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a Department of Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: height • inbreeding depression • outcrossing • Scrophulariaceae • seed mass, seed set • self-compatibility • Verbascum thapsus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There are two main assumptions of the effective pollination hypothesis: (1) competition for pollinator service exists, and (2) increased pollinator service provides fitness benefits in terms of greater pollen quantity and/or higher pollen quality (increased outcrossing and reduced selfing). The effect of pollinator service on selfing rate may depend on the mode of selfing that the plant possesses (Lloyd, 1979). "Prior" self-fertilization involves selfing before a flower opens and before stigmas are exposed for receipt of outcross pollen. "Competing" self-fertilization involves competition between self-pollen and outcross pollen. Finally, in "delayed" self-fertilization, a flower adds self-pollen to the existing pollen load once potential for outcrossing has ceased. Delayed selfing is always advantageous in monocarpic species since it does not interfere with crossing and acts as a "better than nothing" alternative even when inbreeding depression is high.
In the present study, we explored components of the effective pollination hypothesis for apical dominance using Verbascum thapsus L. (common mullein). This species is among the tallest of monocarpic herbaceous species in North America, commonly growing to heights of 2 m or more from a single stem. It rarely branches despite numerous axillary meristems associated with shortly spaced leaves along the stem. Hence, its tall, sparsely branched architecture is a product of very strong apical dominance (Lortie and Aarssen, 1997). The main stalk and any branches that are produced terminate in a single indeterminate inflorescence spike of bright yellow flowers that are visited by pollinators, primarily bees. In a field survey, we analyzed the relationship between plant height and pollinator visitation. Pollen deposition, seed set, and seed mass of emasculated vs. intact flowers were compared to assess the role of pollinators and the importance of "prior" and "delayed" selfing in V. thapsus. Finally, we recorded seed mass and seed set derived from artificial self- and artificial cross-pollinations to evaluate self compatibility and to test for early-acting inbreeding depression.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Allocation to reproductive biomass is small in V. thapsus relative to other monocarpic species (Reinartz, 1984b). A high investment in structural biomass may represent an adaptation to its commonly impoverished habitat conditions, or the tall stalk of V. thapsus may serve to attract pollinators and aid in seed dispersal (Reinartz, 1984b). Flowers mature in a spiral from the bottom of the inflorescence to the top. Each individual flower lasts only until midday, at which time a delayed selfing mechanism is assumed to be implemented when corollas dehisce (Gross and Werner, 1978). Autumn seed dispersal is limited spatially; 75% of the seeds land within 1 m of the parent plant (Gross and Werner, 1978). However, fruits retain many seeds over the winter when dispersal may occur more extensively over ice and snow.
Study site
Quarries and disturbed fields were surveyed for prospective study populations of V. thapsus in the vicinity of Kingston, Ontario during May 1995. Two sites were selected in Kingston Township (population A 44°15' N,76°35' W; population B 44° 23' N, 76° 18' W) based on their proximity to one another (~30 km) and large population size (~500 plants). Verbascum thapsus was the dominant species at both sites. Since it was essential that flower manipulation occurred before arrival of pollinators (see below), both sites had to be treated early in the morning and hence needed to be located nearby. As well, a large population size was desirable to ensure that experimentation on selected plants would not disrupt pollination dynamics of the population as a whole. In a small population, manipulation of all the plants might severely interfere with pollinator activity and natural pollen flow. A large population provided many untreated "buffer" plants.
Both sites were spot sprayed twice with pesticide (Dursban 4E) to eliminate weevil seed predation. Spraying was conducted in early June before flowering and associated pollinator activity was prevalent. Some weevils were sighted throughout the growing season even after two applications of pesticide and were removed from the plant by hand when noticed.
Survey of plant heights in relation to pollinator visitation
A survey of site A was performed on 20 July 1995 and 3 August 1995 between 0730 and 0900. While systematically walking through the site, we observed individuals being visited by pollinators. When a pollinator was sighted on a given (target) individual, its nearest neighbor that did not have a pollinator at the same time was identified. Plant height, inflorescence length, number of branches, and number of open flowers were recorded on both the target plant and the neighbor. The distance between the target and neighbor was also recorded. A total of 50 target/neighbor pairs were recorded on each survey day. Care was taken not to sample the same target/neighbor pair more than once. Observer bias was minimal since sampling was not done from a distance but from within the patch. Hence, height differences could not be readily discerned when identifying pairs. Inflorescences of most plants were near or above eye level, reducing the possibility of pollinators on taller plants being observed preferentially. As well, since flowers open in spiral arrays up the spike, pollinator foraging position on the inflorescence is largely independent of plant height, i.e., a pollinator on a tall plant could be foraging below the height of a neighboring shorter plant and hence would not be necessarily easier to detect.
Survey of pollen deposition
Five individuals were selected randomly and tagged at site A. On each treatment day, a pair of flowers per plant was chosen based on their proximity. This was done to reduce the effect of inflorescence position, which may bias pollinator visitation. One flower was emasculated (just after flower opening) and the other was left intact. Treatments were applied for an average of 8 d per plant, throughout the flowering period (13 July to 8 August 1995). When a plant did not have any flowers open on a given treatment day, treatment was prevented and hence the total number of treatment days for such an individual was reduced. The number of open flowers was counted on each day to test for the effect of this variable on pollen deposition.
Stigmas were harvested once pollinator activity had ended for the day, but before the flowers had closed and hence before delayed selfing occurred. Slides were prepared in the field. A small cube of basic fuschin jelly (Kearns and Inouye, 1993) was placed on a clean slide and a fresh stigma then placed onto the surface of the jelly. A flame was held under the slide to melt the fuschin jelly, a cover slip was placed over the jelly and the stigma was crushed with firm pressure onto the cover slip. Crushing the stigma to a single-cell layer enabled all pollen grains to be distinguished and counted. Pollen grains were counted under a microscope by systematically scanning the complete surface area of the jelly. These treatments parallel those of treatments four (E + O) and five (O) of the pollination experiments (see below) permitting a test of the effects of treatment on both pollen deposition and subsequent seed set.
One stigma was harvested from each of ten haphazardly chosen individuals on 14 August 1995, just after flowers had opened. Stigmas were selected from the flowers that appeared (by visual inspection) to not yet have pollen deposited onto the stigmatic surface (i.e., "clean"). This was done to test the assumption that stigmas possessed little or no pollen prior to the application of treatments (i.e., no "prior" selfing). Stigmas were harvested haphazardly from those that appeared to be "clean." Stigmas were prepared and pollen grains were counted as described above.
Pollination experiments
Pollination experiments were conducted at both field sites and in a greenhouse. In the field, 30 individuals were randomly selected and tagged at each site; five plants were grown from seed for greenhouse use. In the greenhouse, plants were watered daily, nutrients were added intermittently, and plants were placed in full sunlight. Five treatments were designed to control pollen transfer so that the components of pollen sources were regulated. Two treatments involved artificial pollination, whereby either outcross (X) or self (S) pollen was introduced to the stigma of the target flower. Subsequently, anthers were removed (emasculation) (E) immediately (i.e., early morning) to prevent delayed selfing when corollas dehisced. These techniques resulted in artificial self (E + S) and artificial cross (E + X) treatments. The third treatment involved leaving the flower intact, permitting delayed or natural selfing (S) only. These first three treatments were conducted in the absence of pollinator visitation and hence were easily manipulated within the greenhouse. In the field, pollinator exclusion bags were used (made from fine cloth mesh). Wire frames were designed to prevent the bags from brushing against the flowers and interfering with pollination treatments.
All flowers open on a given day in the greenhouse were treated and divided equally among the treatments. This resulted in a total of 18–58 flowers per treatment per plant from 11 May to 9 June 1995. Each plant produced ~90 flowers over the flowering season.
The fourth and fifth treatments involved full exposure to natural pollinators (open) (O) and hence were conducted in the field sites only. Treatment 4 involved emasculation (E + O) and treatment 5 involved leaving the flower intact (O). Flowers were chosen and assigned to treatments haphazardly from open flowers that did not yet appear to have any pollen deposited onto the stigma (i.e., "clean" stigmas).
Each individual in the field was subjected to all five treatments over the peak flowering season (5 July–3 August 1995). If an individual had no open flowers on a given treatment day, treatment was prevented for that plant. The treatments were rotated over 3-d cycles, spanning 16 d for site A and 13 d for site B. Hence, the 30 plants were divided into three groups of ten and a maximum of 25 flowers were treated per plant (i.e., each plant had five flowers affected by each of the five treatments). For example, on day 1, a plant would be bagged and undergo treatments (E + S), (E + X) and (S). On day 2, the bag would be removed and the plant would have a single flower emasculated (E + O). Finally, on day 3, the bag would remain off the plant and the flowers would be left intact (O). The calyx of each flower treated was marked with nontoxic, acrylic paint with each color corresponding to treatment. To prevent pollinator interference, treatments were applied early in the morning, just as flowers were opening (first site, 0515; second site, 0630). The order of site visitation was alternated to reduce the possible effects of delayed treatment. Such a delay could permit pollinator activity before treatments were implemented and hence interfere with regulated pollen deposition. However, once pollinator activity had ceased for the day, bags were transferred to the appropriate plants at the site that would be visited second on the following day. As a result, only the emasculated (E + O) treatment could be affected, in which case pollinator activity may be slightly underestimated (i.e., bags were still on the plants when pollinator activity had commenced). Flowers closed at ~1100, depending on weather conditions. Hence, it was assumed that daily pollinator activity was long enough for the influence of this lag time to be relatively small.
In both experiments, fruits were harvested at maturity, just prior to dehiscence and number of seeds per fruit were counted. For plants in the field experiment, weevil damage (if any) was indexed by recording the number of weevils per fruit (two, one, or none) and the life stage of the weevil (adult, juvenile, or pupae). Due to weevil damage, replication of treatments per plant was reduced to zero or one in some cases. Hence, mean seed number per treatment for each plant was used in the analyses. Plant was not analyzed as a main effect due to this reduced replication.
Fifteen of the 30 plants in each site were haphazardly chosen to compare seed mass from undamaged fruits in the artificial self vs. artificial cross treatments. Twenty seeds were selected randomly from each fruit and weighed. Mean seed mass was calculated by dividing this value by 20.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The interpretation of greater branching in taller plants requires further study. Branching may be energetically affordable in only larger plants or branching may be a compensatory response to fruit infestation by weevils (Lortie and Aarssen, 1997). Branching may also be size dependent in the adaptive sense that fitness may be maximized by investing in only vertical extension when small (to maximize the chances of attaining a dominant vertical position within the population) and adding lateral growth through branching (to maximize floral display and seed production) only after the plant has already reached a tall stature.
Pollen quantity and seed set
Flowers that were artificially pollinated did not produce more seeds than intact flowers with natural pollination (Fig. 6a, b). However, intact naturally pollinated flowers produced significantly more seeds than flowers that were allowed only natural (delayed) selfing (Fig. 6d). Flowers with artificial selfing also produced more seeds than flowers with natural (delayed) selfing (Fig. 6c). Hence, under natural pollination, plants were not pollen limited. However, flowers with only delayed selfing (in the absence of pollinators) were pollen limited producing only 75% of maximum seed set; full seed set was not realized without pollinators.
Under natural pollination, emasculated flowers received a 50% reduction in pollen deposition by the time of flower closure (Fig. 3) but only a 5% reduction in seed set (Fig. 6e) relative to intact flowers. Hence, natural levels of autogamous pollen comprised ~50% of the total pollen load even before delayed selfing. Pollinators, however, may be less attracted to emasculated flowers, particularly when primarily harvesting pollen (Young and Stanton, 1990). This effect may be significant given the low nectar levels in V. thapsus and that pollinators (honey bees and bumble bees) appear to forage for only pollen (S. Donnelly, personal observation). If so, the level of autogamous pollen may have been overestimated and the role of pollinators underestimated.
Pollen quality
Self pollen is of lower quality to a plant than outcross pollen when inbreeding depression ensues. Although inbreeding depression in V. thapsus was not detected in this study through seed set or seed mass, this conclusion has limitations. Clearly, it is important to sample indices of fitness throughout the life cycle of progeny. Inbreeding depression may be detectable only in later life stages (e.g., Belaoussoff and Shore, 1995). Inbreeding depression also may not be detectable unless the plant undergoes stress (Eckert and Barrett, 1994). Environmental conditions in the field sites were not measured, although each population was large, of high density and possessed large individuals; relatively low stress may be generally implied from their success. Conditions within the greenhouse were also benign.
Darwin (1876) detected inbreeding depression in V. thapsus, using height as an estimate of fitness after several generations of selfing. This is of particular interest, under the effective pollination hypothesis for apical dominance (Aarssen, 1995). If tall plants have a fitness advantage via artificial outcrossing, it follows that tall plants that occur naturally may also have been progeny of highly outcrossed parentals. Under the effective pollination hypothesis, the parents may have been tall to gain these outcross pollinations through increased pollinator service. Here, height does not necessarily have to be a heritable trait per se, but simply an index of fitness reflecting levels of inbreeding.
When cross pollinations were performed, anthers were used (for pollen transfer) from individuals within a convenient distance (1–2 m). Hence, we did not account for biparental inbreeding associated with crossing closely spaced individuals; this can produce a conservative estimate of inbreeding depression. However, since seed dormancy enables seed dispersal in time, all of the individuals of a given "cohort" are unlikely to have been produced from the same parental generation. Therefore, distances between individuals do not necessarily reflect degree of relatedness.
Population size and density
The populations selected for this study were atypical as V. thapsus populations are often small and isolated (Gross, 1980). Population size and density probably affect pollinator attraction and degree of plant competition for pollinator service (Sih and Baltus, 1987). The cost of within-plant pollinator movement is low in dense populations (Roberston and Macnair, 1995). As a result, pollinators are able to visit many plants instead of just the most attractive and hence the benefit of attractive features is reduced. This permits an increased number of visitations per plant and the potential to maintain equalized visitation to all flowers, under the ideal free distribution hypothesis (Dreisig, 1995). In this study, pollen deposition was not influenced by flower number, which may be attributed to high population density.
Decreased outcrossing rates may result from decreases in a population size and density, inferred through seed set (Kunin, 1993) and electrophoretic analysis (Farris and Mitton, 1984; Treuren et al., 1993). These studies support the theoretical modeling of Inguarsson and Lundberg (1995), which proposes that pollinator service is not limiting in high-density environments. Pollinator limitation would, however, arise when pollinators are rare. Such circumstances arise when a population is too small to attract pollinators. Hence, the delayed selfing mechanism may be crucial for V. thapsus when growing in small, isolated populations where pollinator service may be limited and reproductive assurance is required. The proportion of autogamous pollen on stigmas in such populations may be expected to be greater than reported in the present study.
Male vs. female reproductive success
Pollinator service may benefit male as well as female reproductive success. In this study, only female RS was estimated via seed set and pollen deposition. However, increased male reproductive success is often positively correlated with attractive floral traits, measured through various indices (e.g., pollen removal). Often only an improvement in male reproductive success is realized (Willson and Price, 1977; Young and Stanton, 1990). Further study is required to test whether the benefits of tall stature under the effective pollination hypothesis for apical dominance are realized through greater male reproductive success.
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FOOTNOTES |
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REFERENCES |
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