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Pollen competition as a reproductive isolating mechanism between two sympatric Hibiscus species (Malvaceae)

Department of Ecology, Evolution, and Organismal Biology, The Ohio State University at Marion, 1465 Mt. Vernon Avenue, Marion, Ohio 43302

Received for publication August 26, 1997. Accepted for publication July 21, 1998.

	ABSTRACT

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Differences in pollen tube growth rates (certation) between heterospecific (foreign) and conspecific pollen may strongly influence whether hybrid offspring are produced after mixed pollen loads are delivered to a stigma. For both members of a sympatric species pair, Hibiscus moscheutos and H. laevis, pollination by pure loads of foreign pollen resulted in fruit set that was not significantly different from conspecific pollination, indicating that pure loads of foreign pollen could readily result in hybrid offspring. However, the number of seeds per fruit from pure foreign pollinations was significantly less than that of pure conspecific pollination. Simultaneous mixed pollination resulted in a proportion of hybrid seeds (detected by an electrophoretic marker enzyme) that was significantly lower than expected based upon the capacity of foreign pollen to effect fertilization when applied in pure pollinations. After these 50/50% pollen mixtures were applied to stigmas, 8.0 and 7.4% hybrids were produced when H. moscheutos and H. laevis were the ovule parents, respectively. For these Hibiscus species, pollen competition appears to function as a barrier to hybridization that is of moderate intensity compared with similar barriers occurring between other recently studied sympatric species pairs.

Key Words: certation • Hibiscus • Malvaceae • pollination • pollen competition • hybridization • reproductive isolation

	INTRODUCTION

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Many closely related plant species will form hybrids after pollination with pure loads of interspecific pollen. If the species are sympatric, have overlapping flowering periods, and share pollinators, hybridization might occur frequently in nature. In instances where hybrid offspring have low fitness compared with purebred ones, the production of hybrid offspring by an ovule parent would waste ovules that might otherwise produce purebred offspring. This is because individuals that produce inviable or sterile hybrids will contribute fewer offspring to future generations than those in the same population that do not hybridize (Grant, 1971 ). Selection has often favored stigmas and styles that have a greater germination and growth of conspecific pollen relative to pollen of another species. Because the number of pollen grains on a stigma often greatly exceeds the number of ovules, certation (differences in pollen tube growth rate) could affect the probability that certain classes of pollen grains will accomplish fertilization instead of others. This effect has been suspected or known for a long time, especially with respect to the formation of conspecific progeny following mixed pollinations (Grant, 1975 ). Charles Darwin thought it to be very widespread, saying "It is well known that if pollen of a distinct species be placed on the stigmas of a flower, and its own pollen be afterward, even after a considerable interval of time, placed on the same stigma, its action is so strongly prepotent that it generally annihilates the effect of the foreign pollen" (Darwin, 1859 ).

There have been several detailed tests of certation. In an early study Smith and Clarkson (1956) directly observed that foreign Iris tenuis pollen took almost twice as long to reach the ovules in I. tenax flowers as did conspecific I. tenax pollen. In a study of five taxa of Haplopappus (Asteraceae) that included separate species as well as conspecific subspecies, Smith (1970) found pollen tube growth rates (assayed by the reduced pollen staining capacity of hybrid plants' offspring) was negatively correlated with phylogenetic distance of relatedness. Recent studies that employ electrophoretic markers to detect hybrids have shown similar results. In an examination of the approximate frequency with which the two members of the "Louisiana Iris" species complex might hybridize, Carney, Cruzan, and Arnold (1994) applied various mixtures of genetically marked Iris fulva and I. hexagona pollen on stigmas of the two species. They found that, in both species, the distribution of hybrid seeds was significantly less than expected under the assumption of random siring by pollen grains in the mixtures. For example, an equal mixture of the two pollen types resulted in 27% hybrid seeds when I. hexagona was the ovule parent, and 0% when it was I. fulva. Rieseberg, Desrochers, and Youn (1995) examined the potential strength of interspecific pollen competition as a reproductive barrier between the annual sunflowers Helianthus annuus and H. petiolaris. As in the preceding studies they used mixtures of foreign and conspecific pollen in various ratios and observed that foreign pollen suffered a strong disadvantage. For example, the 1:1 mixture produced no more than 4% hybrids by either ovule parent instead of the expected 50%.

I studied pollen competition in two species of wetland perennials, the common rose mallow, Hibiscus moscheutos L. and the halberd-leaved rose mallow Hibiscus laevis All. Both are robust marsh perennials that produce large flowers during July through September, which are pollinated by generalist bumble bees and, in some portions of their ranges, by a specialized solitary bee Ptilothrix bombiformis (Hymenoptera, Apidae, Anthophorinae). Although they flower simultaneously and share pollinators, H. moscheutos tends to occur in open marshes, while H. laevis is more common along the banks of slow-moving rivers (Blanchard, 1976 ). These habitats sometimes merge with one another. Although specific data are lacking concerning this aspect of their pollination ecology, it seems likely that occasionally flowers of each species may receive mixed loads of heterospecific and conspecific pollen. Hybrids have been reported but are uncommon, apparently due both to the general tendency not to co-occur on a site and the existence of postpollination barriers such as those examined closely in the present study (Blanchard, 1976 ). Wise and Menzel (1971) noted diminished fruit and seed set in crosses between members of southern United States populations of the two species. Consequently, there is good reason to assume that evolution may have favored the development of prezygotic reproductive isolating mechanisms. On the other hand, the relative importance of habitat selection has not been assessed in detail. If the rarity of hybrids is mainly a result of the parental species living too far apart for mating to occur, pollen competition may not actually effect reproductive isolation at the present time in the evolutionary history of the species. Whatever prezygotic mating barriers exist may have arisen as a by-product of divergence (Grant, 1971 ) and could serve in the future to keep the species separate if the habitat barriers diminish. The purpose of the present study was to determine whether pollen competition potentially acts as a reproductive barrier, by assessing the degree to which hybridization is prevented in mixed pollinations.

	MATERIALS AND METHODS

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Three large multistemmed individuals of each species were dug out of natural wetland areas located 16 km apart in Ottawa County, Ohio, and transplanted into a garden plot in Franklin County, Ohio. Pollinations were performed in a diallelic fashion, wherein each plant received (1) single-species individual pollinations to determine how freely hybrids are formed in the absence of pollen competition, and (2) two-species combination (simultaneous) pollinations using all pairwise combinations (a total of 27 simultaneous pollinations per species acting as the ovule parent). The treatments included self-pollinations, as the species are freely self-compatible (Spira, 1989 ; Klips and Snow, 1997 ).

Like many other members of the Malvaceae, rose mallow flowers are large and possess numerous stamens that are monadelphous, forming a tube that is united with the base of the corolla. The long style (4.9 cm in the H. moscheutos flowers used in this experiment and 3.9 cm in the H. laevis flowers) ends in five capitate stigmas. The fruit is a capsule containing up to 75 ovules in H. laevis and up to 130 ovules in H. moscheutos (estimates based on maximum seed set in hand-pollinations wherein few aborted ovules were observed). Simultaneous two-species pollinations were performed during July and August 1996 by fully coating two stigmas with pure pollen from each species, and clipping off the fifth stigma (Fig. 1). Pollen was applied by gently brushing stigmas with a cluster of 5-10 stamens excised with forceps from the donor flower. I affirmed that pollination of two stigmas was capable of yielding full seed set by comparing seed set of 30 pure two-stigma pollinations with an equal number of four-stigma pollinations. Although seed set appeared to be slightly diminished when only two stigmas are covered with pollen, the difference was not statistically significant (P > 0.1). Accordingly, in seed-set comparisons of pure pollen loads, the two-stigma and four-stigma data are pooled to achieve greater precision.

View larger version (124K): [in this window] [in a new window]   Fig. 1. Hibiscus moscheutos flower after receiving 50/50% mixed-pollination treatment. The fifth stigma has been removed.

The plants used in this study carried diagnostic (to species) alleles of GPI, an enzyme that was readily detected in seeds using the horizontal starch gel electrophoresis described in Wendel and Weeden (1989) . Ten seeds from each of the 27 mixed-pollination fruits were assayed. Hybrids were recognized by their heterozygote banding pattern.

	RESULTS

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In agreement with earlier findings of a partial reproductive barrier between these species (Wise and Menzel, 1971 ), hybrid pollinations resulted in significantly lower seed set than conspecific pollinations (herein regarded as "full" seed set). With H. moscheutos as the ovule parent, 63.7% of full seed set resulted from pure hybrid pollination (t = 27.2, P < 0.001). For H. laevis, the hybrid value was 77.7% of full seed set (t = 42.3, P < 0.001) (Fig. 2). Fruit set did not vary significantly between treatments for either species (N = 30 fruits per treatment; P > 0.05 for both species).

View larger version (44K): [in this window] [in a new window]   Fig. 2. Seed set resulting from pure single-donor conspecific, single-donor foreign pollinations, and equal-mixture simultaneous pollinations on two sympatric Hibiscus species (N = 22–28 fruits per treatment, bars depict +1 SE). Differences between the two single-donor pollen types are highly significant (see text).

In mixed pollinations, foreign pollen was much less effective in setting seeds than expected based on its performance when applied in pure pollen loads. Expected hybrid frequencies after mixed pollinations were computed by multiplying the relative seed set performance of hybrid pollen (derived from the pure pollinations) times 0.5 (since equal mixtures of pollen were applied). This hybrid pollination correction factor was applied because a null hypothesis of 50/50% hybrid/conspecific seed would have incorrectly implied equal seed set potential of the two pollen types. Hibiscus moscheutos set only 7.4% hybrid seeds (chi-squared = 72.5, P < 0.001). and H. laevis 8.8% (chi-squared = 102.3, P < 0.001). For both species most of the 27 mixed pollination fruits yielded zero hybrids among the ten seeds sampled (Fig. 3).

View larger version (33K): [in this window] [in a new window]   Fig. 3. Hibiscus species 50/50% mixed conspecific/foreign pollination results. Number of fruits (of a total of 27 fruits examined) yielding various specified numbers of hybrid seeds (per ten seeds randomly sampled per fruit). Cross-hatching denotes ovule parent.

	DISCUSSION

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The performance of these plants is similar to results obtained by previous studies that used 50/50% mixtures of the two pollen types. Such studies typically have reported extremely low fractions of hybrid seeds: no more than 4% in Helianthus spp. crosses (Rieseberg, Desrochers and Youn, 1995 ) and none whatsoever in Iris fulva (Carney, Cruzan and Arnold, 1994 ). Despite the definite advantage realized by conspecific rose mallow pollen, a sizable fraction of hybrids (7–8%) nonetheless occurred in the rose mallows. Reproductive isolation in these species, therefore, is not especially strong. Given proximity sufficient to allow individual pollinators to visit flowers of the two species, as sometimes occurs where rivers are especially wide with open marsh borders (Klips, personal observation) a small amount of hybridization is likely to occur in nature. Because of the apparent pollen competition detected here, hybrids should be infrequent and the exclusion of foreign pollen may have evolved in response to selection pressure to minimize the waste of gametes. However, given the readiness with which a few (rather than none whatsoever) hybrid progeny are formed, introgression of genes from one species into populations of the other might occur. This has not been detected for the rose mallows, but might be profitably sought after, especially in the few places where these two rose mallow species are known to occur in mixed stands.

	FOOTNOTES

 
¹ The author thanks Theresa Culley, David Inoue, Allison Snow, and an anonymous reviewer for technical advice and comments on the manuscript. This research was supported by an Ohio State University at Marion Faculty Research Grant.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Blanchard, O. J. 1976 A revision of species segregated from Hibiscus sect. Trionum (Medicus) de Candolle sensu lato (Malvaceae). Ph.D. dissertation, Cornell University, Ithaca, NY.

Carney, S. E., M. B. Cruzan, and M. L. Arnold. 1994 Reproductive interactions between hybridizing irises: analyses of pollen-tube growth and fertilization success. American Journal of Botany 81: 1169–1175.[CrossRef][ISI]

Darwin, C. 1859 On the origin of species by means of natural selection, or the preservation of favored races in the struggle for life. Murray, London.

Grant, V. 1971 Plant speciation. Columbia University Press, New York, NY.

———. 1975 Genetics of flowering plants. Columbia University Press, New York, NY.

Klips, R. A., and A. A. Snow. 1997 Delayed autonomous self-pollination in Hibiscus laevis (Malvaceae). American Journal of Botany 84: 48–53.[Abstract]

Rieseberg, L. H., A. M. Desrochers, and S. J. Youn. 1995 Interspecific pollen competition as a reproductive barrier between sympatric species of Helianthus (Asteraceae). American Journal of Botany 82: 551–519.

Smith, E. B. 1970 Pollen competition and relatedness in Haplopappus section Isopappus (Compositae). II. American Journal of Botany 57: 874–880.[CrossRef][ISI]

Smith, H. H., and Q. D. Clarkson. 1956 Cytological studies of interspecific hybridization in Iris, subsection Californicae. American Journal of Botany 43: 582–88.[CrossRef][ISI]

Snow, A. A., and T. P. Spira. 1996 Pollen-tube competition and male fitness in Hibiscus moscheutos. Evolution 50: 1866–1870.

Spira, T. P. 1989 Reproductive biology of Hibiscus moscheutos (Malvaceae). In J. Bock and Y. Linhart [eds.], The evolutionary ecology of plants, 247–255.Westview Press, Boulder, CO.

Wendel, J. F., and N. F. Weeden. 1989 Visualization and interpretation of plant isozymes. In D. E. Soltis and P. S. Soltis [eds.], Isozymes in plant biology, 5–45. Dioscorides Press, Portland, OR.

Wise, D. A., and M. Y. Menzel. 1971 Genetic affinities of the North American species of Hibiscus sect. Trionum. Brittonia 23: 425–437.[CrossRef][ISI]

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This Article Abstract Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (9) Citing Articles via Google Scholar Google Scholar Articles by Klips, R. Search for Related Content PubMed Articles by Klips, R. Agricola Articles by Klips, R.