Pia Mutikainen

HERBIVORE RESISTANCE IN BETULA PENDULA: EFFECT OF FERTILIZATION, DEFOLIATION, AND PLANT GENOTYPE

Plant resistance to herbivores is affected both by genetic and environmental factors. The carbon–nutrient balance hypothesis (CNB) explains environmentally induced variation in both constitutive and delayed herbivore-induced resistance (DIR) in terms of variation in soil fertility and light regime. The CNB hypothesis predicts that an increase in the availability of nutrients (e.g., fertilization) decreases both constitutive and induced resistance against herbivores. We tested the relative roles of plant genotype, defoliation, and soil fertility in determining herbivore resistance of cloned silver birch Betula pendula Roth saplings. As indicators of insect and mammalian resistance we conducted bioassays with a geometrid moth, Epirrita autumnata (Borkhausen), and counted the resin droplets on the shoot of the saplings, respectively. In addition, we measured rapid induced resistance (RIR) against the insect herbivore. Finally, we analyzed leaf secondary chemistry to investigate the correlations of secondary chemicals with the level of resistance measured using the performance of E. autumnata. With respect to the constitutive resistance against an insect herbivore, our results support the CNB hypothesis; the larvae of E. autumnata had a higher relative growth rate and pupal mass on fertilized saplings compared to nonfertilized saplings, i.e., the fertilized saplings had a lower resistance level. However, the relative growth rate of E. autumnata was significantly decreased by defoliation only when the larvae were grown on fertilized saplings. The number of resin droplets increased due to fertilization and, in fertilized saplings, following defoliation, but these responses were highly determined by the genotype of the sapling. Altogether, the results on resin droplets are not in accordance with the CNB hypothesis. The concentration of condensed tannins correlated negatively with E. autumnata growth rate and pupal mass in both fertilization levels, whereas the concentration of total nontannin phenolics correlated positively with the E. autumnata growth rate in nonfertilized saplings. In addition, the concentration of myricetin glycosides correlated negatively with the pupal mass of E. autumnata, whereas the correlations between E. autumnata performance indices and other groups of flavonol glycosides were either significantly positive (kaempferol glycosides) or nonsignificant (quercetin glycosides). Further, the concentration of 3,4′-dihydroxypropiophenone 3-glucoside (DHPPG) correlated positively with the magnitude of induction in E. autumnata growth rate and pupal mass in fertilized saplings, where the significant induction in resistance occurred. The correlations of secondary chemistry and E. autumnata performance indices suggest that the constitutive level of resistance of B. pendula against E. autumnata is mainly determined by the concentration of condensed tannins, whereas the induced resistance is determined by the concentration of nontannin phenolics, such as flavonol glycosides and DHPPG. We observed significant differences among the clones in their insect and mammalian resistance (i.e., genetic basis for the resistance), which indicates that resistance can evolve as a response to herbivory. However, fertilization explained a higher proportion of variance in insect performance indices than the genotype of the plant, whereas the opposite was true for the amount of resin droplets, which we used as an indicator of mammalian resistance.

Trade-offs in phenolic metabolism of silver birch: Effects of fertilization, defoliation, and genotype

We examined the chemical responses of 10 silver birch (Betula pendula) clones to fertilization and defoliation in a field experiment. In defoliation, every second leaf was removed from the saplings. Three days later, two undamaged short-shoot leaves were collected, air-dried, and analyzed for condensed tannins and 34 nontannin phenolic compounds by high-performance liquid chromatography. The clones showed substantial variation in phenolic composition of the leaves and in chemical responses to fertilization and defoliation. A cluster analysis by UPGMA indicated that the phenolic profiles of birch leaves were affected more by genotype than fertilization or defoliation, and the clones could thus be distinguished from each other. In addition, on the basis of their overall phenolic composition, the clones were clustered loosely in three clone groups. The leaves of fertilized saplings contained lower levels of condensed tannins than controls, as predicted by carbon/nutrient balance (CNB) hypothesis. However, fertilization had no effect on the total amount of nontannin phenolics. The concentrations of (+)-catechin, 3,4′-dihydroxypropiophenone 3-glucoside (DHPPG), 3-cinnamoylquinic acids, and flavone aglycones were lower in fertilized saplings, whereas the opposite was true for 5-cinnamoylquinic acids and the total amount of flavonol glycosides. Although our results provide support for the CNB hypothesis, they also show that the accumulation of phenolic compounds in birch leaves is strongly coordinated. Different branches of the biosynthetic pathway of phenolic compounds may compete for substrates, and such internal metabolic trade-offs may explain the differential accumulation of the compounds. In fertilized saplings, the concentration of condensed tannins was also negatively correlated with the amount of triterpenoid resin droplets measured from the same saplings. We suggest that a linkage via malonyl-CoA between the biosynthetic routes to terpenoids and flavonoid derivatives, such as condensed tannins, may explain the different responses to fertilization reported for terpenoids and phenolics. Undamaged leaves of partially defoliated saplings contained more DHPPG and flavone aglycones and less cinnamic acid derivatives and (+)-catechin than did leaves of control saplings. The induction of DHPPG and flavonoid aglycones was significantly and negatively correlated with the concentration of myricetin glycosides in fertilized saplings, which may indicate a trade-off between induced and constitutive defense. Moreover, in fertilized saplings, the three clone groups formed by UPGMA clustering differed significantly in the magnitude of induction of DHPPG and flavone aglycones. Different birch genotypes may thus have different modes of chemical defense, and the magnitude of chemical response of a genotype may partly depend on resource availability. In general, our results show that new insights in the theory of chemical defense can be gained by accomplishing studies on plant–herbivore interaction with high chemical resolution.

Effects of herbivory on male reproductive success in plants

The effects of herbivory on plant fitness have been assessed in terms of seed or fruit production whereas the effects on male reproductive success have received much less attention. This female-biased approach has been based on the assumption that the male and female reproductive success in plants are correlated with each other and that the effects of environmental factors on female and male function are equal or at least parallel. However, several studies have suggested that male and female reproductive success are not closely related and they may even be negatively correlated. Here we review the literature and also present an experimental study of the effect of herbivory on pollen-tube growth rates. The literature reviewed shows that the effects of herbivory on plant fitness may vary in relation to the fitness measures used and that herbivory may have differential effects on female and male reproductive functions. These differential effects may have evolutionary consequences, as well as consequences for estimating the selective effects of herbivory. Our experiment with Lobelia siphilitica showed that removal of 50% of the leaves during flower development caused a significant reduction in pollen-tube growth rates. We suggest that assessment of both male and female fitness components will give more accurate estimates of the effects of herbivory on total plant reproductive success. Consequently, the role of herbivory as a selective force can be more accurately estimated.

Female frequency and relative fitness of females and hermaphrodites in gynodioecious Geranium sylvaticum (Geraniaceae).

We determined female frequency of 23 populations of the gynodioecious Geranium sylvaticum (Geraniaceae) in Finland. We compared our results to previous results on this species from the 1960s in order to reveal putative changes in female frequencies. Because females may be maintained in gynodioecious populations if their seed production or offspring quality is higher than that of hermaphrodites, we explored reproductive success of females and hermaphrodites in detail in 11 populations for two consecutive years. Female frequencies varied from 0.4 to 27.2%; this variation is similar to that observed in the 1960s. Contrary to previous results that indicated lower seed production in females, females produced 1.2 and 1.7 times more seeds per flower than hermaphrodites in 2000 and 2001, respectively. Females also had higher fruit set than hermaphrodites. Thus, higher seed production of females partly explains the maintenance of gynodioecy in this species. Furthermore, female frequency correlated negatively with relative seed fitness of hermaphrodites suggesting that relative seed fitness is related to population sex ratio. Female frequency and the distance of the population from the most southern population also tended to correlate positively, suggesting that harsher environmental conditions in the north may benefit female plants. Given the observed yearly variation, our results also highlight the importance of temporal variation for the relative seed fitness of females and hermaphrodites.

INBREEDING DEPRESSION IN GYNODIOECIOUS LOBELIA SIPHILITICA: AMONG-FAMILY DIFFERENCES OVERRIDE BETWEEN-MORPH DIFFERENCES

If inbreeding depression is caused by deleterious recessive alleles, as suggested by the partial dominance hypothesis, a negative correlation between inbreeding and inbreeding depression is predicted. This hypothesis has been tested several times by comparisons of closely related species or comparisons of populations of the same species with different histories of inbreeding. However, if one is interested in whether this relationship contributes to mating‐system evolution, which occurs within populations, comparisons among families within a population are needed; that is, inbreeding depression among individuals with genetically based differences in their rate of selfing should be compared. In gynodioecious species with self‐compatible hermaphrodites, hermaphrodites will have a greater history of potential inbreeding via both selfing and biparental inbreeding as compared to females and may therefore express a lower level of inbreeding depression. We estimated the inbreeding depression of female and hermaphrodite lineages in gynodioecious Lobelia siphilitica in a greenhouse experiment by comparing the performance of selfed and outcrossed progeny, as well as sibling crosses and crosses among subpopulations. We did not find support for lower inbreeding depression in hermaphrodite lineages. Multiplicative inbreeding depression (based on seed germination, juvenile survival, survival to flowering, and flower production in the first growing season) was not significantly different between hermaphrodite lineages (δ = 0.30 ± 0.08) and female lineages (δ = 0.15 ± 0.18), although the trend was for higher inbreeding depression in the hermaphrodite lineages. The population‐level estimate of inbreeding depression was relatively low for a gynodioecious species (δ = 0.25) and there was no significant inbreeding depression following biparental inbreeding (δ = 0.01). All measured traits showed significant variation among families, and there was a significant interaction between family and pollination treatment for four traits (germination date, date of first flowering, number of flowers, and aboveground biomass). Our results suggest that the families responded differently to selfing and outcrossing: Some families exhibited lower fitness following selfing whereas others seemed to benefit from selfing as compared to outcrossing. Our results support recent simulation results in that prior inbreeding of the lineages did not determine the level of inbreeding depression. These results also emphasize the importance of determining family‐level estimates of inbreeding depression, relative to population‐level estimates, for studies of mating‐system evolution.

RESISTANCE AND TOLERANCE IN A HOST PLANT–HOLOPARASITIC PLANT INTERACTION: GENETIC VARIATION AND COSTS

Abstract Host organisms are believed to evolve defense mechanisms (i.e., resistance and/or tolerance) under selective pressures exerted by natural enemies. A prerequisite for the evolution of resistance and tolerance is the existence of genetic variation in these traits for natural selection to act. However, selection for resistance and/or tolerance may be constrained by negative genetic correlations with other traits that affect host fitness. We studied genetic variation in resistance and tolerance against parasitic infection and the potential fitness costs associated with these traits using a novel study system, namely the interaction between a flowering plant and a parasitic plant. In this system, parasitic infection has significant negative effects on host growth and reproduction and may thus act as a selective agent. We conducted a greenhouse experiment in which we grew host plants, Urtica dioica, that originated from a single natural population and represented 20 maternal families either uninfected or infected with the holoparasitic dodder, Cuscuta europaea, that originated from the same site. We calculated correlations among resistance, tolerance, and host performance to test for costs of resistance and tolerance. We measured resistance as parasite performance (quantitative resistance) and tolerance as the slopes of regressions relating the vegetative and reproductive biomass of host plants to damage level (measured as parasite biomass). We observed significant differences among host families in parasite resistance and in parasite tolerance in terms of reproductive biomass, a result that suggests genetic variation in these traits. Furthermore, we found differences in resistance and tolerance between female and male host plants. In addition, the correlations indicate costs of resistance in terms of host growth and reproduction and costs of tolerance in terms of host reproduction. Our results thus indicate that host tolerance and resistance can evolve as a response to infection by a parasitic plant and that costs of resistance and tolerance may be one factor maintaining genetic variation in these traits.

LOCAL ADAPTATION, RESISTANCE, AND VIRULENCE IN A HEMIPARASITIC PLANT–HOST PLANT INTERACTION

Abstract.— Coevolution may lead to local adaptation of parasites to their sympatric hosts. Locally adapted parasites are, on average, more infectious to sympatric hosts than to allopatric hosts of the same species or their fitness on the sympatric hosts is superior to that on allopatric hosts. We tested local adaptation of a hemiparasitic plant, Rhinanthus serotinus (Scrophulariaceae), to its host plant, the grass Agrostis capillaris. Using a reciprocal cross‐infection experiment, we exposed host plants from four sites to hemiparasites originating from the same four sites in a common environment. The parasites were equally able to establish haustorial connections to sympatric and allopatric hosts, and their performance was similar on both host types. Therefore, these results do not indicate local adaptation of the parasites to their sympatric hosts. However, the parasite populations differed in average biomass and number of flowers per plant and in their effect on host biomass. These results indicate that the virulence of the parasite varied among populations, suggesting genetic variation. Theoretical models suggest that local adaptation is likely to be detected if the host and the parasite have different evolutionary potentials, different migration rates, and the parasite is highly virulent. In the interaction between R. serotinus and A. capillaris all the theoretical prerequisites for local adaptation may not be fulfilled.

Interactive effects of pollination and heavy metals on resource allocation in Potentilla anserina L.

We studied resource allocation between sexual reproduction and clonal prop- agation in a perennial stoloniferous clonal plant, Potentilla anserina, an obligate outcrosser. We manipulated reproductive effort of Potentilla anserine either by hand-pollinating all flowers or by preventing pollination. To test the effect of resource-limiting conditions on resource allocation and reproductive output, we used a control and two levels of heavy metals (copper and nickel) to limit plant growth. The experiment was conducted as a 2 X 3 factorial design to reveal possible interactions between reproductive manipulation and resource limitation. Heavy metals decreased the total biomass of the plants and number of flowers and ramets produced. Only 50% of the plants grown with the higher level of heavy metals produced flowers. Pollination treatment interacted significantly with the heavy-metal treatment. In the metal control (no metals added) and lower heavy-metal treatment, there were no significant differences in total vegetative biomass between the two pollination treatments. Costs of reproduction in terms of subsequent flowering in the later season appeared to be clear, because the number of flowers per whole plant was lower if the plants were hand-pollinated and because the proportion of flowering ramets decreased due to hand- pollination. However, flowering may also be partly hormonally controlled. In contrast, hand- pollinated plants exposed to high concentrations of heavy metals tended to have greater biomass of vegetative plant structures and higher number of flowers compared to nonpol- linated plants. Together, these results suggest that environmental factors may alter costs of reproduction but do not support the idea that these costs are more acute in resource (pho- tosynthate)-limiting conditions.

Pollen and resource limitation in a gynodioecious species.

Differences between plant sex morphs in pollen or resource availability may affect their relative fitness and thereby the sex ratio of dimorphic species. In gynodioecious species, in which hermaphroditic and female plants coexist, a variety of factors (e.g., hermaphrodite self-fertility or rarity or pollinator discrimination against females) might be expected to lead to stronger pollen limitation in females than in hermaphrodites. On the other hand, females have been found to be superior compared to hermaphrodites in low-nutrient conditions. The effects of supplemental hand-pollination and resource addition on the reproductive output of the self-fertile gynodioecious perennial Geranium sylvaticum (Geraniaceae) were tested for several populations that differ in their female frequency (4.4-23.0%). Both pollen and resource availability limited fruit set and the number of seeds produced per plant; however, seed set (i.e., the number of seeds produced per fruit) was limited only by resources. Because pollen limitation in females did not correlate with female frequency, our results suggest that pollen limitation in females does not depend on the frequency of the pollen-producing hermaphrodites. Furthermore, because pollen and resource availability limited reproductive output of both sex morphs, these factors may not contribute significantly to maintenance and evolution of gynodioecy in G. sylvaticum.

Genetic variation in herbivore resistance and tolerance: the role of plant life-history stage and type of damage

Information of the patterns of genetic variation in plant resistance and tolerance against herbivores and genetic trade‐offs between these two defence strategies is central for our understanding of the evolution of plant defence. We found genetic variation in resistance to two specialist herbivores and in tolerance to artificial damage but not to a specialist leaf herbivore in a long‐lived perennial herb. Seedlings tended to have genetic variation in tolerance to artificial damage. Genetic variation in tolerance of adult plants to artificial damage was not consistent in time. Our results suggest that the level of genetic variation in tolerance and resistance depends on plant life‐history stage, type of damage and timing of estimating the tolerance relative to the occurrence of the damage, which might reflect the pattern of selection imposed by herbivory. Furthermore, we found no trade‐offs between resistance and tolerance, which suggests that the two defence strategies can evolve independently.