaves. This view is supported by observations that in small concentrations willow phenolic glucosides act as feeding stimulants even for certain leaf beetles associated with low-glucoside willows (Kolehmainen et al. 1995). different host plant families in the study area (I) are differently adapted to those non-salicylic phenolic glucosides which are specific to plant families (see above). Basically, phenolic glucosides are potent regulators of host selection behaviour in G. lineola (Tahvanainen et al. 1985; Kolehmainen et al. 1995), and Betulaceaespecific non-salicylic phenolic glucosides are known to be potent deterrents for insect herbivores (Mori et al. 1992). Furthermore, Lindroth & Weisbrod (1991) have in a gypsy moth, Lymantria dispar L., demonstrated intra-specific genetic variation in insect ability to metabolize aspen phenolic glucosides. Similarly, it is also possible that G. lineola populations associated with Betulaceous and Salicaceous host species are differently adapted in their physiology to Betulaceaespecific non-salicylic phenolic glucosides (I). This could partially explain the tradeoff found in the survival of naive larvae of four G. lineola populations on two host species, A. incana and S. phylicifolia. At the intra-specific level of plants, among conspecific clones and within individual plants, leaf quality variation for leaf beetles may be more closely related to the variation in leaf nutritive traits than to secondary chemistry (IV; V). This is not surprising since the leaf secondary chemical variation at intra-specific level of plants is less dramatic than among the different plant species. In article V, leaf nitrogen content seems to explain host preferences for adult G. lineola beetles among 60 S. phylicifolia clones growing in three habitats along a soil moisture gradient. Larval performance was most clearly related to leaf water content, whereas the concentrations of main secondary compounds, ampelopsin, (+)catechin and condensed tannins, were not related to any performance parameter. 5.3. Plant hybridization and leaf beetle herbivory Because of the potentially large role of inter-specific plant hybridization both in the evolution of plants and associated insect herbivores, there has recently been increased scientific interest in insect responses to hybrid plants (Soetens et al. 17 caprea), a high-glucoside species (S. repens) and their inter-specific F1 hybrids with intermediate leaf glucoside content did not differ in their total leaf beetle damage or total leaf herbivory. On the study site there were both non-adapted (L. capreae, Ph. polaris and Phratora vulgatissima L.) and glucoside-adapted (Phratora vitellinae L.) leaf beetle species (see Rowell-Rahier & Pasteels 1982; Tahvanainen et al. 1985; Kelly & Curry 1991; Palokangas & Neuvonen 1992; Kendall et al. 1996). Possibly, decreased feeding by non-adapted insect species on plants with high leaf glucoside content was balanced by increased feeding of well-adapted specialists (V). On the other hand, the complex willow hybrids, F2 and back-cross types, were, in the field, more heavily attacked by leaf beetles and other leafeating herbivores than F1 hybrids and two pure parental species. As the susceptibility of F1 hybrids for herbivores did not differ from that of the parental species, the high susceptibility of complex hybrids is difficult to explain mechanistically. Maybe the inheritance patterns of other leaf traits than phenolic glucosides and condensed tannins should also be known. Regardless of this, current results indicate a natural selection against complex willow hybrids provided that leaf herbivory is a significant determinant of plant overall fitness. Thus, leaf-eating herbivores such as leaf beetles may be partially responsible for maintaining a high richness of species in the plant kingdom. 1991; Hanhimäki et al. 1994; Häggström & Larsson 1995; Fritz et al. 1996; Kendall et al. 1996; Hjältén 1997; Häggström 1997; Orians & Floyd 1997; Orians et al. 1997). These studies have formulated many alternative hypotheses to herbivore responses to hybrid plants: 1) the hybrid susceptibility hypothesis predicts that hybrids are attacked more intensively than either of the parental species, 2) the hybrid resistance hypothesis suggests that hybrids have lower herbivore load than the parental species, 3) the additive hypothesis predicts that hybrid susceptility is intermediate of parental species, 4) the dominance hypothesis states that herbivory loads on hybrids and one of the parental species are equal and 5) the no-difference hypothesis states that all plant categories are equally attacked (see Fritz et al. 1996; Hjälten 1997). Responses of individual herbivore species to hybrid plants, however, do not answer the question regarding what is the overall herbivory susceptibility of different plant categories in a certain system of parental species and their intra-specific hybrids. In nature, plants are usually attacked by a multitude of insect herbivore species, each of which may select totally differently among the plant categories of a particular complex of parental species and hybrids (Fritz et al. 1996; Orians & Floyd 1997; Orians et al. 1997). Soetens et al. (1991) have suggested that leaf phenolic glucoside content may largely predict the field distribution of leafeating insects on clones of parental willow species and their hybrids (see also Kendall et al. 1996). In general, glucoside-adapted insect herbivore species can be expected to prefer high-glucoside willow categories, whereas non-adapted insects tend to feed on low-glucoside clones (Orians et al. 1997). Article V shows that a lowglucoside parental willow species (S. 5.4. The importance of variable food quality in relation to other factors Leaves of S. phylicifolia clones growing in three habitats along a soil moisture gradient (wetland, flood zone and dry zone) were very differently palatable and suitable for 18 behavioural differences of conspecific adults of this beetle indicate that Betulaceous and Salicaceous plants favour and require different adaptations in leaf beetles. This probably results from the fact that individual leaf secondary compounds in plants of these two families are different, even though structurally related. Experiments with A. alni show that an association with phenolics of a certain structural group do not necessarily entail a general pre-adaption to all members of a particular group of compounds. Consequently, individual leaf beetles can be properly adapted only to a limited set of secondary chemicals and host plants. Current results show that several lowmolecular weight willow phenolics deter the feeding of non-adapted leaf beetles, making many willow species poorly palatable for these insects. As shown in the field with A. alni, these poorly palatable willow species are avoided even during leaf beetle outbreaks. On the other hand, the distribution of specific stimulants seems to also have an influence on the host plant preferences of leaf beetles. The presence of specific stimulants probably explains why A. alni even prefer grey alder, A. incana, over the deterrent-poor willows. Moreover, intra-specific variation in leaf nutritive traits may cause a remarkable variation in leaf acceptability and suitability for leaf beetles. Overall, only a small fraction of available plant biomass appears to be adequate food for leaf beetles, which sets fundamental limits for leaf beetle populations. This means that resourcebased bottom-up forces are basically important for leaf beetles. Nevertheless, in some cases, variation in abiotic conditions may overcome the effect of variable food quality on leaf beetle host utilization patterns in the field. G. lineola, since in laboratory experiments G. lineola performed best on and preferred clones growing in the dry zone (V). In this case, host plant quality is clearly linked to the highest leaf nitrogen and water content and the lowest content of secondary phenolics in dry zone clones. In nature, G. lineola, however, is not distributed as expected on the basis of food quality. In the field, both adult G. lineola beetles, larvae and eggs were aggregated in wetland habitats. Variation in enemy pressure obviously does not explain this unexpected result. Instead, a wetland habitat appears to provide for G. lineola more favourable abiotic conditions than the flood or dry zones. Probably, high air humidity in the wetlands is an especially important factor for G. lineola, since its larvae perform better in moist rather than in dry conditions (see also Larsson et al. 1997). The dependency of G. lineola on air humidity may result from ancient phylogenetic constraints, as the majority of the species in the genus Galerucella appear to be rather restricted to aquatic or semi-aquatic plants or terrestrial plants growing in moist habitats (Nokkala & Nokkala 1989a, b; Kouki 1991; Koch 1992). Overall, G. lineola adults in the field probably firstly find a favourable abiotic habitat, and host selection behaviour appears to take place secondarily. These results strongly support recent suggestions that field variation in abiotic environmental conditions may sometimes mask the resource-based bottom-up effects placed upon on herbivorous insects (Williams 1983; Willmer et al. 1996; Ritchie 2000; Sipura & Tahvanainen 2000). 6. CONCLUDING REMARKS A performance trade-off found in naive larvae of four G. lineola populations and 19 activity in condensed tannins. Ecology 78: 1696-1712 Barbosa P, Schultz JC (eds) (1987) Insect outbreaks. Academic Press, San Diego Baur R, Binder S, Benz G (1991) Nonglandular leaf trichomes as shortterm inducible defense of the grey alder, Alnus incana (L.), against the chrysomelid beetle, Agelastica alni. Oecologia 87: 219-226 Baur R, Rank NE (1996) Influence of host quality and natural enemies on the life history of the alder leaf beetles Agelastica alni and Linaeidea aenea. Pp 173-194 in Jolivet PHA, Cox ML (eds) Chrysomelidae Biology. Vol 2: Ecological studies. SPB Academic Publishing, Amsterdam Bernays E, Graham M (1988) On the evolution of host specificity in phytophagous arthropods. Ecology 69: 886-892 Bingaman BR, Hart ER (1993) Clonal and leaf age variation in Populus phenolic glycosides: Implications for host selection by Chrysomela scripta (Coleoptera: Chrysomelidae). Environ Entomol 22: 397-403 Bryant JP, Clausen TP, Reichardt PB, McCarthy MC, Werner RA (1987) Effect of nitrogen fertilization upon the secondary chemistry and nutritional value of quaking aspen (Populus tremuloides Michx.) leaves for the large aspen tortrix (Choristoneura conflictana (Walker)). Oecologia 73: 513-517 Cates RG (1980) Feeding patterns of monophagous, oligophagous and polyphagous insect herbivores: The effect of resource abundance and plant chemistry. Oecologia 46: 22-31 Chinery M (1997) Euroopan hyönteisopas. Otava, Helsinki Clancy KM (1992) Response of western spruce budworm (Lepidoptera: ACKNOWLEDGEMENTS First of all, I wish to express my deepest gratitude to Anne and to our dogs Punkku and Roosa who have shared their life with me. Without your endless love I would never have the strength to complete this thesis. I am grateful to my supervisors Prof. Jorma Tahvanainen and PhD Heikki Roininen for their advice and support, provided in many forms, during this work. 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