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ArjenBiere Department of Plant Population Biology, Netherlands Institute of Ecology, NIOO-CTO, PO Box 40, 6666 ZG Heteren, The Netherlands. Tel.: +31-264791212 (or 91111); fax: +31-264723227; e-mail: firstname.lastname@example.org
Plantago lanceolata L. (ribwort plantain) produces two costly terpenoid secondary plant compounds, the iridoid glycosides aucubin and catalpol. We performed an artificial selection experiment to investigate direct and correlated responses to selection on the constitutive level of iridoid glycosides in the leaves for four generations. Estimated realized heritabilities (±SE) were 0.23 ± 0.07 and 0.23 ± 0.04 for upward and downward selection, respectively. The response to upward selection was caused by selection for a developmental pattern characterized by the production of fewer leaves that on average contain more iridoids, and by selection for a development-independent increase in the level of these compounds. Significant correlated responses were observed for plant growth form. Upward selection resulted in plants with larger sized, but fewer leaves, fewer side rosettes, and fewer spikes, corresponding to a previously distinguished ‘hayfield’ ecotype, whereas downward selection produced the opposite pattern, corresponding to a ‘pasture’ ecotype. This indicates that the level of iridoid glycosides is genetically correlated with morphological traits in P. lanceolata, and is part of the complex of genetically correlated traits underlying the two ecotypes. The genetic association between iridoid level and growth forms suggests that there may be constraints to the simultaneous evolution of resistance to generalist insects (by iridoid glycosides) and to larger grazers (by a high production rate of prostrate leaves and inflorescences) in open grazed habitats where the ‘pasture’ ecotype is found.
Artificial-selection experiments are well suited to study the genetic basis of variation in traits, to estimate heritabilities, and to study genetic correlations among different traits ( Falconer & Mackay, 1996). In this paper, we present a study of direct and correlated responses to artificial selection on constitutive levels of a class of terpenoid compounds in leaves of Plantago lanceolata L. (ribwort plantain). P. lanceolata contains amongst others the two iridoid glycosides aucubin and catalpol ( Duff et al., 1965 ; Bowers & Stamp, 1992). These are monoterpene derivatives produced from mevalonic acid in the isoprenoid biosynthetic pathway ( Croteau, 1987; McGarvey & Croteau, 1995). Aucubin is the biosynthetic precursor of catalpol ( Damtoft et al., 1983 ). Both compounds strongly deter generalist insect herbivores, but can be detoxified, utilized, or even sequestered for their own defence by specialist insect herbivores ( Bowers, 1991). A genetic basis for variation in iridoid glycosides in P. lanceolata has previously been shown ( Bowers et al., 1992 ; Fajer et al., 1992 ; Bowers & Stamp, 1993; Adler et al., 1995 ). Biosynthetic costs of terpenoids rank among the highest of all secondary plant compounds ( Gershenzon, 1994), but studies in P. lanceolata have thus far failed to detect significant fitness costs in terms of a lower growth or biomass of plants producing high levels of iridoid glycosides ( Bowers & Stamp, 1992; Adler et al., 1995 ; Darrow & Bowers, 1997).
In this paper, we focus on the genetic basis of constitutive levels of these iridoid glycosides and their genetic association with other plant traits. Specifically, we address the following questions. (1) Is there a direct response to upward and downward selection on the constitutive leaf iridoid glycoside level and what is the estimated heritability? (2) Are there correlated responses of iridoid glycoside levels in tissues that are not subject to direct selection and of other plant traits? We show that selection on iridoid glycosides results in a correlated response in plant growth form. Upward selection leads to plants with larger, but fewer leaves and spikes, whereas downward selection leads to an opposite pattern. We discuss the possible causes and consequences of such associations between levels of secondary compounds and plant growth form.
Materials and methods
A total of 20 plants of P. lanceolata were collected in 1994 from each of two populations, in Wolfheze (N 52°00′ E 5°48′) and in Heteren (N 51°57′ E 5°44′), the Netherlands. The Wolfheze population grows in a roadside with a sandy soil, and it has a low biomass production due to summer dryness (Location 16 in Linders et al., 1996 ). The Heteren plants were collected from a path along a grassland which had a mixed mowing and grazing management during the 10 years before sampling. In the greenhouse, 20 pairwise reciprocal crosses were performed between plants from the two populations. In 17 of these crosses both maternal parents produced seeds, but in three cases one of the reciprocals failed to produce viable seed. Ripe seeds of these crosses were collected and used to generate the base population for an artificial selection experiment. Seeds were germinated on moistened glass beads in a growth cabinet (light : dark: 14 : 10 h; 25 : 15 °C). After 12 days, 200 seedlings (five seeds per maternal parent from the 17 crosses for which both reciprocals were available, and 10 seeds of the seed-producing maternal parent of the remaining three crosses) were transplanted into plastic pots (9 × 9 × 10 cm) with a mixture of compost and sand (4 : 1). The plants were completely randomized and grown in the greenhouse under 16 h light at 22 : 18 °C day : night. After 3 weeks every transplanted plant received 100 mL of a one-fourth strength Hoagland’s solution. At a standardized plant age (5 weeks after transplanting) leaves of an ontogenetically standardized stage (the third and fourth pair of true leaves produced on the main rosette) of each plant were harvested and processed for analysis of the concentration of the iridoid glycosides aucubin and catalpol using high performance liquid chromatography (HPLC). Plants were selected on the basis of the total iridoid glycoside content in the third and fourth leaf pair. Standardization of leaf position within the main rosette was necessary as iridoid glycoside levels within plants decrease from central (young) to basal (old) positions (e.g. Bowers & Stamp, 1992, 1993). The third and fourth leaf pair was chosen because they were the youngest leaf pairs that were fully expanded in all plants within 5 weeks. From the 200 plants of the base population, 50 plants with the highest and 50 with the lowest levels were selected to initiate lines with directional upward (H, ‘high’) and downward (L, ‘low’) selection, respectively, whereas 50 plants with intermediate levels, i.e. a mean value equal to that of the population mean, were selected to initiate a line with stabilizing (M, ‘medium’) selection. Selected plants of each line were transferred to closed plexiglass cages at anthesis. Spikes were well shaken every day to ensure that pollen was well distributed among plants. After 6 weeks, the ripe seeds were harvested and four seeds per maternal plant family of each of the three lines were used as the starting material for the next generation. Unidirectional selection within the L and H line and stabilizing selection within the M line was continued for four generations. The procedure took ≈10 months every generation. Plants for the base population to the third generation were grown in July 1995, June 1996, April 1997 and January 1998, respectively. Seeds of each full-sib family from the base population and of each half-sib family from subsequent generations of the selection lines that were not used to raise plants during the selection procedure, were stored at room temperature for use in the experiment described below.
Direct and correlated responses to selection
After four generations, one seed of 40 randomly chosen female half-sib families from each of the three lines and four generations, and three seeds of the 40 reciprocal full-sib families from the base population (600 plants in total) were used to evaluate the direct and correlated responses to selection on leaf iridoid glycoside content. Seeds per plant family were germinated separately on moistened glass beads in a growth cabinet (light : dark: 14 : 10 h; 25 : 15 °C). After 12 days, on 8 July 1998, the percentage of germination was calculated per plant family and the seedlings were transplanted into plastic pots (9 × 9 × 10 cm) with a mixture of compost and sand (4 : 1). The plants were completely randomized and grown in the greenhouse under 16 h light at 22 : 18 °C day : night. Every plant received 100 mL of a one-fourth strength Hoagland’s nutrient solution 3 weeks after transplantation. Flowering date of the plants was recorded as the day on which the first flower bud appeared. After 5 weeks, the third and fourth leaf pair and stalks that were in the male flowering stage of each individual were harvested for iridoid glycoside analysis. During harvest a number of plant phenotypic traits were also measured to assess correlated responses: the number of leaves, length and width of the longest leaf of the main rosette, number of side rosettes and their number of leaves, and the number of spikes.
Correlated response of iridoid levels in other leaves
A second experiment was set up to assess whether the response of iridoid glycoside levels in the third and fourth leaf pair was accompanied by a similar response in the rest of the leaves. A single plant from 25 randomly chosen half-sib families from the fourth generation of both the H and the L lines was raised under the same conditions as described above. After a period of 5 weeks after transplantation, the third and fourth leaf pair of the main rosette (of which iridoid levels were under direct selection) and the other leaves of the main rosette and side rosettes (not subject to direct selection) were harvested separately. The fresh and dry weights and the concentration of the iridoid glycosides aucubin and catalpol of these fractions were analysed separately using HPLC.
Leaves and stalks used for chemical analysis were stored at –80 °C in aluminium foil immediately following fresh weight measurement, and dried using a freeze dryer (LABCONCO, Kansas, MO). Fine ground dry material of leaves and stalks (25 and 10 mg, respectively) were extracted in 10 mL of 70% MeOH while shaking overnight. The crude extract was filtered and diluted with Milli-Q water. Aucubin and catalpol were detected by HPLC using a Dionex DX 500 HPLC equipped with a GP40 gradient pump, a Carbopac PA1 guard (4 × 50 mm) and analytical column (4 × 250 mm), and an ED40 Electrochemical Detector for Pulsed Amperimetric Detection (PAD). NaOH (1 M) and Milli-Q water were used as eluents (10 : 90%, 1 mL min−1). Retention times were 2.90 and 4.35 min for aucubin and catalpol, respectively. Concentrations of aucubin and catalpol were analysed using Peaknet Software Release 5.1 (DX-LAN module).
An upper limit to the narrow sense heritability of aucubin, catalpol, and total iridoid glycoside content (the sum of these two components) in the leaves (% dw, all ln-transformed) was estimated from ANOVAs (SPSS procedure MANOVA) of cross effects and reciprocal effects (nested within cross effects) using the reciprocal full-sib families of the base population. The variance component due to cross effects is composed of an additive-genetic variance component and a dominance component (1/2 Va + 1/4 Vd, Falconer & Mackay, 1996). If dominance is small, h2 can thus be estimated from twice the ratio of this variance component to the total variance. As the dominance component is basically unknown, this estimate should be regarded as an upper limit. Confidence limits for h2 were calculated according to standard methods ( Sokal & Rohlf, 1995; p. 215). Realized heritabilities of the (ln-transformed) total iridoid glycoside content of leaves were estimated from regressions of the cumulative selection response on the cumulative selection differential over the four generations of selection. Both parameters were first standardized by using the appropriate phenotypic standard deviations ( Falconer & Mackay, 1996). Analyses of variance (SPSS procedure MANOVA) were performed to test effects of line and generation (both fixed effects) on concentrations of aucubin, catalpol and total iridoids per unit dry weight of leaves (all ln-transformed) and per unit of dry weight of stalks (all square-root transformed), and on the ratio of catalpol to the total iridoid glycoside content of leaves and stalks. Orthogonal contrasts for line-effects were used (1) to test the significance of differences between the H and L lines (high vs. low), and (2) to test for a significant asymmetry in the response of the H and L lines to selection (H and L vs. control). Similar analyses were performed to analyse correlated responses in germination percentage, onset of flowering, number of leaves in the main rosette, number of leaves in side rosettes (ln-transformed), total number of leaves (ln-transformed), number of rosettes (ln-transformed), number of stalks, length and width of the longest leaf, and the product of leaf length and leaf width, hereafter referred to as leaf area. For the second experiment, i.e. the response of iridoid glycoside levels in different leaf fractions measured in the fourth generation only, ANOVAS were conducted to test the effects of line (H vs. L) and leaf fraction (third plus fourth leaf pair, used as criterion during the selection procedure, vs. the rest of the leaves on a plant) on ln-transformed iridoid glycoside levels.
Heritabilities and direct response
Using the full-sib family structure of the base population, we obtained an upper estimate of 0.84 for the narrow-sense heritability of total (aucubin + catalpol) iridoid glycoside content in the third and fourth leaf pair, with a lower 95% confidence limit of 0.37 ( Table 1). Estimates were slightly lower for the the two components aucubin (0.76) and catalpol (0.71, Table 1). Realized heritabilities of leaf iridoid glycoside content (±SE) estimated from regressions of the standardized cumulative selection response on the standardized cumulative selection differential based on ln-transformed data ( Fig. 1) were 0.226 ± 0.068 and 0.228 ± 0.039 for upward and downward selection, respectively. Analysis of the leaf iridoid glycoside content of plants after completion of the selection procedure, when lines from all generations were grown simultaneously in a common environment, showed that the direct response to selection was highly significant ( Table 2a, H vs. L line, P < 0.001). Leaf iridoid glycoside content of the H and L lines increasingly diverged over time ( Fig. 2A, Table 2a, interaction between generation and line H vs. L). In the H line, iridoid glycoside content increased on average by 127 mg g−1 leaf dry weight per generation over the M line, whereas a decrease of 73 mg g−1 was observed in the L line ( Fig. 2A). As a result, the average leaf iridoid glycoside level of plants from the H and L lines differed by a factor of 4 in the fourth generation.
Table 1. The ANOVA of the effects of cross and reciprocal cross (nested within cross) on leaf content (%dw) of aucubin, catalpol and total iridoids (aucubin+catalpol) in P. lanceolata. The proportion of variance explained by each effect is indicated (%V). Narrow sense heritabilities (h2) and their 95% confidence limits are calculated from the variance component due to the cross effect. Data were ln-transformed prior to analysis.
Table 2. The ANOVA of the effects of line and generation on (a) leaf and (b) stalk content (% dw) of aucubin, catalpol, total iridoids (aucubin+catalpol) and the ratio of catalpol to total iridoids in P. lanceolata. Orthogonal contrasts for line effects compare (1) the high (H) vs. the low (L) line, and (2) the medium (M) vs. the H and L line.
Correlated responses of iridoid glycosides in other plant parts
Selection on leaf iridoid content of the third and fourth leaf pair led to a significant response of the iridoid content in leaves not subject to direct selection ( Table 3). The magnitude of this correlated response was significantly lower than that of the primary response (interaction between line and leaf fraction: F[1,96]=10.68, P=0.002); the difference between plants from the H and L selection line for leaves not subject to selection was only a factor of 2, compared with a factor of 3.8 for the primary response ( Table 3). Selection on total iridoid glycoside content in the leaves also resulted in a concomitant response of total iridoid-glycoside content in the stalks ( Fig. 2E, Table 2b). Both components of the total iridoid content, aucubin and catalpol, responded to selection. Selection for high total iridoid-glycoside content led to an increase of both aucubin and catalpol ( Fig. 2B–F, Table 2a,b). The response of catalpol was slightly asymmetrical ( Fig. 2C, Table 2a: contrast H and L vs. M); the response to upward selection was somewhat stronger than the response to downward selection. The relative increase in leaf-catalpol content during upward selection was larger than that of the aucubin content, resulting in an increase in the ratio of catalpol to the total iridoid glycosides ( Fig. 2D, Table 2a). The ratio did not change during downward selection ( Fig. 2D). In the stalk tissue the ratio slightly increased in both the upward and downward selected lines ( Fig. 2H, Table 2b). On the whole, the concentration of catalpol was roughly two times lower than that of its precursor aucubin in the leaves ( Fig. 2B,C), whereas the two compounds were present in roughly equal amounts in the stalks ( Fig. 2E,F). Consequently, the ratio of catalpol to the total iridoid glycoside content was higher in the stalks than in the leaves ( Fig. 2D,H).
Table 3. Levels of iridoid glycosides (% dw) in different leaf fractions of plants selected for high and low levels of iridoid glycosides in the third and fourth leaf pair for four generations. Values are back-transformed mean values. In parentheses: 95% confidence intervals. F-values refer to one-way ANOVAs of differences between the H and L lines.
Correlated responses in other traits
Upward selection for leaf iridoid-glycoside content led to a decrease in all measured ‘number’ traits: the number of leaves in the main rosette of the plant, the number of side rosettes, the number of leaves in the side rosettes, the total number of leaves, and the number of stalks ( Fig. 3A–D,H). Effects of downward selection were in the opposite direction. For instance, after four generations of selection, plants from the H line had on average only 17 leaves, plants from the M line 21, whereas plants from the L line had on average 27 leaves ( Fig. 3D). Conversely, ‘size’ traits showed a positive response to upward selection for leaf iridoid glycoside content. Length, width, and leaf area of the longest leaf increased during selection for high leaf iridoid glycoside content ( Fig. 3E–G, Table 4). The germination percentage and onset of flowering did not show a response to selection ( Table 4). Some traits showed a generation effect independent of the effect of selection (e.g. M line, Fig. 3D), i.e. even though they were grown simultaneously in the same environment, plants from different generations showed fluctuations in these traits between generations.
Table 4. The ANOVA of the effects of line and generation on morphological and life-history traits of P. lanceolata plants selected for different levels of iridoid glycosides for four generations. Values are F-values. Orthogonal contrasts for line effects compare (i) the high (H) vs. the low (L) line, and (ii) the medium (M) vs. the H and L line.
Heritabilities and response of iridoids to selection
In the present study, we obtained an upper estimate of 0.84 for the heritability of leaf iridoid glycoside content in the leaves of P. lanceolata, based on a full-sib analysis of the base population. As this estimate includes an unknown dominance component, it should be regarded as an upper estimate of the narrow-sense heritability of this trait. Estimates of the realized heritability (±SE) based on four generations of artificial selection were 0.23 ± 0.07 and 0.23 ± 0.04 for upward and downward selection, respectively. This indicates that under greenhouse conditions, the genetic component of variation in constitutive levels of iridoid glycosides is 23%. The response declined during consecutive generations; estimates based on the first generation only were two and three times higher than the average response over four generations for downward and upward selection, respectively. This decline can only be partly explained by the expected decrease in genetic variance known as the Bulmer effect ( Falconer & Mackay, 1996; p. 203). Insight into possible causes of this decline would require further study. The estimate of 0.23 is within the range of heritability estimates for secondary plant metabolites found in other studies. For instance, narrow-sense heritabilities for furanocoumarins in P. sativa ranged from 0.07 to 0.91 ( Zangerl et al., 1989 ), estimated heritabilities for shoot and root pyrrolizidine alkaloids in S. jacobaea were 0.33 and 0.43 ( Vrieling et al., 1993 ; Van Dam & Vrieling, 1994), for 2′-cinnamoyl salicortin and salicortin in S. sericea 0.59 and 0.20 ( Orians et al., 1996 ), for phenolic resin content in D. aurantiacus 0.32 ( Han & Lincoln, 1994), and for glycosinolates and their myrosinase enzyme in B. rapa 0.17 and 0.35, respectively ( Siemens & Mitchell-Olds, 1998). The presence of the significant heritable component of variation in leaf iridoid glycoside levels in P. lanceolata indicates that selection imposed by generalist insect herbivores could potentially result in an adaptive response of leaf iridoid levels in the field.
The response of iridoid glycoside levels to selection resulted in a four-fold difference in iridoid glycoside content in the leaf pairs that were under direct selection between the upward and downward selected lines. Both components of the total iridoid glycoside content, aucubin and catalpol, responded to selection, but the relative increase of catalpol during upward selection exceeded that of aucubin. The ratio of catalpol to the total iridoid glycosides therefore increased in the H line, whereas it remained relatively constant in the L and in the M lines. Some studies have shown that catalpol is more deterrent to generalist insect herbivores than aucubin ( Puttick & Bowers, 1988). The shift towards a higher proportion of catalpol may therefore result in an even stronger deterrence of plants selected for high levels of iridoids than expected on the basis of their total iridoid content. Selection on leaf iridoid glycosides was accompanied by responses of iridoid glycoside levels in other plant parts. Differences in iridoid glycoside content between the H and L lines were not only observed in the leaf pairs that were subject to direct selection but also in the other leaves, although the magnitude of the response in those leaves was smaller than in the leaves under direct selection. The 2.5 fold higher shoot iridoid level in plants from the H line compared with plants from the L lines clearly indicates that the response to selection was not merely caused by allocating a higher or lower fraction of a total amount of shoot iridoids to the leaf pair subject to selection. Selection on high leaf iridoid glycoside levels also led to an increase in stalk iridoid glycoside content. Our results therefore indicate that iridoid glycoside levels in leaf and stalk tissues are genetically correlated. This suggests that if leaf feeding insects impose selection on host plants for higher leaf iridoid levels, the response of the host plants to these herbivores may be accompanied by increased resistance towards nonadapted generalist insects feeding on reproductive tissues.
Responses of morphological traits to selection
Although we selected on the levels of a secondary plant compound, a suite of morphological traits showed a correlated response, suggesting that there are genetic correlations between physiological and morphological traits. Such genetic correlations between secondary plant compounds and other phenotypic traits have also been found in other studies. In P. sativa, furanocoumarin levels in the seeds showed significant positive genetic correlations with seed length, width, and the number and area of vittae ( Zangerl et al., 1989 ).
A striking result was that selection for high leaf iridoid glycoside content produced plants with few but large leaves, few side rosettes with a corresponding low number of leaves, and few stalks, whereas L line plants showed a consistent pattern in the opposite direction. The observed differences in growth form between plants from the H and L lines are remarkably similar to differences between two previously described ‘hayfield’ and ‘pasture’ ecotypes, respectively, in P. lanceolata ( Van Groenendael, 1986; Wolff, 1987; Van Tienderen & Van der Toorn, 1991a, b; Van Hinsberg & Van Tienderen, 1997). Hayfield ecotypes (subvar. latifolia Wimm. et Grab.; Pilger, 1937) are characterized by rosettes with few but long, erect leaves and inflorescences. They rarely produce side rosettes and their inflorescences have elongated spikes and long scapes. Pasture ecotypes (subvar. sphaerostachya Mert. et Koch f. minor;Pilger, 1937) form small prostrate rosettes with many small leaves and ascending inflorescences with small roundish spikes. Hayfield ecotypes are found in high, dense vegetation such as hayfields whereas pasture ecotypes occur in open, often grazed vegetation. Reciprocal transplant studies have shown local adaptation of these ecotypes to their natural habitats ( Van Tienderen & Van der Toorn, 1991a), which may be partly related to the ability of hayfield plants to intercept more light in their competitive native habitat with a high and dense vegetation, and to the higher incidence of flowering and maturation of spikes of pasture plants in their native habitat ( Van der Toorn & Van Tienderen, 1992). Studies by Wolff (1987), Wolff & Van Delden (1987, 1989) and Van Hinsberg (1996) have shown that there are strong genetic associations between traits involved in these two complexes. For instance, upward selection on leaf length under simulated sunlight (high R/FR-ratio) ( Van Hinsberg, 1996) and downward selection on leaf angle ( Wolff & Van Delden, 1989) in P. lanceolata produced plants showing a suite of traits consistent with the hayfield ecotype. In the present study, the plants used in the crosses to produce seeds for the base population were collected from two populations that were neither explicitly hayfield nor pasture types. But even if they would have been, the emergence of hayfield and pasture types when selecting on iridoid glycoside content is not self evident. Apparently, leaf iridoid glycoside content is genetically correlated with the complex of traits underlying these two growth forms.
The association between growth form, including leaf production, and iridoid level raises the question whether differences in iridoid levels between plants from the H and the L lines simply reflect an inherent allocation pattern of iridoids that is linked to development, or that there are development-independent differences in iridoid levels between these lines. In agreement with the general pattern observed for within-plant distribution of secondary plant compounds ( Zangerl & Bazzaz, 1992), previous studies have shown that levels of iridoid glycosides in P. lanceolata decrease with leaf age, either because of reallocation or because of breakdown of these compounds in older leaves (e.g. Bowers & Stamp, 1992). This means that in plants with a high rate of leaf production (e.g. that have produced 18 leaves within 5 weeks), the iridoid level in an ontogenetically defined leaf stage (such as the third and fourth leaf pair) may be low simply because these leaves are relatively ‘old’ compared with the third and fourth leaf pair produced on plants with a lower rate of leaf production (e.g. that have produced only 14 leaves in that period of time). Indeed, in the base population, at a fixed plant age, a significant part (39.9%) of the variation in iridoid level among full-sib families could be explained by the average number of leaves produced in the main rosette ( Fig. 4A, P < 0.001). Families that had produced on average 18 leaves in the main rosette, had only half the concentration of iridoids in their third and fourth leaf pair compared with families that had produced on average only 14 leaves in the main rosette ( Fig. 4A). Nevertheless, the response to selection could only be partly ascribed to such developmentally related variation in iridoids. During subsequent generations, the among-family correlation between number of leaves and iridoid level disappeared within lines ( Fig. 4B,C), and there was a large difference in iridoid level between plant families with a similar number of leaves between the two lines. Apparently, continued selection also resulted in an increase in the average level of iridoids in plants with a high rate of leaf production in the H line, and in a decrease in the average iridoid level in plants with a low rate of leaf production in the L line. Analysis of plants from the third generation (the last generation for which replicates at the family level were available) using leaf number as a covariate ( Table 5), confirms that a large part of the differences in iridoid level among families from the H and L lines are developmentally independent (F[1,96]=282.5, P < 0.001). Apparently, the selection response has been based on both development-related and on development-independent variation in iridoid levels.
Table 5. The ANOVA of the effects of selection line and leaf number in the main rosette (covariate) on the level of iridoid glycosides (ln-transformed) and the ratio of catalpol to the total iridoids in the third and fourth leaf pair of P. lanceolata. Analyses based on family mean values for female half-sib families from lines selected for H and L levels of leaf iridoid glycosides that constituted the third generation of selection.
By contrast, one of the correlated responses, the higher ratio of catalpol to the total iridoids in the H line, mainly reflected developmental differences between the two lines ( Fig. 4F, Table 5). Old leaves generally contain a lower ratio of catalpol to total iridoids than new leaves (e.g. Bowers & Stamp, 1992). Accordingly, plants with a high rate of leaf production tended to have a lower catalpol-to-total ratio in the relatively ‘old’ third and fourth leaf pair ( Fig. 4D, P < 0.10). In the third generation, differences in the catalpol-to-total iridoid ratio between families appeared to be mainly due to differences in the number of leaves in the main rosette ( Fig. 4F, P < 0.001). Differences between families from the H and L lines that were independent of development were small ( Fig. 4F, Table 5, F[1,96]=4.93, P=0.029).
The association between iridoid level and growth form has a number of interesting consequences. First, we expect that this association has consequences for plant attack by generalist and specialist insect herbivores in natural populations. From our results, we would predict that hayfield plants show higher overall levels of iridoid glycosides in their leaves than pasture plants. This may result in a higher overall deterrence of hayfield plants to nonadapted generalist insects than pasture plants, but higher palatability to adapted specialists that use iridoid glycosides as feeding or oviposition stimulants ( Bowers, 1991). These predictions will be tested in the near future. Second, a genetic correlation between low leaf iridoid glycoside levels and traits associated with the ‘pasture’ ecotype could in principle impose constraints on the simultaneous evolution of resistance to generalist insects (by increased levels of secondary metabolites) and to larger grazing herbivores (by a high production rate of small procumbent leaves and inflorescences) in open, grazed habitats. In that case we would predict that in open, grazed habitats, patches that suffer higher pressure from generalist insect herbivores show increased levels of iridoid glycosides, associated with a shift towards more ‘hayfield’ like traits.
In conclusion, we have found significant heritable variation for leaf iridoid glycoside content in P. lanceolata. The levels of iridoid glycosides in the leaves are genetically correlated with the levels of these compounds in the reproductive tissues and with plant growth form. These results suggest, first, that selection for high levels of these compounds in the leaves by generalist, leaf feeding insects may result in increased resistance to generalist insects that feed on reproductive tissues. Secondly, in open, grazed habitats, selection for increased levels of these compounds in the leaves may be hampered by their association with a set of morphological traits that has previously been shown to be unfavourable in this type of habitat.
We like to thank Slavica Ivanovic and Sonja Honders for their invaluable help during harvests and with chemical analyses, and Deane Bowers for comments on an earlier version of the manuscript.