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Population Biology |
Wuhan Botanical Garden/Wuhan Institute of Botany, Chinese Academy of Sciences, Wuhan, Hubei 430074 China
Received for publication September 29, 2005. Accepted for publication November 16, 2005.
ABSTRACT
Myricaria laxiflora is restricted to the riverbanks of the Yangtze River valley and will be completely lost owing to the construction of the Three Gorges Dam. Genetic diversity and structure of nine natural and one ex situ populations were investigated using amplified fragment length polymorphisms (AFLPs). A moderate level of gene diversity was found in natural populations, while the ex situ population had the highest. The F statistics calculated by different approaches consistently revealed a high genetic differentiation among natural populations, contributing >45% of the total gene diversity. The Bayesian-based analysis differentiated nine independent populations in accordance with the sites sampled. Estimates of gene flow by FST and coalescent-based simulation analysis indicated a restricted recurrent gene exchange among populations (Nm = 0.290–0.401), whereas genetic distance-based clustering and coalescent-based assignment analyses revealed significant genetic isolation among populations. The migration pattern in M. laxiflora is best explained by a classical metapopulation model, but with a unique unidirectional direction underlined by hydrochoric force that drove dispersal of seeds and propagules from upstream toward downstream populations. Previous efforts in preserving genomic integrity in ex situ conservation were evaluated, and the results provide valuable information to formulate conservation guidelines for successfully reintroducing M. laxiflora to the wild.
Key Words: conservation • gene flow • hydrochory • Myricaria • population structure • riparian plant • unidirectional migration • Yangtze River valley
The Three Gorges Dam (TGD) on the Yangtze River, the world's largest hydroelectric dam, is being built in the middle of a biodiversity hot spot in central China. The TGD will create a 58 000 km2 Three Gorges Reservoir Area (TGRA) and will undoubtedly have a significant impact on biodiversity and ecosystems in this region. Construction will disrupt the natural hydrochory regime of the Yangtze River and alter the evolutionary processes of the existing biodiversity. It poses an extreme environmental impact on plant adaptability and sustainability in the TGRA (Shen et al., 2004). Many riparian plants have adapted to the natural dynamics of the unregulated river over a long period of evolution (Poff et al., 1997
; Stromberg, 2001a
). Natural hydrochory dynamics of seasonal fluctuations in water level, flood disturbance, and other biotic and abiotic factors are important in determining the persistence of riparian plant species and communities (Stromberg, 2001b
; Johnson, 2002
). The long-term impacts of such dramatic environmental changes on riparian plants in a new habitat of regulated water bodies are not yet understood and deserve thorough investigation; various biological losses caused by environmental changes are inevitable (Nilsson et al., 1997
; Johnson, 1998
). For example, if flood disturbance becomes infrequent or if the temporal and spatial population diversity created by flood disturbance declines, species diversity will decrease (Pollock et al., 1998
).
Myricaria laxiflora (Franch.) P.Y. Zhang & Y.J. Zhang is a narrowly distributed species restricted to riverbank and shore habitats within the water-level fluctuation zone in 12 counties from Chongqing to Yichang along the Yangtze River valley. Recently, an exhaustive survey identified 31 natural populations and approximately 90 000 plants over the natural range of the species (Wang et al., 2003
). The entire natural range of the species will be submerged when the TGD is completed in 2009 and the water level rises to 175 m. Myricaria laxiflora will be the first plant to become extinct in the wild due to TGD construction, evoking great concern within the Chinese conservation community and attracting enthusiastic attention among the public and government agencies for rescuing this species.
Myricaria laxiflora is an evergreen shrub, belonging to the genus Myricaria Desv. of the family Tamaricaceae, which comprises four genera present in the Paleo-Mediterranean sea region since the Tertiary (Zhang et al., 1984
, 2001
). Thirteen species have been described in the genus Myricaria, of which 10 occur naturally in China. Except for M. laxiflora, all species are distributed in the Qinghai-Tibet Plateau and adjacent regions. The Himalayas are considered the center of origin. Myricaria laxiflora is the only species in the genus that occurs entirely in the low-altitude region of the Yangtze River valley, with a reversed seasonal growth habit of summer dormancy and winter growth, which is due to long evolutionary adaptation to the natural dynamics of seasonal fluctuations in water level, particularly summer flooding in the Yangtze River valley. Myricaria laxiflora normally undergoes summer dormancy when the water level rises from May to October, but resumes rapid growth when the water level falls in October until April the following year. It is highly tolerant to river flooding and water submergence with characteristics of mixed sexual and asexual reproduction, excessive seed production, and highly clonal propagation, probably as a result of long adaptive evolution in the riverbank habitat within the water-level fluctuating zone (Wu et al., 1998
; Wang et al., 2003
).
Progress has been made in understanding the biology of M. laxiflora before its whole range of natural habitat is completely submerged, mostly by collecting data on morphological characters, natural distribution, habitat, and coexisting plant community structure, plant ecological adaptability, and propagation method for ex situ conservation (Xiong et al., 1996
; Wu et al., 1998
; Shen et al., 1999
; Xu et al., 1999
; Wang et al., 2003
). Apparently, the sustainability of a riparian plant is strongly influenced by the uniqueness of its habitat niche and its ecological adaptability. Rapid winter growth, excessive seed production, and highly efficient wind and water dispersal, combined with highly clonal propagation in alluvial sandy soil, are characteristics that make M. laxiflora well adapted to its riverbank habitat along the Yangtze River valley (Wu et al., 1998
; Shen et al., 1999
). A recent review also suggested that the ancestral population of M. laxiflora possibly originated in the Paleo-Mediterranean sea region, and the subsequent geological formation of the Himalayas and the Yangtze River gorges shaped its current pattern of distribution, which differs from other species in the genus (Wang et al., 2003
). However, little is known about the genetic variation and population structure of the species, which is of critical importance for retaining the genetic integrity of the whole species in any attempts to formulate a conservation strategy (Frankham et al., 2002
). In our previous study (Li et al., 2003
), we examined the genetic diversity of several populations using isozyme electrophoresis and detected high genetic diversity in M. laxiflora, but the study was of limited value in determining conservation strategies because several recently discovered upstream populations (Wang et al., 2003
) were not included in that study. Understanding the genetic diversity and population structure of a species in its full natural range is a fundamental goal of ecological genetic studies (Jørgensen and Mauricio, 2004
). Having only limited population genetic information would have a profound impact on conservation success and would hamper reintroduction efforts for M. laxiflora over the long term. An entire range of population sampling and extensive investigation of genetic diversity and population structure are urgently needed for formulating a thorough conservation strategy to rescue this disappearing species (Robichaux et al., 1997
; Hogbin et al., 1999). When conservation efforts are taken to rescue a species that will be extinct in the wild, an ex situ conservation and subsequent restoration program ultimately becomes the only option. Because the entire natural habitat of M. laxiflora is destined to be submerged as the TGD project progresses, tremendous efforts have been undertaken since the middle 1990s (Wang et al., 2003
). During the past 5 years, several field surveys and collections have been conducted. Most individual plants in the main natural populations have been collected, and an extensive collection of natural populations has been assembled into a specialized ex situ conservation section at Wuhan Botanical Garden (WBG). Two satellite ex situ conservation sites have also been established locally in Lanling Brook and Si Brook that are both branches of the Yangtze River and are close to the natural range of M. laxiflora in Zigui County. Improving the conservation of genetic integrity and ex situ conservation management efficiency is a part of our long-term commitment to ex situ conservation and restoration of M. laxiflora. Ultimately, our goal is to restore self-sustaining populations of M. laxiflora to the wild with their genetic integrity similar to the natural populations after the TGD is completed in 2009. A parallel study to locate suitable restoration sites close to the natural distribution of M. laxiflora is also currently underway, although the underlying adaptive flexibility of ecological adaptation of M. laxiflora to water-level fluctuation zones in the TGRA is poorly understood and requires more ecogenetic investigation (Wang et al., 2003
).
The amplified fragment length polymorphisms (AFLPs) technique provides many advantages, including technical simplicity and large numbers of markers spanning the whole genome without requiring any prior genomic information (Vos et al., 1995
; Gaudeul et al., 2000
). AFLPs are one of the most suitable molecular marker systems to study population genetics with reliable repeatability. Furthermore, only small quantities of DNA template are required, which is especially important for rare and geographically remote species (Hsiao and Lee, 1999
; Keiper and McConchie, 2000
; Tero et al., 2003
; Prentis et al., 2004
).
The purposes of the present study were threefold: (1) to determine and partition the genetic diversity of M. laxiflora populations through its entire natural distribution, (2) to examine the geographical patterns of genetic differentiation among natural populations along the Yangtze River valley, and (3) to assess the genetic integrity of the ex situ conserved population vs. that of natural populations. This valuable information is critically important to our current efforts in formulating better strategies for conservation and reintroduction of M. laxiflora in the future.
MATERIALS AND METHODS
Plant materials and population sampling
In an exhaustive survey of the enitre natural distribution of M. laxiflora in 2002, nine main populations were identified (Table 1 and Fig. 1). We exhaustively sampled all individuals from each population and transplanted them to WBG in the winter of 2002 just before the TGD project progressed to the second phase of construction, which raised the water level to 135 m in October 2003, submerging all populations except one at the upstream end. Each collection site was recorded using a global positioning system (GPS), and geographic distances between populations were estimated on the basis of Chinese regional maps and Yangtze River navigation data. In the spring of 2003, we randomly sampled c. 25–30 plants from each population for AFLP analysis, depending on the availability of the transplanted individuals surviving in WBG. Within each population, the individuals chosen were originally at least 10 m apart based on our field collecting records, to avoid sampling the same plant, except for population MSZ with limited individuals that survived transplanting (Table 1). To evaluate our previous ex situ collection transplanted during 1994 to 1999, the ex situ population in WBG was also included in this study.
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. The AFLP analysis was performed essentially as described by Vos et al. (1995)
. Restriction-ligation was conducted using EcoRI /MseI endonuclease mixture (MBI Fermentas, Vilnius, Lithuania) and double-stranded adaptors. Following incubation at room temperature overnight, the restriction-ligation reactions were diluted with Tris-EDTA buffer for preselective amplification. The preamplified DNA was diluted to serve as template DNA for selective amplification. A total of 48 primer pairs were first screened for selective amplification and eight E + 3 and M + 3 primer pairs were chosen and used in this study (Table 1). The selectively amplified products were mixed with tracking dye, denatured at 94°C, and electrophoresed on 8% denaturing polyacrylamide gels on a 40 x 45 cm EC160 DNA sequencing system (Thermo, New York, New York, USA). Silver staining was performed following the protocol of SILVER SEQUENCE (Promega, Madison, Wisconsin, USA) with slight modification. AFLPs genotypes were manually scored as 1 (present) or 0 (absent) of the DNA fragments ranging in size from 60 base pairs (bp) to 300 bp and assembled in a binary data matrix comprising a total of 257 bands for each of the 280 individuals sampled. Each DNA sample was amplified at least twice to verify the reproducibility of the AFLP technique and only intense and unambiguous bands were manually scored.
Data analyses
To estimate allele frequencies and gene diversity of populations from dominant markers can be problematic (Zhivotosky, 1999
; Alexander et al., 2004
). However, Krauss (2000)
demonstrated that the statistical biases could be eliminated in highly polymorphic marker data sets. It has also been shown that for accurate estimates of the parameters of population genetics, large numbers of AFLP loci and about 30 individuals per population should be used (Tero et al., 2003
). Accordingly, we used the following methods to estimate a set of intra- and interpopulation genetic statistics.
The percentage of polymorphic loci within-population (Pp) was calculated using the POPGEN program (Yeh et al., 2000
). Bands that were monomorphic across all individuals were then discarded from further analyses to reduce statistical bias (Keiper and McConchie, 2000
). Nei's (1973) unbiased expected gene diversity (HeN) assuming Hardy-Weinberg (HW) equilibrium and Shannon and Weaver's (1949)
index (I) were estimated using the same program. The relative magnitude of genetic differentiation among populations was estimated by Nei's GST (Nei, 1987
), assuming the population is either in HW equilibrium (FIS = 0) or completely selfing (FIS = 1), using the POPGEN program. We also used the Bayesian approach of Holsinger et al. (2002)
, which allows a direct estimate of FST from dominant markers without prior knowledge of the FIS, to estimate the heterozygosity (HeH) within populations and to assess genetic differentiation. The f (analogous to FIS) and 
B (analogous to FST) statistics were calculated under different models implemented in the HICKORY version 1.0 program (Holsinger and Lewis, 2003
). Several runs were conducted with default sampling parameters (burn-in = 50 000, sample = 250 000, thin = 50) to ensure consistency of results (Tero et al., 2003
). Genetic variation within and among populations was further partitioned by analysis of molecular variance (AMOVA), and linkage disequilibrium between AFLPs was tested using ARLEQUIN 2.0 (Schneider et al., 2000
).
The assignment analysis that assigns individuals into subpopulations and identifies which individuals are immigrants was conducted using STRUCTURE (Pritchard et al., 2000
). The number of populations (K) was tested and determined assuming no prior information on the number of sites sampled or the site to which each individual belonged. Markov's chain Monte Carlo simulation (MCMC) parameters were set for a burn-in period of 30 000 and a run length of 105 iterations under the model that included no population admixture and the assumption that the allele frequencies are correlated within populations, and several independent runs and tests for robustness of the applied model were performed to ensure consistent results (Tero et al., 2003
). Sampled sites were grouped into the obtained K populations, and individual membership likelihoods were calculated. In addition, the average level of gene flow (Nm) among populations was indirectly estimated by a traditional method based on FST value [Nm = (1 - FST)/4 FST] (Slatkin et al., 1989
). We also tested the possibility (F) that two genes share a common ancestor within a population and the relative likelihood of migration–drift equilibrium (gene flow model vs. drift model) using the program 2MOD (Ciofi et al., 1999
). A simulation with 100 000 iterations was carried out, and the first 10% of the output was discarded to avoid bias resulting from the starting values.
The geographical distances among populations were calculated using the GEODETIC II program (L. S. William-Giel, at http://www.infoairports.com/freeservices/worlddistance.html). The Mantel test (Mantel, 1967
) was used to assess possible relationships between genetic and geographical distances among the nine natural populations along the Yangtze River valley. Furthermore, a standard linear regression between Nei's gene diversity (HeN) of each natural population and population size was calculated to assess the possible impact of genetic drift on populations inversely related to population size. We also hypothesized that the population gene diversity should be highly influenced by the geographic location along the Yangtze River valley because dispersal of propagules of M. laxiflora is likely to be unidirectional, from upstream to downstream. To test this hypothesis, relationships between Nei's gene diversity (HeN) of each natural population and its geographic distance from the population furthest upstream were evaluated. Principle coordinate (PCO) analysis was performed based on Nei's (1978) genetic distance (DNEI) matrix of populations, implemented in the program NTSYS pc2.1 (Rohlf, 2000
).
RESULTS
Eight primer pairs resolved a total of 257 unambiguous bands, of which 106 (41.25%) were polymorphic. A total of 39.69% of polymorphic loci were revealed in the nine natural populations, with two downstream populations and one midstream population containing a higher percentage of polymorphic loci (>20.0%) (Table 1). The ex situ population MWH had a higher Pp of 29.18% higher than the average (18.04%) across the nine natural populations and higher than that of each individual population (Table 1). All individuals of the 10 populations had unique AFLP multilocus genotypes, but population-specific AFLP fragments were detected in five natural populations, MLL, MHS, MTT, MHP, MZT, which were located across the entire natural range. Similarly, of the nine natural populations examined, two downstream populations, MLL and MWJ, and one midstream population, MTT, had higher levels of average gene diversity (HeN > 0.200), while other populations had lower average gene diversity (HeN < 0.200) (Table 1, Fig. 2). It is interesting to note that the ex situ population MWH had the higher gene diversity (HeN: 0.260) than all natural populations. A similar result was also revealed by the Bayesian estimates (HeH) not assuming that the populations are in HW equilibrium, with the HeH values ranging from 0.100 to 0.226 (Table 1, Fig. 2).
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B (analogous to FST) in the f-free model was 0.463 (SD = 0.024) among natural populations, agreeing well with the value of GST obtained when it was assumed that populations were not in HW equilibrium (FST = 0.463 vs. GST = 0.459). Results of the AMOVA analysis are presented in Table 3. Of total genetic variation partitioned in the nine M. laxiflora natural populations, 45.32% was attributed to the differences among populations, 54.68% to the differences among individuals within populations. Clearly, all three different statistical approaches were consistent in indicating that >45% of the genetic diversity occurred among the populations, while <55% occurred within each population. Estimation of gene flow (Nm) suggested a low gene exchange rate among the natural populations along the Yangtze River valley, ranging from 0.290 (derived from B) to 0.355 (derived fromGST).
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The estimates of the posterior probabilities of values of K (number of independent populations) with different values of v (the probability that an individual is an immigrant to a given population, v = 0.01, 0.05, and 0.1) gave similar results that suggested the data did not contain one panmictic population and that the most probable number of inferred clusters was K = 9 (P 
1.000). The assignment analysis revealed that on average 95.6% of the individuals were assigned to the population from which they were sampled, suggesting that each sample site had a distinct population. The composition of each population is shown in Table 4. Of nine natural populations, four populations had 100% assignment success, while the other four populations had >96% assignment. The only exception was the population MTT with the lowest assignment of 66.8%. Interestingly, the individuals in ex situ population MWH were assigned to two natural populations, MLL (18.6%) and MWJ (81.4%). The result of likelihood tests for the gene flow vs. drift model using the 2MOD program was slightly in favor of the gene flow model (P [gene flow] = 0.5634, Bayes factor = 1.2904), but it was impossible to sufficiently distinguish between the two models, suggesting that the populations of M. laxiflora were not completely isolated, but that a certain amount of migration occurred between populations. In the gene flow model, the probabilities of common ancestry (F) within populations were highly different, ranging from 0.147 to 0.649, while the number of migrants per generation (M) in each population estimated as (1 – F)/(4F) varied from 0.135 to1.451, with a mean value of Nm = 0.401 (Table 4).
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Genetic diversity within populations
In the present study, M. laxiflora appeared to possess a moderate level of gene diversity in natural populations along the Yangtze River valley (Nei's 0.262 and Bayesian's 0.255, Table 1, Fig. 2), as compared to the AFLP gene diversity of plants with a similar habitat and range distribution (Russell et al., 1999
; Tero et al., 2003
; Prentis et al., 2004
). A recent investigation of the endangered plant Silene tatarica growing along periodically disturbed riverbanks in Europe revealed little more than half the gene diversity detected in M. laxiflora (Tero et al., 2003
). A similar low level of AFLP gene diversity was also found recently in a riparian perennial plant (stream lily, Helmholtzia glaberrima) in a river range in Australia (Prentis et al., 2004
). However, in a case study of nine populations of the riverine tree Calycophyllum spruceanum over a wide geographical range along river tributaries in the Peruvian Amazon Basin, Nei's unbiased estimate of gene diversity revealed relatively higher gene diversity, ranging from 0.261 to 0.349 (Russell et al., 1999
).
Genetic diversity in plants depends on species-specific characters such as life form, breeding system, geographical range, mode of reproduction, and propagules dispersal (Hamrick and Godt, 1989
; Gaudeul et al., 2000
). As a riparian and narrowly distributed species endemic to the Yangtze River valley, M. laxiflora was not a typical threatened species before the construction of the TGD. The species has a restricted linear distribution along riverbanks in the Yangtze River valley. The populations sampled in this study and their habitats were originally intact with little anthropogenic activity because of the inaccessibility of the Three Gorges along the Yangtze River. Myricaria laxiflora has been characterized as a long-lived perennial plant with free, localized pollination by wind and insects, high seed production, and effective seed dispersal by wind and water (Wu et al., 1998
; Xu et al., 1999
; Li et al., 2003
). According to previous observations in our field surveys and common garden experiments of ex situ conservation (Y. Wang, unpublished data), flowering of M. laxiflora extends from December to June the following year. Insect pollination is usual. Ethereal seeds mantled with white plush could be spread by wind and water (Li et al., 2003
). These characteristics are probably responsible for the moderate diversity retained in M. laxiflora populations.
A relatively high level of linkage disequilibrium between AFLPs detected at the entire natural population level suggested a possible source–sink metapopulation effect of the linearly arranged population structure of M. laxiflora along the Yangtze River valley, with probable dynamics of founder effects in shaping the population structure of this species. Individual migrants among populations mostly result from unidirectional, downstream dispersal. Founder effects in riverbank-inhabited populations by long-term dispersal events would be conjectural and are the probable reason for observed linkage disequilibrium within populations. Furthermore, a limited number of population-specific AFLP fragments detected in five M. laxiflora natural populations (Table 1) may also suggest recent genetic drift occurred owing to population bottlenecks or founder events because the loss of rare alleles is the primary loss of genetic variation resulting from population bottleneck (Nei et al., 1975
).
Population differentiation and gene flow
The FST values estimated by different approaches indicated consistently high genetic differentiation among natural populations and the proportion of genetic differentiation among populations accounted for >45% of the total genetic diversity in M. laxiflora (Tables 2 and 3). A relatively separated distribution of populations within a three-dimensional space in the PCO analysis (Fig. 3) further revealed strong genetic differentiation among the nine natural populations. Good agreement between the GST value assuming populations not in HW equilibrium and Bayesian estimate under a free model (GST = 0.459 vs. FST = 0.463) suggested that M. laxiflora populations had departed slightly from HW equilibrium. A high percentage of genetic variation within populations is commonly found in outcrossing plant species (Hsiao and Lee, 1999
). On the contrary, species with predominantly self-fertilizing and vegetative clonal reproduction usually had a higher percentage of genetic variation among populations (Black-Samuelsson et al., 1997
; Gaudeul et al., 2000
). In M. laxiflora, there is a certain degree of selfing and asexual reproduction. In a controlled selfing experiment, viable seeds were obtained and germinated successfully (S.-B. Liu, Wuhan Botanical Garden, unpublished data), while several other studies have confirmed that M. laxiflora could be easily propagated by shoot cuttings, indicating that M. laxiflora is highly capable of asexual reproduction (Xiong et al., 1996
; Xu et al., 1999
). Based on these results and the unique topographic habitat conditions along the Yangtze River valley, we can infer that M. laxiflora had a mixed reproduction system of insect outcrossing pollination, selfing, and vegetative clonal reproduction. This mixed breeding system has probably played an important role in shaping the current population structure. Moreover, in the case of plants with an aggregate of linearly distributed populations such as M. laxiflora, high interpopulation genetic differentiation is a phenomenon (Hogbin and Peakall, 1999
; Gaudeul et al., 2000
; Prentis et al., 2004
), compared with the taxa that do not have this type of distribution (Jørgensen and Mauricio, 2004
; Odat et al., 2005). For example, in the Australian plant Zieria prostrata growing along a 3-km stretch of coastline, a high level of population divergence (37% among populations) was revealed by AMOVA analysis (Hogbin and Peakall, 1999
). In Eryngium alpinum, a mean FST = 0.40 was found among 14 populations along the French Alps (Gaudeul et al., 2000
). On the contrary, as a nonlinearly distributed species in North America, the level of genetic variation among 53 wild populations of Arabidopsis thaliana was only 20% (Jørgensen and Mauricio, 2004
). Similarly, an AMOVA analysis of Ranunculus acris, which is widely distributed in Europe indicated low genetic variation (10.97%) between populations (Odat et al., 2005).
The magnitude of genetic differentiation among populations is almost solely determined by the ability of a plant species to disperse its seeds or propagules. Restricted migration among geographically separated sites has often been used to explain this pattern of variation (Hogbin and Peakall, 1999
; Gaudeul et al., 2000
). According to the assignment analysis, the proportion of migrants was 0.044 (Table 4) among 250 plants of the nine natural populations sampled. Assuming unidirectional migration between all nine natural populations, there are 36 (9 x 8/2) possible migration routes, and the estimate of the average migration rate among the nine populations would be m = 0.0012. Assuming that sampled population size in this study (N = 250) is a proximate estimate of the effective size (Ne) of the populations (all individuals are mature plants and contribute to reproduction), we find an estimated average of 0.300 migrants (Nm) per generation between populations. This is well in accordance with the traditional indirect estimate of gene flow based on F statistics (Nm = 0.290 derived from Bayesian B and Nm = 0.355 derived from GST assuming no HW equilibrium). The estimate of gene flow based on common ancestry analysis also gave a similar migration rate of mean value M = 0.401(Table 4). Thus, it appears that restricted gene flow has occurred among M. laxiflora populations linearly distributed along the Yangtze River valley and underlies an evolutionary process for population differentiation in M. laxiflora.
Most individuals (mean 95.6%) of the nine natural populations were assigned to the population from which they were sampled; again this suggests genetic isolation and limited gene flow between populations. Obviously pollen-mediated gene flow seems to be effective only within each population but diminishes logarithmically with increasing distance (Tero et al., 2003
). Thus the observed migration events of long-distance dispersal were probably due to dispersal of seeds or asexual propagules. The topography of the riverbank along the Yangtze River valley is complicated, and water flow should be the only driving force for dispersal of seeds or vegetative propagules because high mountains and sudden, steep gorges have isolated the entire natural range of M. laxiflora. Steep cliffs and torrential water currents made its original habitat almost inaccessible to people and animals, making dispersal of seeds or vegetative propagules by anthropochory or zoochory rare or impossible. Migration movements were mainly driven by hydrochoric dispersal, and the movements should be unidirectional downstream. Observed migration events revealed by the assignment analysis strongly supported this scenario. Of five populations in which immigrants were identified, four populations had immigrants from upstream populations, indicating that most migration events occurred via the unidirectionally from upstream to downstream sites (Table 4). For example, 0.5% and 1.7% of the individuals identified as immigrants in population MWJ came from the upstream population MHS and MTT, respectively. Similarly, 1.7% and 1.5% of individuals in population MHP came from the upstream populations MSZ and MZT, while at the highest, 33.1% of individuals sampled in population MTT were assigned to the upstream population MSZ (Table 4).
Under restricted gene flow, an isolation-by-distance (IBD) pattern of population structure probably developed (Hutchison and Templeton, 1999
). The significantly positive correlation between genetic and geographic distances detected in M. laxiflora (r = 0.6294, P = 0.002) indicated this IBD pattern influenced population differentiation in M. laxiflora. The IBD model usually suggests a metapopulation structure (Gaudeul et al., 2000
). Moreover, M. laxiflora populations seemed to be demographically independent (see results, K = 9), but could nevertheless be linked by occasional exchange of migrants and slow extinction-colonization dynamics along the Yangtze River valley. A low level of successful long-distance dispersal could also lead to strong founder effects that influenced intrapopulation diversity in different populations (Table 1, Fig. 2). Demographic processes such as different germination success at different population sites could probably create the conditions for occasional and restricted dispersal distances, depending on uniqueness of the locations along the riverbank. Seed germination experiments indicated that the seed life of M. laxiflora for germination remained for only ca. 48 h, that germination peaked after ca. 24 h in free-flowing water, and that the most suitable environment for seed germination is in static or slow water or in moist sand (Y. Tao, Wuhan Botanical Garden, unpublished manuscript). Availability of river bays with shores along the Yangtze River valley was a critical factor for water-carried M. laxiflora propagules to reach a certain dispersal distance within the seed viability period. All of these restricted conditions for seed germination suggested a potentially low level of successful seed dispersal, and thus restricted gene flow and founder events should result in significant differentiation among populations.
Linear migration pattern
In an aggregate of linearly arranged populations, Tero et al. (2003)
have recently discussed five basic hypotheses of migration patterns among populations, including: (1) a genetically uniform population model with free gene flow across the populations, (2) a fragmented population model without recurrent gene flow between populations, (3) a stepping-stone population model, (4) a source–sink model and (5) the classical metapopulation model. These different models of population structure gave different testable hypotheses about the distribution of genetic variation within and among populations. If unidirectional hydrochory were the primary factor for gene flow, the downstream populations functioning as a confluence would contain higher gene diversity than would upstream populations (Russell et al., 1999
). In M. laxiflora, the two most downstream populations and one midpopulation were found to harbor much higher genetic diversity than other populations (Fig. 2). A significantly positive correlation (r = 0.4157, P = 0.004) detected between Nei's gene diversity (HeN) in each natural population and the geographic distance of each population located away from the population furtherest upstream, MZT, suggest that the distribution of genetic variation of M. laxiflora populations was highly influenced by hydrochory that shaped a unique unidirectional migration pattern among the populations (Fig. 4). Furthermore, we inferred from assignment analysis the existence of nine independent populations (K = 9, P 1.000), and likelihood tests for gene flow vs. drift models by the 2MOD program revealed a certain amount of migrations between populations (P [gene flow] = 0.5634, Bayes factor = 1.2904), indicating that the populations of M. laxiflora were neither separated at the same time from a formerly continuous population nor completely isolated without recurrent gene flow between populations. Thus, the genetically uniform and the fragmented population models did not fit the observed migration pattern in M. laxiflora.
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). High interpopulation differentiation and significant levels of linkage disequilibria are often associated with typical nonequilibrium dynamics of classical metapopulations (Prentis et al., 2004
). The observed migration pattern in M. laxiflora is probably best explained by the classical metapopulation model, but with a unique unidirectional migration pattern underlined by a dominant hydrochoric force that drove dispersal of seeds and asexual propagules from the upstream to the downstream populations. We thus propose a new metapopulation model termed unidirectional connection for this uniquely distributed M. laxiflora within an aggregate of linearly and narrowly arranged populations (Fig. 4). Theoretically, in this model, each population or subpopulation is an independent breeding unit, and gene movement should be dominantly regulated by unidirectional hydrochoric dispersal with minimal or without other directional gene movement. A gradual increase of intrapopulation genetic diversity would be found in a linear distribution from the upstream toward the downstream populations. However, some exceptions would be expected depending on the topographic structures and rip current in rivers. For M. laxiflora populations along the Yangtze River valley, the gene movement driven by hydrochoric dispersal would result in a gradual accumulation of gene diversity from the upstream toward the downstream populations. But a higher intrapopulation genetic diversity found in one midstream population, MTT (Table 1 and Fig. 2), probably resulted from a slow current and curved bay-like topography with a wider river shore at the site that was favorable for depositing flowing seeds and vegetative propagules along the river. On the other hand, the lowest intrapopulation genetic diversity, found in the other midstream population MXQ, could be caused by a rip current and/or a narrow river shore. Obviously, topography, habitat environment, and hydrochory played an important role in regulating the gene movement among M. laxiflora populations along the Yangtze River valley. This scenario was further supported by PCO analysis, which revealed that populations are clustered by geographic distances along the Yangtze River valley, except for one population, MGJ, which was located midstream, but clustered with the upstream populations.
Implications for conservation and reintroduction
When a plant species faces the complete destruction of its entire natural habitat, immediate action should be taken for conservation and reintroduction. The ultimate goal for conserving an endangered plant is to eventually reintroduce the species back into the wild while retaining the genetic integrity of the whole species. Maintaining existing levels of genetic variation of endangered plants is a vital component in formulating ex situ conservation strategies and planning subsequent reintroduction programs (Oostermeijer et al., 2003
). In this study, the ex situ population in WBG revealed the highest genetic diversity (HeN = 0.260; HeH = 0.223; I = 0.385) compared to the nine natural populations, suggesting that the previous ex situ conservation sufficiently encompassed overall genetic diversity retained in the species. In addition, the PCO analysis and assignment analysis consistently provided evidence that individuals in the ex situ population were collected from the MWJ population and adjacent area, in which relatively high genetic diversity existed. The subsequent exhaustively sampled collections from all populations during 2002 and the established network of ex situ conservation sites should have further enhanced our efforts in conservation of the genomic integrity in the ex situ conserved populations so that random loss of genetic variants could be avoided due to loss of individual ecotypes in the practice of ex situ conservation. The previous efforts should greatly contribute to our long-term commitment to the reintroduction of M. laxiflora to its natural habitat.
While reintroduction programs are underway, recovery of M. laxiflora in the wild is more challenging. Because the natural hydrochory dynamics of seasonal fluctuations in the water level along the Yangtze River valley are now regulated by the river dam (TGD) water system for generating hydroelectricity, the natural habitats for M. laxiflora no longer exist. Several considerations need to be emphasized. First, sites to be selected for reintroduction should maximize the chance of successful reestablishment of self-sustaining populations in the wild. The environment should match, as closely as possible, the environment to which the population was adapted. Several sites located downstream from the TGD and Yangtze River branches were selected and rigorously evaluated; reintroductions are now underway. Secondly, the evidence of genetic differentiation among the natural populations obtained in the present study should be taken into account, and the translocated individuals should be selected from the populations with individuals that are most likely to adapt to the reintroduction sites. The geographically closest natural populations would be the best choice. Finally, the reintroduction procedure should save as many different populations as possible to maximize genetic diversity and to increase evolutionary potential for coping with the new environment. Populations that are genetically most diverse and/or differentiated (e.g., MWJ and MTT) should be considered top priority for reintroduction. Although many other issues such as inbreeding or outbreeding depression, ecological adaptability, and reproduction fitness are not fully understood, reintroduction of the species is currently underway and substantial progress has been made. The results in the present study provide valuable information for formulating conservation guidelines to successfully reintroduce M. laxiflora to the wild.
FOOTNOTES
The authors thank M. Kang, Z.-Z. Li, and F.-Y. Tang for their technical assistance and J. Wu and S.-B. Liu for their help in collecting samples. This paper has also benefited from comments made by J.-Q. Li and T. Wang. This research was funded by a key initiative grant of the Chinese Academy of Sciences (KSCX2-SW-104) and NSF of China grants (30370153 and 30470185); by WZ no. 050618 of the Conservation Genetic Laboratory in Wuhan Botanical Garden, Chinese Academy of Sciences and grant no. 05053935 of the Office for Migration, Hubei Province. 
2Author for correspondence (hongwen{at}wbgcas.cn
), fax: +86-27-87510331)
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