Araki & Kadono 2003 - Restricted Seed Contribution and Clonal Dominance in a Free-floating Aquatic...

Ecological Research

(2003)

18

, 599–609

Blackwell Science, LtdOxford, UKEREEcological Research0912-38142003 Ecological Society of JapanSeptember 2003185599609Original Article

Clonal dominance in aquatic bladderwortS. Araki and Y. Kadono

*Author to whom correspondence should beaddressed. Present address: Research Center for CoastalLagoon Environments, Shimane University, Matsue690-8504, Japan. Email: [email protected]

Received 30 October 2002. Accepted 26 March 2003.

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


R. Br. is an aquatic angiosperm species common in natural and irrigation pondsin temperate regions. This species reproduces both sexually and vegetatively, but in southwesternJapan the occurrence of male-sterile populations, in which plants produce no pollen and propagateonly vegetatively, has been recorded. We studied the reproductive contribution of seeds in normalpollen-producing populations using isozyme analyses, a pollination experiment under culture andfield observations. Although seedlings obtained from controlled mating indicated segregation ofisozyme, polymorphism of the isozyme genotype was detected mainly among populations, but rarelywithin each pond population. This suggested clonal dominance and rarity of seed or seedling survivalin natural populations. In the pollination experiment, the mean seed set ratio in cross-pollinationbetween 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 lowratio in crossing between the same genotype plants was ascribed to the clonality of the parents. Ingeneral, these results corresponded with the low ratios in seed setting observed in natural popula-tions (7.9–13.7%). All the male-sterile populations we surveyed showed the same genotype, thusmale sterility in the study area was considered to have the same origin.

Key words:

isozyme; male sterility; sexual reproduction;


; vegetativereproduction.

INTRODUCTION

Vegetative reproduction by the root system orother organs occurs in various taxa of angiosperms(Klimes

et al

. 1997). Most of these species alsoreproduce in a sexual way through seed produc-tion. The relative contribution of vegetative andsexual modes to reproduction has been a subject ofecological studies because it is essential to knowthis information to adequately explain the repro-ductive system of the species (Ellstrand & Roose1987; Eriksson 1997). The amount of seedlingrecruitment varies among species and/or habitats

(Eriksson 1989), thus the relative importance andbiological role of the vegetative and sexual systemsare considered to vary among species and theirenvironments. In particular, a strong reliance onvegetative propagation is found in aquatic habi-tats. Many studies on aquatic macrophytes havesuggested single, or very few, genet compositionsin a population or through neighboring popula-tions, although most of these species produce seedor at least have the ability to produce seed throughsexual processes (Les 1988; Barrett

et al

. 1993;Grace 1993). This remarkable clonality is, in part,because these seedlings have a higher risk of dyingin inadequate light and dissolved oxygenconditions in bottom water or sedimenthabitats and the chance of seedling recruitment isrestricted (Barrett 1980; Smolders

et al

. 1995a,1995b; Barrat-Segretain 1996).

In some groups, the occurrence of sterile strains,such as species hybrids (Waterway 1994; Holling-sworth

et al

. 1996a) or triploids (Nakamura &

600 S. Araki and Y. Kadono

Kadono 1993; Nakamura

et al

. 1998), that prop-agate only vegetatively have been reported. Ifseeds cannot contribute to reproduction because ofhigh mortalities of seeds or seedlings, mutationsthat prevent seed production may be maintainedin these species because seed loss has no disadvan-tage. Furthermore, it may be advantageous if thereis a trade-off between seed production and vegeta-tive reproduction. Irrespective of whether such atrade-off exists (Muir 1995; Verburg & During1998), it is predicted that sterile mutants can beeasily maintained in populations that reproducemainly vegetatively (Eckert

et al

. 1999; Eckert2002).

The aquatic bladderwort,


R.Br. (Lentibulariaceae), includes both fertile andsterile plants. This species inhabits natural andirrigation ponds in a submerged, free-floatingform. In summer, well-grown plants developscapes above the water surface and bear approxi-mately four to six flowers per scape. These flowersare self- or cross-pollinated by small insects, suchas aphids or small dipterous species (Yamamoto &Kadono 1990; S. Araki and Y. Kadono, pers. obs.,1996). In autumn, the plants form a vegetativehibernating organ, turion, on the tips of the mainshoot and each branch. Poorly grown plants do notdevelop scapes during the flowering season andreproduce only vegetatively by turions. Turionsand seeds remain dormant until the followingspring, whereas other parts of the plant body dis-appear in winter. Yamamoto and Kadono (1990)reported the occurrence of male-sterile populationsof this species, in which plants produce no pollengrains, in the southern part of Hyogo Prefecture,southwestern Japan. Plants in those populationsreceive no pollen and produce no seed, thus it isnot a case of gynodioecy. If seeds are essential forreproduction of this species, it is difficult toexplain how male-sterile plants could spread. Butif seeds give no or rare reproductive contributionin this species, abnormality in pollen production isnot disadvantageous.

In the present study, we examined whether ornot seeds contribute to reproduction in normal,pollen-producing populations of

U. australis

through field observations of fruit and seed pro-duction, pollination experiments under cultureand an investigation of the population geneticstructure using isozyme analysis.

In the present paper we describe the study spe-cies as

U. australis

following Kadono (1993),although there has been some confusion in the tax-onomy of this species. Formerly two taxa,

U. japonica

(Makino 1914) and

U. tenuicaulis

(Miki1935), were reported as novel species in Japan.They differ from each other in some morphologicalfeatures of the turion (Miki 1935; Yamamoto &Kadono 1988). The former occurs mainly innorthern Japan, especially in Hokkaido, and rarelyin other regions of Japan, whereas the latter is dis-tributed widely in Japan and rarely in Hokkaido(Kadono 1994). These two taxa are now treated inthree ways according to different taxonomic opin-ions. Komiya and Shibata (1980) regard both ofthem as synonyms of

U. australis

and describe

U. australis

form.

australis

and

U. australis

form.

tenuicaulis

, respectively. However, Tamura (1981)describes the former as

U. vulgaris

var.

japonica

andthe latter as

U. tenuicaulis

. Kadono (1993)describes the former as

U. vulgaris

var.

japonica

andthe latter as

U. australis

. To date, no consensus hasbeen achieved in nomenclature of these taxa. Weconsider, from the features mentioned above, andrecently reported isozyme differences, that thesetwo taxa are genetically differentiated from eachother (Araki 2000). Thus, our present study doesnot include plants that have been referred to as

U. japonica

or

U. vulgaris

var.

japonica

, althoughthey are sometimes included in

U. australis.

METHODS

Field observations

We define a population as all the plants growing ina pond and refer to each population using the nameof the place in which the pond is located. If morethan two ponds are located in the same locality,each of the populations in the area is numbered(e.g. Yutani-1 and Yutani-2).

We conducted field observations to confirmflowering and pollen production for 32 popula-tions, namely Yutani-1 to Yutani-29 in YokawaTown, Heisou in Kakogawa City, and Biwakoh-1and Biwakoh-3 in Kasai City, Hyogo Prefecture.We visited these irrigation ponds weekly orbiweekly in the flowering seasons of 1995 and1996, and recorded the occurrence or absence offlowering. Pollen production was checked for

Clonal dominance in aquatic bladderwort 601

selected plants by observing anthers under micro-scope for each population.

Fruit and seed set ratios were investigated in fivepopulations, Yutani-3, Yutani-25, Heisou, Biwa-koh-1 and Biwakoh-3. We visited those popula-tions on one to four occasions in the floweringseason of 1996, and counted fruits and pedunclesthat had already dropped their corollas. The fruitset ratio was calculated for each population as thenumber of fruits divided by the total number ofpeduncles without corolla. Next, we sampled all orsome of the fruit-setting plants and dissectedfruits. The seed set ratio was calculated for eachmature fruit as the number of seeds divided by thetotal number of ovules (approx. 100 per flower). Incases where plants could not be collected, only thefruit set ratio was determined in the field.

Pollination experiment

For the purposes of isozyme analysis, plants col-lected from 47 populations in Hyogo Prefecturewere cultivated in containers placed in the campusof Kobe University. Among them, plants fromnine populations, Yutani-3, -8, -11, -12, -23, -25,Heisou, Biwakoh-1 and -3, flowered during culti-vation and were used in the pollination experi-ment. We paid attention to the possibility of cross-pollination between the same clonal individuals,which is equal to self-pollination. To avoid mixingtrue cross-pollination and pseudo-cross-pollina-tion, we employed two types of cross-pollination,namely, mating with the plant which indicated thesame genotype from an isozyme study and with aplant that had a different genotype. Plants weretreated in one of the following four ways whenthey were in bloom. The treatments were:

1 ‘Bagging’. Each scape was bagged during theflowering period.

2 ‘Selfing’. The flower was self-pollinated artifi-cially and the whole scape was bagged.

3 ‘Crossing with the same genotype’. The flowerwas cross-pollinated with pollen grains obtainedfrom an individual of the same genotype and thewhole scape was bagged.

4 ‘Crossing with different genotype’. The flowerwas cross-pollinated with pollen grainsobtained from an individual with a differentgenotype and the whole scape was bagged.

Plants collected from Yutani included male-sterileplants. Those plants were used only in the fourthtreatment as maternal plants. Seed set ratio in eachfruit was transformed to its arcsine square root forstatistical analysis.

Isozyme analysis

We collected plants for isozyme analysis from 47irrigation ponds located in the southern part ofHyogo Prefecture. These included the 32 popula-tions that were used for the field observations,except for Yutani-16 and Yutani-20. Each of thefloating plant bodies was picked up intact from thewater. Sample sizes between populations rangedfrom 1 to 146 (mean 13) because the amount ofgrowing plant differed among ponds. Samplescould include ramets of the same genet becauseeach individual can produce plural turions andconsequently plural ramets. When genotypicvariation within a population was detected in theisozyme study, many plants were sampled addi-tionally from the pond and the population geneticstructure was intensively examined. Seedlingsobtained from the pollination experiment werealso analyzed.

We used horizontal starch gel electrophoresisfollowing the method of Soltis

et al

. (1983). Tris-HCl buffer–PVP solution was employed as agrinding buffer with a modification of mercapto-ethanol concentration to 0.3%. Histidine-citratebuffer, corresponding to system 9 in Soltis

et al

.(1983), and its one-fourth dilution was used as anelectrode buffer and gel buffer, respectively. ThepH of the electrode buffer was adjusted to 6.5 byan alteration in the amount of citric acid. A 1–5 cm length of vegetative shoot tip was ground in0.3 ml grinding buffer and centrifuged at50 000

g

for 3 min. The supernatant was soakedinto wicks and inserted into the slit of the gel,which was then electrophoresed for 15 min. Afterthe wicks have been drawn, the electrophoresis wascarried out again under constant current (35 mA)for 6 h. The staining method of the gel followedthe method outlined by Soltis

et al

. (1983).In preliminary tests, we analyzed the following

14 enzymes: aconitase (ACN), adenylate kinase(ADK), alcohol dehydrogenase (ADH), formatedehydrogenase (FDH), glutamate dehydrogenase(GDH), hexokinase (HK), isocitrate dehydrogenase


(IDH), malate dehydrogenase (MDH), malicenzyme (ME), mannose-6-phosphate isomerase(MPI), 6-phosphogluconate dehydrogenase (6PG),phosphoglucoisomerase (PGI), phosphoglucomu-tase (PGM), and shikimate dehydrogenase (S kDH).Among these, clear and stable banding patternswere detected in PGI and PGM of adult plants andin PGI of seedlings. Phosphoglucomutase activityin seedlings was low and could not be detected asa banding pattern. Thus, we used electrophoreticpatterns of PGM and PGI in the genetic analysis ofadult plants and PGI for seedlings.

RESULTS

Pollen production

Among the 29 populations at Yutani, no flowerswere observed in 17 populations during the twoflowering seasons under natural conditions. How-ever, plants collected from two populations,Yutani-12 and -23, in which no flowers occurredin natural conditions, bloomed in culture for thepollination experiment (Table 1). All the floweringplants sampled in Yutani-3 and some from Yutani-25 were confirmed to produce pollen grains. Plantsin Heisou, Biwakoh-1 and -3 bloomed and pro-duced pollen grains. In contrast, all the plantssampled from Yutani-7, -8, -11, -17, -19, -21,-24, -27, -28 and -29, and the majority of plants inYutani-25, did not produce pollen grains, thusshowing male sterility. Also plants of Yutani-12and -23, in which flowers were observed only incultivation, showed male sterility.

Fruit and seed production in natural populations

In natural populations, fruit setting was observedonly at Yutani-3, -25, Heisou and Biwakoh-1.Fruit and seed set ratios are shown in Table 2.Fruit set ratio in these populations ranged from 0to 50%. In Heisou, fruit set ratio varied signifi-cantly with time (

G-

test:

G

=

25.0, d.f.

=

2,

P

<

0.005). A significant difference among popu-lations on the same date was also observed. Thefruit set ratio on 24 September differed signifi-cantly between Biwakoh-1 and -3 (

G

=

22.8,d.f.

=

1,

P

<

0.005), although both were pollen-producing populations. Seed set ratio was approx-

imately 10% (7.9–13.7%) in the three populationsinvestigated (Yutani-25, Heisou and Biwakoh-1)and did not differ significantly (

ANOVA

:

F

3,19

=

0.262,

P

>

0.05).

Pollination experiment

The results of the pollination experiment areshown in Table 3. There was no fruit set in thebagging treatment. In contrast, more than 80% offruit set was observed in the three types of handpollination. There was no significant difference infruit set ratios among the three types of selfingtreatments, crossing with the same type and cross-ing with different types (

G

-test:

G

=

2.21,d.f.

=

2,

P

>

0.05). However, mean seed set ratiosdiffered significantly among the three types ofhand pollination (

ANOVA

:

F

2,100

=

77.7,

P

<

0.01).Multiple comparisons using Tukey tests indicatedthat the mean seed set ratios in the cases of selfingand crossing with the same type, were approxi-mately 10% and did not differ significantly fromeach other (

q

100,3

=

0.157,

P

>

0.05). In contrast,the mean seed set ratio in the case of crossing witha different type, which was more than 40%, dif-fered significantly from selfing (

q

100,3

=

16.4,

P

<

0.01) and from crossing with the same type(

q

100,3

=

16.3,

P

<

0.01).

Isozyme analysis

Six (A–F) and three (1–3) types of electrophoreticpatterns were detected in PGM and PGI, respec-tively, from plants in natural populations (Fig. 1).Although the ploidy level of

Utricularia

species isnot clear (Taylor 1989), two staining bands ofPGM in the anodal side can be interpreted as anallozymic pattern because PGM is considered to bea monomer enzyme (Weeden & Wendel 1989).Thus, the pattern of types A and F indicate het-erozygotes and the other four patterns indicatehomozygotes about the locus (PGM-1). In addi-tion, the two bands in the cathodic side, which aredetected in types D, E, and F, may indicate alloz-ymes at another locus (PGM-2). However, plantsindicating a homozygotic pattern, which has onlythe nearest band to the cathode, were not found inour study, thus, the two bands in the cathodic sideshown in types D, E, and F may be a consequenceof gene duplication. The electrophoretic patterns


Table 1

The occurrence or absence of flowering (Fl), pollen production (PP) and genetic types (GT) of two enzymes,phosphoglucomutase and phosphoglucoisomerase, in the aquatic bladderwort,


R. Br., in eachpond studied

Localities of the ponds Fl PP GT

Yokawa Town (34

∞

52

¢

N, 135

∞

06 ¢ E) Yutani-1 – nd A1 (5)Yutani-2 – nd A1 (3)Yutani-3 + + B2 (5)Yutani-4 – nd B2 (5)Yutani-5 – nd A1 (7)Yutani-6 – nd A1 (5)Yutani-7 + – A1 (14)Yutani-8 + – A1 (8)Yutani-9 – nd A1 (5)Yutani-10 – nd A1/B2 (5)Yutani-11 + – A1 (10)Yutani-12 + – A1 (10)Yutani-13 – nd A1 (10)Yutani-14 – nd B3 (5)Yutani-15 – nd E3 (12)Yutani-16 – nd ndYutani-17 + – A1 (10)Yutani-18 – nd A1 (9)Yutani-19 + – A1 (10)Yutani-20 – nd ndYutani-21 + – A1 (1)Yutani-22 – nd A1 (5)Yutani-23 + – A1 (5)Yutani-24 + – A1 (10)Yutani-25 + –/+ A1/A3 (88)Yutani-26 – nd A1 (7)Yutani-27 + – A1 (10)Yutani-28 + – A1 (19)Yutani-29 + – A1 (5)

Kakogawa City (34∞48 ¢ N, 134∞51 ¢ E) Heisou + + C2/F3 (146)Kasai City (34∞57 ¢ N, 134∞52 ¢ E) Biwakoh-1 + + A2 (10)

Biwakoh-2 nd nd A2 (5)Biwakoh-3 + + A2 (5)Abiki nd nd A1 (10)

Akashi City (34∞40 ¢ N, 134∞57 ¢ E) Ohkubo nd nd D1 (10)Takarazuka City (34∞55 ¢ N, 135∞20 ¢ E) Oharano nd nd A1 (10)

Sakaino nd nd A1 (25)Kamisasori-1 nd nd A1 (10)Kamisasori-2 nd nd A1 (17)

Sanda City (34∞58 ¢ N, 135∞15 ¢ E) Ono-1 nd nd A1 (5)Ono-2 nd nd A1 (24)Yamada nd nd A1 (10)Aimoto-1 nd nd A1 (15)Aimoto-2 nd nd A1 (9)Ochibara-1 nd nd A1 (4)Ochibara-2 nd nd A1 (15)

(+), Flowers occurred or pollen production was confirmed; (–), no flowering or pollen grains were produced; (nd), no data. Thenotations of GT are the same as in Fig. 1 and the sample sizes in the enzyme studies are in parentheses.


of PGI could not be interpreted for genotype, butvariation in the banding pattern was considered toreflect genotypic variation. The multilocus geno-type of each plant was referred to as ‘type A1’,‘type C2’ etc. based on the combination of electro-phoretic patterns of the two enzymes.

The genotypes detected in each population areshown in Table 1. Intrapopulational variations ofmultilocus genotype were only detected in three

populations (i.e. Heisou, Yutani-10 and Yutani-25). In Heisou, 122 plants were type F3 and 24were type C2 among the 146 plants tested. InYutani-25, 71 plants were type A1 and 17 were A3among the 88 plants tested. In Yutani-10, thecoexistence of plants of type A1 and B2 was con-firmed in a preliminary survey, but all plants in thepond died out in the summer of 1995 for unknownreasons, thus further data were unavailable. To the

Yashiro Town (34∞56 ¢ N, 135∞02 ¢ E) Simokume nd nd A3 (5)Ureshino nd nd A3 (5)Manose nd nd A3 (5)

Localities of the ponds Fl PP GT

(+), Flowers occurred or pollen production was confirmed; (–), no flowering or pollen grains were produced; (nd), no data. Thenotations of GT are the same as in Fig. 1 and the sample sizes in the enzyme studies are in parentheses.

Table 1 Continued.

Table 2 Percentage of fruit and seed set under natural conditions

Population Date† Fruit set (%)

Seed set (%)

Range Mean

Yutani-3 5 September 3.3 (30) – –20 September 50.0 (4) – –25 September 0.0 (4) – –2 October 0.0 (8) – –

Yutani-25 22 August – 2.0–13.0 (4) 9.3Heisou 6 August 48.1 (27) 0.0–29.2 (12) 7.9

20 August 0.0 (11) – –18 September 4.8 (63) 0.0–30.0 (3) 13.7

Biwakoh-1 24 September 18.0 (61) 5.4–10.6 (4) 8.2Biwakoh-3 24 September 0.0 (109) – –

†All dates are in 1996.Numbers in parentheses are sample sizes. (–), data unavailable.

Table 3 Percentages of fruit and seed set in the pollination experiment under cultivation

Treatment Fruit set (%)

Seed set (%)

Range Mean

Bagging 0.0 (56) – –Selfing 93.1 (58) 0.0–17.2 (45) 7.6Crossing-same† 84.3 (51) 0.0–29.4 (41) 7.6Crossing-different‡ 85.7 (21) 10.8–91.0 (17) 45.7

†Crossing with plants of the same genotype.‡Crossing with plants of a different genotype.Numbers in parentheses are sample sizes. (–), data unavailable.


contrary all the plants collected from male-sterilepopulations were shown to be type A1. Plants ofother types that flowered (A2, A3, B2, C2 and F3)produced pollen grains (Table 1).

The geographic distribution of each genotype inthe southern part of Hyogo Prefecture is shown inFig. 2. Different patterns of distribution werefound for each genotype. For example, populations

of type A2 were distributed only in Kasai City, andthree of the four populations of type A3 were inYashiro Town. In eastern areas (Sanda and Takara-zuka) all the studied populations were type A1. InYutani, where small ponds were densely located,five types were found to occur. Among these fivetypes, type A1 was the most common, and theother types (A3, B2, B3, and E3) were restricted toseveral ponds (see Table 1).

Three types of polymorphism were detectedfrom 10 seedlings originated in self- or cross-pol-lination among plants of type 2 in PGI (Fig. 3).One of these types, which is referred to as type 4,was a type that was not found in natural popula-tions. Figure 2 does not show a typical segregationpattern of dimeric enzyme, although PGI is con-sidered to be a dimer (Weeden & Wendel 1989).There may be invisible or overlapping bands.

Fig. 2. Geographical distri-bution of Utricularia australiscategorized by the genotype intwo enzyme systems, phospho-glucomutase and phosphoglu-coisomerase. (�), A1; (�), A2;(�), A3; (�), B2; (�), B3; (▼),C2; (�), D1; (▲), E3; (�), F3.The symbol characters of gen-otypes follow Fig. 1. Neigh-boring populations with thesame genotype are representedby a single mark. (Kas.), Kasai;(Kak.), Kakogawa; (Ak.),Akashi; (Ya.), Yashiro; (Yo.),Yokawa; (Sa.), Sanda; (Ta.),Takarazuka.

Fig. 1. Variations in the electrophoretic pattern oftwo enzymes, phosphoglucomutase (PGM) and phos-phoglucoisomerase (PGI) of Utricularia australis col-lected from 47 populations in the southern part ofHyogo Prefecture, Japan. Each staining pattern isreferred to as type A–F for PGM and type 1–3 for PGI.The Rf values of each band in type F of PGM are 1.18,1.12, 1.00 and 0.94, and in type 1 of PGI are 1.00,0.88, 0.75, 0.63 and 0.50, respectively, in order fromthe anode to the cathode.

Fig. 3. Zymograms of phosphoglucoisomerasedetected in seedlings obtained from mating among type2 parents. The symbolic number of each pattern is thesame as in Fig. 1. A pattern that was not found in thenatural populations is expressed as type 4 here.


Zymograms of seedlings that have parents of typesother than type 2 could not be determined becauseof the limited availability of seedlings.

DISCUSSION

As all the male-sterile plants were type A1 in theenzyme study, we inferred that male sterility inthe study area has the same origin and that all themale-sterile plants belong to a single clonalgroup. In addition, isozyme analysis suggestedclonal dominance not only in male-sterile popula-tions, but also in seed-producing populations.Seedlings of types 2, 3 and 4 of PGI occurred frommating between type 2 parents. However, plantsof type 3 or 4 were not found in six of the sevenponds in which plants of type 2 occurred(Table 1). This suggests that recruitment fromseeds is rare and only clonal propagation fromturions has been prevalent in those populations. InHeisou, plants of both type F3 and type C2occurred. However it was not possible that thetype F3 plants had originated from mating amongtype C2 plants because type F of PGM has twoalleles that type C does not share. In the same way,type C2 cannot be the descendant of type F3because type 2 of PGI cannot derive from recom-bination of the genotype of type 3. Furthermore, ifmating had occurred between these two types,plants other than types C2 and F3 would haveoriginated, but such plants were not found in thepopulation. These results suggest that geneticpolymorphism in the Heisou population is not aconsequence of sexual reproduction, but of inde-pendent colonization of two different clonal lin-eages into the pond.

Clonal dominance in this species can be inferredfrom genetic variation of PGM. Plants of types Aand F were heterozygotes in PGM-1. However,coexistence of plants of type A and other homozy-gotic types was not found in our study. The coex-istence of plants of type F and homozygotic type Cwas found in Heisou, but we have already dis-cussed above that type C2 cannot be a descendantof type F3 in this population. Thus, the trace ofsegregation of the two alleles of PGM-1 was notfound within each of the populations, althoughpolymorphism among populations may be a con-sequence of sexual recombination.

Why is recruitment from seeds rare? Clonaldominance occurs in many aquatic macrophytepopulations (Verkleij et al. 1983; Van Wijk 1988,1989; Les 1991; Barrett et al. 1993; Grace 1993;Hollingsworth et al. 1996b). The reasons for thisare not necessarily clear, but some explanationshave been proposed, including inadequate lightintensity or dissolved oxygen concentration forseedlings. In the case of U. australis, most of theturions in cultivation remained floating through-out winter, whereas the seeds sunk when they weresuspended in water (S. Araki and Y. Kadano, pers.obs., 1996). If seeds are buried in sediment at thebottom of the pond, seed and/or seedling survivalmay be reduced because of low light and oxygen.Even if seeds are not buried, seedlings will be lessvigorous because the resource storage capabilitiesof a seed are far less than a turion. The dry weightof a turion varies according to its size. The smallestturion, which is approximately 1 mm long, weighsapproximately 0.07 mg, whereas the largest one,approximately 10 mm, weighs approximately40 mg. However, all seeds are approximately1 mm in size and weigh approximately 0.04 mg(S. Araki & Y. Kadono, unpubl. data, 1996), whichis 1/1000 in comparison with a turion.

A mosaic pattern in the geographic distributionof the multilocus genotype was found in the south-ern part of Hyogo Prefecture. It is considered thatthis pattern has been derived by clonal dominancein reproduction and the colonization processes ofthe species. Waterfowl movement may play a rolein colonization of aquatic plants by trapping seedsin feathers (Cook 1990; Vivian-Smith & Stiles1994). In the case of U. australis, the occurrence ofthe same genotype in nearby populations and thespread of the male-sterile type A1 suggest that dis-persal of vegetative propagules has occurred. It ishighly likely that each genet succeeds in establish-ment as a founder spreads into neighboring habi-tats by vegetative means. Thus, metapopulationbecomes a mosaic of genets as illustrated by gen-otype distribution.

In addition to the rarity of seed or seedling sur-vival, the chance of sexual reproduction isrestricted by low seed production. The fruit setratio in natural populations is low when comparedwith hand pollination and sometimes decreases tozero. More intensive research on the relationshipbetween the amount of fruit production and


pollinator activity or weather conditions is neededfor clarification of the reasons for fruit set ratiofluctuation in natural populations.

Seed set ratio appears to be diminished for somegenetic reasons. The pollination experimentshowed that seed set ratios were low in the cases ofselfing and crossing between plants of the samemultilocus genotype. In contrast, crossingbetween plants of different genotypes resulted inhigher seed set ratios. If we assume that plantswith the same multilocus genotype belong to thesame clonal lineage, crossing with the same typecorresponds to intraclonal pollination, which isequal to self-pollination. Crossing with a differenttype corresponds to interclonal pollination. Thus,the results of the pollination experiment indicate alow seed set ratio in intraclonal mating and a highseed set ratio in interclonal mating. Low seed setratios observed in natural populations are also con-sidered to be consequences of the clonality of thepopulation. There are two explanations for lowseed set ratio in intraclonal pollination:

1 Inbreeding depression. Inbreeding depressionsmay have prevented normal seed development(Seavey & Bawa 1986; Husband & Schemske1996; Manicacci & Barrett 1996; Mahy &Jacquemart 1998). Even if the parent plantsgrow normally, homozygosis that prevents seeddevelopment may occur through mating.

2 Self-incompatibility. Self-incompatibilities inmany plant species are genetically controlled bya single multi-allelic S locus (Haring et al.1990; Lee et al. 1994; Murfett et al. 1994;Richman et al. 1995). If U. australis is self-incompatible, mating within the same clonalindividuals results in low seed set ratios becausethe parents have the same S allele.

Because habitats such as ponds and lakes are iso-lated from each other, various types of genetic fea-tures of the population are easily affected byfounder effects or genetic bottlenecks (Hedrick &Parker 1998; Johnson & Black 1998; Schug et al.1998). Genetic bottlenecks affect the number ofgenets in a population and/or the number of S alle-les within a population (Imrie et al. 1972; Reinartz& Les 1994). In the case of U. australis, it is prob-able that the number of genets in one population isone or a few at the time of establishment of a newpopulation. If such a founder effect is repeated dur-

ing the process of colonization, the number of gen-ets in a population decreases further and finallybecomes one. In this situation, all the individualsin a population belong to the same genet and havethe same S allele, thus cross-pollination within thepopulation is equal to self-pollination, which mayresult in inbreeding or incompatible effect.

ACKNOWLEDGEMENTS

We are grateful to Mr T. Suzuki, Museum ofNature and Human Activities, for his suggestionsfor improving the techniques of isozyme detectionand to the owners of the irrigation ponds investi-gated.

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