Genetic relationship and genetic diversity among Typha taxa from East Asia basedon AFLP markers

Hye Ryun Na, Changkyun Kim, Hong-Keun Choi *

Department of Biological Sciences, Ajou University, 5 San, Woncheon-dong, Youngtong-gu, Suwon City, Gyeonggi-do 443-749, Republic of Korea

Aquatic Botany 92 (2010) 207–213

A R T I C L E I N F O

Article history:

Received 8 April 2009

Received in revised form 2 December 2009

Accepted 3 December 2009

Available online 5 December 2009

Keywords:

Typha

AFLP

Genetic diversity

Genetic relationship

East Asia

A B S T R A C T

The genetic relationship and diversity among four Typha taxa in East Asia were evaluated using amplified

fragment length polymorphism (AFLP) markers. Three AFLP selective primer combinations generated a

total of 707 amplification products, of which 704 (99.6%) were polymorphic. The unweighted pair-group

method with arithmetic mean (UPGMA) dendrogram and principal component analysis (PCA) plot

confirmed the taxonomic status of four separate species. East Asian Typha taxa separated into two

groups: the first with Typha angustifolia and the second with T. orientalis, T. laxmanni, and T. latifolia with

a high bootstrap value for UPGMA (93%) and a low first score for PCA (25%). The two clusters

corresponded with two sections based on the bracteoles in the female flower: section Bracteolatae and

section Ebracteolatae. T. angustifolia showed the highest genetic diversity among the four Typha taxa

(percentage of polymorphic loci [PPL] = 71%, Ho = 0.157), whereas T. latifolia had the lowest genetic

diversity (PPL = 40%, Ho = 0.117). Genetic diversity was related to the presence of the gap between male

and female inflorescences. A positive correlation between genetic distance and geographic distance was

clearly found in the two species with continuous inflorescences (T. latifolia and T. orientalis). This positive

correlation was not observed in the other species with discontinuous spikes (T. angustifolia and T.

laxmanni).

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1. Introduction

Typha L. (Typhaceae) is a genus of monocotyledonous clonalemergent plants with a nearly worldwide distribution (Kronfeld,1889; Smith, 1987). Typha species are highly productive aquaticweeds and rapid colonizers in the littoral zone of lakes, along riversand in marshes (Suda et al., 1977; Smith, 1987). Speciesidentification of Typha has generally depended upon character-istics of floral and inflorescence morphology including the lengthof the male and female inflorescences, the contiguous or separateposition of inflorescences, presence or absence of bracteoles, shapeof the stigma in the female flower, and the number of pollen grainsunited together (Kronfeld, 1889; Graebner, 1900; Smith, 1987;Kadono, 1996; Kim et al., 2003). However, the delimitation ofTypha species is confused because of frequent hybridizationincluding introgression (Lee, 1975; Smith, 1987; Kuehn et al.,1999).

Four taxa, Typha latifolia L., T. angustifolia L., T. orientalis Presl,and T. laxmanni Lepechin are generally recognized in eastern Asia,which includes South Korea, Japan and Far East Russia (Fedchenko,

* Corresponding author. Tel.: +82 31 219 2618; fax: +82 31 219 1795.

E-mail address: hkchoi@ajou.ac.kr (H.-K. Choi).

0304-3770/$ – see front matter � 2009 Elsevier B.V. All rights reserved.

doi:10.1016/j.aquabot.2009.12.001

1934; Kadono, 1996; Kim et al., 2003). T. latifolia is readilydistinguished from the other species by its wide leaves and femaleinflorescences and tetrad pollen. T. angustifolia is distinguishedfrom T. orientalis and T. laxmanni by the long male inflorescencesand a large gap between male and female inflorescences. T.

laxmanni is distinguished from T. orientalis by a shorter femaleinflorescence (Kadono, 1996; Kim et al., 2003). Using 25quantitative morphological characteristics, the individuals ofTypha species from South Korea and Far East Russia formedunique clusters according to traditionally recognized species (Kimet al., 2003). Furthermore, a UPGMA dendrogram of themorphological characteristics for Typha species revealed twoclusters representing: (1) T. angustifolia, T. orientalis, and T.

laxmanni and (2) T. latifolia (Kim et al., 2003). Although thephenetic relationship of these species is relatively well defined interms of morphological characteristics, the genetic relationshiphas not yet been well addressed. To resolve this problem, weadopted molecular analyses which have proven useful to deter-mine species delimitation and in the understanding of geneticrelationships (Kuehn et al., 1999; Lamote et al., 2005; Tsyuskoet al., 2005).

The genetic diversity of Typha is reportedly lower than in otherherbaceous plants (Suda et al., 1977; Mashburn et al., 1978; Sharitzet al., 1980; Keane et al., 1999; Lamote et al., 2005). In previous

H.R. Na et al. / Aquatic Botany 92 (2010) 207–213208

studies involving geographic regions in different countries, T.

latifolia commonly showed lower genetic variation than otherTypha species, especially T. angustifolia (Lee, 1975; Lamote et al.,2005; Tsyusko et al., 2005). The difference in genetic diversitybetween T. latifolia and T. angustifolia was shown to be related totheir different reproductive characteristics (Lee, 1975; Keane et al.,1999; Lamote et al., 2005; Tsyusko et al., 2005).

In East Asian, especially in South Korea, T. angustifolia is morewidely distributed than T. latifolia, T. orientalis and T. laxmanni (Kimet al., 2003). The observed relationships between genetic distanceand geographic distance may be partly explained by the pattern ingenetic variation in each Typha species (Tsyusko et al., 2005). Forthis reason, we became curious as to whether T. angustifolia

presented the highest genetic diversity among Typha speciesdistributed in this geographic region, as has been previouslyreported (Lee, 1975; Lamote et al., 2005; Tsyusko et al., 2005).

Our first objective was to clarify the taxonomic identities andgenetic relationships for the four Typha taxa from East Asia. Byexamining intraspecific and interspecific variation, we hoped togenerate molecular signatures for the accurate identification ofTypha species that could be reliably used for studies of speciesphylogeny and evolution. The second objective was to assess therelationship between genetic diversity and reproductive char-acteristics among the Typha taxa. We were also interested inexamining the correlation between genetic and geographicdistances to detect genetic isolation by distance. While large-and small-scale genetic variation patterns for Typha in NorthAmerica and Europe have been elucidated, the genetic variation forthe rest of the world is not so well known.

The amplified fragment length polymorphism (AFLP) markermethod was chosen here for the analysis of genetic relationshipand genetic diversity among Typha taxa. AFLP fragments are spreadover the entire nuclear genome, as expected from the distributionof restriction enzyme sites (Hansen et al., 1999). Thus, AFLPgenerates large data sets that reliably reflect discriminationbetween entire individual genomes (Hedren et al., 2001; Kimet al., 2008, 2009).

2. Materials and methods

2.1. Plant materials

A total of 69 individual plants including T. angustifolia (36), T.

latifolia (13), T. orientalis (8), and T. laxmanni (12) were collectedfrom South Korea and in neighboring regions such as China, Japanand Far East Russia (Appendix A). Sparganium erectum L. was usedas an outgroup for UPGMA analysis. One complete shoot for eachpopulation was sampled and the distance among populationsvaried from 0.5 to 1700 km. Species identification was based onmorphological characteristics (Kim et al., 2003). Disparity in thenumber of samples was due to a different geographic distributionand abundance (Kim et al., 2003). Most voucher specimens of eachsample are deposited at AJOU except for one T. orientalis that wascollected in Japan (Appendix A).

Table 1Summary of statistics for the bands obtained with three AFLP primer combinations.

Primer combination Total bands/polymorphic bands No. of speci

T.a.

ECO-CAAC/MSE-ACTG 228/228 (100.0%) 36 (25.6%)

ECO-CAAC/MSE-ACGT 224/221 (98.7%) 37 (23.9%)

ECO-CACC/MSE-ACGT 255/255 (100.0%) 52 (36.0%)

Sum 707/704 (99.6%) 125

T.a., Typha angustifolia; T.l., Typha latifolia; T.o., Typha orientalis; T.x., Typha laxmanni; S.e.,

in total bands of each species.

2.2. DNA extraction and AFLP analysis

The top of leaves were clipped from fresh or dried specimensand stored at�80 8C until DNA extraction. Total genomic DNA wasisolated from 0.5 g of fresh leaf tissue and 0.1 g of dried leaf tissueusing the modified CTAB protocol of Drabkova et al. (2002) asoutlined by Doyle and Doyle (1987). Briefly, extracted DNA wasresuspended in Tris–EDTA buffer (10 mM Tris, pH 8.0; and 1 mMEDTA, pH 8.0), and the DNA concentration for each sample wasdetermined using a spectrophotometer (Geneflow Ltd., Stafford-shire, UK).

AFLP analysis was performed essentially as described by Voset al. (1995), except that florescence technology was used forimaging the labeled DNA bands to obtain high resolution (Kimet al., 2008, 2009). Total genomic DNA (2 mg) was digested with 5units of an EcoRI and MseI endonuclease mixture in a total volumeof 50 mL for 6 h at 37 8C; digestion was checked by electrophoresison a 1.2% agarose gel. 10 mL of ligation mixture contained 5 pM ofEcoRI adaptor, 50 pM of MseI adaptor, 1� T4 DNA ligase buffer and1 unit of T4 DNA ligase (Promega, Madison, WI). This mixture wasadded to the digests in a total volume of 40 mL, and the ligationmixture was incubated overnight at 4 8C in a cold chamber. Thereaction primer pair EcoRI + A/MseI + C was used, and amplifica-tion was carried out for 30 cycles of denaturation (1 min at 94 8C),annealing (1 min at 56 8C), and extension (1 min at 72 8C). Asample run on a 1.5% agarose gel showed a visible smear in therange of 100–1000 bp. The pre-amplification products werediluted 10-fold with distilled water as template for furtherselective amplification, which was performed with an EcoRI-primer tagged with a 6-FAM dye and MseI-primer (see Table 1 forcombinations). PCR conditions included one cycle at 94 8C for3 min; then 13 cycles of 94 8C for 30 s, 65 8C for 30 s (with areducing ramp of 0.7 8C per cycle), and 72 8C for 1 min; followed by23 cycles at 94 8C for 30 s, 56 8C for 30 s, and 72 8C for 1 min.Reaction products were fractionated with an ABI 3100 DNAsequencer (Applied Biosystems, Foster City, CA). AFLP-GeneScanfiles were further scored with Genotyper 3.7 (Applied Biosystems).

2.3. Data analysis

AFLP bands were scored as ‘0’ (absent) or ‘1’ (present) in abinary matrix for each primer. Scored fragment sizes ranged from50 to 500 bp. The binary matrix was subjected to phenetic analysis,with S. erectum as an outgroup. The Nei–Li restriction site distance(Nei and Li, 1979) was calculated for genetic distance withPAUP*4.0 program (Swofford, 2003) and the UPGMA tree wasconstructed. In the same program bootstrap support wascalculated with 1000 re-samples. To show the relationship amongTypha taxa, principal components analysis (PCA) was carried outusing S-plus6 (Venables and Ripley, 2002) with exception for theoutgroup.

To estimate genetic diversity, we used the following computerprograms: TFGPA 1.3 (Miller, 1997) for percentages of polymorphicloci (PPL at the 0.99 level), Nei’s (1973) gene diversity (Ho), and

fic bands

T.l. T.o. T.x. S.e.

12 (12.1%) 15 (13.4%) 7 (6.2%) 23 (35.9%)

15 (11.7%) 8 (8.6%) 15 (14.2%) 0 (0.0%)

5 (4.5%) 15 (10.3%) 9 (6.6%) 4 (12.9%)

32 38 31 27

Sparganium erectum. The number in parentheses is the percentage of specific bands

Fig. 1. Unrooted UPGMA dendrogram based on Nei–Li distance of 69 Typha

individuals and one Sparganium erectum. The tree was constructed with 707 AFLP

markers. Bootstrap values of 60 and above are shown for the major nodes and are

linked to species names (based on 1000 replicates).

Fig. 2. Relationship of 69 individuals from four Typha taxa visualized by PCA based

on 680 AFLP markers. The first three principal components represent 43% of the

total variation (PC 1 = 25%, PC 2 = 10%, and PC 3 = 8%).

H.R. Na et al. / Aquatic Botany 92 (2010) 207–213 209

Nei’s (1978) unbiased expected heterozygosity (He); and POPGENE1.31 (Yeh et al., 1997) for Shannon and Weaver’s (1949) index (I).The Bayesian approach described by Holsinger et al. (2002) wasalso used to determine genetic diversity (hs, analogous to He) usingHickory 1.0.4 (Holsinger and Lewis, 2005). To ensure consistency ofresults (nBurnin = 50,000, nSamples = 250,000, thin = 50 in eachrun), we used five runs for each of three models (full model, f = 0model, and f-free model). Model selection was based on thedeviance information criterion (DIC) (Spiegelhalter et al., 2002).Models with smaller DICs are preferred. However, a difference of>6 DIC units among different models is required to select onemodel over another (Holsinger and Lewis, 2005).

To assess the partitioning of AFLP genotypic variation amongand within taxa, analysis of molecular variance (AMOVA) was usedwith WinAMOVA 1.55 (Excoffier et al., 1992; Excoffier, 1993).Input files for this program were generated by the programAMOVA-PREP (Miller, 1998). Significant levels of variance wereobtained with tests that included 1000 permutations per analysis.

Linear regression analysis was used to assess the correlationbetween the amount of genetic diversity (PPL, Ho, He, hs, I) andsample size (N), using SAS 9.1 (SAS Institute, 2004). And thisanalysis was also used to determine the relationship between thegenetic diversity and the 10 quantitative reproductive character-istics (Kim et al., 2003) (Table S3).

A Mantel test (Mantel, 1967) was conducted to identify anycorrelations between genetic distance (Nei–Li distance) andgeographic distance for each Typha taxa. One sample of T. orientalis

collected from Japan was excluded from a Mantel test due to lack ofgeographical data. Significance of that test was determined byTFGPA 1.3, with 1000 permutations (Miller, 1997).

3. Results

3.1. AFLP polymorphism

The three chosen AFLP primer combinations yielded 707detectable bands, of which 704 were polymorphic (Table 1).Numbers of unequivocally detectable band positions varied from224 to 255, depending on the primer pairs. On average, 235polymorphic bands were amplified by each primer combination.Among the total fragments, 27 bands were specific for S. erectum.125 bands specifically occurred in one or more individuals of T.

angustifolia, whereas 32, 38, and 31 bands were specific for one ormore taxa of T. latifolia, T. orientalis and T. laxmanni, respectively(Table 1).

3.2. Species delimitation and genetic relationship

Genetic distance (Nei–Li distance) was calculated both withinand between Typha taxa. Average genetic distance within speciesvaried from 0.053 in T. latifolia to 0.071 in T. angustifolia. Averagegenetic distance among the four taxa ranged from 0.097 between T.

latifolia and T. laxmanni to 0.149 between T. angustifolia and T.

laxmanni (Tables S1 and S2).All taxa have their own distinct branches in the UPGMA

dendrogram. T. angustifolia was strongly supported by a bootstrapvalue of 94% and the cluster consisting of T. latifolia, T. laxmanni andT. orientalis was well supported by a bootstrap value of 93%. Withinthis cluster, T. latifolia and T. laxmanni were weakly supported by abootstrap value of 67% and 62%, respectively (Fig. 1).

Relationships among the taxa were further illustrated by thethree-dimensional scatter plot of a PCA (Fig. 2). The first, secondand third principal components accounted for 25%, 10% and 8% ofthe total variance, respectively, and distinguished four speciesgroups, in agreement with the branches distinguished in theUPGMA tree.

3.3. Genetic diversity among taxa

With AFLP makers occurring in Typha, the percentage ofpolymorphic loci (PPL) for the four taxa ranged from 40% (T.

latifolia) to 71% (T. angustifolia). T. angustifolia exhibited the highestheterozygosity in the other parameters (Ho = 0.157; He = 0.159;

Table 2Genetic diversity of the four Typha taxa in East Asia.

Taxon N PPL Ho He hs I

T. angustifolia 36 70.7 0.157 0.159 0.164 0.256

T. latifolia 13 39.9 0.117 0.122 0.138 0.183

T. orientalis 8 41.6 0.133 0.142 0.161 0.206

T. laxmanni 12 43.7 0.143 0.149 0.160 0.217

N, sample size; PPL, percentage of polymorphic loci; Ho, Nei’s (1973) gene diversity;

He, unbiased expected heterozygosity (Nei, 1978); hs, genetic diversity using

Bayesian approach; I, Shannon and Weaver’s (1949) index.

Table 3Analysis of molecular variance (AMOVA).

Level of

variation

df SSD MSD Variance

component

p value*

Absolute %

Among taxa 3 2109.5 703.2 43.7 45.6 <0.001

Within taxa 65 3383. 5 52.1 52.1 54.4

SSD, sum of squared deviations; MSD, mean squared deviations.* Levels of significance are based on 1000 permutations.

H.R. Na et al. / Aquatic Botany 92 (2010) 207–213210

hs = 0.164; I = 0.256) (Table 2). A highly significant correlation wasdetected between levels of genetic variation (PPL value) andsample size (r = 0.98, p = 0.02), but no significant correlation wasdetected between sample size and the other four parameters (Ho,He, hs and I). These four parameters were strongly inter-correlated(r = 0.79–0.99, 0.01 < p < 0.05).

AMOVA test indicated a highly significant (p < 0.001) geneticvariation among taxa (54%) as well as within taxa (46%), indicatingstrong genetic differentiation among species (Table 3). For the 10quantitative reproductive characteristics of Typha species, only onecharacteristic, the gap length between male and female inflor-

Fig. 3. Correlation between genetic and geographic distances among Typha individuals. Th

permutations). (a) T. angustifolia; r = �0.110, p = 0.772, (b) T. latifolia; r = 0.564, p = 0.00

escences, positively correlated with genetic diversity (r = 0.93,p = 0.07; Table S3).

3.4. Relationship between genetic and geographic distances among

individuals

Genetic distance significantly correlated with the geographicdistance in T. latifolia and T. orientalis by Mantel test (r = 0.564,p = 0.002 and r = 0.840, p = 0.001, respectively) (Fig. 3b and c). In T.

angustifolia and T. laxmanni, no correlation was observed betweengenetic and geographic distances (Fig. 3a and d).

4. Discussion

4.1. AFLP polymorphism

The level of AFLP polymorphism (99.6%) provided a highresolution for the delimitation of the species and the detectionof genetic diversity within the genus Typha in East Asia (Table 1).The amount of genetic variation detected by AFLP markers in thefour Typha taxa is higher than previously shown for isozymes(Lee, 1975; Suda et al., 1977; Mashburn et al., 1978; Sharitzet al., 1980) or variable number tandem repeat (VNTR) loci(Keane et al., 1999), but lower than for microsatellite loci(Tsyusko et al., 2005).

The level of genetic variation detected for each Typha taxa in thepresent study is not significantly different (p = 0.156) from aprevious AFLP marker study (Lamote et al., 2005). While the totalnumber of bands (680) yielded by three AFLP primer combinationsin our study is considerably more than the bands (177) scored bythe six primer combinations from the previous study, almost halfof total genetic variation (46%) detected presently contributed todistinguishing the four Typha species. Both studies using the samemolecular marker showed almost similar genetic diversity forTypha species, in comparison with other studies (Keane et al.,

e correlation coefficients (r) and p values are given from Mantel test (based on 1000

2, (c) T. orientalis; r = 0.840, p = 0.001, and (d) T. laxmanni; r = �0.236, p = 0.841.

H.R. Na et al. / Aquatic Botany 92 (2010) 207–213 211

1999; Tsyusko et al., 2005) using different markers, despite havingdifferent sample sizes and different geographic regions (Table S4).

4.2. Species relationships

The UPGMA dendrogram (Fig. 1) based on 707 AFLP bandsindicated that all four Typha taxa form a genetically distinct group.This means that recent gene flow among Typha species in East Asiawas likely infrequent, and that the species represents well-definedentities. Indeed, Typha species breeding is reported to be mainlyinbreeding or vegetative reproduction (Krattinger, 1975). Further-more, Typha species are generally well defined by morphologicaland ecological characteristics in East Asia (Kim et al., 2003),although most of the morphologically discriminating character-istics are only available in mature and fertile individual plants(Fedchenko, 1934; Hotchkiss and Dozier, 1949; Kadono, 1996). ThePCA (Fig. 2) distinguished the Typha taxa from one other by the firstthree components, which accounted for 43% of the total variance.The first principal component (25%) clearly distinguished T.

angustifolia from the other three taxa. T. latifolia, T. orientalis andT. laxmanni were well-separated from each other by the second(10%) and third (8%) principal components.

T. angustifolia formed a cohesive group in cluster analysis(Fig. 1) and PCA (Fig. 2). Furthermore, this species showed thehighest numbers of specific markers among all the Typha taxa inEast Asia (Table 1). Individuals of T. angustifolia are readilydistinguished from the other Typha species by the linear shape ofstigma, the presence of bracteoles in female flower, the long gapbetween male and female inflorescences, and the long maleinflorescence (Kim et al., 2003).

It is remarkable that T. orientalis individuals formed a singlegroup in both analyses (Figs. 1 and 2). This species was weaklyseparated with T. angustifolia and T. laxmanni in a PCA plot based onthe morphological characteristics (Kim et al., 2003). The geneticisolation in T. orientalis is probably due either to its narrowdistribution and small population size, or to directional selectionfavoring one or very few genotypes in an isolated range ofenvironments (Hamrick and Godt, 1996).

East Asian Typha taxa separated into two clusters in the UPGMAdendrogram and PCA plot (Figs. 1 and 2): (1) T. angustifolia and (2)T. latifolia, T. orientalis and T. laxmanni. The two clusters aresupported by two sections based on the bracteoles in female flower(Kronfeld, 1889; Graebner, 1900): section Bracteolatae (T. angu-

stifolia) and section Ebracteolatae (T. latifolia, T. orientalis and T.

laxmanni). The genetic relationship is discordant with thephenogram of the Typha species using morphological character-istics (Kim et al., 2003). This discordance seems to be linked withdifferences between phenotypic plasticity and genetic variation inthe genus Typha (Lee, 1975).

4.3. Genetic diversity among taxa

T. angustifolia displayed the highest genetic diversity among thefour Typha taxa, whereas T. latifolia showed the lowest. Geneticdiversity (PPL, Ho, He and I) measured within T. angustifolia wasobserved to be about 1.5-fold greater than T. latifolia. The presentlyindicated greater genetic diversity of T. angustifolia versus T.

latifolia is supported by previous studies (Lamote et al., 2005;Tsyusko et al., 2005). In the present study, populations wererepresented by only one sample. Although we did not comparevariation levels within and between populations, it is meaningfulto compare our results with previous studies for genetic diversityat the species level.

Different levels of genetic diversity shown for each Typha

species likely partly reflect the different breeding systems relatedto inflorescence and pollen characteristics (Lee, 1975). Among the

four Typha taxa, T. angustifolia and T. laxmanni displayed highgenetic diversity (Table 2). Both species have a distinctive gapbetween male and female inflorescences, whereas T. latifolia and T.

orientalis have continuous spikes. Presently, we observed asignificant relationship between genetic diversity (Ho) and thegap distance at the species level (r = 0.93, p = 0.07) (Table S3). Theinflorescence characteristics seem to be related to the pollinationsystem in Typha species (Krattinger, 1975). T. angustifolia shows ahigher cross-pollination rate due to the higher positioned maleinflorescence and lighter pollen in comparison to T. latifolia

(Krattinger, 1975). The gap is largest in T. angustifolia (4.4 � 1.5 cm)and second largest in T. laxmanni (2.5 � 0.8 cm) (Kim et al., 2003).Outcrossing species tend to be more genetically diverse (Hamrick andGodt, 1996). We suggest that T. angustifolia shows the highest geneticdiversity related to inflorescence characteristics due to outcrossing.

4.4. Correlation between genetic distance and geographic distance

Positive correlation between genetic and geographic distancesamong individuals within Typha taxa represents isolation bydistance, which may result from more intensive gene flow amongsites that are closely related geographically, and reduced gene flowover longer distances (Tsyusko et al., 2005; Fer and Hroudova, 2009).Correlation between genetic and geographic distances for T. latifolia

and T. orientalis was presently observed, but was not evident for T.

angustifolia and T. laxmanni (Fig. 3). The isolation of T. latifolia wasobserved in Ukraine (Tsyusko et al., 2005). The lack of correlationbetween genetic and geographic distances in T. angustifolia alsocorresponds to Tsyusko’s study. We suggest that heavier pollen (i.e.T. latifolia) is effectively dispersed only by strong wind, whereaslighter pollen (i.e. T. angustifolia) probably remains suspended in theair for a long time (Krattinger, 1975). Seed production of T.

angustifolia is higher than T. latifolia, but the fruits of both speciesare effectively distributed by wind (Krattinger, 1975).

Considering pollen and seed dispersal in Typha, lack of isolationby distance in T. angustifolia can be inferred from a largelyreticulate distribution pattern, which may result from extensiveoutbreeding and wind-pollinated species such as Phragmites

australis (Lambertini et al., 2008; Fer and Hroudova, 2009). Thehigh capability of T. angustifolia for growth in various habitats(McMillan, 1959; McNaughton, 1966; Lee, 1975; Grace andWetzel, 1982) may partially explain the lack of isolation bydistance as well as a wide distribution. T. angustifolia grows in bothfresh and brackish marshes, whereas T. latifolia grows in only freshmarshes (McMillan, 1959; Lee, 1975).

Our results show that genetic variation as assessed with AFLPsupports the taxonomic status of East Asian Typha taxa includingthe two sections of Bracteolatae and Ebracteolatae based on thebracteoles in the female flower. Higher genetic diversity of T.

angustifolia than T. latifolia, as was previously reported in NorthAmerica and Europe (Lee, 1975; Lamote et al., 2005; Tsyusko et al.,2005), is presently supported by the data collected in East Asia. Ourstudy also suggests that the inflorescence characteristics arerelated to the breeding system of Typha species, which results inthe different levels of genetic diversity and the distributionalpatterns.

Acknowledgements

This work was supported by the Program for Collection,Management and Utilization of Biological Resources (grantM10867010003) from the Ministry of Education, Science &Technology (MEST) of the Republic of Korea. We thank Dr. W.Till (WU) for providing specimen, Drs. W. Li and F. Liu of the WuhanBotanical Garden, and Mr. Y. Ito, University of Tokyo, for their helpsin field survey.

Appendix A

List of samples used in the AFLP analysis (most voucher specimens are deposited in AJOU except one of T. orientalis in WU).

Taxon, locality, collectors and vouchers

Typha angustifolia L. Korea: Gangwon Hwacheon Georyeri, 38.1299 N, 127.7680 E, H. Kim, 2003.07.19; Korea: Gangwon Hwacheon Nonmiri, 38.0858 N,

127.6870 E, C. Ahn, 2003.07.10; Korea: Gangwon Yangyang Yeomgye, 38.0345 N, 128.7028 E, H.R. Na 80181, 2008.07.14; Korea: Gangwon Yeongwol Ssangyoungri,

37.1786 N, 128.3159 E, C. Kim 240, 241, 1998.07.29; Korea: Gyeonggi Goyang Naeyudong, 37.7250 N, 126.8380 E, C. Kim 492, 493, 1999.09.16; Korea:

Gyeonggi Namyangju Joanri, 37.5340 N, 127.3032 E, C. Ahn, 2003.07.11; Korea: Gyeonggi Suwon Woncheon-dong, 37.2808 N, 127.0603 E, C. Kim, 1999.06.24;

Korea: Gyeonggi Yangpyeong Yangsuri, 37.5337 N, 127.3165 E, C. Ahn, 2003.07.11; Korea: Gyeonggi Yeoju Gulamri 37.2361 N, 127.7033 E, C. Ahn, 2003.07.24;

Korea: Seoul Gildong 37.5401 N, 127.1519 E, C. Kim 372, 1999.07.10; Korea: Chungbuk Boeun Bocheongcheon 36.4807 N, 127.7241 E, C. Ahn, 2003.07.09; Korea:

Chungbuk Jecheon Guhakri 37.165 N, 128.1077 E, C. Kim 235, 1998.07.29; Korea: Chungnam Asan Shinajeongho 36.7667 N, 126.975 E, C. Kim 173, 1998.07.30;

Korea: Chungnam Taean Naeri 36.9073 N, 126.3092 E, C. Kim 555, 556, 1999.10.27; Korea: Chungnam Taean Seungeonri 36.5065 N, 126.3614 E, C. Kim 178, 466,

475, 1999.08.31; Korea: Gyeongbuk Gunwi Segyeongri 36.2404 N, 128.4866 E, H.R. Na 80262, 2008.07.24; Korea: Gyeongnam Sacheon Changchonri 35.1799 N,

127.9244 E, H.R. Na 80370, 2008.07.27; Korea: Jeonbuk Jangsu Jangpanri 35.6766 N, 127.5204 E, H.R. Na 80374, 2008.07.29; Korea: Jeonbuk Kimje Daedongri

35.86 N, 126.8569 E, H.R. Na, 2007.06.27; Korea: Jeonbuk Kimje Seoamdong 35.8083 N, 126.875 E, C. Kim 264, 1998.07.31; Korea: Jeonnam Gangjin Inmcheonri

34.6071 N, 126.7346 E, H.R. Na 37128-1, 37128-2, 2006.08.09; Korea: Jeonnam Jangheung Hangwonri 34.6887 N, 126.8957 E, H.R. Na 37189, 2006.08.10; Korea:

Jeonnam Muan Dalsanri 34.8848 N, 126.4738 E, H.R. Na 80378-1, 80378-2, 2008.08.20; Korea: Jeju Yongdangri 33.3148 N, 126.185 E, H.-K. Choi, 2006.07.12;

China: Anhui Hefei 31.8775 N, 117.2 E, C. Kim C1, C2, 2002.07.12; China: Hubei Wuhan Baoan Lake 30.197 N, 114.7339 E, H.R. Na 80591-1, 80591-2, 2008.10.10;

Japan: Shizuoka Abe River 34.9922 N, 128.3597 E, H.-K. Choi 551, 2000.07.23; Russia: Primorski Krai Khanka Lake 44.5283 N, 132.4497 E, H.-K. Choi 83, 91,

1997.07.26; T. latifolia L. Korea: Gyeonggi Gwacheon Juamdong 37.4575 N, 127.0247 E, H.-K. Choi 891, 892, 893, 2008.09.18; Korea: Gyeonggi Yeoju Deokpyeongri

37.1939 N, 127.663 E, H.R. Na 40016, 2007.07.25; Korea: Incheon Gangwhado Gilgeongri 37.6492 N, 126.4663 E, J. Jung 0806018, 2008.06.11; Korea: Jeonbuk

Jangsu Jangpanri 35.6766 N, 127.5204 E, H.R. Na 80373, 2008.07.29; China: Jilin Duman River 42.1987 N, 129.1904 E, H.-K. Choi 771, 772, 773, 2007.07.25; Japan:

Hokkaido Nemuro 43.1562 N, 145.2425 E, H.R. Na 7J095, 2007.09.15; Japan: Hokkaido Sarufutsumura 45.2078 N, 142.2228 E, H.R. Na 7J125, 2007.09.18; Japan:

Shizuoka Abe River 34.9922 N, 128.3597 E, H.-K. Choi 550, 2000.07.23; Russia: Primorski Krai Khanka Lake 44.5283 N, 132.4497 E, H.-K. Choi 101, 1997.07.26;

T. orientalis Presl Korea: Gangwon Gosung Hawjinpo 38.4736 N, 128.4248 E, J. Jung, 2008.08.27; Korea: Gangwon Yeongwol Ssangyoungri 37.1786 N, 128.3159 E,

C. Kim 55,56, 1998.07.29; Korea: Chungnam Taean Banggangri 36.8842 N, 126.2167 E, C. Kim 468, 1999.09.01; Korea: Jeonbuk Kimje Seoamdong 35.8083 N,

126.875 E, C. Kim 10, 18, 1998.07.31; Korea: Jeonnam Yeongam Sanhori 34.7623 N, 126.5028 E, J. Jung 0808025, 2008.08.21; Japan: M. Togashi 3382 (WU);

T. laxmanni Lepechin Korea: Gyeonggi Ansan Daebudo 37.2657 N, 126.5886 E, Y. Ryu 0806071, 2008.06.12; Korea: Gyeonggi Ansan Sadong 37.281 N, 126.8397 E,

C. Kim 484, 486, 1999.09.20; Korea: Gyeonggi Hwaseong Bibong 37.2667 N, 126.8167 E, H.-K. Choi 287, 1997.07.11; Korea: Incheon Yeonghengdo 37.2433 N,

126.4544 E, C. Kim 221, 1998.06.06; Korea: Seoul Gildong 37.5401 N, 127.1519 E, C. Kim 367, 1999.07.10; Korea: Chungnam Asan 36.8111 N, 126.95 E, C. Kim 232,

1998.07.03; Korea: Chungnam Sapsido 36.3325 N, 126.3617 E, C. Kim, 1999.06.10; Korea: Chungnam Taesan Naeri 36.9073 N, 126.3092 E, C. Kim 558, 560, 562,

1999.10.27; Russia: Primorski Krai Khanka Lake 44.5283 N, 132.4497 E, H.-K. Choi 304, 1997.07.26. Sparganium erectum L. Korea: Jeju Hagari 33.4542 N,

126.3528 E, C. Kim 212, 1998.06.27.

H.R. Na et al. / Aquatic Botany 92 (2010) 207–213212

Appendix B. Supplementary data

Supplementary data associated with this article can be found, in

the online version, at doi:10.1016/j.aquabot.2009.12.001.

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