Genetic diversity of an amphicarpic species, Amphicarpaea edgeworthii Benth. (Leguminosae) based on RAPD markers

Biochemical Systematics and Ecology 33 (2005) 1246e1257 www.elsevier.com/locate/biochemsyseco

Genetic diversity of an amphicarpic species, Amphicarpaea edgeworthii Benth. (Leguminosae) based on RAPD markers Yi Zhang, Ji Yang, Guang-Yuan Rao* College of Life Sciences, Peking University, Beijing 100871, PR China Received 10 October 2004; accepted 5 July 2005

Abstract Amphicarpaea edgeworthii Benth. is an amphicarpic legume widespread in China. Amphicarpy describes the phenomenon that a plant produces aerial as well as subterranean fruits. A. edgeworthii can reproduce via three kinds of flowers: aerial chasmogamous flowers, aerial cleistogamous flowers, and subterranean cleistogamous flowers. Although there are some studies on the population genetic structure of species with both chasmogamous and cleistogamous flowers, none has been done for that of an amphicarpic species so far. The present study uses random amplified polymorphic DNA (RAPD) to assess level and pattern of genetic variation in 15 natural populations of A. edgeworthii. A total of 131 stable and clearly scored RAPD bands were achieved from 13 primers. The average genetic diversity within populations estimated by Shannon’s information index was 0.218 at the population level, but ranged from 0.119 to 0.302, which was significantly different (P ! 0.01). Different statistical analyses revealed a high level of genetic differentiation among populations (GST Z 0.473e 0.527). Thus, the pattern of genetic structure of A. edgeworthii is consistent with that of an inbreeding species. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Amphicarpy; Genetic variation; RAPD; Amphicarpaea edgeworthii Benth.

* Corresponding author. Tel.: C86 10 62753035; fax: C86 10 62751526. E-mail address: [email protected] (G.-Y. Rao). 0305-1978/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2005.07.009

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1. Introduction Studies of the spatial distribution of genetic variation within and between populations, or population genetic structure, have greatly facilitated our understanding of speciation, adaptation and population dynamics. The population genetic structure of a species can be influenced by many factors, but the breeding system of a plant species is one of the major factors (Hamrick and Godt, 1989, 1996; Bussell, 1999). As shown by empirical studies based on allozymes and DNA markers, inbreeding plant species tend to have less genetic variation, whereas greater differentiation among populations than the outcrossers (Hamrick and Godt, 1989; Liu et al., 1998). Even though early theoretical studies suggested that only complete selfing and complete outcrossing can be evolutionarily stable (e.g. Lande and Schemske, 1985), it has been documented that intermediary selfing rates can be selected under certain conditions (Holsinger, 1991; Cheptou and Mathias, 2001; Cheptou and Dieckmann, 2002). Empirical evidences also showed that plants with mixed mating systems are relatively common in nature (Waller, 1986; Holsinger, 1988). Good examples can be found in Impatiens capensis (Schemske, 1978), Lamium amplexicaule (Lord, 1979, 1982), and Amphicarpaea bracteata (Schnee and Waller, 1986), in which an individual plant bears morphologically and developmentally distinct cleistogamous and chasmogamous flowers. Many plant species can produce two or more kinds of flowers in the same individual, and some produce more than one morphologically distinct type of fruit. Customarily, amphicarpy describes the phenomenon in which a plant produces aerial as well as subterranean fruits (Schnee and Waller, 1986). The characters shared by most amphicarpic plants include the presence of (i) cleistogamous subterranean flowers that produce much large fruits with limited dispersal, and (ii) aerial flowers that are potentially open pollinated and set many fruits suited for long distance dispersal (Kaul et al., 2000). Such life history traits of amphicarpic species obviously have great effect on the gene flow among and within populations, and then on the genetic structure of the population. Except for the breeding system, the population genetic structure of a species depends on its evolutionary history, population history, and on the level of environmental heterogeneity (Boody et al., 2000). Therefore, knowledge of the population genetic structure of a species is essential to make valid biological interpretations about its breeding system, reproductive biology and microevolutionary history (Martin et al., 1997; Bussell, 1999). Although there are some studies on the population genetic structure of species with both chasmogamous (CH) and cleistogamous (CL) flowers (Culley and Wolfe, 2001, and references therein), little is known about amphicarpic species. Random amplified polymorphic DNA (RAPD) markers have been widely applied in population genetics (Lynch and Milligan, 1994; Wolfe and Liston, 1998). The main drawback with RAPDs, however, is their dominant nature, which cannot distinguish genotypes between the dominant homozygote and heterozygote at individual loci. Nevertheless, efficient degree of statistical power can be achieved from dominant markers using different models (Holsinger et al., 2002). For instance, the Bayesian hierarchical model constructed in terms of the classical F-statistics can

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be used for data derived from dominant markers (Holsinger et al., 2002; Holsinger and Wallace, 2004). However, the data can be difficult to reproduce as RAPDs are sensitive to changes in reaction conditions and DNA concentration (Wolfe and Liston, 1998). Nevertheless, recent comparative studies between allozymes, RAPDs and AFLPs (Sun and Wong, 2001; Kjølner et al., 2004) showed they had similar analytical efficiency as markers for a population genetic study, and suggested that RAPDs could still be used as reliable markers under precautious laboratory conditions. Amphicarpaea edgeworthii Benth. is an amphicarpic species widespread in China. This species produces aerial cleistogamous and chasmogamous flowers as well as subterranean flowers. Its interesting reproductive traits, large distribution range, and divergent habitats make A. edgeworthii a good model for examining evolutionary scenario of plants with mixed mating system. In the present study, we employed RAPD markers to investigate the genetic diversity in A. edgeworthii occurring in different habitats over a large geographical range. Our objectives are to determine the population genetic structure of this amphicarpic species, and to compare the levels and patterns of genetic variation with the theoretical predictions based on the biological and distributional attributes associated with the species.

2. Materials and methods 2.1. Study species and population sampling A. edgeworthii is a slender, twining annual legume occurring in damp, disturbed habitats such as shaded woodland and moist roadside. Three types of flowers are produced in an individual of the species: aerial chasmogamous (ACH) flowers, aerial cleistogamous (ACL) flowers, and subterranean cleistogamous (SCL) flowers. Both aerial flowers produce typical legume containing one to three indurate seeds. Subterranean flowers are cleistogamous, and they are produced on the runners (usually 0.1e0.7 m long) from the axils of the underground cotyledons. The pod of SCL flowers is ellipsoid and indehiscent, and usually contains one fleshy seed, which is around 10 times larger than aerial ones. The subterranean seeds have a much higher germination rate than aerial seeds according to our field and greenhouse experiments, ca. 87.2% vs. 7.5%. Dispersal through pollen seems limited because in our field studies we have not found an efficient pollinator, e.g. insects, that could cover the distance between ACH flowers. The pod of aerial flowers is compressed and 2-valved. The valves coil after dehiscence of the ripen legume. Using this mechanism, seeds can disperse to 0.5 m. Seed dispersal through birds is not found. Subterranean seeds could extend 1.2 m by rhizomes, with the average of 0.45 m based on the field investigation of a population, NAN of the species. Fifteen populations of A. edgeworthii were sampled throughout its distribution range in China (Fig. 1). They represent different habitats, and have nearly equal

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Fig. 1. Map showing the geographic distribution of the 15 sampled populations of A. edgeworthii.

sample size (G22) as shown in Table 1. Healthy young leaves were taken from randomly selected individuals (at least 4 m apart) and dried in silica gel. 2.2. DNA extraction and RAPD protocols Genomic DNA was extracted from 0.03 to 0.04 g pieces of silica gel desiccated leaf material following the 2! CTAB method (Doyle, 1991). Initially, we carried out an RAPD primer trial with 243 primers from the Operon 10-mer Kit (Table 2). As a result, 13 primers were selected and used for the present study because they yielded the highest number of clear, reproducible and unambiguous polymorphic bands which reveal both intra- and interpopulation variations.

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Table 1 Locality and habitats of 15 populations sampled of A. edgeworthii Population code

N

Locality and habitat

Size

Collectors and vouchersa

HAN

22

O5000

Zhang et al. (2002)

NAI

22

ca. 2000

Zhang et al. (2002)

TAI

22

ca. 1000

Zhang et al. (2002)

YING

22

ca. 800

Zhang et al. (2002)

JI

21

O5000

Zhang et al. (2002)

ZHU

22

ca. 500

Zhang et al. (2002)

LAO

22

ca. 400

Zhang et al. (2002)

DONG

22

O2000

Zhang et al. (2002)

XI

22

O5000

Zhang et al. (2002)

NAN

22

ca. 3000

Zhang et al. (2002)

JIN

22

ca. 1000

Zhang et al. (2002)

JING

22

ca. 700

Zhang et al. (2002)

LI

20

ca. 400

Rao et al. (2002)

GAN

21

ca. 3000

Rao et al. (2002)

FO

22

Mt. Changbaishan, Jilin Province; in moist grasses along roadside, Alt. 735 m Mt. Changbaishan, Jilin Province; in mesic grassland along roadside, Alt. 680e720 m Mt. Taibaishan, Shaanxi Province; in moist grass- and bushland along stream, Alt. 2000 m Mt. Taibaishan, Shaanxi Province; forest margins along riverside, Alt. 1200 m Mt. Jigongshan, Henan Province; on mesic slope, Alt. 480 m Mt. Zhushan, Hebei Province; under trees, Alt. 550 m Mt. Laoshan, Shandong Province; gravel hillside in open forest, Alt. 100 m Mt. Lingshan, Beijing City; in mesic hillside, Alt. 1100 m Mt. Lingshan, Beijing City; under trees, Alt. 1100 m Mt. Lingshan, Beijing City; under trees, Alt. 1150 m Mt. Jinshan, Beijing City; in open forest, Alt. 280 m Jingdong County, Yunnan Province; on open and moist hillside, Alt. 1640 m Lijiang County, Yunnan Province; in bushland, Alt. 2400 m Hui County, Gansu Province; on moist hillside, Alt. 1700 m Mt. Jingfoshan, Sichuan Province; in grasses, Alt. 700 m

O5000

Rao et al. (2002)

N Z number of individuals sampled, size Z population size estimated. a All vouchers are deposited in the Herbarium of Peking University.

DNA concentration was determined by comparing the sample with a reference DNA in 1% agarose gel. Polymerase chain reaction (PCR) was carried out in a volume of 20 mL, containing 1 mL of DNA (20 ng/mL), 1 mL of primer (8 pm/mL), 1.6 mL of dNTP (2.5 mM of each dATP, dCTP, dGTP and dTTP), 2 mL of the 10! PCR buffer (200 mM TriseHCl pH 8.4, 200 mM KCl, 2.5 mM MgCl2), 0.2 mL Taq plus polymerase (5 U/mL), and 14.2 mL sterile water. The amplification reaction was performed in a PTC-100TM Thermal Controller (MJ Research, Inc.) with the following cycles: (1) 94  C/5 min; (2) 45 cycles of 94  C/ 30 s, 36  C/1 min, 72  C/1.5 min; (3) 72  C/10 min. Random samples of 80 individuals (25%) were amplified at least twice to ensure that the PCR profiles were reproducible. Amplified products were resolved in 1.5% agarose gel at 100 V for 3 h, stained with ethidium bromide, and photographed with a digital camera under UV light.

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Table 2 Primer trial for the present RAPD analysis Operon primer set

Primers tested

B H K N P R T U V W X Y Z

1 1 1 1 1 1 1 1 2 1 2 1 1

2 2 2 2 2 2 2 2 3 2 3 2 2

3 3 3 3 3 3 3 3 4 3 4 3 3

4 4 4 4 4 4 4 4 5 4 5 4 4

5 5 5 5 5 5 5 5 6 5 6 5 5

7 6 6 6 6 6 7 7 7 6 8 6 6

8 7 7 7 7 7 8 8 8 7 9 7 7

9 10 11 12 13 16 17 18 19 8 9 10 11 12 13 14 15 16 17 18 19 20 8 9 10 12 13 14 15 16 17 18 19 20 8 9 10 11 12 13 14 15 16 17 18 19 20 8 9 10 11 12 13 14 15 16 17 18 19 20 8 9 10 11 12 13 14 15 17 18 19 20 9 10 11 12 13 14 16 17 18 19 20 9 10 11 12 13 14 15 16 17 18 19 20 9 10 12 13 14 15 16 17 18 19 20 8 9 10 11 12 13 14 15 16 17 18 19 20 10 11 12 13 14 16 17 18 19 20 8 9 12 13 15 16 17 18 19 20 8 9 10 11 12 13 14 15 16 17 18 19 20

The primers selected and used for the final analyses are italicized.

2.3. Data analysis Amplified bands were scored in a size range from 0.2 to 2 kb. The resultant presence/absence matrix was imported to different programs for analyses. The application of diverse statistical methods may clarify the problem occurring in the analysis of RAPD data and allows a better understanding of the biological phenomenon being study (Palcios and Gonza´lez-Candelas, 1997). With POPGENE (version 1.31, Yeh et al., 1997), polymorphism parameters within populations was estimated, i.e. the number of polymorphic loci (Np), percentage of polymorphic loci ( p), number of alleles per locus (A), effective number of alleles per locus (Ae), Nei’s (1973) P gene diversity (H ), and Shannon’s information index (Lewontin, 1972, S Z ÿ Pi log2Pi, where Pi is the frequency of a given RAPD band). To compare the level of genetic diversity between populations, analysis of variance for Shannon’s information index and Nei’s (1973) gene diversity (H ) was conducted (randomized block design, two fixed factors: primer and S/H, testing main effects) (SPSS 10.0, 1999). Nei’s (1973) total gene diversity (HT), Shannon’s information index at the species level, coefficient of gene differentiation (GST), and Nei’s (1972) genetic identity and genetic distance between populations were also calculated with POPGENE. The additional measure for partitioning genetic variation was estimated based on Shannon’s information index, or obtained with AMOVA (Arlequin version 2.0, Schneider et al., 2000) and Bayesian hierarchical model (Hickory version 0.8, Holsinger and Lewis, 2003), respectively. Principal coordinates (PCo) were computed to ordinate relationships among individuals and among populations with Euclidean distance matrix (MVSP version 3.13f, Kovach, 2001). Neighbour-joining cluster analysis was performed to demonstrate the relationships among populations using Nei’s genetic distance (PAUP* 4.0, Swofford, 1998). A Mantel test was done to evaluate the correlation between genetic distance and geographical distance (NTSYSpc version 2.02, Rohlf, 1997).

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3. Results From 326 individuals of 15 populations, 13 selected primers yielded 131 clearly identifiable bands in a size range from 230 to 1700 bp. All of them were polymorphic at the species level. Each primer produced 5e13 bands (with an average number of 10.77). The primers were significantly different in their ability to detect genetic diversity within populations (P ! 0.01, Table 4). The percentage of polymorphic bands ( p) for a single population ranged from 24.43 (population ZHU) to 60.31% (population GAN) (Table 3). The intrapopulation variation differed greatly among populations in this species, the highest (H Z 0.200 G 0.192; S Z 0.302 G 0.278) in population JI, and the lowest (H Z 0.079 G 0.161; S Z 0.119 G 0.233) in ZHU (Table 3). The analyses of variance for S and H both showed that the level of the intrapopulation genetic diversity differed significantly among populations (P ! 0.01, Table 4). The total genetic diversity at the species level was high (HT Z 0.273 G 0.154; S Z 0.425 G 0.200). The coefficient of genetic differentiation between populations (GST) was 0.473 (estimated by partitioning of the total gene diversity), 0.488 (Shannon’s information index), 0.497 (qB, Bayesian method), and 0.527 (Fst, AMOVA; Table 5). All the estimates of genetic differentiation were very similar and indicated a high level of genetic differentiation among populations. Population pairwise relationships showed the lowest genetic distance between the populations NAN and XI (0.038), and the highest between GAN and ZHU (0.388). Principal coordinates analysis (PCoA) illustrated a similar distribution pattern of the Table 3 Genetic variation within populations Population HAN NAI TAI YING JI ZHU LAO DONG XI NAN JIN JING LI GAN FO

Np 54 72 64 68 78 32 35 39 57 64 59 48 72 79 74

p 41.22 54.96 48.85 51.91 59.54 24.43 26.72 29.77 43.51 48.85 45.04 36.64 54.96 60.31 56.49

A

Ae

H

S

1.412 G 0.494 1.550 G 0.499 1.489 G 0.502 1.519 G 0.502 1.595 G 0.493 1.244 G 0.431 1.267 G 0.444 1.298 G 0.459 1.435 G 0.498 1.489 G 0.502 1.450 G 0.499 1.366 G 0.484 1.550 G 0.499 1.603 G 0.491 1.565 G 0.498

1.220 G 0.337 1.277 G 0.348 1.278 G 0.361 1.253 G 0.350 1.331 G 0.347 1.136 G 0.296 1.154 G 0.289 1.155 G 0.296 1.250 G 0.359 1.281 G 0.362 1.211 G 0.314 1.205 G 0.353 1.282 G 0.335 1.310 G 0.366 1.312 G 0.380

0.130 G 0.185 0.166 G 0.190 0.162 G 0.198 0.150 G 0.190 0.200 G 0.192 0.079 G 0.161 0.093 G 0.167 0.092 G 0.165 0.145 G 0.195 0.165 G 0.197 0.129 G 0.178 0.116 G 0.188 0.171 G 0.190 0.182 G 0.197 0.180 G 0.203

0.197 G 0.267 0.254 G 0.272 0.243 G 0.284 0.228 G 0.271 0.302 G 0.278 0.119 G 0.233 0.139 G 0.245 0.141 G 0.241 0.217 G 0.279 0.247 G 0.284 0.199 G 0.258 0.173 G 0.268 0.260 G 0.276 0.276 G 0.281 0.270 G 0.287

Average 59.7 G 15.4 45.55 G 11.73 1.455 G 0.117 1.244 G 0.061 0.144 G 0.036 0.218 G 0.055 Species level 131 100 2.000 1.440 0.273 0.425 The standard deviation was shown in mean G SD. Np Z number of polymorphic loci, p Z percentage of polymorphic loci, A Z number of alleles per locus, Ae Z effective number of alleles per locus, S Z Shannon’s information index (Lewontin, 1972), and H Z Nei’s (1973) gene diversity.

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Y. Zhang et al. / Biochemical Systematics and Ecology 33 (2005) 1246e1257 Table 4 Analysis of variance for S/H within populations Source of variation

d.f.

SS

MS

F

Between populations Between primers Error

14 12 168

0.562/0.245 0.314/0.142 1.311/0.627

0.040/0.018 0.026/0.012 0.008/0.004

5.145**/4.690** 3.355**/3.175**

Total variation

194

2.187/1.015

**The level of significance is P ! 0.01.

RAPD variations (Fig. 3): about 30.8% of the total variation was described by the first two PCo; the genetic diversity structure was shown generally in line with the geographical structure of the species at small geographical scale; and the highest levels of genetic similarity were found between individuals from the same population. But exceptions were also presented: the JIN population was genetically more similar to ZHU and LAO than to the geographically close populations DONG, XI and NAN (Fig. 2); individuals from the JI population did not cluster together (Fig. 3). No significant correlation between Nei’s genetic distance and the geographic distance was found among populations of A. edgeworthii based on Mantel test (Mantel test, 1000 permutations; r Z 0.144, P Z 0.143).

4. Discussion The population genetic structure of several CH/CL species in which the same individual produced both aerial chasmogamous (CH) and cleistogamous (CL) flowers has been studied (Culley and Wolfe, 2001). Apart from a few studies for this kind of plants, no data were available on the population genetic structure of species that reproduce via ACH/ACL and cleistogamous subterranean (SCL) flowers. Therefore, our results on A. edgeworthii provide a first survey on the population genetic structure of an amphicarpic species using molecular markers. Sun and Wong (2001) argued that bias could be avoided in assessing genetic diversity when primers revealing different levels of polymorphism are used. Out of 243 primers examined for this study, the 13 selected ones exhibited varying ability for detecting genetic variation within and among populations (Tables 3 and 4). Of 326 individuals investigated, each had a unique RAPD profile. All these showed that the primers used in this study were both efficient to detect a usually low level Table 5 Hierarchical analysis of molecular variance (AMOVA) of RAPD haplotypes using the Fst method of estimating genetic differentiation among 15 populations of A. edgeworthii Source of variation

d.f.

%Variation

P

Among populations Within populations

14 311

52.71 47.29

!0.001

d.f., degree of freedom; %Variation, percentage of the total variance; P, levels of significance for the distribution for that hierarchical level being different from random.

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Fig. 2. Neighbour-joining tree of Nei’s genetic distance among 15 populations of A. edgeworthii.

polymorphism in the suspected inbreeding populations, and proper to analyze the population structure of A. edgeworthii. Our results revealed a high level of total genetic variation (HT Z 0.273 G 0.154; S Z 0.425 G 0.200) in A. edgeworthii. The interpopulation differentiation was also very high (GST Z 0.473 for the partitioning of the total genetic diversity, 0.488 for the Shannon’s information index, qB Z 0.497 from Bayesian analysis, and Fst Z 0.527 from AMOVA). The genetic differentiation value is usually !20% for outbreeding and O50% for inbreeding species based on allozyme data (Hamrick and Godt, 1989, 1996). Available data show that the CH/CL species usually have high inbreeding, little or no genetic variability within populations, and large genetic differences among populations (Fst(q) Z 0.46e0.92), with the exception of Viola pubescens (q) Z 0.29) (Culley and Wolfe, 2001, and references therein). The same pattern of the population genetic structure was found in A. edgeworthii. This species, 3.0 2.5 2.0 1.5 1.0

Axis 2

0.5 0.0 -0.5 -1.0 -1.5 -2.0

-2.43

-1.62

-0.81

0.00

0.81

1.62

2.43

3.24

-2.5 4.05

HAN NAI TAI YING JI ZHU LAO DONG XI NAN JIN JING LI GAN FO

Axis 1 Fig. 3. Principal coordinates analysis of 326 individuals of A. edgeworthii. Axis 1 extracted 21.84%, and axis 2 extracted 8.96% of the variance.

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however, morphologically differs from the CH/CL species by having three types of flowers: open aerial chasmogamous flower, close aerial cleistogamous flower, and subterranean cleistogamous flower. Anyhow, the amount and distribution of its genetic variation found in this species was consistent with that of a selfing species. The genetic variation within populations differs in selfing species (Schoen and Brown, 1991; Bonnin et al., 1996). Comparable patterns are apparent in populations of A. edgeworthii (Table 3), for example, the highest level of genetic diversity occurs in population JI ( p Z 59.54%, H Z 0.200 G 0.192 and S Z 0.302 G 0.278), whilst the lowest occurs in population ZHU ( p Z 24.43%, H Z 0.079 G 0.161 and S Z 0.119 G 0.233). The findings can be attributed to many factors, such as mating system, genetic drift and genotypeeenvironment interactions. A possible explanation for this in A. edgeworthii is that different habitats function on the formation of the following generation through affecting a variety of life history traits, such as outcrossing rate, seed dispersal, and seed germination. The genetic relationships between populations in a species do not often accord with their geographical distance, especially for the species with large distribution area (Irwin, 2001; Qiu et al., 2004). The same pattern occurred in A. edgeworthii. Cluster analysis demonstrated that most populations clustered in accordance to the geographic distribution of populations, while population JIN was not close to population DONG, XI and NAN although they were less than 200 km apart from each other. In addition, PCo analysis showed that individuals from the same population grouped together generally. However, individuals from population JI did not, implying that there is high local differentiation within the population. The relationship between genetic and geographical distance might be limited at certain geographical distance because the population history, the action of random genetic drift, or the presence of nonneutral variation in a species have a great influence on it (Le Corre et al., 1997). The mating system of A. edgeworthii has not been studied, but our present RAPD analyses, together with the field observations showed that it is obviously a predominantly selfing species. Therefore, the breeding system in this species played a central role in determining the amount and distribution of genetic variation within and between populations. As suggested by theoretical studies (e.g. Schoen and Lloyd, 1984), the plasticity in reproduction strategy of a species is a response to different selective pressures in spatially and temporally heterogeneous environments. The mixed mating system of A. edgeworthii could be a reproduction strategy to allow individuals to adjust their reproduction in response to their habitats by producing genetically similar and dissimilar seeds varying in sizes and dispersal capability. More intense studies including common garden experiments, quantitative analysis of phenotypic traits, characterization of its mating system etc. are necessary for a better understanding of the evolutionary history and population dynamics of A. edgeworthii.

Acknowledgements Thanks are due to Zhan-Jiang Zhong for his help during the fieldwork. We are also grateful to Professor Monique Simmonds, Dr. Yan-Ping Guo and an

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anonymous reviewer for their constructive comments on an earlier version of this paper. The work was supported by the National Natural Science Foundation of China (NSFC, grant no. 30070052 and 30370091).

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