Aquatic Botany 80 (2004) 227–240 www.elsevier.com/locate/aquabot

Genetic diversity of maackianus in the Yangtze River Wei Lia,*, Li-Qun Xiaa,b, Jian-Qiang Lia, Guang-Xi Wanga,c

aLaboratory of Aquatic Biology, Wuhan Botanical Garden, The Chinese Academy of Sciences, Wuhan 430074, Hubei, PR China bCollege of Fisheries, Zhanjiang Ocean University, Zhanjiang 524025, Guangdong, PR China cGraduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

Received 2 July 2003; received in revised form 8 March 2004; accepted 8 July 2004

Abstract

The genetic diversity and genetic structure of Potamogeton maackianus A. Benn. in seven lakes of the middle reaches of the Yangtze River were studied using random amplified polymorphic DNA (RAPD). The gene flow and genetic relationships between populations were analyzed in combination with the geographic distribution and the river system of the lakes. A total of 112 bands were amplified and 59 bands (52.7%) were polymorphic. Each of the 86 individuals investigated exhibited a unique genotype. The Shannon index was used to measure genetic diversity, and the total genetic diversity was 0.414 and the mean genetic diversity of populations was 0.148. P. maackianus showed a relatively high level of genetic diversity. Analyses of molecular variance (AMOVA) revealed that 63.8% of the total genetic diversity existed among populations and 36.2% within them, which was consistent with the genetic structure computed by the Shannon index: among-population variation and within population variation accounted for 64.4 and 35.7%, respectively. The gene flow among populations was very limited, and genetic isolation among populations occurred even though they were connected through the Yangtze River. Cluster analysis divided the seven populations into two groups, and the genetic relationships among the populations had no obvious association with their geographic distribution, or the historical relations with the river system of the lakes where they occurred. Mantel tests revealed that distance was an important factor affecting the genetic structure in

* Corresponding author. Tel.: +86 27 87510140; fax: +86 27 87510251. E-mail addresses: [email protected], [email protected] (W. Li).

0304-3770/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2004.07.003 228 W. Li et al. / Aquatic Botany 80 (2004) 227–240 populations. The development history of P. maackianus populations in Honghu Lake had an obvious effect on its genetic structure. # 2004 Elsevier B.V. All rights reserved.

Keywords: Potamogeton maackianus A. Benn.; RAPD; Genetic diversity; Genetic structure; Gene flow; The Yangtze River

1. Introduction

The middle-lower reaches of the Yangtze River is the largest floodplain in China. Thousands of shallow lakes exist in this enormous area and almost all are inter-connected via the river system. This connection accounts for the high similarities of macrophyte compositions in different lakes (Li, 1995; Li and Zheng, 1998). The genetic diversity of a macrophyte in such a habitat is the subject of the present paper. Allozyme methods have been widely used to analyze the genetic variation in aquatic . Many studies considered that macrophytes in general had relatively low levels of genetic variation, higher rates of fixation and lower levels of sexual recombination compared with terrestrial plants (Les, 1988; Laushman, 1993; Schlueter and Guttman, 1998). High levels of genetic diversity in aquatic plants have also been reported in some species (Harris et al., 1992; Lokker et al., 1994; Mader et al., 1998). Compared to the allozyme method, random amplified polymorphic DNA (RAPD) can detect more polymorphic loci, and can quantify the genetic diversity and genetic structure more accurately, especially when genetic variation is low (Furnier and Mustaphi, 1992; Esselman et al., 1999; Kerher et al., 2000; Comes and Abbott, 2000). Recently, RAPD has been applied in the studies of aquatic plants, e.g. population structure (Waycott, 1995, 1998; Angel, 2002); relationships among different accessions (Madeira et al., 1997) and evaluation of genetic variation of endangered species (San Mart´ın et al., 2003). Potamogeton maackianus A. Benn. () is widely distributed in East Asia and can be found in almost all kinds of freshwater habitat (Sun, 1992). It is one of the dominant species in many lakes of the middle-lower reaches of the Yangtze River, and also the main founder species of submerged vegetation of this area (Li, 1995). P. maackianus is wind-pollinated and self-compatible, but mainly reproduces vegetatively. The recruitment and expansion of its population depend mainly on the clonal growth of stolons. In this study, the genetic diversity of P. maackianus in lakes of the middle reaches of the Yangtze River was evaluated by RAPD with the following aims: (1) to determine the genetic variation level of the species: the recent rapid decline of P. maackianus in many lakes has been ascribed to its possible low genetic diversity; (2) to determine the genetic relationships among different populations in relation to their degree of connectivity.

2. Materials and method

2.1. Sample collection

The river section from Yichang, Hubei to Hukou Jiangxi, about 938 km long, comprises the middle course of the Yangtze River with a catchment area about 680,000 km2. In the W. Li et al. / Aquatic Botany 80 (2004) 227–240 229 more than 30 lakes larger than 10 km2 investigated during June 1999–November 2000, six natural lakes, namely Changhu Lake (CL), Honghu Lake (HH), Xiliang Lake (XL), Niushan Lake (NS), Bao’an Lake (BA) and Chihu Lake (CH), together with a reservoir Mulan Lake (ML) had large populations of P. maackianus. In each lake, the geographical coordinates of sampling stations were pre-calculated with the aid of GIS. The actual sampling sites for P. maackianus depended on its distribution in the lakes. Each site was located using GPS. At each site, 10 random samplings were grabbed to get the plant materials. The material (mainly healthy leaves) was carefully washed and dried by placing it immediately in a plastic bag with desiccant. All the material collecting from one site was treated as one individual. Samples from one lake were treated as one population. In all, 86 individuals were collected in seven populations. The distances between the adjacent sample sites in a lake were 0.5–2 km.

2.2. DNA extraction

The total genomic DNA was extracted with the modified CTAB method of (Rogers and Bendich, 1985), where 0.03–0.05 g dried plant tissue was ground in liquid nitrogen and incubated with 600 mLof2 CTAB extraction buffer (2% CTAB, 100 mM Tris–HCl pH 8.0, 20 mM EDTA, 1.4 M NaCl, 0.1% b-mercaptoethanol) for 20–30 min at 56 8C, DNA was extracted with 500 mL of chloroform/isoamyl alcohol (24:1) two or three times and precipitated with 600 mL1 CTAB precipitated buffer (1% CTAB, 50 mM Tris–HCl pH 8.0, 10 mM EDTA). The solution was centrifuged for 10 min at 3500 rpm.The DNA pellet was dissolved in 400 mL 1 M NaCl and re-precipitated with 800 mL ethanol at 20 8C overnight. After 2 min centrifugation at 13,000 rpm and a wash with 76% ethanol, the pellet was dried and resuspended in 100 mLTE(10mMTris and 1 mM EDTA). The quality of the isolated DNA was examined on a 0.8% agarose gel and the concentration was estimated by DNA/Protein Analyzer (Beckman DU530).

2.3. RAPD polymerase chain reaction (PCR)

The RAPD technique has been criticized for its low reproducibility. However, the reproducibility of this method can be satisfactory if standard and strict experimental steps can be established and implemented (Zou et al., 2001), with special attention to: (a) shape of the temperature profile; (b) type of polymerase; and (c) Mg2+, Taq and DNA concentration (Hoelzel and Green, 1998). All the experiments in the present work were carried out on the same set of instruments. First, three samples from Honghu Lake were used to select primers and set up a RAPD procedure that can produce clear, stable and reproducible bands in at least three replicates. Primers and RAPD procedure were then evaluated on all samples from Honghu Lake and Niushan Lake with three replicates. Nineteen primers that produced clear and reproducible bands were selected from 60 primers (Operon kit A, B and R). The following procedure was strictly followed in the final experiment. DNA extracts were diluted to 10–20 ng/mL for use in the PCR reaction. The RAPD PCR was carried out in 20 mL reaction volumes consisting of 2.0 mLof10 PCR reaction buffer 230 W. Li et al. / Aquatic Botany 80 (2004) 227–240

(TaKaRa Inc.), 0.125 mM of each dNTP, 0.25 mM of primer, 0.5 U of Taq DNA polymerase (TaKaRa) and 2 mL of template DNA. PCR reactions were carried out in a MJ Research PTC-100 thermocycler programmed for an initial denaturation step of 94 8C, followed by 40 cycles of 1 min at 94 8C, 1 min at 36 8C, 1.5 min at 72 8C. The reaction was completed with a final run at 72 8C for 6 min. A negative control reaction that had template DNA omitted was included with every run to check for contamination of stock chemicals. Amplification products were separated on 1.4% agarose gels in 1 TAE buffer, stained with ethidium bromide and visualized and photographed under UV light (UVP GDS- 7600). The graphs were scored by two people, and their uncommon bands (less than 5%) were re-checked and discussed before making a final decision.

2.4. Data analysis

RAPD bands were scored as present (1) or absent (0) for each DNA example (range of band sizes: 300–2000 bp), and treated as phenotypic data in the analysis. The total genetic diversity of P. maackianus and the genetic diversity of each population were measured by the proportion of polymorphicP bands (PPB) and Shannon’s information index of diversity (Lewontin, 1972), h ¼ pi log2 pi (pi is the frequency of a haplotype). WINAMOVA software (ver. 1.55) (Excoffier et al., 1992) was used to perform analyses of molecular variance (AMOVA) with a Euclidean distance matrix calcul- ated from the RAPD profile of each individual (Huff et al., 1993). In the analysis, the genetic variation was partitioned into two levels: among individuals within populations versus among populations. Permutations (9999) were computed to obtain the significance level. Cluster analysis was used to describe the relationships among and within different P. maackianus populations. First, a Manhattan distance matrix (Wright, 1978) between populations was calculated and a 1000 times bootstrapping was performed by the RAPDDIST programme in the RAPDistance package (Black, 1997). Then, the cluster analysis tree was produced with the NEIGHBOUR and CONSENSUS program of the PHYLIP package (ver. 3.5) using UPGMA method (Felsenstein, 1993). Second, the Euclidean distance matrix of the 86 individuals was used to generate the dendrogram with NTSYS software using the UPGMA method (Rohlf, 1993). Mantel tests (Mantel, 1967) were performed to analyze the effects of geographical distance on genetic variation by using NTSYS software. First, on the population level, correlation analysis was performed on the matrix of Manhattan distances (the same as used in cluster analysis) and the geographical distances of all pairs of populations. The geographical distances among pair of populations were calculated from the coordinates of the geographical centers of lakes. Second, on the individual level, the matrix of Euclidean distances (the same as used in cluster analysis) and the geographical distances of all pairs of individuals in each population were analyzed separately. The geographical distances among pairs of individuals were calculated from the coordinates of the sampling stations in a lake as long as there was no land on their straight-line link; otherwise, the distance between the pair of individuals was calculated through a common nearest individual between them. W. Li et al. / Aquatic Botany 80 (2004) 227–240 231

3. Result

3.1. Genetic diversity of Potamogeton maackianus

A total of 112 bands were amplified by 19 primers, in which 52.7% (59 bands) were polymorphic. The primers produced 5.9 bands and 3.1 polymorphic bands on average (Table 1). Fig. 1 shows a RAPD profile from primer OPB-05 of all individuals. Each individual had a unique genotype. PPB for each population is shown in Table 2. The variability levels of the seven populations measured by PPB were sequenced as: HH > NS > XL > CH > ML > BA > CL. In the 86 individuals analyzed, samples from station 5 and 6 in Changhu Lake, samples from station 11 and 12 in Xiliang Lake had only one band difference. The distance between the two adjacent sites were about 2 and 2.4 km, respectively. The genetic diversity estimated by Shannon’s information index of diversity is shown in Table 3. The total genetic diversity (Hsp) was 0.41, and the mean genetic diversity of populations (Hpop) was 0.15. The genetic variability within populations (Hpop/Hsp) accounted for 35.7% of total genetic diversity. Most of the variability (64.3%, (Hsp Hpop)/ Hsp) occurred among populations. The genetic diversity in the seven populations varied between 0.19 and 0.11 and was ordered: HH > XL > NS > CH > BA > ML > CL. AMOVA also showed that the genetic diversity mainly existed among populations, with 63.8% of the variance among and 36.2% within population, respectively (Table 4).

Table 1 RAPD primers used in final RAPD analysis, their sequence of nucleotides, and the number of polymorphic bands produced by each primer Primer code Sequence (50!30) Total number of Percentage of polymophic bands polymophic bands OPA-01 CAGGCCCTTC 6 (3) 50.0 OPA-02 TGCCGAGCTG 3 (2) 66.7 OPA-03 AGTCAGCCAC 5 (3) 60.0 OPA-04 AATCGGGCTG 7 (3) 42.9 OPA-08 GTGACGTAGG 5 (3) 60.0 OPA-09 GGGTAACGCC 5 (3) 60.0 OPA-10 GTGATCGCAG 5 (2) 40.0 OPA-11 CAATCGCCGT 4 (3) 75.0 OPA-13 CAGCACCCAC 6 (2) 33.3 OPA-19 CAAACGTCGG 5 (3) 60.0 OPB-01 GTTTCGCTCC 7 (4) 57.1 OPB-04 GGACTGGAGT 6 (3) 50.0 OPB-05 TGCGCCCTTC 7 (5) 71.4 OPB-06 TGCTCTGCCC 8 (5) 62.5 OPB-10 CTGCTGGGAC 7 (4) 57.1 OPB-11 GTAGACCCGT 7 (5) 74.1 OPB-12 CCTTGACGCA 2 (0) 0 OPB-17 AGGGAACGAG 10 (4) 40.0 OPB-18 CCACAGCAGT 7 (2) 28.6 Total 112 (59) Mean 5.9 (3.1) 51.9 232 W. Li et al. / Aquatic Botany 80 (2004) 227–240 60 are the – 48, 58 – 86 are the samples of XL, N is the – 32 are the samples of NS, and 33 – 74 are the samples of CL, 75 – 10 are the samples of CH, 11 – 68 are the samples of BA, 69 – 57 are the samples of ML, 61 – HH19 of HH, 49 – cation products generated with OPB-05 (M as the marker, 1 fi HH16, HH17 – Fig. 1. RAPD ampli samples HH01 negative control reaction that had template DNA omitted). W. Li et al. / Aquatic Botany 80 (2004) 227–240 233

Table 2 Locations of the seven Potamogeton maackianus populations and their proportion of polymorphic bands (PPB) in RAPD analysis Population Lakes of Latitude (N) Longitude (E) Sample Band Polymorphic Proportion of collection no. no. band no. polymorphic bands (%) CL Changhu Lake 308220–308320 1128150–1128300 6 89 16 18.0 HH Honghu Lake 298380–298590 1138110–1138280 19 97 28 28.9 XL Xiliang Lake 298510–308020 1148000–1148100 12 92 25 27.2 ML Mulan Lake 318050 1148280 9 80 17 21.3 BA Bao’an Lake 308120–308180 1148390–1148460 8 86 18 20.9 NS Niushan Lake 308190 1148320 22 95 26 27.4 CH Chihu Lake 298440–298500 1158370–1158440 10 88 23 26.1 Mean 12.3 89.6 21.9 24.2 Total 86 112 59 52.7

Table 3 Genetic diversities of the seven populations of Potamogeton maackianus estimated by Shannon’s information index Population Genetic diversity BA 0.13 CL 0.11 XL 0.18 ML 0.12 CH 0.14 NS 0.17 HH 0.19

Mean genetic diversity of populations (Hpop) 0.15 Total genetic diversity (Hsp) 0.41 Genetic diversity within populations (Hpop/Hsp) 0.36 Genetic diversity among populations (Hsp Hpop/Hsp) 0.64

Table 4 Analyses of molecular variance (AMOVA) with Euclidean distance matrix of 86 individuals of Potamogeton maackianus using 112 RAPD bands Source of variation d.f. SS MS Variance component Percentage total P-value Among population 6 363.99 60.67 4.885 63.78% <0.0001 Within population 79 218.78 2.77 2.77 36.22% Total 85 582.77 63.44 7.65 d.f.: Degree of freedom; MS: mean square; SS: sum of square. The P-value was estimated computing 9999 permutations.

3.2. Relationships among and within populations

The seven populations were separated into two groups by cluster analysis with population NS and HH in one group, and the rest in the other (Fig. 2). Bootstrap supports 234 W. Li et al. / Aquatic Botany 80 (2004) 227–240

Fig. 2. Dendrogram of the seven populations of Potamogeton maackianus using UPGMA. The numbers marked on the branches are bootstrap values (%) out of 1000 bootstrapping. the result with values all higher than 50%. The genetic relationship was not consistent with the geographic distribution of lakes along the Yangtze River. The population ML and CL clustered first despite having no direct river link and a long distance between them. The two populations then clustered with the farthest downriver population CH. The distance between the strongly supported group HH and NS was about 130 km, with Xiliang Lake in- between but clustered to another group. A cluster analysis (UPGMA) of the 86 individuals xwith NTSYS separated populations clearly (Fig. 3). The individuals from one lake grouped together inclusively first before clustering with other populations. Lakes have obvious isolation effects on the exchanges between different populations. On the population level, the pattern that lakes were grouped was similar to that shown in Fig. 2.

3.3. Geographic patterns of genetic variability of Potamogeton maackianus

The Mantel test (Table 5) showed a significant negative correlation among the seven populations (r = 0.46, P = 0.96). While on the individual level, significant correlations

Table 5 Mantel tests for correlations between the genetic distances and geographical distances of all pairs of individuals in each population of Potamogeton maackianus Population rP(random Z < observed Z) BA 0.39 93.50%* CL 0.14 71.99% XL 0.04 61.35% ML 0.35 96.82%** CH 0.29 89.64% NS 0.13 90.90%* HH 0.18 95.15%** * P > 90%. ** P > 95%. W. Li et al. / Aquatic Botany 80 (2004) 227–240 235

Fig. 3. Dendrogram of the 86 individuals of Potamogeton maackianus using UPGMA. 236 W. Li et al. / Aquatic Botany 80 (2004) 227–240 were found in population ML (r = 0.35, P = 0.97) and HH (r = 0.18, P =0. 95), and population BA and NS only showed weak significant correlations. The relationships among the remaining three populations were not significant.

4. Discussion

4.1. Genetic diversity of Potamogeton maackianus

4.1.1. The clone diversity of the populations of Potamogeton maackianus Clonal growth is a common character of macrophytes, which may strongly influence the growth and genetic diversity of their populations (Waycott, 1995). Populations of aquatic plants might be composed of one or several clones (Les, 1988; Schlueter and Guttman, 1998). The population of P. maackianus might be composed of many clones, as each of its 86 individuals sampled had a unique genotype based on the RAPD results. The large sampling scale was considered the main reason for the higher genetic variation of P. maackianus as the shortest distance between adjacent sample locations in a lake was at least 0.5 km. Sampling the same genet in different stations was almost impossible on such a large spatial scale. Of cause, RAPD itself can produce more abundant genet markers, and detect higher genet diversity compared to allozyme method (Ayres and Ryan, 1999; Keller, 2000). However, some clones in a P. maackianus population might be very large. With samples from station 5 and 6 in Changhu Lake, station 11 and 12 in Xiliang Lake for example, only one band difference was detected despite the large distance between the two adjacent sites. Few studies on the clone size, the clone number and the clone structure in submerged plants have been carried out compared to terrestrial clonal plants. It is difficult to ascertain the ramet connection when sampling under water. Such studies will be helpful to reveal the character and the effect of vegetative reproduction of submerged plants if suitable sample methods and sample scale are used.

4.1.2. The genetic diversity of Potamogeton maackianus Before the present work, the genetic variation of P. maackianus was expected to be low, as vegetative reproduction by rhizomes was considered to be the most important means of maintaining and expanding natural populations, and all the lakes where its populations occurred had close relationships with the river systems. However high genetic diversity was found in P. maackianus populations in the middle reaches of the Yangtze River, as detected by PPB and Shannon’s index (Tables 2 and 3). We think the following three reasons might be responsible for the high genetic diversity of P. maackianus. First, sexual reproduction might play a more important role than formerly expected. Although low seed production ratio was observed in natural populations (Jin and Guo, 2001), a great number of seeds were produced every year because of its usually huge population size in lakes. The fate of seeds after they ripen is not clear as no seedlings were reported although it could be detected in seed banks in relatively higher number (Li, 1995). The unclear seed storage might contribute a lot to its genetic variations. This phenomenon was also reported in P. pectinatus, the unexpected higher levels of genetic variation was ascribed to sexual reproduction (Hangelbroek et al., 2002). W. Li et al. / Aquatic Botany 80 (2004) 227–240 237

Second, P. maackianus usually covers large areas in a lake, even the whole lake basin. The enormous population will help to maintain genetic diversity and avoid diversity loss resulting from genetic drift. In addition, the large area could also provide enough habitats to avoid intraspecific competition. Third, in one of its main distribution areas, P. maackianus has been present for a long time and might accumulate a lot of somatic mutations through clonal growth. Somatic mutations can contribute more to standing genetic variation in populations than do gametic mutations and thereby can increase plant evolutionary rates (Gill et al., 1995). The long history of P.maackianus in this area will also allow genetic diversity to accumulate through low rates of sexual reproduction.

4.2. Geographic pattern of genetic variations

4.2.1. The genetic relationship between populations and gene flow Higher flora similarity in lakes of the middle-lower reaches of the Yangtze River was attributed to their close connections with the river systems, which was also expected to explain the geographic pattern of their genetic variations, and also a directional transport pattern from up to downstream was expected to be found. However, cluster result of lakes showed no relation with their geographic distribution (Fig. 2), and even a significant negative correlation between genetic diversity and distance was found. In addition, each population seemed to have a very strong tying force as all the individuals from a lake were inclusively clustered together (Fig. 3), which was also reported in the seagrass Halodule wrightii from Christmas Bay (Angel, 2002). The cluster pattern both on the population and individual levels, together with the distribution pattern of genetic variation among and within populations (Table 4) suggested that large genetic differentiation had happened among P. maackianus populations. One main reason was considered the lack of gene exchange among populations, which can be evaluated by values of Fst and Nm calculated from RAPDistance software. With the RAPD data, Fst = 0.611, Nm =(1 Fst)/4 Fst = 0.16,

Fig. 4. Effect of population history on genetic distribution pattern within Potamogeton maackianus populations in Honghu Lake. Left: distribution of 19 individuals. Right: dendrogram of the 10 individuals using UPGMA. Dashed line is the combine-line. 238 W. Li et al. / Aquatic Botany 80 (2004) 227–240 too small to prevent population subdivision (Wright, 1931). The lack of highly mobile propagules has been considered to be the main reason for limited gene exchanges among lakes. However, individuals in a lake showed high similarity. This suggests that the population in a lake developed from an initial population, which defines the genetic characteristics of the latter populations. Owing to the lack of high mobile vegetative propagule, little gene flow could occur between different lakes. That is why the genetic variation was mainly distributed among populations. As to the genetic relationships among populations, despite the direct transformation of propagules, other factors such as birds and man’s fishery activities could also be responsible for the similarity between distant populations.

4.2.2. The genetic structure within the population of Potamogeton maackianus A common development history of P. maackianus was obvious and suggested in lakes of the middle-lower reaches of the Yangtze River (Li, 1995): This development pattern also supports the single origin of P. maackianus in a lake. From this development pattern, it was thought that P. maackianus populations in a lake were composed of subpopulations with different development time, and a spatial pattern was obvious. This suggestion was partly supported by Table 5, which shows that distance has an important role in determining the genetic geographic patterns of P. maackianus in some lakes. Of course, distance was not the only,or the most important, factor that affected the genetic structure in a population, especially when interference from man’s activities had become more and more frequent and severe. Owing to the clear history of vegetation in Honghu Lake, we use it as the example to discuss the effect of vegetation history on its genetic variations. P. maackianus was first reported in the northwest part of Honghu Lake in the 1950s, just shortly after several dams were built for flood control. From then on, P. maackianus expanded swiftly to the whole lake, and had become a dominant submersed macrophyte by the 1980s (Li, 1995). Fig. 4 shows the distribution of the 19 individuals in Honghu Lake (left), and the cluster dendrogram of individuals (right). For conciseness, the dendrogram was grouped by means of the combined-line method (Zhong et al., 1990) and defined the different groups as different genotypes. Type 1 was composed of the following individuals: HH1–HH14, which were distributed in the whole lake. Type 2 and 3 were composed of HH17, 18, 19 and HH15, 16, respectively, and all distributed in the northwest part of the lake. The genotype distribution of P.maackianus in Honghu Lake coincided quite well with the development history of the population: In the northwest part where the earliest population was found, the genetic diversity of P. maackianus was higher than in the other part. With the expansion of P. maackianus to other parts, gene differentiation occurred simultaneously. The reasons that were considered to contribute to the higher genetic diversity of P. maackianus were also responsible for the genetic variation within the population.

Acknowledgement

The study was funded by the following projects: the Innovation Project of The Chinese Academy of Sciences (KZCX1-SW-12), The Natural Science Foundation of China (grant W. Li et al. / Aquatic Botany 80 (2004) 227–240 239 no. 39970065), and Special Foundation of the Chinese Academy of Sciences. Dr. S.P. He, P. Nie and X.D. Xu were greatly acknowledged for their kind help in experiments. Dr. Q.H. Cai was greatly thanked for his help in GIS calculation.

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