Our study first addressed whether there was heritable genetic variation for the diverse suite of functional traits measured from O. biennis, and we found significant genetic variance for all traits (Table 1). Clonal heritabilities ranged from 0.05 to 0.92 (mean = 0.57) and the coefficient of genetic variance ranged between 2.4 and 408 (mean = 50.0). The amount of herbivory on plants, measured as the proportion of leaves damaged, exhibited low heritability (H2 = 0.19) compared to other traits. Traits associated with the physiology of leaves (sla, C:N ratio, % water) exhibited heritability values (mean = 0.26) that were 28% lower than those for life-history traits (bolting and flowering date, life-history strategy and plant biomass) (mean = 0.36) (Table 1). The concentrations of secondary compounds exhibited the highest heritability values (mean = 0.68). Lifetime fruit production was also heritable (Table 1), indicating genetic variance for fitness.
Covariation among plant traits
Our second question sought to understand whether plant traits genetically covaried with one another, and we found that the 276 pairwise tests among the 24 traits revealed extensive covariation among traits, measured as either genetic Pearson correlation coefficients or genetic covariances (Supplementary Table 1). The high frequency of significant pairwise associations (128 of 276 significant tests at 0.05 level) was unlikely to have been due to chance (Binomial expansion test: P = 4.2 x 10-89). The average correlation between traits was positive (mean rgenotype = 0.11), although the distribution of correlation coefficients was approximately normal (Figure 2a), with many statistically significant positive (88 tests with P<0.05) and negative (40 tests with P<0.05) pairwise correlations and covariances (Supplementary Table 1). Many of the negative correlations occurred between physiological traits and secondary compounds, or among different types of secondary compounds, principally ellagitannins (Supplementary Table 1).
We used hierarchical cluster analysis on pairwise genetic correlations to better understand the relationship among traits (Figure 2b). Cluster analysis identified four groups of covarying traits in which traits positively covaried within these groups. Covariation among groups was typified by significantly negative or non-significant correlations (Figure 2b). Groups did not clearly separate according to the types of traits associated with them (i.e. ecophysiological, life-history, resistance, or morphology). The largest groups (1 and 3) contained all main trait types, and smaller groups of traits either contained only secondary compounds (group 2), or a mix of trichome density (a morphological trait) and secondary compounds (group 4).
Our third research objective was to determine whether individual plant traits or multivariate suites of traits predicted resistance to herbivory. Five traits explained 73% of the total variation in herbivory by outbreaking P. japonica beetles – the dominant herbivore on plants (Overall model: F5,33 = 17.66, P < 0.001, r2 = 0.73; Table 2, Fig. 3a). The most important traits among these were several phenolic compounds. Specifically, quercetin glucuronide (a flavonoid glycoside) was negatively associated with herbivory (Fig. 3b), while the cumulative concentrations of several minor flavonoids (“other flavonoids”, Fig. 3c) and several ellagitannins (“other ET’s”, Fig. 3d) were positively associated with herbivory (Fig. 3d). Life-history strategy (rosettes versus flowering plants) and an ellagitannin (peak 1) were also positively related to herbivory, but the effects of these compounds were only marginally significant (P = 0.06, Table 2). The variance-covariance matrix revealed no trade-offs among these traits, while several traits positively covaried with one another (Supplementary Table 1).
A multivariate distillation of all 24 traits identified two principal components (PCs) that explained 45% of the variation in herbivory (F2,36 = 14.5, P < 0.001, r2 = 0.45). Each significant PC summarized variation in multiple traits (see Table 2).
Natural selection on plant traits
Finally, we asked whether there was selection on plant traits and we found that natural selection acted on multiple life-history and chemical traits of O. biennis. Significant selection was detected on 17 of the 24 traits, as measured by selection differentials (Supplementary Table 2). Conventional multivariate genotypic selection analyses (Rausher, 1992) revealed that selection acted on just four plant traits. There was positive directional selection for an increase in plant biomass (Fig. 4a), flowering during the first year (Fig. 4b), and greater concentrations of the ellagitannin oenothein A (Fig. 4c, Table 3). We also detected directional selection for decreases in the concentration of quercetin glucuronide (Fig. 4d), a compound associated with decreased P. japonica herbivory. We found quadratic selection on biomass, which reached a maximum within the range of data at 1.55 standardized biomass units, suggesting the presence of stabilizing selection (Fig. 4a). There was also quadratic selection on quercetin glucuronide, where weak disruptive selection acted with a fitness minimum at 0.89 standardized units (Fig. 4d).
Somewhat counterintuitively, damage by herbivores was positively associated with plant fitness (Supplementary Table 2), suggesting that this relationship is indirectly mediated by one or more plant traits that jointly influenced both herbivory and plant fitness. Consistent with this idea, quercetin glucuronide and life-history strategy were subject to positive directional selection and also associated with increased herbivory. When we re-examined the relationship between relative fitness and herbivory after accounting for variation in fitness explained by traits under selection (see Table 3), the significant relationship between herbivory and relative plant fitness disappeared (F1,31 = 0.11, P = 0.91). We can infer from this result that herbivory had no direct effect on O. biennis fitness. However, that is not to say that resistance to P. japonica cannot evolve due to correlated selection.