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Outcrossing rates as affected by pollinators and the heterozygote advantage of Monochoria korsakowii
Aquatic Botany 62 (1998) 135±143
Outcrossing rates as affected by pollinators and the heterozygote advantage of Monochoria korsakowii Guangxi Wanga,*, Yuji Yamasueb, Kazuyuki Itoha,1, Tokuichi Kusanagib a
Department of Lowland Farming, Tohoku National Agricultural Experiment Station, Ministry of Agriculture, Forestry and Fisheries, Omagari, Akita 014-0102, Japan b Faculty of Agriculture, Kyoto University, Kyoto 606-8502, Japan Received 15 June 1997; accepted 16 April 1998
Abstract Monochoria korsakowii shows somatic enantiostyly as each plant bears two kinds of flowers: left- and right-handed flowers, which differ in style deflection. Since the capsules of M. korsakowii and M. vaginalis contain hundreds of seeds, we detected heterozygotes with two isozyme markers, Adh-1 and Pgi-2 locus, and measured outcrossing rates for single fruits in each of the target genotypes. Outcrossing rates were measured on an experimental population of Adh-11/Adh-11 homozygotes, in which target plants of the Adh-10/Adh-10 homozygote were planted at three different positions. Outcrossing rates for M. korsakowii ranged from 37% to 80% with pollinators and were 0% without pollinators. The variation in the outcrossing rates was significantly affected by the species of pollinators but was not affected by the position of target in the experimental population. When the flowers were visited by both Apis cerana japonica and Xylocopa circumvolans, the mean outcrossing rate was 72.3%, and when visited by Apis cerana japonica only, the mean outcrossing rate was 49.2%. The heterozygous seeds, detected by Adh-1 locus in the present experiment, had greater weight, and produced larger seedlings than those from homozygous seeds. In a comparative experiment, the outcrossing rate for M. vaginalis, was zero because it had been self-pollinated before the flowers opened, or it was cleistogamous. # 1998 Elsevier Science B.V. All rights reserved. Keywords: Monochoria korsakowii; Pontederiaceae
* Corresponding author. Tel.: +81 187 66 2771; fax: +81 187 66 2639; e-mail: [email protected] 1 Present address: Lab. of Plant Ecology, National Institute of Agro-Environmental Sciences, Ministry of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki 305-8604, Japan. 0304-3770/98/$ ± see front matter # 1998 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 3 7 7 0 ( 9 8 ) 0 0 0 7 6 - X
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G. Wang et al. / Aquatic Botany 62 (1998) 135±143
1. Introduction Mating systems largely determine the genetic structure of populations and the evolutionary significance has long intrigued biologists (Darwin, 1878; Mather, 1943; Stebbins, 1957; Jain, 1976; Clegg, 1980; Motten and Antonovics, 1992). Jain and Allard (1960) proposed three mating systems, that is, exclusive or predominant outcrossing, predominant selfing, and mixed selfing and outcrossing. However, there are diverse mechanisms governing mating systems in flowering plants. In plants with entomophilous flowers, such mechanisms include flower morphology and the insect pollinators visiting flowers. Monochoria korsakowii Regel et Maack is a summer annual weed found in pools, ditches, canals and rice-fields of eastern Asia (Cook, 1989; Wang et al., 1995). M. korsakowii produces two kinds of enantiomorphic flowers which develop alternatively on each branch of the inflorescence; the style curves sideways either to the left or right (L- or R-handed flowers, respectively). The flowers also have dimorphic stamens (heteranthery). Each flower has five small stamens with yellow anthers as well as one large stamen with a purplish-blue anther. The large anther and the stigma are symmetrical with respect to the median plane of the flower and the small anthers are at the central upper position in the flower (Wang et al., 1995). The artificial pollination study (Wang et al., 1995) suggested that since both large and small stamens were fertile and the plant was self-compatible, self-fertilization could occur both through autogamy and geitonogamy, but its frequency varied depending on the numbers of the enantiostylic L- and R-handed flowers opening on each individual on a particular day and the frequency of insect pollencarrying vectors visiting the flowers. The objective of our present study was to quantitatively determine the outcrossing rates of M. korsakowii between individual plants in a population and to determine whether the heterozygote has advantages in seed weight and seedling vigor. M. vaginalis (Burm.f.) Kunth was used as a reference. This species also has somatic enantiostyly, but is often cleistogamous or self-pollinated before the flowers open. 2. Materials and methods 2.1. Genetic markers About 10 000 seeds were collected from two populations in Mikata, Fukui Prefecture and Kurashiki, Okayama Prefecture, Japan for M. korsakowii, and from two populations in Takatsuki, Osaka Prefecture and Sakurai, Nara Prefecture for M. vaginalis in 1992, which were used to find some genetic markers. Starch-gel electrophoresis was employed following the procedures described by Glaszmann et al. (1988). A crude extract of one seed germinating at 30C in the dark for 4 days provided us with a zymogram sufficiently resolved. 2.2. Experimental populations With the seeds of M. korsakowii collected in 1992, we screened the two isozyme homozygotes, Adh-11/Adh-11 from the Mikata population and Adh-10/Adh-10 from the
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Kurashiki population. The isozyme homozygotes of M. vaginalis were similarly prepared: Pgi-21/Pgi-21 from the Takatsuki population and Pgi-22/Pgi-22 from the Sakurai population. In early June of 1994, we set up two experimental populations, one for M. korsakowii and the other for M. vaginalis, at an irrigated paddy field of the Kyoto Experimental Farm of Kyoto University. In the population of M. korsakowii, 50 plants of the Adh-11/Adh-11 homozygote were planted with 0.5±0.7 m spacing with a rectangular design and three target plants of the Adh-10/Adh-10 homozygote were also planted at the center, corner and edge of the population. The population of M. vaginalis consisted of 50 plants of the Pgi22/Pgi-22 homozygote with 0.4±0.5 m spacing and three Pgi-21/Pgi-21 target plants. We carefully observed the flowers, the insects visiting the flowers and their behavior during flowering periods of these species from late August to the middle of October. In November, all the capsules of the target plants were collected, and samples of seeds from each capsule were germinated after breaking their dormancy. We estimated the outcrossing rate, t, using the expression tH/p, where H is the frequency of heterozygotes and p is the frequency of the marker allele. 2.3. Heterozygote seed weight and seedling vigor Fruits of each target plant of M. korsakowii were collected in the fall of 1994, and stored in a pool outdoors for 3 months to release seed dormancy. The seeds were then air dried under room conditions for 10 days. All the seeds from each fruit were weighed and placed on a germination bed at 30C in the dark for 4 days. A previous study (Wang et al., 1996a) showed that M. korsakowii had a high germination rate, c.a. 95%, under these conditions. Thus, in order to eliminate the effects of photosynthetic assimilates on the result of electrophoresis, we grew seedlings in the dark instead of in the light, so that a clear electrophoregram could be obtained (Wang et al., 1996b). Shoot length of the germinating seed was measured and then they were subjected to an electrophoretic analysis to determine whether the seedling was heterozygous or homozygous. 3. Results 3.1. Genetic markers Using the seeds collected from Fukui and Okayama Prefecture for M. korsakowii, and from Osaka and Nara Prefecture for M. vaginalis, we obtained zymograms of various enzymes to find a suitable genetic marker and found a set of isozymes, ADH-1, of alcohol dehydrogenase (EC 1.1.1.1) and PGI-2 of phosphoglucose isomerase (EC 5.3.1.9) for M. korsakowii and M. vaginalis, respectively. Our hybridization experiment revealed that the sets of the isozymes were those allozymes specified by a polymorphic locus in each species. The homozygosity of the plants used to set up two experimental populations was confirmed by selfing the plants in 1993.
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Fig. 1. Diagram of plant arrangements in the experimental populations. Solid circles represent Adh-11/Adh-11 individuals in M. korsakowii and Pgi-22/Pgi-22 individuals in M. vaginalis, and hollow circles represent Adh-10/ Adh-10 individuals in M. korsakowii and Pgi-21/Pgi-21 individuals in M. vaginalis.
3.2. Natural outcrossing rates Fig. 1 and 2 show the experimental populations. In the population of M. korsakowii, observed proportions of heterozygous seeds, Adh-10/Adh-11, per fruit of the target Adh10/Adh-10 plants ranged from 0 to 75%. Estimated outcrossing rates ranged from 37% to 80% with pollinators, but no outcrossing was observed without a pollinator (Table 1). The variation in outcrossing rates was significantly affected by the species of pollinator. When the flowers were visited by both Xylocopa circumvolans and Apis cerana japonica, the mean outcrossing rate was 72.3%, while the mean was 49.2% when the flowers were visited by only Apis cerana japonica. But the outcrossing rates did not significantly differ between flowers of the target plants placed at three different positions of the experimental population; the target plants placed on the edge, center, and corner showed similar mean outcrossing rates (Table 1). In the population of M. vaginalis, the flowers were observed to be often cleistogamous or self-pollinated before they opened (Fig. 3). All of the seeds
Table 1 Effects of pollinators and target individual's positions on outcrossing rates, t (calculated as H/p), in the experimental population of M. korsakowii Pollinators Xylocopa circumvolans Apis cerana japonica Only Apis cerana japonica No pollinator Target individual's positions Edge of the population Center of the population Corner of the population
Outcrossing rates, t Mean
S.D.
0.72 c 0.49 b 0a
0.06 0.11 0
0.51 a 0.48 a 0.46 a
0.33 0.30 0.28
Means within a column followed by the same letter are not significantly different at the 5% level according to Duncan's multiple range test.
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Fig. 2. The experimental populations of M. korsakowii (upper) and M. vaginalis (below).
Fig. 3. A cleistogamous flower (A) and a sideview of a self-pollinated flower (perianth removed) (B) of M. vaginalis.
Fig. 4. Starch gel electrophoresis of Adh in M. korsakowii (A) and Pgi in M. vaginalis (B).
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in the fruits of the target plants had an identical zymogram to those of the parental target plants, Pgi-21/Pgi-21, and therefore, no outcrossing was observed. Fig. 4A, lanes 1±4 and 5±8 are Adh-11/Adh-11 and Adh-10/Adh-10 respectively, and lanes 9±28 show progeny genotypes derived from Adh-10/Adh-10 (target genotype) individual. Among these, lanes 9±11 and 13±27 are heterozygotes (Adh-10/Adh-11) by outcrossing, and lanes 12 and 28 are homozygotes (Adh-10/Adh-10) by selfing. Fig. 4B, lanes 1 and 2 are Pgi-22/Pgi-22 and Pgi-21/Pgi-21 respectively, and lanes 3±6 show the progeny genotypes derived from Pgi-21/Pgi-21 (target genotype) individual. All of them are homozygotes (Pgi-21/Pgi-21) by selfing. 3.3. Heterozygote seed weight and seedling vigor Seed weight of both heterozygote and homozygote in respect to Adh-1 locus showed a normal distribution with a range from 0.56 to 0.82 mg and from 0.38 to 0.68 mg, respectively. The mean weight of heterozygotous seeds of M. korsakowii in respect of Adh-1 locus was 0.71 mg and apparently larger than the homozygote seed with 0.54 mg. This large seed weight supported more seedling vigor and shoots of the heterozygotous seedlings grew longer than the homozygotes during the 4-day period in the germination bed (Fig. 5). The mean seedling length of heterozygotous seeds was 0.91 cm and the homozygote one was 0.46 cm.
Fig. 5. Relationship between seeds produced by outcrossing or selfing and weight of seed or length of seedling sampled for isozyme analysis by starch gel electrophoresis 4 days after seeding in a fruit.
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4. Discussion The comparative study of plant mating systems is a powerful means to identify the factors which determine natural outcrossing rates (Vasek, 1965; Ganders et al., 1977). The large differences in outcrossing rates between M. korsakowii and M. vaginalis detected in the present study, appear to be due to their modes of pollination. We previously studied, flowering behavior of these species and insect pollinators in detail (Wang et al., 1995). In M. korsakowii, the anthers do not dehisce until the flowers open. After the flowers open, many insect individuals visit them until the anthers become empty. Two bee species, Xylocopa circumvolans and Apis cerana japonica, were observed on the flowers of the target plants of the experimental population. These two bees forage pollen of the small stamens of both L- and R-handed flower with no preference. When they visit L- or R-handed flowers, the large anther and stigma touch the left and right side or the right and left side of the insect's hairy abdomen, respectively. Therefore, self-fertilization through geitonogamy is promoted on a plant with both flower morphs open on same day. However, outcrossing is predominant when flowers of only one morph are open on a plant. In the present experimental population, a flower of the target plant was visited about 80 times by only Apis cerana japonica or by both Apis cerana japonica and Xylocopa circumvolans during a fine day of September, 1994, but no pollinators were observed on a rainy day. Xylocopa circumvolans appeared to be the more effective pollinator than Apis cerana japonica (Table 1). The former bee could carry more pollen than the latter, since it has a hairy abdomen with its width covering the distance between the stigma and large anther. Moreover, the large size of Xylocopa circumvolans forces the flower to be bent down and the pollen becomes scattered on its hairy abdomen. No outcrossing was observed in the experimental population of M. vaginalis. On the chasmogamous flowers, no pollinator was observed and the anther of large stamen starts shedding pollen onto the stigma before the flower opens. We conclude that, its smaller flower size was unsuitable to the pollinators. Moreover, the tendency of many M. vaginalis flowers to self-pollinate before anthesis also precludes outcrossing: in flowers with overlapping stigma and anther of large stamen, the anther starts shedding pollen onto the stigma before the flower opens. The genetic variability of M. korsakowii within and among populations was previously studied using five loci of three enzyme systems in ten natural populations (Wang et al., 1996b), from two of which seed material for the present experiments originated. The total genetic diversity (HT) and intrapopulational genetic diversity (HS) obtained were 0.333 and 0.227, respectively. These values were larger than those found for other outcrossing species reported by Loveless and Hamrick (1984). It is likely that this large genetic diversity of M. korsakowii is due not only to its high outcrossing rate, but also to a heterozygote advantage. Heterozygous seeds, detected by Adh-1 locus in the present experiment, had larger weight and produced larger seedlings than homozygote ones. When compared to the greater genetic diversity of M. korsakowii, particularly within populations, the diversity of M. vaginalis was extremely small (Wang et al., 1996b). We concluded that the degree of genetic variability in populations of the two Monochoria species was affected by their mating systems. As observed in the present experiment, the
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mating system of M. korsakowii operates through both selfing and outcrossing while that of M. vaginalis is exclusively selfing. M. korsakowii and M. vaginalis are self-compatible (Uchida, 1955; Wang et al., 1995). From the work described above, it is clear that at least in self-compatible species the mating system is extremely flexible. Though, the two Monochoria species both have somatic enantiostyly, the differences in specific properties of pollination biology may give rise to very large differences in outcrossing rates. Furthermore, in some species large stores of genetic variation affecting the outcrossing rate exist. Since the mating system is so flexible and plays such a crucial role in determining genotype frequencies of every sexual generation and the rate of breakdown of associations between loci, the quantitative analysis of the mating system should form a part of any study of selection in plant populations (Ennos, 1981). Acknowledgements We are indebted to Dr. M. Kato of Kyoto University for his identification of insect specimens and advice on pollination biology. We wish to thank T. Enomoto and T. Mineta of Okayama University and Dr. Y. Nakayama of Kyoto University for their help in collecting materials. We also thank two anonymous reviewers for their careful comments. References Clegg, M.T., 1980. Measuring plant mating systems. BioScience 30, 814±818. Cook, C.D.K., 1989. A revision of the genus Monochoria. In: Tan, Mill and Elias (Eds.), Plant Taxonomy, Phytogeography and Related Subjects. The Davis and Hedge Festschrift, Edinburgh University Press, Edinburgh, 149±184. Darwin, C., 1878. The Effects of Cross and Self Fertilization in the Vegetable Kingdom, 2nd ed. J. Murray, London. Ennos, R.A., 1981. Quantitative studies of the mating system in two sympatric species of Ipomoea (Convolvulaceae). Genetica. 57, 93±98. Ganders, F.R., Cariy, K., Griffiths, A.J.F., 1977. Outcrossing rates in natural populations of Plectritis brachystemon (Valerianaceae). Can. J. Bot. 55, 2070±2074. Glaszmann, J.C., de los Reyes, B.G., Khush, G.S., 1988. Electrophoretic variation of isozymes in plumules of rice (Oryza sativa L.) ± a key to the identification of 76 alleles at 24 loci. IRRI Research Paper Series No. 134, 2±14. Jain, S.K., 1976. The evolution of inbreeding in plants. Ann. Rev. Ecol. Syst. 7, 469±495. Jain, S.K., Allard, R.W., 1960. Population studies in predominantly self-pollinated species. I. Evidence for heterozygote advantage in a closed population of barley. Proc. Natl. Acad. Sci. USA 46, 1371±1377. Loveless, M.D., Hamrick, J.L., 1984. Ecological determinants of genetic structure in plant populations. Ann. Rev. Ecol. Syst. 15, 65±69. Mather, K., 1943. Polygenic inheritance and natural selection. Biol. Rev. 18, 32±64. Motten, A.F., Antonovics, J., 1992. Determinants of outcrossing rate in a predominantly self-fertilizing weed Datura stramonium (Solanaceae). Am. J. Bot. 79, 419±427. Stebbins, G.L., 1957. Self fertilization and population variability in the higher plants. Am. Naturalist 91, 337± 354. Uchida, M., 1955. On the self pollination by means of copulatory action in Monochoria vaginalis Presl. Collecting and Breeding. 17, 34±37 (in Japanese).
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Vasek, F.C., 1965. Outcrossing in natural populations II Clarkia unguiculata. Evolution 19, 152±156. Wang, G.X., Miura, R., Kusanagi, T., 1995. The enantiostyly and the pollination biology of Monochoria korsakowii (Pontederiaceae). Acta Phytotax. Geobot. 46, 55±65. Wang, G.X., Kusanagi, T., Itoh, K., 1996a. Environmental Factors Relating to Dormancy Breaking, Germination and Emergence of Seeds in Monochoria korsakowii Regel et Maack and M. vaginalis (Burm. f.) Kunth. Weed Res. Japan 41, 247±254 (in Japanese with English summary). Wang, G.X., Kusanagi, T., Itoh, K., 1996b. Isozyme variation in Monochoria korsakowii Regel et Maack and M. vaginalis (Burm. f.) Kunth (Pontederiaceae). Weed Res. Japan 41, 255±263 (in Japanese with English summary).
Outcrossing rates as affected by pollinators and the heterozygote advantage of Monochoria korsakowii Guangxi Wanga,*, Yuji Yamasueb, Kazuyuki Itoha,1, Tokuichi Kusanagib a
Department of Lowland Farming, Tohoku National Agricultural Experiment Station, Ministry of Agriculture, Forestry and Fisheries, Omagari, Akita 014-0102, Japan b Faculty of Agriculture, Kyoto University, Kyoto 606-8502, Japan Received 15 June 1997; accepted 16 April 1998
Abstract Monochoria korsakowii shows somatic enantiostyly as each plant bears two kinds of flowers: left- and right-handed flowers, which differ in style deflection. Since the capsules of M. korsakowii and M. vaginalis contain hundreds of seeds, we detected heterozygotes with two isozyme markers, Adh-1 and Pgi-2 locus, and measured outcrossing rates for single fruits in each of the target genotypes. Outcrossing rates were measured on an experimental population of Adh-11/Adh-11 homozygotes, in which target plants of the Adh-10/Adh-10 homozygote were planted at three different positions. Outcrossing rates for M. korsakowii ranged from 37% to 80% with pollinators and were 0% without pollinators. The variation in the outcrossing rates was significantly affected by the species of pollinators but was not affected by the position of target in the experimental population. When the flowers were visited by both Apis cerana japonica and Xylocopa circumvolans, the mean outcrossing rate was 72.3%, and when visited by Apis cerana japonica only, the mean outcrossing rate was 49.2%. The heterozygous seeds, detected by Adh-1 locus in the present experiment, had greater weight, and produced larger seedlings than those from homozygous seeds. In a comparative experiment, the outcrossing rate for M. vaginalis, was zero because it had been self-pollinated before the flowers opened, or it was cleistogamous. # 1998 Elsevier Science B.V. All rights reserved. Keywords: Monochoria korsakowii; Pontederiaceae
* Corresponding author. Tel.: +81 187 66 2771; fax: +81 187 66 2639; e-mail: [email protected] 1 Present address: Lab. of Plant Ecology, National Institute of Agro-Environmental Sciences, Ministry of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki 305-8604, Japan. 0304-3770/98/$ ± see front matter # 1998 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 3 7 7 0 ( 9 8 ) 0 0 0 7 6 - X
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1. Introduction Mating systems largely determine the genetic structure of populations and the evolutionary significance has long intrigued biologists (Darwin, 1878; Mather, 1943; Stebbins, 1957; Jain, 1976; Clegg, 1980; Motten and Antonovics, 1992). Jain and Allard (1960) proposed three mating systems, that is, exclusive or predominant outcrossing, predominant selfing, and mixed selfing and outcrossing. However, there are diverse mechanisms governing mating systems in flowering plants. In plants with entomophilous flowers, such mechanisms include flower morphology and the insect pollinators visiting flowers. Monochoria korsakowii Regel et Maack is a summer annual weed found in pools, ditches, canals and rice-fields of eastern Asia (Cook, 1989; Wang et al., 1995). M. korsakowii produces two kinds of enantiomorphic flowers which develop alternatively on each branch of the inflorescence; the style curves sideways either to the left or right (L- or R-handed flowers, respectively). The flowers also have dimorphic stamens (heteranthery). Each flower has five small stamens with yellow anthers as well as one large stamen with a purplish-blue anther. The large anther and the stigma are symmetrical with respect to the median plane of the flower and the small anthers are at the central upper position in the flower (Wang et al., 1995). The artificial pollination study (Wang et al., 1995) suggested that since both large and small stamens were fertile and the plant was self-compatible, self-fertilization could occur both through autogamy and geitonogamy, but its frequency varied depending on the numbers of the enantiostylic L- and R-handed flowers opening on each individual on a particular day and the frequency of insect pollencarrying vectors visiting the flowers. The objective of our present study was to quantitatively determine the outcrossing rates of M. korsakowii between individual plants in a population and to determine whether the heterozygote has advantages in seed weight and seedling vigor. M. vaginalis (Burm.f.) Kunth was used as a reference. This species also has somatic enantiostyly, but is often cleistogamous or self-pollinated before the flowers open. 2. Materials and methods 2.1. Genetic markers About 10 000 seeds were collected from two populations in Mikata, Fukui Prefecture and Kurashiki, Okayama Prefecture, Japan for M. korsakowii, and from two populations in Takatsuki, Osaka Prefecture and Sakurai, Nara Prefecture for M. vaginalis in 1992, which were used to find some genetic markers. Starch-gel electrophoresis was employed following the procedures described by Glaszmann et al. (1988). A crude extract of one seed germinating at 30C in the dark for 4 days provided us with a zymogram sufficiently resolved. 2.2. Experimental populations With the seeds of M. korsakowii collected in 1992, we screened the two isozyme homozygotes, Adh-11/Adh-11 from the Mikata population and Adh-10/Adh-10 from the
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Kurashiki population. The isozyme homozygotes of M. vaginalis were similarly prepared: Pgi-21/Pgi-21 from the Takatsuki population and Pgi-22/Pgi-22 from the Sakurai population. In early June of 1994, we set up two experimental populations, one for M. korsakowii and the other for M. vaginalis, at an irrigated paddy field of the Kyoto Experimental Farm of Kyoto University. In the population of M. korsakowii, 50 plants of the Adh-11/Adh-11 homozygote were planted with 0.5±0.7 m spacing with a rectangular design and three target plants of the Adh-10/Adh-10 homozygote were also planted at the center, corner and edge of the population. The population of M. vaginalis consisted of 50 plants of the Pgi22/Pgi-22 homozygote with 0.4±0.5 m spacing and three Pgi-21/Pgi-21 target plants. We carefully observed the flowers, the insects visiting the flowers and their behavior during flowering periods of these species from late August to the middle of October. In November, all the capsules of the target plants were collected, and samples of seeds from each capsule were germinated after breaking their dormancy. We estimated the outcrossing rate, t, using the expression tH/p, where H is the frequency of heterozygotes and p is the frequency of the marker allele. 2.3. Heterozygote seed weight and seedling vigor Fruits of each target plant of M. korsakowii were collected in the fall of 1994, and stored in a pool outdoors for 3 months to release seed dormancy. The seeds were then air dried under room conditions for 10 days. All the seeds from each fruit were weighed and placed on a germination bed at 30C in the dark for 4 days. A previous study (Wang et al., 1996a) showed that M. korsakowii had a high germination rate, c.a. 95%, under these conditions. Thus, in order to eliminate the effects of photosynthetic assimilates on the result of electrophoresis, we grew seedlings in the dark instead of in the light, so that a clear electrophoregram could be obtained (Wang et al., 1996b). Shoot length of the germinating seed was measured and then they were subjected to an electrophoretic analysis to determine whether the seedling was heterozygous or homozygous. 3. Results 3.1. Genetic markers Using the seeds collected from Fukui and Okayama Prefecture for M. korsakowii, and from Osaka and Nara Prefecture for M. vaginalis, we obtained zymograms of various enzymes to find a suitable genetic marker and found a set of isozymes, ADH-1, of alcohol dehydrogenase (EC 1.1.1.1) and PGI-2 of phosphoglucose isomerase (EC 5.3.1.9) for M. korsakowii and M. vaginalis, respectively. Our hybridization experiment revealed that the sets of the isozymes were those allozymes specified by a polymorphic locus in each species. The homozygosity of the plants used to set up two experimental populations was confirmed by selfing the plants in 1993.
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Fig. 1. Diagram of plant arrangements in the experimental populations. Solid circles represent Adh-11/Adh-11 individuals in M. korsakowii and Pgi-22/Pgi-22 individuals in M. vaginalis, and hollow circles represent Adh-10/ Adh-10 individuals in M. korsakowii and Pgi-21/Pgi-21 individuals in M. vaginalis.
3.2. Natural outcrossing rates Fig. 1 and 2 show the experimental populations. In the population of M. korsakowii, observed proportions of heterozygous seeds, Adh-10/Adh-11, per fruit of the target Adh10/Adh-10 plants ranged from 0 to 75%. Estimated outcrossing rates ranged from 37% to 80% with pollinators, but no outcrossing was observed without a pollinator (Table 1). The variation in outcrossing rates was significantly affected by the species of pollinator. When the flowers were visited by both Xylocopa circumvolans and Apis cerana japonica, the mean outcrossing rate was 72.3%, while the mean was 49.2% when the flowers were visited by only Apis cerana japonica. But the outcrossing rates did not significantly differ between flowers of the target plants placed at three different positions of the experimental population; the target plants placed on the edge, center, and corner showed similar mean outcrossing rates (Table 1). In the population of M. vaginalis, the flowers were observed to be often cleistogamous or self-pollinated before they opened (Fig. 3). All of the seeds
Table 1 Effects of pollinators and target individual's positions on outcrossing rates, t (calculated as H/p), in the experimental population of M. korsakowii Pollinators Xylocopa circumvolans Apis cerana japonica Only Apis cerana japonica No pollinator Target individual's positions Edge of the population Center of the population Corner of the population
Outcrossing rates, t Mean
S.D.
0.72 c 0.49 b 0a
0.06 0.11 0
0.51 a 0.48 a 0.46 a
0.33 0.30 0.28
Means within a column followed by the same letter are not significantly different at the 5% level according to Duncan's multiple range test.
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Fig. 2. The experimental populations of M. korsakowii (upper) and M. vaginalis (below).
Fig. 3. A cleistogamous flower (A) and a sideview of a self-pollinated flower (perianth removed) (B) of M. vaginalis.
Fig. 4. Starch gel electrophoresis of Adh in M. korsakowii (A) and Pgi in M. vaginalis (B).
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in the fruits of the target plants had an identical zymogram to those of the parental target plants, Pgi-21/Pgi-21, and therefore, no outcrossing was observed. Fig. 4A, lanes 1±4 and 5±8 are Adh-11/Adh-11 and Adh-10/Adh-10 respectively, and lanes 9±28 show progeny genotypes derived from Adh-10/Adh-10 (target genotype) individual. Among these, lanes 9±11 and 13±27 are heterozygotes (Adh-10/Adh-11) by outcrossing, and lanes 12 and 28 are homozygotes (Adh-10/Adh-10) by selfing. Fig. 4B, lanes 1 and 2 are Pgi-22/Pgi-22 and Pgi-21/Pgi-21 respectively, and lanes 3±6 show the progeny genotypes derived from Pgi-21/Pgi-21 (target genotype) individual. All of them are homozygotes (Pgi-21/Pgi-21) by selfing. 3.3. Heterozygote seed weight and seedling vigor Seed weight of both heterozygote and homozygote in respect to Adh-1 locus showed a normal distribution with a range from 0.56 to 0.82 mg and from 0.38 to 0.68 mg, respectively. The mean weight of heterozygotous seeds of M. korsakowii in respect of Adh-1 locus was 0.71 mg and apparently larger than the homozygote seed with 0.54 mg. This large seed weight supported more seedling vigor and shoots of the heterozygotous seedlings grew longer than the homozygotes during the 4-day period in the germination bed (Fig. 5). The mean seedling length of heterozygotous seeds was 0.91 cm and the homozygote one was 0.46 cm.
Fig. 5. Relationship between seeds produced by outcrossing or selfing and weight of seed or length of seedling sampled for isozyme analysis by starch gel electrophoresis 4 days after seeding in a fruit.
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4. Discussion The comparative study of plant mating systems is a powerful means to identify the factors which determine natural outcrossing rates (Vasek, 1965; Ganders et al., 1977). The large differences in outcrossing rates between M. korsakowii and M. vaginalis detected in the present study, appear to be due to their modes of pollination. We previously studied, flowering behavior of these species and insect pollinators in detail (Wang et al., 1995). In M. korsakowii, the anthers do not dehisce until the flowers open. After the flowers open, many insect individuals visit them until the anthers become empty. Two bee species, Xylocopa circumvolans and Apis cerana japonica, were observed on the flowers of the target plants of the experimental population. These two bees forage pollen of the small stamens of both L- and R-handed flower with no preference. When they visit L- or R-handed flowers, the large anther and stigma touch the left and right side or the right and left side of the insect's hairy abdomen, respectively. Therefore, self-fertilization through geitonogamy is promoted on a plant with both flower morphs open on same day. However, outcrossing is predominant when flowers of only one morph are open on a plant. In the present experimental population, a flower of the target plant was visited about 80 times by only Apis cerana japonica or by both Apis cerana japonica and Xylocopa circumvolans during a fine day of September, 1994, but no pollinators were observed on a rainy day. Xylocopa circumvolans appeared to be the more effective pollinator than Apis cerana japonica (Table 1). The former bee could carry more pollen than the latter, since it has a hairy abdomen with its width covering the distance between the stigma and large anther. Moreover, the large size of Xylocopa circumvolans forces the flower to be bent down and the pollen becomes scattered on its hairy abdomen. No outcrossing was observed in the experimental population of M. vaginalis. On the chasmogamous flowers, no pollinator was observed and the anther of large stamen starts shedding pollen onto the stigma before the flower opens. We conclude that, its smaller flower size was unsuitable to the pollinators. Moreover, the tendency of many M. vaginalis flowers to self-pollinate before anthesis also precludes outcrossing: in flowers with overlapping stigma and anther of large stamen, the anther starts shedding pollen onto the stigma before the flower opens. The genetic variability of M. korsakowii within and among populations was previously studied using five loci of three enzyme systems in ten natural populations (Wang et al., 1996b), from two of which seed material for the present experiments originated. The total genetic diversity (HT) and intrapopulational genetic diversity (HS) obtained were 0.333 and 0.227, respectively. These values were larger than those found for other outcrossing species reported by Loveless and Hamrick (1984). It is likely that this large genetic diversity of M. korsakowii is due not only to its high outcrossing rate, but also to a heterozygote advantage. Heterozygous seeds, detected by Adh-1 locus in the present experiment, had larger weight and produced larger seedlings than homozygote ones. When compared to the greater genetic diversity of M. korsakowii, particularly within populations, the diversity of M. vaginalis was extremely small (Wang et al., 1996b). We concluded that the degree of genetic variability in populations of the two Monochoria species was affected by their mating systems. As observed in the present experiment, the
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mating system of M. korsakowii operates through both selfing and outcrossing while that of M. vaginalis is exclusively selfing. M. korsakowii and M. vaginalis are self-compatible (Uchida, 1955; Wang et al., 1995). From the work described above, it is clear that at least in self-compatible species the mating system is extremely flexible. Though, the two Monochoria species both have somatic enantiostyly, the differences in specific properties of pollination biology may give rise to very large differences in outcrossing rates. Furthermore, in some species large stores of genetic variation affecting the outcrossing rate exist. Since the mating system is so flexible and plays such a crucial role in determining genotype frequencies of every sexual generation and the rate of breakdown of associations between loci, the quantitative analysis of the mating system should form a part of any study of selection in plant populations (Ennos, 1981). Acknowledgements We are indebted to Dr. M. Kato of Kyoto University for his identification of insect specimens and advice on pollination biology. We wish to thank T. Enomoto and T. Mineta of Okayama University and Dr. Y. Nakayama of Kyoto University for their help in collecting materials. We also thank two anonymous reviewers for their careful comments. References Clegg, M.T., 1980. Measuring plant mating systems. BioScience 30, 814±818. Cook, C.D.K., 1989. A revision of the genus Monochoria. In: Tan, Mill and Elias (Eds.), Plant Taxonomy, Phytogeography and Related Subjects. The Davis and Hedge Festschrift, Edinburgh University Press, Edinburgh, 149±184. Darwin, C., 1878. The Effects of Cross and Self Fertilization in the Vegetable Kingdom, 2nd ed. J. Murray, London. Ennos, R.A., 1981. Quantitative studies of the mating system in two sympatric species of Ipomoea (Convolvulaceae). Genetica. 57, 93±98. Ganders, F.R., Cariy, K., Griffiths, A.J.F., 1977. Outcrossing rates in natural populations of Plectritis brachystemon (Valerianaceae). Can. J. Bot. 55, 2070±2074. Glaszmann, J.C., de los Reyes, B.G., Khush, G.S., 1988. Electrophoretic variation of isozymes in plumules of rice (Oryza sativa L.) ± a key to the identification of 76 alleles at 24 loci. IRRI Research Paper Series No. 134, 2±14. Jain, S.K., 1976. The evolution of inbreeding in plants. Ann. Rev. Ecol. Syst. 7, 469±495. Jain, S.K., Allard, R.W., 1960. Population studies in predominantly self-pollinated species. I. Evidence for heterozygote advantage in a closed population of barley. Proc. Natl. Acad. Sci. USA 46, 1371±1377. Loveless, M.D., Hamrick, J.L., 1984. Ecological determinants of genetic structure in plant populations. Ann. Rev. Ecol. Syst. 15, 65±69. Mather, K., 1943. Polygenic inheritance and natural selection. Biol. Rev. 18, 32±64. Motten, A.F., Antonovics, J., 1992. Determinants of outcrossing rate in a predominantly self-fertilizing weed Datura stramonium (Solanaceae). Am. J. Bot. 79, 419±427. Stebbins, G.L., 1957. Self fertilization and population variability in the higher plants. Am. Naturalist 91, 337± 354. Uchida, M., 1955. On the self pollination by means of copulatory action in Monochoria vaginalis Presl. Collecting and Breeding. 17, 34±37 (in Japanese).
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Vasek, F.C., 1965. Outcrossing in natural populations II Clarkia unguiculata. Evolution 19, 152±156. Wang, G.X., Miura, R., Kusanagi, T., 1995. The enantiostyly and the pollination biology of Monochoria korsakowii (Pontederiaceae). Acta Phytotax. Geobot. 46, 55±65. Wang, G.X., Kusanagi, T., Itoh, K., 1996a. Environmental Factors Relating to Dormancy Breaking, Germination and Emergence of Seeds in Monochoria korsakowii Regel et Maack and M. vaginalis (Burm. f.) Kunth. Weed Res. Japan 41, 247±254 (in Japanese with English summary). Wang, G.X., Kusanagi, T., Itoh, K., 1996b. Isozyme variation in Monochoria korsakowii Regel et Maack and M. vaginalis (Burm. f.) Kunth (Pontederiaceae). Weed Res. Japan 41, 255±263 (in Japanese with English summary).