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2Department of Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0116
Received for publication July 9, 1998. Accepted for publication December 22, 1998.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: behavior; hummingbirds • Mimulus • pollination • Scrophulariaceae • stigma sensitivity
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). All species with touch-sensitive stigmata have hermaphroditic flowers with tubular corollas, a single pistil, and a stigma composed of two lobes. Time to closure of the stigma following tactile stimulation is quite variable among species, taking anywhere from 1 or 2 s to over an hour (Bertin, 1982
; A. E. Fetscher, unpublished data). There appear to be no species with touch-sensitive stigmata outside the Scrophulariales. Ipomopsis aggregata (Polemoniaceae) has a trilobed stigma, which also closes, but receipt of pollen, rather than touch, is necessary to elicit this response (Waser and Fugate, 1986
).
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; Lloyd, 1911
; Newcombe, 1922
, 1924
; Guttenberg and Reif, 1958
; Linskens, 1976
), mostly focusing on the role of turgor as a proximate explanation of the mechanics of closure. Various hypotheses addressing the adaptive significance of stigma closure have been proposed: stigma closure aids pollen capture and receipt (Linskens, 1976
; Thieret, 1976
), prevents intrafloral selfing (Kerner von Marilaun, 1895
; Newcombe, 1922
, 1924
; Lloyd and Yates, 1982
; Ritland and Ritland, 1989
), and reduces interference between pollen receipt and export functions within the flower (Webb and Lloyd, 1986
). However, no empirical tests of these hypotheses have been published.
This study asks three main questions. First, what triggers stigma closure in Mimulus aurantiacus and what factors determine whether the stigma reopens? We test the separate effects of touch and pollen on stigma closure. In addition, we use experiments to determine the effects of pollen load and fertilized ovules on the frequency and timing of stigma reopening. Second, does stigma closure enhance female fitness? One hypothesis for the adaptive significance of stigma closure is that it aids capture, adherence, or germination of pollen and thus enhances seed set (Linskens, 1976
; Thieret, 1976
). We compare pollen receipt and seed set of flowers with stigmata manipulated to remain open with that of flowers with stigmata that close. Finally, is stigma closure costly to female fitness? Stigmata that close and do not reopen prevent the flower from receiving additional pollen and, conceivably, pollen from additional donors. Stigma closure after receipt of insufficient pollen for full seed set would appear costly to female function. Even if full seed set were achieved, failure to receive additional pollen might be costly to female fitness if higher quality offspring result from larger pollen loads or pollen from more donors (Willson, 1979
). We use experimental manipulations to examine whether additional pollen is received if stigmata do not close and how receipt of additional pollen affects seed number and quality.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Factors determining stigma closure and reopening
Reopening time of stigmata not receiving pollen was determined on 22 first-day flowers (across six bushes) by lightly touching stigmata with a finger free of pollen. Corollas and anthers had been removed the day before in order to discourage pollinator visitation. Preliminary studies show that removal of these organs has no effect on stigma behavior. All stigmata closed when touched, after which flowers were observed at half-hour intervals to determine whether their stigmata had reopened.
To examine the effect of pollen alone on stigma closure, pollen was gently sprinkled onto the stigmata of 28 first-day flowers across seven bushes. This technique does not induce the immediate closure characteristic of response to touch, presumably because stigmata are insensitive to touch below a certain amount of pressure. The stigmata were observed at half-hour intervals following addition of pollen and scored for whether they had closed.
The time lag to reopening of pollinated stigmata was estimated in 1998 at the UC San Diego field station in La Jolla, California, by hand pollinating (using uniformly light pollen loads) the stigmata of 77 first-day flowers, across 11 bushes, from which corollas and stamens had been removed. Unless otherwise noted, all hand pollinations used a roughly equal mixture of pollen from five randomly chosen donors mixed on a microscope slide. The pollen mixture was then applied to stigmata by pressing them gently against the slide. All stigmata closed during hand pollinations. The stigmata were then observed at 2-h intervals from 20 to 32 h following pollination and scored for whether they had reopened.
To determine the effects of pollen receipt and seed production on stigma reopening and reclosing over a span of several days, stigmata of all first-day flowers across 20 bushes were hand pollinated in May through July 1997 with variable pollen loads. Corollas and stamens had been removed prior to pollination. All stigmata closed in response to contact during the application of pollen. Stigmata were then observed at either 1 and 3 d, 1 and 6 d, 2 and 4 d, 2 and 5 d, or 3 and 5 d following pollination and scored for whether they were open or closed. Upon senescence, stigmata were collected and stored in FAA (Kearns and Inouye, 1993
). To count the number of pollen grains that had been applied during hand pollinations, stigmata were stained in 0.1% aniline blue in 0.15 mol/L K2HPO4 (pH 9.0) and mounted on microscope slides for viewing under ultraviolet illumination. Each pollen grain on the slide was counted by eye. Mature fruits were harvested for seed counts.
The time it takes pollen to access ovules was determined in 1998 by hand pollinating 155 flowers across six bushes, then removing each style at its junction with the ovary using a sharp forceps at some hourly timepoint between 14 and 31 h. Ovaries were observed 1 mo after pollination in order to deduce whether pollen tubes had reached the ovary as evidenced by development of fruits.
Costs and benefits of stigma closure for fitness through female function
In 1996, manipulations preventing stigma closure were performed in order to examine the effect of stigma closure on female fecundity and offspring quality. Stamens were removed, before anther dehiscence, from all flowers used in the experiment in order to avoid any possible confounding effects of intrafloral self pollen on seed number and quality. The possible effect of stigma closure on intrafloral pollen transfer was not being examined in the present study. Within each bush, stigmata from half of all first-day flowers (one from each nodal pair), across 16 bushes, were forced to remain permanently open, in a natural position, by anchoring the nonreceptive surface of each stigmatic lobe to the style with Elmer's® School Glue. This manipulation leaves the receptive surface of the stigma intact and in the same orientation as an unmanipulated, open stigma as depicted in Fig. 1. The remainder of the flowers were controls with unmanipulated stigmata. Natural pollination was allowed to ensue, and mature fruits from all flowers in the experiment were collected for seed counts. To estimate an early component of offspring quality, groups of 50 seeds/fruit were placed in petri dishes containing 5 mL sterile water, set on a laboratory shelf under ambient light, and every 2 d for a period of 14 d, positions of petri dishes were randomly rearranged. After 14 d, seeds were scored for germination.
The effect of stigma closure on the amount of pollen received was determined in 1997 by repeating the manipulation to prevent stigma closure as described above on one first-day flower at each node across 13 bushes, leaving the remaining flowers to serve as controls. As before, stamens were removed prior to anther dehiscence. Following natural pollination, stigmata were collected from manipulated and control flowers upon senescence, and pollen counts were made as described above.
Whether or not pollen arriving at a stigma subsequent to a previous pollen load is capable of contributing to seed set has important implications for whether stigma closure is costly to female fitness. Late-arriving pollen might be incapable of siring seed if earlier fertilized ovules outcompete later ones for maternal resources. To determine whether pollen loads received subsequent to the first would be able to sire seed, all first-day flowers (across ten bushes), from which stamens and corollas had been removed, were hand pollinated with small, relatively uniform, pollen loads. A second load of pollen was added to a subset of these stigmata randomly with respect to node on each bush either 2, 4, 8, 10, 22, 28, or 48 h after the first addition of pollen. For all stigmata that had not reopened prior to the second addition of pollen, it was necessary to spread open the closed stigmatic lobes with a forceps and insert pollen with another forceps. Upon senescence of the flowers, stigmata were collected for pollen counts and fruits were harvested for seed counts.
If stigmata were to remain open following pollen deposition they could receive multiple loads of pollen and, potentially, pollen from additional sires. To determine whether multiple sires contributing to the total pollen received by a flower result in fitter offspring, all first-day flowers on 35 bushes were hand pollinated using roughly equal, saturating amounts of pollen from either one, or an equal mixture of five, randomly chosen donors. Corollas and stamens were removed from these flowers prior to hand pollination. Stamens only were removed from other first-day flowers that were otherwise unmanipulated to serve as naturally pollinated controls. Upon maturation, fruits were harvested, seeds were counted, and germination rates of seeds from each fruit were determined as described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). All stigmata touched in the absence of pollen reopened between 2.5 and 4.5 h (
= 3.14 h, N = 22) after closure. Time to reopening was generally much longer, and the frequency of reopening much lower, when stigmata had been pollinated than when they had been touched but no pollen was transferred. Of the 77 stigmata that were hand pollinated and observed at 2-h intervals from 20 to 32 h after pollination, one reopened sometime before 20 h after pollination, five reopened between 20 and 22 h, two reopened between 22 and 24 h, two reopened between 24 and 26 h, and two reopened between 26 and 28 h. The remaining 55 stigmata stayed closed for the duration of the experiment. For the long-term stigma behavior experiment in which stigmata were hand pollinated with variable loads and observed at two timepoints over a span of several days, 33 of 199 stigmata were found to be open at least at one observation timepoint. Twelve of these stigmata underwent a secondary closure, which occurred between 1 and 3 d after pollination in one flower and sometime between 3 and 6 d in all others (Table 1). Of the flowers that were hand pollinated and then had their styles removed at some timepoint between 14 and 31 h after pollination, the ovaries of 14.2% showed signs of development into fruits a month later. Some of the ovaries whose styles had been removed as early as 15 h after pollination showed signs of development into fruits, indicating that pollen can reach ovules as soon as 15 h following deposition onto the stigma.
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650 pollen grains, the slope of the regression relating pollen to seeds was indistinguishable from zero, indicating that receipt of pollen beyond 650 grains would not be expected to result in an increase in seed set. The mean seed set for flowers receiving 650 pollen grains was 247 (SE =13.9; N = 44). Even below these numbers of pollen grains and seeds, stigmata usually failed to reopen. Pollen and seed numbers separately were each highly significant determinants of stigma reopening in simple logistic regressions (P < 0.0001 for both variables). However, multiple logistic regression showed that seed count remained a highly significant determinant of stigma reopening (P = 0.0005) when the effect of pollen was removed, whereas pollen count was not significant (P = 0.0784) when seed number was present in the model.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). What triggers secondary closure in some (nearly half) of the stigmata that reopen after hand pollination, a phenomenon that has been observed in several species in the Scrophulariales (Newcombe, 1922
; Bertin, 1982
), remains unclear. There was no obvious relationship between secondary closure and pollen or seed counts. So long as some pollen was received, stigma reopening was not a direct function of the size of the pollen load, but was strongly dependent on the number of seeds produced (Figs. 2, 5). The relationship between seed count and stigma reopening suggests that the stigma may remain closed permanently in response to the onset of development of a sufficient number of fertilized ovules and reopen only when the number of ovules fertilized is low. When a pollinated stigma does reopen, it takes longer to do so than the amount of time it takes pollen to reach ovules (within 15 h), providing additional support for the hypothesis that reopening of a pollinated stigma is a response to the number of ovules fertilized.
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130 seeds set, stigmata never reopen. Even below this number, 72% of stigmata failed to reopen. The tendency of the stigma to reopen when touched in the absence of pollen, yet to remain closed after receiving pollen, has also been noted in Proboscidea louisianica (Martyniaceae) (Thieret, 1976
), Catalpa speciosa (Bignoniaceae) (Stephenson and Thomas, 1977
), and Mimulus guttatus (Dudash and Ritland, 1991
).
One hypothesis for the adaptive significance of stigma closure is that it improves capture and germination of, and fertilization by, pollen (Linskens, 1976
; Thieret, 1976
). We found no support for this hypothesis. Stigma closure did not increase seed set nor germination rate. However, we have not assessed potential benefits of stigma closure to offspring quality post germination.
Stigma closure would seem likely to result in pollen limitation if closure were permanent even after receiving insufficient pollen for full seed set, which is often the case in M. aurantiacus. Despite this, we found no reduction in seed set for flowers whose stigmata closed, relative to flowers with stigmata manipulated to remain permanently open. Our failure to detect a female fecundity cost of stigma closure could not have been attributable to the inability of secondary pollen loads to sire seed. Pollen added up to 48 h after the first application of pollen can sire additional seed (Fig. 4). Stigma reopening following pollen deposition could therefore allow a second opportunity for the receipt of pollen that could fertilize additional ovules.
Several factors could account for our failure to detect a cost of stigma closure in terms or reduced seed set. First, limited visitation, such that flowers cannot reliably expect a second pollen-depositing visit could partially explain the lack of cost. Stigmata tacked permanently open with glue and exposed to pollinators in a natural setting did, on average, receive more pollen than unmanipulated control stigmata. However, pollen receipt was highly variable across nodal pairs of treatment and control flowers. Only six of 22 of the glued open stigmata received more than twice the number of pollen grains found on control flowers, while at four nodes, the control flowers received a twofold or greater excess of pollen (Fig. 2). Second, much of the difference in mean pollen loads received by the two treatments occurred in flowers that received >650 grains. We can detect no increase in seed set from pollen loads above this value. Third, based on pollen loads found on naturally pollinated glued open and control stigmata, the regression line in Fig. 2 predicts mean seed sets of 164 for manipulated flowers and 142 for controls. This difference, though it potentially represents a significant cost of stigma closure to female function on an evolutionary scale, might not be detectable given the variation in the data and our sample sizes.
We found no measurable cost nor benefit of stigma closure in M. aurantiacus to seed production or quality. Two other hypotheses for the adaptive significance of this behavior are being examined. First, intrafloral selfing might be prevented if stigmata close before the pollinator has an opportunity to reach the anthers and move pollen back onto the stigma. Second, Webb and Lloyd (1986)
suggest that stigma closure could represent "movement herkogamy" in which female organs, after pollen receipt, move to reduce interference with pollen dissemination. The architecture of M. aurantiacus flowers with tubular form, approach herkogamy, broad stigma lobes and anthers closely appressed to the style appears to make interference between female and male functions likely. The fact that pollinated stigmata are capable of reopening, but tend not to, also makes this a reasonable hypothesis. Such behavior suggests that, if the open stigma interferes with pollen removal or export, it may benefit the plant to keep stigmata closed (and switch to a functionally male state) after receiving pollen, even though maximal seed set may not always be achieved.
Since pollen alone causes the stigma to close, as it does in I. aggregata (Waser and Fugate, 1986
), rapid response to touch suggests that there is something to be gained by immediate response to pollinator visitation. We have shown that stigma closure is not necessary for pollen capture nor adherence, making such a function an unlikely explanation for rapid stigma closure. The other hypotheses for the adaptive significance of stigma sensitivity to touch, that it may prevent intrafloral selfing or reduce pollen–pistil interference, are both good candidates to explain the need for rapid closure of the stigma, because both hypotheses provide a context in which the advantage of stigma closure can be realized within a single visit by a pollinator to a flower. In fact, it appears that rapid stigma closure would be necessary for preventing intrafloral selfing. Stigma closure in M. aurantiacus usually occurs rapidly enough that closure could often be complete before the forager leaves the flower. It must also be taken into account, however, that many Scrophulariales with touch-sensitive stigmata do not respond as rapidly to touch. In many Bignoniaceae, for instance, stigma closure takes between 10 and 60 s, longer than a pollinator visit (Bertin, 1982
; A. E. Fetscher, unpublished data), so prevention of intrafloral selfing is unlikely to be the universal explanation of stigma closure. Experiments are underway to test both the male–female interference and the intrafloral selfing hypotheses for the adaptive significance of stigma closure.
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FOOTNOTES |
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3 Author for correspondence (fetscher@biomail.ucsd.edu).
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Burck, W. 1902 On the irritable stigmas of Torenia Fournieri and Mimulus luteus and on the means to prevent the germination of foreign pollen on the stigma. Koninklijke Akademie van Wetenschappen te Amsterdam. Proceedings of the Section of Sciences 4: 184.
Dudash, M. R., and K. Ritland. 1991 Multiple paternity and self fertilization in relation to floral age in Mimulus guttatus (Scrophulariaceae). American Journal of Botany 78: 1746–1753.[CrossRef][ISI]
Guttenberg, H. V., and B. Reif. 1958 Der Mechanism der Narbenbewegung von Mimulus sp. in seiner Abhangigkeit von der oxydativen Atmung. Planta 50: 498–503.[CrossRef][ISI]
Kearns, C. A., and D. W. Inouye. 1993 Techniques for pollination biologists. University Press of Colorado, Niwot, CO.
Kerner von Marilaun, A. 1895 The natural history of plants, their forms, growth, reproduction, and distribution. Henry Holt, New York, NY.
Linskens, H. F. 1976 Stigmatic responses. In K. H. Sheikh and Y. Vardar [eds.], Proceedings of the third MPP Meeting, 1–12. Ege University, Izmir.
Lloyd, D. G., and J. M. A. Yates. 1982 Intrasexual selection and the segregation of pollen and stigmas in hermaphrodite plants, exemplified by Wahlenbergia albomarginata (Campanulaceae). Evolution 36: 903–913.[CrossRef][ISI]
Lloyd, F. E. 1911 Certain phases of the behavior of the stigma-lips in Diplacus glutinosus Nutt. Plant World 14: 257.
Mitchell, R. J. 1997 Effects of pollination intensity on Lesquerella fendleri seed set: Variation among plants. Oecologia 109: 382–388.[CrossRef][ISI]
Newcombe, F. C. 1922 Significance of the behavior of sensitive stigmas I. American Journal of Botany 9: 99–120.[CrossRef][ISI]
———. 1924 Significance of the behavior of sensitive stigmas II. American Journal of Botany 11: 85–93.[CrossRef][ISI]
Ritland, C., and K. Ritland. 1989 Variation of sex allocation among eight taxa of Mimulus guttatus species complex (Scrophulariaceae). American Journal of Botany 76: 1731–1739.[CrossRef][ISI]
Stephenson, A. G., and W. W. Thomas. 1977 Diurnal and nocturnal pollination of Catalpa speciosa (Bignoniaceae). Systematic Botany 2: 190–199.
Thieret, J. W. 1976 Floral biology of Proboscidea louisianica (Martyniaceae). Rhodora 78: 169–179.
Waser, N. M., and M. L. Fugate. 1986 Pollen precedence and stigma closure: a mechanism of competition for pollination between Delphinium nelsonii and Ipomopsis aggregata. Oecologia 70: 573–577.
Webb, C. J., and D. G. Lloyd. 1986 The avoidance on interference between the presentation of pollen and stigmas in angiosperms. II. Herkogamy. New Zealand Journal of Botany 24: 163–178.[ISI]
Willson, M. F. 1979 Sexual selection in plants. American Naturalist 113: 777–790.[CrossRef][ISI]
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