Overcompensation in Response to Mammalian Herbivory
.... why do some plants benefit from the direct effects of herbivory?
Over the past decade there has been a heated debate as to whether or not plants can benefit from being eaten. The basis of this controversy has focused on whether the direct effects of herbivory can enhance plant fitness. Until recently, resolution has been hampered by a lack of supportive evidence. Our long-term studies of deer and elk browsing on scarlet gilia, Ipomopsis aggregata, in north central Arizona near Flagstaff have shown that herbivory results in a 2-3 fold increase in both maternal and paternal fitness. Our ultimate long-term goal is directed toward understanding overcompensation at the ecological, evolutionary, genetic, biochemical, and physiological levels.
Life Cycle of Scarlet Gilia
Scarlet gilia is a monocarpic, hermaphroditic, biennial/perennial herb that flowers from July through late September. Following germination, scarlet gilia develops into a leafy rosette and after 1-8 years in a vegetative state, it sends up a single stalk that flowers, reproduces once, and then dies. Hence it is reproductive for only 1 year making it ideal for acquiring measures of lifetime fitness.
Scarlet gilia is self-incompatible and pollinated by two species of hummingbird - the rufous hummingbird and the broad-tailed hummingbird - and a single species of hawkmoth, the white-lined sphinx.
Maternal Fitness Following Ungulate Herbivory
During the period of stem elongation, on the average 77% of all plants are browsed by mule deer and elk. Of these 80% have approximately 95% of their aboveground biomass consumed, being browsed down to a height of 1-2 cm. Removal of the single inflorescence stimulates the production of, on the average, five new flowering stalks from dormant lateral buds along the remaining portion of the plant's stem. This change in plant architecture results in an increase in flower and fruit production. Because there are no significant differences between browsed and unbrowsed individuals in the number of seeds produced per fruit, seed mass, germination success, or subsequent seedling survival, an increase in total fruits produced by browsed plants results in an increase in maternal fitness. This has been further substantiated by tracking the long-term success of paired plots of browsed and unbrowsed plants.
Paternal Fitness Following Ungulate Herbivory
More recent studies have shown that ungulate herbivory also results in an increase in paternal fitness. Although browsed plants produced approximately 2 times as many flowers per plant as plants that were not browsed, no reductions in either the number of pollen grains produced per flower or pollen viability were found. In addition, even though browsed plants produced narrower corollas and shorter styles than plants that were not browsed, hand- and hummingbird-pollination experiments demonstrated that these differences did not impede the transport of pollen, such that browsed and unbrowsed plants sired and set equal numbers and qualities of seed per cross, whether crossed with a browsed or an unbrowsed "female". Furthermore, visitation rates on a per flower basis were not significantly different between browsed and unbrowsed plants. An additional experiment (and genetic analysis), which was designed to naturally assess the paternal contribution of browsed versus unbrowsed individuals, confirmed that higher flower, and hence, pollen production by browsed plants translates into an approximate two-fold increase in paternal fitness.
Effects of Secondary Herbivory
Studies of natural and simulated herbivory were conducted to assess the effects of secondary herbivory on the reproductive success of Ipomopsis aggregata. Over the 5 year period of this study 77% of all plants were browsed by ungulate herbivores. Of these, 33% were subsequently browsed. Removal of the single inflorescence stimulated the production of, on average, five new flowering stalks from dormant lateral buds along the remaining portion of the plant's stem. Although regrowth shoots were initially avoided by ungulates following the removal of scarlet gilia's single inflorescence, plant tips were secondarily browsed following stem elongation and flower bud formation.
Secondary herbivory had no effect on the compensatory outcome. Plants that were naturally browsed produced significantly higher numbers of flowers and fruits than plants that were not eaten, even when plants were secondarily browsed. Because there were no significant differences in numbers of seeds produced per fruit or seed mass, an increase in total fruits produced by browsed plants resulted in an increase in fitness through seed production.
Observational and experimental results indicate that I. aggregata switches from a "mutualistic" to an "antagonistic" interaction with its ungulate herbivores in order to achieve its greatest fitness. Results of experimental clipping showed that high levels of secondary herbivory on I. aggregata would be detrimental, decreasing fitness by approximately 70%. An apparent change in plant quality following the initial bout of herbivory, however, deters high levels of subsequent herbivory, restricting tissue removal to the tips of the plant. Although the phytochemistry of I. aggregata has not been extensively investigated, it is known to contain a number of phenolic compounds, including hydroquinone, scopoletin, related coumarin derivatives, and cucurbitacins, primarily cucurbitacin B. Cucurbitacins, in particular, are bitter triterpenes that are highly toxic to vertebrate herbivores; these compounds are known to be induced in other systems by herbivore feeding.
Effects of Competition
When plants were found in close association with either pines or grasses (to add in the potential negative effects of competition), browsed plants still outperformed control plants, producing significantly more flowers and fruits than uneaten control plants.
Selective Agents in the Removal of Apical Dominance
Before we can begin to understand the evolutionary interactions between plants and herbivores it is necessary to ascertain whether there are other selection pressures, both past and present, involved in generating the observed plant response patterns. Apical damage, for example, can be caused by a number of factors other than herbivory, including frost damage, fire, pathogen attack, trampling and breakage by wind.
Prior to fire suppression programs beginning in the early 1900's, fire was an important historical component of the forest/grassland community of which scarlet gilia is a part. Low intensity surface fires occurred at regular intervals of 2-10 years, burning through all parts of the community. These lightning-induced fires predominate during the monsoon season and coincide with the period of stem elongation and flowering in scarlet gilia. Thus fire represents a relatively predictable and potentially important selection pressure.
Nonetheless, experiments designed to test the effects of fire demonstrated that fire was not an important selective agent involved in shaping scarlet gilia's compensatory response (see the Effects of Fire section). Two alternative candidates, frost damage and ungulate trampling, can cause the removal of apical damage and a response similar to that generated by ungulate and insect herbivores. However, they are probably minor factors favoring selection toward growth compensation; experimental and observational results demonstrate that apical dominance was removed in only 3% of plants exposed to freezing temperatures and ungulate trampling caused breakage and release of apical dominance in only 0.2% of plants. Long term field observations suggest that neither pathogen attack or wind damage play any role in the release of apical dominance in these sites.
Effects of Fire
The most common response to fire was the production of one or more new clonally derived rosettes. This was an unexpected result; this typically monocarpic herb rarely produces clonal offspring. Although rosette production lessened the detrimental effects of fire by giving plants that cloned a second chance to flower, these newly formed rosettes delayed flowering for at least one year and had significantly higher overwinter mortality rates than rosettes from unburned control plots. In addition, significantly fewer individuals from the burn treatments flowered and there was significantly higher immediate mortality. There was, however, no detrimental effect on the reproductive success of individuals that flowered following the burn. Overall, cumulative estimates of plant performance suggest that at the population level fire results in a 4.5-fold decrease in relative plant fitness. However, fire-induced seed germination could negate the detrimental effects of fire on the population dynamics of scarlet gilia. In the year of the burn there was a 116-fold increase in the number of germinating seeds and by the second year this translated into an approximate 6-fold difference in the number of surviving rosettes.
Geographic Evidence for Overcompensation
Studies recently completed have also shown that overcompensation is more widespread than previously thought. Evidence for overcompensation was found in more than half of the populations studied throughout Colorado.
Evolution of Overcompensation - Is There a Heritable Basis?
We are presently interested in studying the evolution of overcompensation using experimental and quantitative genetic approaches within and among populations of scarlet gilia. These studies will be conducted within each of four populations that have been subjected to very different historical levels of ungulate herbivory; two of these overcompensate (populations in Arizona) and the other two do not (populations in Colorado). Specifically we are interested in:
- Measuring the relative importance of environmental versus genetic factors in determining variation within populations in regrowth potential following ungulate herbivory.
- Determining whether there are genetic constraints on the evolution of traits associated with fitness compensation in either the Arizona or Colorado populations.
- Estimating selection gradients on traits associated with fitness compensation within each of the populations.
- Measuring the relative importance of environmental versus genetic factors in determining variation in regrowth potential between four populations of scarlet gilia, two that overcompensate and two that do not.
How Common is Overcompensation?
We are also interested in the generalizability of the phenomenon of overcompensation. Although there are an increasing number of cases of oovercompensation being uncovered, it is possible that we are continually overlooking many cases of overcompensation. To date, the majority of studies on plant-herbivore interactions have focused on the maternal, as opposed to the paternal, side of fitness. Because there are many plants that produce more flowers following apical damage where fruit and seed set is reduced ar unaffected, the paternal component of fitness could be enhanced, resulting in a increase in total fitness. We have provided the first example of such a case. Using a combination of experimental and genetic approaches, we have shown that herbivory on Ipomopsis arizonica results in an overall increase in reproductive success through the paternal as opposed to the maternal component of fitness.
Studies incorporating physiological, biochemical and molecular genetic mechanisms of over compensation are of primary future interest.
Functional Genomics of Overcompensation
With cDNA microarrays it is now possible to examine the simultaneous expression of thousands of genes permitting analysis of a plants response to any environmental cue of interest (biotic or abiotic). Experimental comparisons will enable one to not only better assess the behavior of genes previously implicated in a given process, but also to assist in the discovery and identity of novel genes and gene pathways associated with particular biological phenomena. Changes in the multi-gene patterns of expression can provide clues about regulatory mechanisms and broader cellular functions and biochemical pathways leading to phenotypic differences. In addition, an added advantage of using arrays, especially those that contain probes for thousands of different genes, is that it is not necessary to guess what the important genes or mechanisms are in advance. To date, several informative microarray analyses have been carried out on Arabidopsis including studies on coordinated plant defense responses, differential gene expression in response to mechanical wounding and insect feeding, seed development, discovery of nitrate regulatory mechanisms and gene discovery and metabolic pathway analysis. These studies point out the utility and power of microarray analyses in gaining new and exciting information.
In this study we are using cDNA microarrays to begin to assess the molecular basis of overcompensation in Arabidopsis thaliana. We have screened and characterized over 50 ecotypes acquired both from the Arabidopsis Biological Resource Center located in Columbus, Ohio and from wild populations where genetic variation for tolerance has been previously characterized. These plants demonstrate a range of responses, from those that undercompensate to those that overcompensate following removal of apical dominance during the early stages of stem elongation (i.e., those that increase flower, fruit and seed production as a result of architectural changes in the plant following apical damage).
Using the Affymetrix GeneChip System we are examining the differential expression of over 25,000 Arabidopsis genes comparing plants that undercompensate to those that overcompensate following early season clipping (i.e., removing apical dominance at approximately 2 cm of growth) chosen from the variation observed among maternal lineages within a single ecotype. We will also compare clipped plants to their associated unclipped controls (for an overall total of 4 treatment groups; 2 clipped and 2 unclipped controls from, 1 clipped and 1 unclipped undercompensating set of plants and 1 clipped and 1 unclipped overcompensating set of plants).
Comparisons between these groups will be made from three sets of tissues in three separate sets of arrays (4 chips/tissue X 3 tissues = 12 chips) including 1) tissues taken from the entire aboveground plant, 2) tissues taken from the entire root system and, 3) meristematic tissue taken at an early stage following clipping (control tissues will be taken at a similar stage of growth as the regrowth tissues from the clipping experiments). As a first step, we chose to screen entire aboveground and belowground tissues because growth compensation is undoubtedly an integrated whole plant response; i.e., new meristems are activated, architectures are altered, regrowth rates are enhanced, photosynthetic rates are enhanced, flower, fruit and seed production is enhanced, reallocation patterns are altered, roots become sinks for a period of time, increased root secondary vascular tissue, etc. Such an approach is not unreasonable, given that genes associated with many of these traits and pathways have already been characterized in Arabidopsis.
We are also going to look at one tissue type most closely associated with growth compensation early in development meristematic tissue. Although ideally we would like to look at several tissue types through time to assess an integrated picture of growth compensation and the phenomenon of overcompensation, costs would be prohibitive at such an early stage. Instead we hope to characterize the system to gain some preliminary data that will help guide future work and form the basis of future proposals to State and Federal agencies that would clearly be interested in this line of research.