You are on page 1of 11

Blackwell Science, LtdOxford, UKEREEcological Research0912-38142003 Ecological Society of JapanSeptember 2003185599609Original ArticleClonal dominance in aquatic bladderwortS.

Araki and Y. Kadono

Ecological Research (2003) 18, 599–609

Restricted seed contribution and clonal dominance in a
free-floating aquatic plant Utricularia australis R. Br. in
southwestern Japan
Satoru Araki* and Yasuro Kadono

Department of Biology, Faculty of Science, Kobe University, Nada, Kobe, 657-8501, Japan
Utricularia australis R. Br. is an aquatic angiosperm species common in natural and irrigation ponds
in temperate regions. This species reproduces both sexually and vegetatively, but in southwestern
Japan the occurrence of male-sterile populations, in which plants produce no pollen and propagate
only vegetatively, has been recorded. We studied the reproductive contribution of seeds in normal
pollen-producing populations using isozyme analyses, a pollination experiment under culture and
field observations. Although seedlings obtained from controlled mating indicated segregation of
isozyme, polymorphism of the isozyme genotype was detected mainly among populations, but rarely
within each pond population. This suggested clonal dominance and rarity of seed or seedling survival
in natural populations. In the pollination experiment, the mean seed set ratio in cross-pollination
between plants of the same isozyme genotype (7.6%) did not differ significantly from self-pollination
(7.6%), but was lower than cross-pollination between plants of different genotypes (45.7%). The low
ratio in crossing between the same genotype plants was ascribed to the clonality of the parents. In
general, these results corresponded with the low ratios in seed setting observed in natural populations (7.9–13.7%). All the male-sterile populations we surveyed showed the same genotype, thus
male sterility in the study area was considered to have the same origin.
Key words: isozyme; male sterility; sexual reproduction; Utricularia australis; vegetative

Vegetative reproduction by the root system or
other organs occurs in various taxa of angiosperms
(Klimes et al. 1997). Most of these species also
reproduce in a sexual way through seed production. The relative contribution of vegetative and
sexual modes to reproduction has been a subject of
ecological studies because it is essential to know
this information to adequately explain the reproductive system of the species (Ellstrand & Roose
1987; Eriksson 1997). The amount of seedling
recruitment varies among species and/or habitats

*Author to whom correspondence should be
addressed. Present address: Research Center for Coastal
Lagoon Environments, Shimane University, Matsue
690-8504, Japan. Email:
Received 30 October 2002.
Accepted 26 March 2003.

(Eriksson 1989), thus the relative importance and
biological role of the vegetative and sexual systems
are considered to vary among species and their
environments. In particular, a strong reliance on
vegetative propagation is found in aquatic habitats. Many studies on aquatic macrophytes have
suggested single, or very few, genet compositions
in a population or through neighboring populations, although most of these species produce seed
or at least have the ability to produce seed through
sexual processes (Les 1988; Barrett et al. 1993;
Grace 1993). This remarkable clonality is, in part,
because these seedlings have a higher risk of dying
in inadequate light and dissolved oxygen
conditions in bottom water or sediment
habitats and the chance of seedling recruitment is
restricted (Barrett 1980; Smolders et al. 1995a,
1995b; Barrat-Segretain 1996).
In some groups, the occurrence of sterile strains,
such as species hybrids (Waterway 1994; Hollingsworth et al. 1996a) or triploids (Nakamura &

In summer. Utricularia australis R. They differ from each other in some morphological features of the turion (Miki 1935. But if seeds give no or rare reproductive contribution in this species. australis through field observations of fruit and seed production. in the southern part of Hyogo Prefecture. whereas other parts of the plant body disappear in winter. from the features mentioned above. and rarely in other regions of Japan. japonica (Makino 1914) and U. However. it is predicted that sterile mutants can be easily maintained in populations that reproduce mainly vegetatively (Eckert et al. were reported as novel species in Japan. Eckert 2002). tenuicaulis (Miki 1935). Formerly two taxa. australis and describe U. japonica or U. We visited these irrigation ponds weekly or biweekly in the flowering seasons of 1995 and 1996. In autumn. tenuicaulis. japonica. Tamura (1981) describes the former as U. japonica and the latter as U. 1996). S. Heisou in Kakogawa City. The aquatic bladderwort. Irrespective of whether such a trade-off exists (Muir 1995. (Lentibulariaceae). 1999. vulgaris var. in which plants produce no pollen grains. on the tips of the main shoot and each branch. These flowers are self. The former occurs mainly in northern Japan. that propagate only vegetatively have been reported. Nakamura et al. If more than two ponds are located in the same locality. Verburg & During 1998). tenuicaulis. Kadono (1993) describes the former as U. In the present study. Pollen production was checked for . Hyogo Prefecture. respectively. Poorly grown plants do not develop scapes during the flowering season and reproduce only vegetatively by turions. Araki and Y. We conducted field observations to confirm flowering and pollen production for 32 populations. it is difficult to explain how male-sterile plants could spread. southwestern Japan. pollen-producing populations of U. vulgaris var. To date. If seeds are essential for reproduction of this species. thus it is not a case of gynodioecy. australis form. Yutani-1 and Yutani-2). such as aphids or small dipterous species (Yamamoto & Kadono 1990. especially in Hokkaido. it may be advantageous if there is a trade-off between seed production and vegetative reproduction. turion. Thus. mutations that prevent seed production may be maintained in these species because seed loss has no disadvantage. the plants form a vegetative hibernating organ. Plants in those populations receive no pollen and produce no seed. our present study does not include plants that have been referred to as U. Yamamoto and Kadono (1990) reported the occurrence of male-sterile populations of this species. australis following Kadono (1993). we examined whether or not seeds contribute to reproduction in normal. Yamamoto & Kadono 1988). 1998). obs. includes both fertile and sterile plants. and recorded the occurrence or absence of flowering. Turions and seeds remain dormant until the following spring. pollination experiments under culture and an investigation of the population genetic structure using isozyme analysis. These two taxa are now treated in three ways according to different taxonomic opinions.600 S. australis and U. whereas the latter is distributed widely in Japan and rarely in Hokkaido (Kadono 1994). Araki and Y. Br.. each of the populations in the area is numbered (e.or cross-pollinated by small insects. abnormality in pollen production is not disadvantageous. U. australis. Kadono Kadono 1993. japonica and the latter as U. australis. although there has been some confusion in the taxonomy of this species. In the present paper we describe the study species as U. METHODS Field observations We define a population as all the plants growing in a pond and refer to each population using the name of the place in which the pond is located.g. although they are sometimes included in U. We consider. pers. Kadono. australis form. free-floating form. well-grown plants develop scapes above the water surface and bear approximately four to six flowers per scape. namely Yutani-1 to Yutani-29 in Yokawa Town. no consensus has been achieved in nomenclature of these taxa. Komiya and Shibata (1980) regard both of them as synonyms of U. vulgaris var. and recently reported isozyme differences. This species inhabits natural and irrigation ponds in a submerged. Furthermore. and Biwakoh-1 and Biwakoh-3 in Kasai City. that these two taxa are genetically differentiated from each other (Araki 2000). If seeds cannot contribute to reproduction because of high mortalities of seeds or seedlings.

Fruit and seed set ratios were investigated in five populations. Histidine-citrate buffer. 100 per flower). Each scape was bagged during the flowering period. Seed set ratio in each fruit was transformed to its arcsine square root for statistical analysis. flowered during cultivation and were used in the pollination experiment. we sampled all or some of the fruit-setting plants and dissected fruits. -23. In preliminary tests. -8. mating with the plant which indicated the same genotype from an isozyme study and with a plant that had a different genotype. we analyzed the following 14 enzymes: aconitase (ACN).5 by an alteration in the amount of citric acid. Each of the floating plant bodies was picked up intact from the water.3 ml grinding buffer and centrifuged at 50 000 g for 3 min. only the fruit set ratio was determined in the field. The staining method of the gel followed the method outlined by Soltis et al. (1983). The supernatant was soaked into wicks and inserted into the slit of the gel. The flower was cross-pollinated with pollen grains obtained from an individual of the same genotype and the whole scape was bagged. adenylate kinase (ADK). TrisHCl buffer–PVP solution was employed as a grinding buffer with a modification of mercaptoethanol concentration to 0. Pollination experiment For the purposes of isozyme analysis. hexokinase (HK). plants from nine populations. except for Yutani-16 and Yutani-20. The seed set ratio was calculated for each mature fruit as the number of seeds divided by the total number of ovules (approx. To avoid mixing true cross-pollination and pseudo-cross-pollination. Biwakoh-1 and -3. Heisou. The pH of the electrode buffer was adjusted to 6. -25. These included the 32 populations that were used for the field observations. Yutani-3. glutamate dehydrogenase (GDH). and its one-fourth dilution was used as an electrode buffer and gel buffer. the electrophoresis was carried out again under constant current (35 mA) for 6 h. -12. formate dehydrogenase (FDH). We visited those populations on one to four occasions in the flowering season of 1996. When genotypic variation within a population was detected in the isozyme study. plants collected from 47 populations in Hyogo Prefecture were cultivated in containers placed in the campus of Kobe University. In cases where plants could not be collected. many plants were sampled additionally from the pond and the population genetic structure was intensively examined. After the wicks have been drawn. isocitrate dehydrogenase . 601 Plants collected from Yutani included male-sterile plants. and counted fruits and peduncles that had already dropped their corollas. -11. (1983). Among them. which is equal to self-pollination. Yutani-25. corresponding to system 9 in Soltis et al. Seedlings obtained from the pollination experiment were also analyzed. The flower was self-pollinated artificially and the whole scape was bagged. 3 ‘Crossing with the same genotype’. Yutani-3.3%. alcohol dehydrogenase (ADH). We used horizontal starch gel electrophoresis following the method of Soltis et al. 2 ‘Selfing’. A 1– 5 cm length of vegetative shoot tip was ground in 0. Isozyme analysis We collected plants for isozyme analysis from 47 irrigation ponds located in the southern part of Hyogo Prefecture. namely. (1983). Biwakoh-1 and Biwakoh-3. The flower was cross-pollinated with pollen grains obtained from an individual with a different genotype and the whole scape was bagged. Next. which was then electrophoresed for 15 min. respectively.Clonal dominance in aquatic bladderwort selected plants by observing anthers under microscope for each population. Heisou. We paid attention to the possibility of crosspollination between the same clonal individuals. Samples could include ramets of the same genet because each individual can produce plural turions and consequently plural ramets. The fruit set ratio was calculated for each population as the number of fruits divided by the total number of peduncles without corolla. 4 ‘Crossing with different genotype’. Sample sizes between populations ranged from 1 to 146 (mean 13) because the amount of growing plant differed among ponds. The treatments were: 1 ‘Bagging’. Plants were treated in one of the following four ways when they were in bloom. we employed two types of cross-pollination. Those plants were used only in the fourth treatment as maternal plants.

-25. In addition. Multiple comparisons using Tukey tests indicated that the mean seed set ratios in the cases of selfing and crossing with the same type.3 = 16. which are detected in types D. Pollination experiment The results of the pollination experiment are shown in Table 3. Plants in Heisou. phosphoglucomutase (PGM).9–13. There was no significant difference in fruit set ratios among the three types of selfing treatments. RESULTS Pollen production Among the 29 populations at Yutani. fruit set ratio varied significantly with time (G-test: G = 25. which has only the nearest band to the cathode. A significant difference among populations on the same date was also observed.3 = 16.7%) in the three populations investigated (Yutani-25. All the flowering plants sampled in Yutani-3 and some from Yutani25 were confirmed to produce pollen grains. from plants in natural populations (Fig.7.f. plants indicating a homozygotic pattern. Among these. P < 0. However. The electrophoretic patterns .3 = 0. However. Yutani-12 and -23. P < 0. phosphoglucoisomerase (PGI). mean seed set ratios differed significantly among the three types of hand pollination (ANOVA: F2. we used electrophoretic patterns of PGM and PGI in the genetic analysis of adult plants and PGI for seedlings.05). respectively. may indicate allozymes at another locus (PGM-2). malate dehydrogenase (MDH).005). crossing with the same type and crossing with different types (G-test: G = 2.01). -21.01).3.05). the two bands in the cathodic side shown in types D. E. and the majority of plants in Yutani-25.100 = 77. malic enzyme (ME). Heisou and Biwakoh-1) and did not differ significantly (ANOVA: F3. Araki and Y. clear and stable banding patterns were detected in PGI and PGM of adult plants and in PGI of seedlings. bloomed in culture for the pollination experiment (Table 1). Although the ploidy level of Utricularia species is not clear (Taylor 1989). Biwakoh-1 and -3 bloomed and produced pollen grains. all the plants sampled from Yutani-7. mannose-6-phosphate isomerase (MPI). Fruit and seed set ratios are shown in Table 2. Isozyme analysis Six (A–F) and three (1–3) types of electrophoretic patterns were detected in PGM and PGI. P < 0. The fruit set ratio on 24 September differed significantly between Biwakoh-1 and -3 ( G = 22. plants collected from two populations. differed significantly from selfing (q100. P > 0. Phosphoglucomutase activity in seedlings was low and could not be detected as a banding pattern. the mean seed set ratio in the case of crossing with a different type. P > 0. -24. There was no fruit set in the bagging treatment. thus. P < 0. E. 1).01) and from crossing with the same type (q100. did not produce pollen grains. fruit setting was observed only at Yutani-3. Seed set ratio was approx- imately 10% (7.f. d. were not found in our study. -28 and -29. = 1. which was more than 40%. Fruit set ratio in these populations ranged from 0 to 50%. -17. -19.f. P < 0.262. thus showing male sterility. showed male sterility. Thus.157.005). -8. d. and F may be a consequence of gene duplication. = 2. d. although both were pollenproducing populations.05). and shikimate dehydrogenase (S kDH).19 = 0. the pattern of types A and F indicate heterozygotes and the other four patterns indicate homozygotes about the locus (PGM-1). in which no flowers occurred in natural conditions. Heisou and Biwakoh-1. In contrast. and F. in which flowers were observed only in cultivation. were approximately 10% and did not differ significantly from each other (q100. -27. Kadono (IDH).21. the two bands in the cathodic side. P > 0. no flowers were observed in 17 populations during the two flowering seasons under natural conditions. Thus. In Heisou.602 S. -11. In contrast. two staining bands of PGM in the anodal side can be interpreted as an allozymic pattern because PGM is considered to be a monomer enzyme (Weeden & Wendel 1989).8. = 2. Also plants of Yutani-12 and -23. However. more than 80% of fruit set was observed in the three types of hand pollination.4. In contrast. 6-phosphogluconate dehydrogenase (6PG). Fruit and seed production in natural populations In natural populations.0.

(nd). 134∞51 ¢ E) Kasai City (34∞57 ¢ N. 135∞06 ¢ E) Kakogawa City (34∞48 ¢ N.. Flowers occurred or pollen production was confirmed. phosphoglucomutase and phosphoglucoisomerase. 134∞57 ¢ E) Takarazuka City (34∞55 ¢ N. 134∞52 ¢ E) Akashi City (34∞40 ¢ N. 135∞20 ¢ E) Sanda City (34∞58 ¢ N.Clonal dominance in aquatic bladderwort 603 Table 1 The occurrence or absence of flowering (Fl). . 1 and the sample sizes in the enzyme studies are in parentheses. no data. in each pond studied Localities of the ponds Yokawa Town (34∞52 ¢ N. in the aquatic bladderwort. Br. 135∞15 ¢ E) Yutani-1 Yutani-2 Yutani-3 Yutani-4 Yutani-5 Yutani-6 Yutani-7 Yutani-8 Yutani-9 Yutani-10 Yutani-11 Yutani-12 Yutani-13 Yutani-14 Yutani-15 Yutani-16 Yutani-17 Yutani-18 Yutani-19 Yutani-20 Yutani-21 Yutani-22 Yutani-23 Yutani-24 Yutani-25 Yutani-26 Yutani-27 Yutani-28 Yutani-29 Heisou Biwakoh-1 Biwakoh-2 Biwakoh-3 Abiki Ohkubo Oharano Sakaino Kamisasori-1 Kamisasori-2 Ono-1 Ono-2 Yamada Aimoto-1 Aimoto-2 Ochibara-1 Ochibara-2 Fl PP GT – – + – – – + + – – + + – – – – + – + – + – + + + – + + + + + nd + nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd + nd nd nd – – nd nd – – nd nd nd nd – nd – nd – nd – – –/+ nd – – – + + nd + nd nd nd nd nd nd nd nd nd nd nd nd nd A1 (5) A1 (3) B2 (5) B2 (5) A1 (7) A1 (5) A1 (14) A1 (8) A1 (5) A1/B2 (5) A1 (10) A1 (10) A1 (10) B3 (5) E3 (12) nd A1 (10) A1 (9) A1 (10) nd A1 (1) A1 (5) A1 (5) A1 (10) A1/A3 (88) A1 (7) A1 (10) A1 (19) A1 (5) C2/F3 (146) A2 (10) A2 (5) A2 (5) A1 (10) D1 (10) A1 (10) A1 (25) A1 (10) A1 (17) A1 (5) A1 (24) A1 (10) A1 (15) A1 (9) A1 (4) A1 (15) (+). The notations of GT are the same as in Fig. no flowering or pollen grains were produced. (–). Utricularia australis R. pollen production (PP) and genetic types (GT) of two enzymes.

6 (4) – – – – – 9.0 (11) 4. Kadono Table 1 Continued.0–13. Numbers in parentheses are sample sizes.4 (41) 10. 135∞02 ¢ E) Simokume Ureshino Manose Fl PP nd nd nd nd nd nd GT A3 (5) A3 (5) A3 (5) (+).9 – 13.0–29.7 8.0 (61) 0. (–).7 (21) – 0. Localities of the ponds Yashiro Town (34∞56 ¢ N. (–). ‘type C2’ etc.6 7.e.7 † Crossing with plants of the same genotype.604 S. In Yutani-25.0 (4) 0. Table 3 Percentages of fruit and seed set in the pollination experiment under cultivation Seed set (%) Treatment Bagging Selfing Crossing-same† Crossing-different‡ Fruit set (%) Range Mean 0. (–). no data. but all plants in the pond died out in the summer of 1995 for unknown reasons. In Yutani-10. Araki and Y. The multilocus genotype of each plant was referred to as ‘type A1’. data unavailable. 122 plants were type F3 and 24 were type C2 among the 146 plants tested. The genotypes detected in each population are shown in Table 1.0–29. Yutani-10 and Yutani25). the coexistence of plants of type A1 and B2 was confirmed in a preliminary survey. but variation in the banding pattern was considered to reflect genotypic variation. Heisou. The notations of GT are the same as in Fig.0 (4) 0.3 7.0–30. Flowers occurred or pollen production was confirmed. ‡ of PGI could not be interpreted for genotype.2 – † All dates are in 1996.6 45.2 (12) – 0. 71 plants were type A1 and 17 were A3 among the 88 plants tested. thus further data were unavailable.2 (45) 0.1 (27) 0. To the .0 (17) – 7. Intrapopulational variations of multilocus genotype were only detected in three populations (i.8 (63) 18.4–10. data unavailable.1 (58) 84.0 (3) 5. Crossing with plants of a different genotype. based on the combination of electrophoretic patterns of the two enzymes.3 (51) 85.0 (56) 93.3 (30) 50. In Heisou.0 (109) – – – – 2. 1 and the sample sizes in the enzyme studies are in parentheses. no flowering or pollen grains were produced. Table 2 Percentage of fruit and seed set under natural conditions Seed set (%) Population Yutani-3 Yutani-25 Heisou Biwakoh-1 Biwakoh-3 Date† Fruit set (%) Range Mean 5 September 20 September 25 September 2 October 22 August 6 August 20 August 18 September 24 September 24 September 3.0 (8) – 48.0–17. Numbers in parentheses are sample sizes. (nd).0 (4) 0.8–91.

B3.Clonal dominance in aquatic bladderwort contrary all the plants collected from male-sterile populations were shown to be type A1. There may be invisible or overlapping bands.). in order from the anode to the cathode.50. 0. C2.75. One of these types. B2. and E3) were restricted to several ponds (see Table 1). The symbol characters of genotypes follow Fig. Japan. 1. A pattern that was not found in the natural populations is expressed as type 4 here. The Rf values of each band in type F of PGM are 1. 1. 0. (). Yashiro.).). phosphoglucomutase (PGM) and phosphoglucoisomerase (PGI) of Utricularia australis collected from 47 populations in the southern part of Hyogo Prefecture. (). D1. B2. Fig. 605 of type A2 were distributed only in Kasai City. 1. A3. Three types of polymorphism were detected from 10 seedlings originated in self.18. and the other types (A3.94. (Sa.or cross-pollination among plants of type 2 in PGI (Fig.). Variations in the electrophoretic pattern of two enzymes.00. (Ya.88. was a type that was not found in natural populations. A2. Zymograms of phosphoglucoisomerase detected in seedlings obtained from mating among type 2 parents. 1. (Yo. A3. 2. B2.). B3. For example. 1. (Kas.). where small ponds were densely located. In eastern areas (Sanda and Takarazuka) all the studied populations were type A1. Takarazuka. Figure 2 does not show a typical segregation pattern of dimeric enzyme.00 and 0.). (). E3. Kakogawa. A1. Neighboring populations with the same genotype are represented by a single mark. (▼). The symbolic number of each pattern is the same as in Fig. Sanda. which is referred to as type 4. Plants of other types that flowered (A2. respectively.63 and 0. and in type 1 of PGI are 1. In Yutani. (). (Kak. Different patterns of distribution were found for each genotype. Kasai. Fig.12. Geographical distribution of Utricularia australis categorized by the genotype in two enzyme systems. (). (). (Ak. although PGI is considered to be a dimer (Weeden & Wendel 1989). populations Fig. C2 and F3) produced pollen grains (Table 1). 2. (Ta. Akashi. Each staining pattern is referred to as type A–F for PGM and type 1–3 for PGI. type A1 was the most common. F3. Yokawa. . (). The geographic distribution of each genotype in the southern part of Hyogo Prefecture is shown in Fig. Among these five types. five types were found to occur. 3). 0. (▲). 3. phosphoglucomutase and phosphoglucoisomerase. and three of the four populations of type A3 were in Yashiro Town.

but such plants were not found in the population.606 S. Kadano. Araki & Y. whereas the seeds sunk when they were suspended in water (S. which is 1/1000 in comparison with a turion. most of the turions in cultivation remained floating throughout winter. coexistence of plants of type A and other homozygotic types was not found in our study. we inferred that male sterility in the study area has the same origin and that all the male-sterile plants belong to a single clonal group. In the case of U. Clonal dominance in this species can be inferred from genetic variation of PGM. weighs approximately 40 mg. pers.. However. Les 1991. The reasons for this are not necessarily clear. 1989. A mosaic pattern in the geographic distribution of the multilocus genotype was found in the southern part of Hyogo Prefecture. Barrett et al. australis. but we have already discussed above that type C2 cannot be a descendant of type F3 in this population. but some explanations have been proposed. In addition to the rarity of seed or seedling survival. weighs approximately 0. However. Grace 1993. obs. 3 and 4 of PGI occurred from mating between type 2 parents. approximately 10 mm. all seeds are approximately 1 mm in size and weigh approximately 0. plants of both type F3 and type C2 occurred. Plants of types A and F were heterozygotes in PGM-1. Why is recruitment from seeds rare? Clonal dominance occurs in many aquatic macrophyte populations (Verkleij et al. seed and/or seedling survival may be reduced because of low light and oxygen. but also in seed-producing populations. Hollingsworth et al. seedlings will be less vigorous because the resource storage capabilities of a seed are far less than a turion. Van Wijk 1988. However it was not possible that the type F3 plants had originated from mating among type C2 plants because type F of PGM has two alleles that type C does not share. The dry weight of a turion varies according to its size. data. if mating had occurred between these two types. These results suggest that genetic polymorphism in the Heisou population is not a consequence of sexual reproduction. Kadono. plants of type 3 or 4 were not found in six of the seven ponds in which plants of type 2 occurred (Table 1). australis. plants other than types C2 and F3 would have originated. It is considered that this pattern has been derived by clonal dominance in reproduction and the colonization processes of the species.04 mg (S. In the case of U. metapopulation becomes a mosaic of genets as illustrated by genotype distribution. Araki and Y. More intensive research on the relationship between the amount of fruit production and . The fruit set ratio in natural populations is low when compared with hand pollination and sometimes decreases to zero. 1996b). but of independent colonization of two different clonal lineages into the pond. whereas the largest one. 1996). In the same way. This suggests that recruitment from seeds is rare and only clonal propagation from turions has been prevalent in those populations. It is highly likely that each genet succeeds in establishment as a founder spreads into neighboring habitats by vegetative means. Waterfowl movement may play a role in colonization of aquatic plants by trapping seeds in feathers (Cook 1990. the occurrence of the same genotype in nearby populations and the spread of the male-sterile type A1 suggest that dispersal of vegetative propagules has occurred. the trace of segregation of the two alleles of PGM-1 was not found within each of the populations. DISCUSSION As all the male-sterile plants were type A1 in the enzyme study. Even if seeds are not buried. The coexistence of plants of type F and homozygotic type C was found in Heisou. In addition. In Heisou. isozyme analysis suggested clonal dominance not only in male-sterile populations. Furthermore. which is approximately 1 mm long. The smallest turion. 1996). including inadequate light intensity or dissolved oxygen concentration for seedlings. Kadono Zymograms of seedlings that have parents of types other than type 2 could not be determined because of the limited availability of seedlings. Seedlings of types 2.07 mg. Thus. Thus. However. unpubl. Araki and Y. Vivian-Smith & Stiles 1994). 1983. type C2 cannot be the descendant of type F3 because type 2 of PGI cannot derive from recombination of the genotype of type 3. 1993. the chance of sexual reproduction is restricted by low seed production. If seeds are buried in sediment at the bottom of the pond. although polymorphism among populations may be a consequence of sexual recombination.

Seed production in natural populations. Richman et al. If U. If such a founder effect is repeated dur- 607 ing the process of colonization. B ARRETT S. (1987) Patterns of genotypic diversity in clonal plant species. various types of genetic features of the population are easily affected by founder effects or genetic bottlenecks (Hedrick & Parker 1998. Because habitats such as ponds and lakes are isolated from each other. in a former river channel. (2000) Isozyme differentiation between two infraspecies taxa of Utricularia australis R. Crossing with a different type corresponds to interclonal pollination. Low seed set ratios observed in natural populations are also considered to be consequences of the clonality of the population. (1997) Clonal life histories and the evolution of seed recruitment. (1990) Seed dispersal of Nymphoides peltata. Backhuys Publishers.) Sm. Br. A. E CKERT C. (1989) Seedling dynamics and life histories in clonal plants. which may result in inbreeding or incompatible effect. Decodon ferticillatus (Lythraceae). crossing with the same type corresponds to intraclonal pollination. the results of the pollination experiment indicate a low seed set ratio in intraclonal mating and a high seed set ratio in interclonal mating. (Lentibulariaceae) in Japan. E LLSTRAND N. (1996) Germination and colonisation dynamics of Nuphar lutea (L. De Kroon & J. Aquatic Botany 55: 31–38. Groenendael) pp. D. Leiden. ACKNOWLEDGEMENTS We are grateful to Mr T. Mahy & Jacquemart 1998). There are two explanations for low seed set ratio in intraclonal pollination: 1 Inbreeding depression. E CKERT C. Manicacci & Barrett 1996. Oikos 55: 231–238. (1993) Evolutionary processes in aquatic plant populations. G. American Journal of Botany 74: 123–131. E RIKSSON O. If we assume that plants with the same multilocus genotype belong to the same clonal lineage. In contrast. australis is selfincompatible. In the case of U. In: The Ecology and Evolution of Clonal Plants (eds H. & H USBUND B. australis. 1995). Aquatic Botany 44: 105–145. & M ITCHELL S. Husband & Schemske 1996. D ORKEN M. 1994. Inbreeding depressions may have prevented normal seed development (Seavey & Bawa 1986. thus cross-pollination within the population is equal to self-pollination. G. REFERENCES A RAKI S. B ARRETT S. Suzuki. the number of genets in a population decreases further and finally becomes one. Evolutionary Ecology 15: 501–520. H. C. it is probable that the number of genets in one population is one or a few at the time of establishment of a new population. 2 Self-incompatibility. for his suggestions for improving the techniques of isozyme detection and to the owners of the irrigation ponds investigated. Reinartz & Les 1994). Johnson & Black 1998. 211–226. (1999) Loss of sex in clonal populations of a flowering plant. 1990. C. Murfett et al. all the individuals in a population belong to the same genet and have the same S allele. Seed set ratio appears to be diminished for some genetic reasons. The pollination experiment showed that seed set ratios were low in the cases of selfing and crossing between plants of the same multilocus genotype. Journal of Applied Ecology 17: 113–124. Even if the parent plants grow normally. homozygosis that prevents seed development may occur through mating. Lee et al. Evolution 53: 1079–1092. C. E CKERT C. C OOK C. Genetic bottlenecks affect the number of genets in a population and/or the number of S alleles within a population (Imrie et al. 1972. H. Acta Phytotaxonomica et Geobotanica 51: 31–36. 1998). crossing between plants of different genotypes resulted in higher seed set ratios. E RIKSSON O.. & R OOSE M. K. In this situation. II. Schug et al. E. (1980) Sexual reproduction in Eichhornia crassipes (water hyacinth). mating within the same clonal individuals results in low seed set ratios because the parents have the same S allele. Aquatic Botany 37: 325–340. Self-incompatibilities in many plant species are genetically controlled by a single multi-allelic S locus (Haring et al. L. C. (2002) Loss of sex in clonal plants.Clonal dominance in aquatic bladderwort pollinator activity or weather conditions is needed for clarification of the reasons for fruit set ratio fluctuation in natural populations. which is equal to self-pollination. G. . Museum of Nature and Human Activities. B ARRAT -S EGRETAIN M.. 1994. Thus.

Van Groenendael) pp. S UZUKI T. Nature 367: 563–566. N AKAMURA T. (1993) Lentibulariaceae. K LIMES L. 400–404. (1994) Bottleneckinduced dissolution of self-incompatibility and breeding system consequences in Aster furcatus (Asteraceae). pectinatus and P. General Education 9: 163–212. & K AO T. M AKINO T... M OU B. R ICHMANN A. Heredity 80: 163–172. Canadian Journal of Botany 76: 37–42. Tokyo (in Japanese). R. pectinatus ¥ P. (1994) S-RNase expressed in transgenic Nicotiana causes S-allelespecific pollen rejection... De Kroon & J. P.. Bunichi Sogo Shuppan. L EE H. A THERTON T.. M AHY G. 1–29. M IKI S. filliformis. (1986) Late-acting self-incompatibility in angiosperms. & V AN G ROENENDAEL J. D. Kadono G RACE J. L ES D. Aquatic Botany 51: 259–268.) Royle in Japan. A.. P. (1998) Mating system of Calluna vulgaris: self-sterility and outcrossing estimations. f. I MRIE B. B. The Botanical Magazine. (1995) The cost of reproduction to the clonal herb Asarum canadense (wild ginger). & C LARKE A. (1993) The adaptive significance of clonal reproduction in angiosperms: an aquatic perspective. S CHUG M. J OHNSON M. & R OSS D. Journal of Plant Research 111: 581–585. J. In: The Ecology and Evolution of Clonal Plants (eds H. A NDERSON M. Iwatsuki. M. E. H USBAND B. M. Bahamas.. & B ARRETT S. Aquatic Botany 44: 159–180.. (1990) Selfincompatibility: a self-recognition system in plants. American Journal of Botany 78: 1070–1082. F. H.) Royle. American Journal of Botany 81: 446– 455. (1996) Fertility differences among floral morphs following selfing in tristylous Eichhornia paniculata (Pontederiaceae): inbreeding depression or partial incompatibility? American Journal of Botany 83: 594– 603. N AKAMURA T. (1998) Increased genetic divergence and reduced genetic variation in populations of the snail Bembicium vittatum in isolated tidal ponds. The Botanical Magazine. K AO T. K OMIYA S. K. Heredity 75: 405–415. G RAY J. Plant Systematics and Evolution 202: 233–254. (1935) New water plants in Asia Orientalis III.. K LIMESOVA J. R. A.. J. M ANICACCI D.. & L ES D. P RESTON C. H ARING V. Volume IIIa (eds K. & S HIBATA C. & K ADONO Y. C.. M UIR A. D. & F UERST P. S EAVEY S. M. . M. H EDRICK P. Canadian Journal of Botany 73: 1683–1686. Botanical Review 52: 195–219. H. W. Araki and Y. & M C C LURE B. (1998) A comparative study of isoenzyme patterns of Hydrilla verticillata (L. population structure. In: Flora of Japan. L ES D. G. & G ORNALL R. & R OELOFS J... B.608 S. G ASSER C. (1998) MHC variation in the endangered Gila topminnow. L. (1998) Isolation and genetic diversity of Gambusia hubbsi (mosquitofish) populations in blueholes on Andros Island. Leiden. Tokyo 28: 1–30. M.. K ADONO Y. Nature 367: 560–563. D.. (Hydrocharitaceae) in Japan. Tokyo 49: 847–852. & K ADONO Y. Heredity 80: 336–346. E. K IRKMAN C. (1991) Genetic diversity in the monoecious hydrophile Ceratophyllum (Ceratophyllaceae). C. W. S. (1997) Clonal plant architecture: a comparative analysis of form and function. (1995) S-allele sequence diversity in natural populations of Solanum carolinense (Horsenettle). D OWNHOWER J. Tokyo. M URFETT J. J. Kodansha. Ohba) pp. Potamogetonaceae). Australian Journal of Biological Sciences 25: 343–349.. D. (1914) Observations on the flora of Japan. H OLLINGSWORTH P. B ROWN L. & B AWA K. H. S CHAEFFER S. P. H ENDRIKS R. A. S MOLDERS A. Bulletin of Nippon Dental University. A. P RESTON C. H OLLINGSWORTH P. & J ACQUEMART A. Acta Phytotaxonomica et Geobotanica 44: 123–140. (1996a) Isozyme evidence for the parentage and multiple origins of Potamogeton ¥ suecicus (P. & P ARKER K. (1980) Distribution of the Lentibulariaceae in Japan. Science 250: 937–941. D EN H ARTOG C. Evolution 52: 194–199. & G ORNALL R. A. H UANG S. S. and evolution in hydrophilous angiosperms. Boufford & H. C. & B LACK R. Annals of Missouri Botanical Garden 75: 819–835. M C C LURE B. H. (1994) Aquatic Plants of Japan. (1995a) Observations on fruiting and seed-set of Stratiotes aloides L. R EINARTZ J. Evolution 50: 54–70. (1988) Breeding systems. f. E. in the Netherlands. (1996) Evolution of the magnitude and timing of inbreeding depression in plants. (1972) Computer simulation of a sporophytic self-incompatibility breeding system. S EARS D. Yamazaki. D. J. (1993) Chromosome number and geographical distribution of monoecious and dioecious Hydrilla verticillata (L. (1994) S proteins control rejection of incompatible pollen in Petunia inflata. & U YENOYAMA M. (1996b) Genetic variability in two hydrophilous species of Potamogeton. W. K ADONO Y. T. filiformis (Potamogetonaceae).. Plant Systematics and Evolution 202: 219– 232. Backhuys Publishers. & S CHEMSKE D.

. (1988) Distribution of Utricularia vulgaris L..Clonal dominance in aquatic bladderwort S MOLDERS A. T AMURA M. J. Dioscorides Press. Tominari) pp. & K ADONO Y. Aquatic Botany 17: 43–59. 137–139. 46–72. E. V IVIAN -S MITH G. T. American Fern Journal 73: 9–27. III. London. Y AMAMOTO I. J. Tokyo (in Japanese). In: Isozymes In Plant Biology (eds D. V ERBURG R. S. P. Canadian Journal of Botany 72: 860–871. J. Portland. In: Wild Flowers of Japan III (eds Y. gel and electrode buffers. Wetlands 14: 316–319. . W EEDEN N. Aquatic Botany 31: 211–258. I. T AYLOR P. S OLTIS D. var japonica (Makino) Tamura and Utricularia tenuicaulis Miki in the southern part of Hyogo Prefecture. A. (1988) Ecological studies on Potamogeton pectinatus L. biomass production and life cycles under field conditions. & K ADONO Y. Aquatic Botany 33: 271– 299. pp. D EN H ARTOG C. H AUFLER C. F. & S TILES E. (1990) A study on the reproductive biology of aquatic Utricularia species in southwestern Japan. G. W ATERWAY M. Reproductive strategies and germination ecology. J. Satake.. & G ASTONY G. (1998) Vegetative propagation and sexual reproduction in the woodland understorey pseudo-annual Circaea lutetiana L. Y AMAMOTO I. E. H. W. Her Majesty’s Stationery Office. D ARROW D.. (1989) The Genus Utricularia: A Taxonomic Monograph. Plant Ecology 134: 211–224. & T ORENBEEK M. and staining schedules. (1994) Dispersal of salt marsh seeds on the feet and feathers of waterfowl. Aquatic Botany 51: 269–279. H ORNEMAN G. The Journal of Phytogeography and Taxonomy 36: 72–75 (in Japanese with English summary). C. J. Heibonsha. S. (1995b) Germination and seedling development in Stratioites aloides L. P IETERSE A.f. Soltis & P. C. S. & R OELOFS J. Watari & T. Ohwi.. J. & D URING H.) Royle. Acta Phytotaxonomica et Geobotanica 41: 189–200 (in Japanese with English abstract). V AN W IJK R. (1989) Ecological studies on Potamogeton pectinatus L. Soltis). (1981) Lentibulariaceae. (1994) Evidence for the hybrid origin of Carex kunieskernii with comments on hybridization in the genus Carex (Cyperaceae). J. & W ENDEL J. southwestern Japan. M. Kitamura. (1983) Comparative study of the morphology and isoenzyme patterns of Hydrilla verticillata (L. V AN W IJK R. F. General characteristics. W. 609 V ERKLEIJ J. (1989) Genetics of plant isozymes. H. (1983) Starch gel electrophoresis of ferns: a compilation of grinding buffers.