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Reproductive Biology |
School of Biological Sciences, Washington State University, Pullman, WA, 99164-4236
Received for publication April 8, 2003. Accepted for publication August 5, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: Asclepias • geitonogamy • inflorescence design • plant's dilemma • pollination
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Geitonogamy is likely the most widespread mode of self-pollination (Lloyd, 1992
), and natural populations of many hermaphroditic taxa experience substantial geitonogamous pollination (de Jong, Waser, and Klinkhamer, 1993
). Geitonogamy bears potentially significant mating costs to all plant species in which individuals present multiple flowers at once (Snow et al., 1996
). In self-compatible species, geitonogamy will result in greater inbreeding depression (Holsinger and Thomson, 1994
; Harder and Barrett, 1995
). In self-incompatible species, geitonogamy will result in pollen discounting and outcross-pollen interference (Holsinger, Feldman, and Christiansen, 1984
; Lloyd and Webb, 1986
; Schoen and Lloyd, 1992
; Klinkhamer and de Jong, 1993
). Due to its widespread and largely deleterious nature, geitonogamy has been hypothesized as a major selective force in plant reproductive ecology (Wyatt and Broyles, 1994
). Here, we investigate the impacts of geitonogamy on inflorescence design evolution.
Inflorescence design, the manner in which total flower number is apportioned between the size, arrangement, spacing, and phenology of inflorescence units (Fishbein and Venable, 1996
), is at the core of angiosperm reproductive success. The majority of flowering plants participate in pollination mutualisms (Buchmann and Nabhan, 1996
), a coevolutionary relationship that has shaped how, when, and where angiosperms present their flowers to insect pollinators. However, the adaptive significance of inflorescence design is still not well understood (Wyatt, 1976
, 1982
; Wyatt and Broyles, 1994
; Fishbein and Venable, 1996
), particularly the role of geitonogamy in constraining evolution of inflorescence size. Here, we focus on two major hypotheses that have emerged concerning inflorescence design evolution: the pollen donation and plant's dilemma hypotheses.
The pollen donation hypothesis emphasizes that large inflorescences enhance male fitness (Willson and Rathcke, 1974
; Broyles and Wyatt, 1995
; Burd and Callahan, 2000
). A central assumption of the pollen donation hypothesis, stemming from the common observation that most flowers do not produce fruit (Sutherland and Delph, 1984
), is that maternal resources, not pollen, limit fruit set. On the other hand, the plant's dilemma hypothesis emphasizes that the evolution of inflorescence design likely reflects a trade-off (or a plant's dilemma, Klinkhamer and de Jong, 1993
; Ohashi and Yahara, 1999
) between pollinator attraction selecting for large inflorescences and geitonogamy selecting for smaller inflorescences (Holsinger, 1996
).
Much of the pioneer work on inflorescence design evolution has been conducted on milkweeds (Asclepias). Asclepiads possess a unique pollen packaging system in which pollen is dispersed in discrete packets containing hundreds of pollen grains known as pollinia. Pollinia greatly facilitate the estimation of male and female fitness because of the relative ease of counting the number of pollinia removed (an estimate of male fitness) or inserted (estimate of female fitness) (Willson and Price, 1977
; Broyles and Wyatt, 1990
, 1991
; Pleasants, 1991
; Fishbein and Venable, 1996
; Morgan and Schoen, 1997
). Willson and colleagues (Willson and Rathcke, 1974
; Willson and Price, 1977
) found that individuals of A. syriaca having larger displays attracted more pollinators and had a greater number of pollinia removed, but did not produce higher fruit set. Willson concluded that female fitness must be resource limited (not pollen limited), and that large floral displays therefore primarily benefit male fitness—the pollen donation hypothesis. However, Wyatt (1980)
noted that high selfing rates in the self-incompatible milkweeds could also explain these observations. Thus, large displays both attract pollinators and promote geitonogamy—the plant's dilemma hypothesis.
This study investigates the impact of geitonogamy on the evolution of inflorescence design in Asclepias speciosa. Specifically, we evaluate the significance of geitonogamy for female fertility in natural populations of A. speciosa. Our goals are to investigate whether the following predictions of the plant's dilemma hypothesis occur in natural milkweed populations: (1) female fertility is pollen limited and (2) self-pollinations increase abortion rates and decrease fruit set. Our experimental results suggest that geitonogamy increases abortion rates and reduces female fitness, and thus likely plays an important role in the evolution of inflorescence design in milkweeds.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). It spreads clonally by rhizomes, and each ramet produces several (2–7, mean = 5; M. S. Finer, unpublished data) temporally staggered umbels during the summer flowering season. Umbels typically consist of 15–25 flowers (M. S. Finer, unpublished data), which all share a common period of anthesis (flowers open within 2 d of each other and wilt simultaneously; Bookman, 1984
). Each flower has five stigmatic slits; three slits lead to one ovary and the other two slits lead to the second ovary. Asclepias speciosa appears to be largely self-incompatible, with rare self-compatible individuals (Bookman, 1984
). Self-pollination rates in natural Asclepias populations appear to be extremely high (Wyatt and Broyles, 1994
), with estimates ranging from 66 to 97% (Morse, 1982
; Pleasants, 1991
; Shore, 1993
). In addition, milkweeds possess a unique late-acting self-incompatibility mechanism in which all selfed ovules abort during early seed development (Bookman, 1983
; Kahn and Morse, 1991
). This abortion mechanism is likely to significantly reduce female fitness, since each selfed pollinium contains enough pollen grains to usurp all ovules within the ovary. Further, male fitness is completely reduced, since all pollinia are inherently discounted (i.e., removed from the outcross pollen pool). Experimental populations are from locations in eastern Washington, selected during the summer of 1999. We selected three undisturbed populations from Turnbull National Wildlife Refuge near Spokane, WA, and three disturbed populations along roadsides of Whitman County, Washington. The undisturbed populations consist of 500–800 ramets, and disturbed populations consist of fewer ramets (200–300). The disturbed populations were near roads, but buffered by substantial vegetation. Disturbance is not a central focus of this study, but the distinction between disturbed and undisturbed sites is maintained to accurately reflect sampling strategy in statistical analysis.
Experimental design
To investigate the consequences of geitonogamy for fruit production, each population received three experimental pollination treatments (Bag, Open, Control; described in detail below). Treatments were replicated in a blocked design; each block consisted of one ramet for each of the three treatments and was replicated 10 times in the population. Location of blocks in each population were chosen to evenly sample the spatial extent of the population. Within each block, the three treatments were randomly assigned to ramets that were similar in size and flowering phenology and within 1 m of one another. Whenever possible, the blocks were chosen from the same root crown in order to minimize genetic variation between treatments. We recorded total number of umbels produced per experimental plant to assess ramet similarity between treatments. The umbel closest to anthesis (flowers all still in bud) on each ramet was designated the experimental umbel. All three treatments within a block were always initiated, hand-pollinated (except the Control), and scored for fruit set at the same time.
Bag treatment umbels were covered in pollinator-excluding bridal veil while all the flowers were still in bud. Approximately 2–3 d after all flowers on the umbel had fully opened, the bag was temporarily removed and six flowers received one hand-pollinated outcross pollinium each. Hand pollinations followed the protocol described by Bookman (1984)
and attempted to mimic the natural pollination process. Six hand pollinations per umbel exceed the maximum number of fruits observed on any naturally pollinated umbels (four). Pollen always came from a local source in order to minimize outbreeding depression, but from a different population in order to guarantee that it was outcross pollen. Pollinations within a block shared a pollen pool consisting of 3–4 flowers from different individuals from the local pollen source. Each block then had a different pollen pool from the other blocks. The bridal veil was replaced immediately after the hand-pollinations.
Open treatment umbels were not covered in bridal veil. Umbels were allowed to open with full exposure to natural pollination. Following 2–3 d of this open pollination, six flowers received one hand-pollinated outcross pollinia each, as in the Bag treatment. The same pollen source was always used for the Bag and Open treatment of a given block. Following hand-pollination, the experimental umbel was again left open to natural pollinators. Thus, the Bag treatment was protected from geitonogamy before and after outcross hand-pollinations, while the Open treatment experienced natural rates of geitonogamy before and after outcross hand-pollinations.
Control treatment umbels received neither bridal veil nor hand-pollinations. The Control thus provides an estimate of natural levels of fruit set.
Experimental umbels were tracked periodically throughout development. Bridal veil from the Bag treatment was removed after the flowers began to wither to allow for natural pod development. We recorded number of pods initiated, pods aborted, and mature pods for all treatments.
Statistical analysis
The overall design is a two-way ANOVA, with additional covariates included to accommodate details of the experiment. Fixed main effects include disturbance (undisturbed, disturbed), pollination treatment (Bag, Open, and Control) and their interaction. The design includes two levels of nesting, both involving random effects. Populations nest within each disturbance class, and blocks (the basic unit of replication) nest within populations. We fitted the complete mixed model using the SAS GLIMMIX macro, using restricted maximum likelihood with Poisson error. GLIMMIX iteratively calls PROC MIXED to fit the overall model. The levels of nesting are included as covariates using the RANDOM statement. Since the covariates explain variation but are not our primary focus, we report only the main effects and their interaction. The post-hoc Tukey-Kramer adjustment compares treatment means.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Fruit set (pod maturation) differed significantly between the Bag, Open, and Control treatments (Table 2). The interaction term and differences between sites were not statistically significant (Table 2). Fruit set in umbels of the Control treatment averaged 0.71 ± 0.09 pods per umbel (Fig. 1, lower), indicating that under natural pollination conditions approximately 3.5% of flowers mature as fruit (0.71 pods per umbel, average umbel size 
20 flowers). The Open treatment produced 1.76 ± 0.13 fruits per umbel, significantly more than the Control treatment (P < 0.0001). Supplemental hand pollinations therefore increased fruit set. The Bag treatment averaged 2.36 ± 0.13 fruits per umbel, significantly higher than both the Open (P < 0.003) and Control (P < 0.0001). Plants receiving only hand outcross pollen therefore produced approximately three times more fruits per flower than plants receiving natural levels of pollination.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Importance of geitonogamy
The Bag and Open treatments each received equal amounts of hand-pollinated outcross pollen (6 pollinia), but the Bag treatment excluded pollinators while the Open treatment experienced natural outcross and selfing rates. Plants in both treatments initiated similar numbers of fruit (e.g., Fig. 1), but fruit abortion was significantly higher in the Open than in the Bag treatment. Thus, a fraction of natural pollinations results in fruit abortion.
We hypothesize that fruit abortion following natural pollination is due to the effects of geitonogamy. Mating system studies on A. syriaca suggest that rates of geitonogamy in natural Asclepias populations are extremely high (Wyatt and Broyles 1994
). Morse (1982
) estimated that 97% of pollinations in large clones result from selfing. Pleasants (1991)
, using radioactively labeled pollinia, demonstrated that 37% of inserted pollinia originated from within the umbel and 71% originated from within 1 m of the umbel. Shore (1992), using electrophoretic methods, estimated the rate of self-pollination to be 66% in a natural population. Although the level of geitonogamy is not known in A. speciosa, these results from other milkweed studies suggest that the Open treatment likely experienced many geitonogamous pollinations both before and after the hand outcross pollinations.
Milkweed pollination studies strongly suggest that self-pollinations greatly reduce the success of cross-pollinations. Once a selfed pollinium is deposited, the pollen germinates and penetrates ovaries as quickly as (or even quicker than) outcross-pollen (Bookman, 1983
; Kahn and Morse, 1991
), only to abort during seed development. Moreover, this abortion appears to function at an umbel-wide level (Morse, 1994
), so geitonogamous pollinations occurring before, simultaneously, or soon after outcrossing may seriously compromise the outcross pollen in that umbel. Broyles and Wyatt (1993
) demonstrated that simultaneously inserting self- and outcross pollen decreases fruit set in A. exaltata by 49% and by 81% when self-pollen preceded outcross-pollen by 24 hours. In addition, Wyatt (1980)
demonstrated that early resource competition among ovaries within umbels likely influence fruit set in A. tuberosa, as did Bookman (1983
) for A. speciosa. Even if flowers receive plentiful outcross-pollen in the Open treatment, geitonogamy may cause considerable abortion at the umbel level, and significantly decrease fruit set. No self-pollinations interfere with the hand outcross pollinia in the Bag treatment, resulting in comparatively high fruit set.
Reduced fruit set in the Open compared to Bag treatment is consistent with previous studies that suggest geitonogamous pollinations in milkweeds reduce female reproductive success. Bookman (1983)
found that the majority of failures to produce fruits in natural populations of A. speciosa was due to post-fertilization abortion. Over 60% of ovaries aborted after fertilization, presumably due largely to self-incompatibility reactions. Morse (1994
) found that in A. syriaca, bagged, hand cross-pollinations produced 30 times more follicles than unbagged, hand cross pollinations exposed to natural pollinators.
Compatible pollen limitation
Figure 1 illustrates that female fitness is compatible pollen limited. We reach this conclusion because both fruit initiation and fruit set were significantly lower in the Control than in the hand-pollinated Bag and Open treatments. This indicates that plants are capable of producing more fruits (i.e., are not resource limited) if supplemented with outcross pollen. Therefore, large inflorescences may be serving to enhance both female and male fitness. It appears that fruit set is not pollen limited, but compatible pollen limited; large inflorescences are likely causing incompatible pollinations via geitonogamy. Bookman (1983)
found that mature pods of A. speciosa form from only 2 to 3% of all flowers in the field, yet over 80% received pollen. That fruit set is compatible pollen limited and that large inflorescences may have a negative effect due to geitonogamy violate the major assumptions of the pollen donation hypothesis, i.e., that fruit set is resource limited and that large inflorescences have positive fitness effects.
Evolution of inflorescence design
This study provides evidence that natural rates of geitonogamy significantly reduce female fitness in A. speciosa by increasing abortion rates. High natural rates of geitonogamy would also compromise male fitness due to complete pollen discounting (Burd and Callahan, 2000
). There is expected to be a cost to larger inflorescences, which often have the highest rates of geitonogamy. The plant's dilemma hypothesis recognizes this cost by predicting that the increasing rates of geitonogamous pollination may place an upper limit on inflorescence size (Holsinger, 1996
). The pollen donation hypothesis, on the other hand, does not recognize the cost of geitonogamy in larger inflorescences. As Burd and Callahan (2000)
clarify, there are several different variations of the pollen donation hypotheses, each with varying predictions. However, all predict increasing male fitness with increasing flowers per inflorescence or plant.
This study also demonstrates that female fitness in A. speciosa is compatible pollen limited. The pollen donation hypothesis, which is an explanation for the evolution of excess flowers (Burd and Callahan, 2000
), assumes resource-limited female fitness. However, our results, and those of Bookman (1984)
suggest that low fruit set in A. speciosa may be the cost of geitonogamy. There is support in the literature that milkweeds (Broyles and Wyatt, 1997
), and indeed plants in general (Burd, 1994
), are pollen limited.
We conclude that both of these findings support the plant's dilemma hypothesis and recommend that theories dealing with inflorescence design evolution be broadened to recognize the cost of geitonogamy.
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FOOTNOTES |
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Bookman S. S. 1984 Evidence for selective fruit production in Asclepias. Evolution 38: 72-86[CrossRef][ISI]
Broyles S. B. R. Wyatt 1990 Paternity analysis in a natural population of Asclepias exaltata: multiple paternity, functional gender, and the ‘pollen-donation’ hypothesis. Evolution 44: 1454-1468[CrossRef][ISI]
———. 1991 Effective pollen dispersal in a natural population of Asclepias exaltata: the influence of pollinator behavior, genetic similarity, and mating success. American Naturalist 138: 1239-1249[CrossRef][ISI]
———. 1993 The consequences of self-pollination in Asclepias exaltata, a self-incompatible milkweed. American Journal of Botany 80: 41-44
———. 1995 A reexamination of the pollen-donation hypothesis in an experimental population of Asclepias exaltata. Evolution 49: 89-99[CrossRef][ISI]
———. 1997 The pollen donation hypothesis revisited: a response to Queller. American Naturalist 149: 595-599[CrossRef][ISI]
Buchmann S. G. Nabhan 1996 The Forgotten Pollinators. Island Press, Washington, D.C., USA
Burd M. 1994 Bateman's principle and plant reproduction: the role of pollen limitation in fruit and seed set. Botanical Review 60: 83-139[CrossRef][ISI]
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de Jong T. J. N. M. Waser P. G. L. Klinkhamer 1993 Geitonogamy: the neglected side of selfing. Trends in Ecology and Evolution 8: 321-325
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Kahn A. P. D. H. Morse 1991 Pollinium germination and putative ovule penetration in self- and cross-pollinated common milkweed Asclepias syriaca. American Midland Naturalist 126: 61-67[CrossRef][ISI]
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Morse D. H. 1982 The turnover of milkweed pollinia on bumble bees, and implications for outcrossing. Oecologia 53: 187-196[CrossRef][ISI]
———. 1994 The role of self-pollen in the female reproductive success of common milkweed (Asclepias syriaca, Asclepiadaceae). American Journal of Botany 81: 322-330[CrossRef][ISI]
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Schoen D. J. D. G. Lloyd 1992 Self- and cross-fertilization in plants. III. Methods for studying modes and functional aspects of self-fertilization. International Journal of Plant Sciences 153: 381-393[CrossRef][ISI]
Shore J. S. 1993 Pollination genetics of the common milkweed, Asclepias syriaca L. Heredity 70: 101-108[ISI]
Snow A. A. T. P. Spira R. Simpson R. A. Klips 1996 The ecology of geitonogamous pollination. In D. G. Lloyd and S. C. H. Barrett [eds.], Floral biology: studies on floral evolution in animal-pollinated plants, 191–216. Chapman & Hall, New York, New York, USA
Sutherland S. L. F. Delph 1984 On the importance of male fitness in plants: patterns of fruit set. Ecology 65: 1093-1104[CrossRef][ISI]
Willson M. F. P. W. Price 1977 The evolution of inflorescence size in Asclepias (Asclepiadaceae). Evolution 31: 495-511[CrossRef][ISI]
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Wyatt R. 1976 Pollination and fruit set in Asclepias: a reappraisal. American Journal of Botany 63: 845-851[CrossRef][ISI]
———. 1980 The reproductive biology of Asclepias tuberosa: I. Flower number, arrangement, and fruit set. New Phytologist 85: 119-131[CrossRef][ISI]
———. 1982 Inflorescence architecture: how flower number, arrangement, and phenology affect pollination and fruit set. American Journal of Botany 69: 585-594[CrossRef][ISI]
Wyatt R. S. B. Broyles 1994 Ecology and evolution of reproduction in milkweeds. Annual Review of Ecology and Systematics 25: 423-441
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