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Laboratory of Forest Ecology and Physiology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan; Kansai Research Center, Forestry and Forest Products Research Institute, Momoyama, Fushimi, Kyoto 612-0855, Japan; Tree Genetics Laboratory, Department of Forest Genetics, Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 305-8687, Japan
Received for publication February 7, 2006. Accepted for publication November 11, 2006.
ABSTRACT
We examined genetic differentiation among eight local populations of a metapopulation of Magnolia stellata using 10 nuclear and three chloroplast microsatellite (nSSR and cpSSR) markers and evaluated the influence of historical gene flow on population differentiation. The coefficient of genetic differentiation among populations for nSSR (FST = 0.053) was less than half that for cpSSR (0.137). An isolation-by-distance pattern was detected for nSSRs, but not cpSSRs. These results suggest that pollen flow, as well as seed dispersal, has significantly reduced genetic differentiation among populations. We also examined patterns of contemporary pollen flow by paternity analysis of seeds from nine seed parents in one of the populations using the nSSR markers and found it to be greatly restricted by the distance between parents. Although most pollen flow occurred within the population, pollen flow from outside the population accounted for 2.5% of the total. When historical and contemporary pollen flows among populations were compared, the levels of pollen flow seem to have declined recently. We conclude that to conserve M. stellata, it is important to preserve the whole population by maintaining its metapopulation structure and the gene flow among its populations.
Key Words: conservation • gene dispersal • genetic diversity • Japan • Magnolia tomentosa • metapopulation • simple sequence repeat • star magnolia
The extensive use of natural resources to meet the needs of expanding human populations has led to growing concerns about the effects of deforestation and habitat fragmentation on plant and animal species (Lande, 1998
). The disappearance and fragmentation of natural populations could lead to reductions in the rate of gene flow among populations and consequent increases in genetic differentiation among populations and genetic structuring and reductions in genetic variation within populations due to genetic drift (Hartl and Clark, 1997
). Gene flow among populations heavily influences the degree to which they remain independent evolutionary units and thus plays a major role in the microevolution of species (Husband and Barrett, 1996
). Gene flow is also important in conservation contexts (Frankham et al., 2002
) because when a species is confined to small populations, its genetic characteristics are strongly influenced by genetic drift and inbreeding (Barrett and Kohn, 1991
; Ellstrand and Elam, 1993
). A metapopulation is an assemblage of local populations that usually are small and are linked by loose relationships, i.e., there is some gene flow among them. Many species have such metapopulation characteristics (Hanski and Simberloff, 1997
), and in such cases, gene flow among local populations could mitigate losses of genetic variation caused by genetic drift in local populations and thus save them from extinction via so-called "genetic rescue" (Richards, 2000
). Many studies have shown that even very minor amounts of gene flow can have profound effects on recipient populations, e.g., increasing their fecundity (Heschel and Paige, 1995
; Madsen et al., 1999
; Sheridan and Karowe, 2000
; Dick, 2001
; Newman and Tallmon, 2001
; Ingvarsson, 2002
).
The conventional approach used to quantify gene flow is to transform estimates of population genetic structure (FST) into indirect estimates of the average number of migrants exchanged per generation among populations (Nm) (Neigel, 1997
). However, the resulting estimates are rarely accurate because the underlying mathematical model makes many biologically unrealistic assumptions (Whitlock and McCauley, 1999
). Furthermore, the model may not reflect contemporary variation in gene exchange among populations or current changes in dispersal processes because it gives historical estimates (Sork et al., 1999
). However, indirect estimation of gene flow has a great advantage if cytoplasmic (chloroplast and mitochondrial DNA) markers, as well as nuclear DNA markers, are employed (Ennos, 1994
). In a species whose cytoplasmic genomes display maternal or maternally biased inheritance, as in many angiosperms (Mogensen, 1996
), gene flow in the cytoplasmic genomes is restricted to seed movement. Because gene flow for nuclear DNA can occur via the dispersal of both seeds and pollen, information on the relative influence of the two sources of gene flow on genetic structure can be obtained by comparative analysis of genetic markers derived from the two genomes.
An alternative way to estimate gene flow is to use parentage analysis to identify parents (usually fathers) and then directly quantify the patterns of gene flow (usually pollen flow). Gene flow estimated in this way is contemporary, not historical. Direct measurements of contemporary gene flow can elucidate pollen and seed dispersal parameters more precisely than indirect measures of historical gene flow and can provide important conservation-relevant information indicating whether recent habitat changes are causing population isolation by decreasing pollen flow (Sork et al., 1999
). Using advances in genetic marker technology, we can monitor contemporary gene flow using microsatellite markers with very high polymorphism and codominant expression (Chase et al., 1996
).
For effective conservation, we must elucidate present gene flow parameters, i.e., whether the amount of contemporary gene flow is sufficient to prevent population decline. We must also elucidate historical gene flow and evaluate changes in levels of gene flow by comparing it with contemporary gene flow. It is important to know whether the amount of gene flow has remained constant or has changed because of habitat alterations. In this study, we examined the genetic differentiation among local populations within a metapopulation of a threatened tree species, Magnolia stellata, using nuclear microsatellite (nuclear single sequence repeat, nSSR) markers and maternally inherited chloroplast microsatellite (cpSSR) markers, then evaluated the influence of historical gene flow on the population differentiation. We also examined patterns of contemporary pollen flow by paternity analysis of seeds collected from known seed parents in one of the local populations using nSSR markers. Furthermore, we compared historical and contemporary pollen flow among local populations and inferred changes in pollen flow levels. Finally, we addressed the importance of gene flow for the persistence of this species.
MATERIALS AND METHODS
Study species
Magnolia stellata Maxim. (Nooteboom, 1994
; synonym, M. tomentosa Thunb., Ueda, 1986
), a deciduous tree of the Magnoliaceae, is endemic to the area around the Ise Bay of central Japan and is now a threatened species, classed as "vulnerable" in the Red Data Book of Japanese vascular plants because of urban development (Environment Agency of Japan, 2000
). Local populations of this species are usually small and occur intermittently or in isolation in swampy places such as small rivers and bogs, but sets of adjacent local populations may represent hierarchical metapopulation structures. The species reaches heights of up to 10 m. It blossoms in early spring, forming protogynous, insect-pollinated flowers. The mean flowering duration for individual trees is 15.2 d, and individual flowers bloom for 10.2 d on average (S. Setsuko et al., unpublished manuscript). The showy flowers of Magnolia are thought to be mainly pollinated by beetles (Thien, 1974
; Bernhardt and Thien, 1987
; Kikuzawa and Mizui, 1990
; Ishida, 1996
), which may be less efficient pollinators than bees (Ramsey, 1988
). The fruits are aggregated, with at most two red seeds per follicle, and may be dispersed by birds (Callaway, 1994
).
Study site and field methods
The research site, located in the Kaisho Forest, near Nagoya City, Aichi Prefecture, Japan (35°11'25'' N, 137°06'55'' E), supports a secondary forest mainly composed of Pinus densiflora, Ilex pedunculosa, Quercus serrata, and Clethra barbinervis (nomenclature follows Ohwi and Kitagawa, 1992
) (Tamaki et al., 2005
). The potential, natural vegetation of this area is evergreen broad-leaved forest, but this forest is now mainly dominated by deciduous trees because the trees were used for fuel wood over long periods in the past. However, because the wood is no longer used for fuel, evergreen broad-leaved trees have been increasing via secondary succession (Aichi Prefecture, 1998
).
A local population of M. stellata along the Yato River (local population Y) and the seven local populations around it were surveyed (Fig. 1; hereafter population refers to a local population, defined as an aggregation of genets separated by a watershed). The nearest population outside the study area is about 1000 m away from population C. Only adult genets (defined as genets that were observed to flower at least once in 2002–2004) within these populations were used in this study. Since M. stellata genets usually consist of several ramets due to sprouting and layering (Goto et al., 1998; Setsuko et al., 2004
), different genets had to be distinguished by examining the connections between flowering ramets above the ground in conjunction with microsatellite analysis. For DNA analysis, leaf samples were collected from all adult genets (i.e., one of each set of connected flowering ramets and every flowering ramet that did not have confirmed connections to other flowering ramets) in all populations. The spatial coordinates and diameter at breast height (dbh) of each flowering ramet were measured.
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DNA extraction and microsatellite genotyping
Genomic DNA was extracted from leaves of all sampled flowering ramets and germinated seedlings using a modified CTAB method (Murray and Thompson, 1980
). Genotypes of all sampled flowering ramets and germinated seedlings were determined using 10 nSSR markers: nine (stm0148, stm0184, stm0191, stm0222, stm0223, stm0251, stm0334, stm0353, and stm0423) developed for M. stellata (Setsuko et al., 2005
), and one, M6D8, developed for M. obovata (Isagi et al., 1999
). Three cpSSR markers for M. stellata were used: trnL intron, trnH-trnK, and trnG intron (Ueno et al., 2005
). From our preliminary experiments, the mode of inheritance of cpDNA in M. stellata was inferred to be maternal, as it is for many angiosperms. PCR fragments were separated using a 3100 Genetic analyzer and GeneScan software (Applied Biosystems, Foster City, California, USA) (see Setsuko et al., 2005
and Ueno et al., 2005
for details).
Data analysis
For nSSRs, the number of alleles, allelic richness (El Mousadik and Petit, 1996b
), observed heterozygosity (HO), gene diversity (HE; Nei, 1987
), and fixation index (FIS = 1 – HO/HE) were calculated for each locus and each population using FSTAT version 2.9.3.2 (Goudet, 1995
). Departures from Hardy–Weinberg equilibrium at each locus and linkage disequilibrium between loci were tested by an exact test using a Markov chain method implemented in GENEPOP version 3.3 (Raymond and Rousset, 1995
), with Bonferroni corrections. We evaluated the power of marker sets using the exclusion probability (Marshall et al., 1998
), i.e., the probability of excluding a randomly chosen non-father on the basis of allele frequencies. Paternity analysis was performed by a likelihood-based approach based on multilocus genotypes for all adult genets and offspring using CERVUS version 2.0 (Marshall et al., 1998
). In this study, the simulation parameters required by the program were set as follows: 10 000 cycles, 308 candidate parents (= all adult genets identified across the study population), 0.99 as the proportion of candidate parents sampled, and 1.00 and 0.001 as the proportions of loci typed and mistyped, respectively. For cpSSR, haplotype diversity (= gene diversity for haploid genomes) was calculated for each population according to Nei and Tajima (1981)
.
For nSSRs and cpSSRs, HT and HS (Nei, 1987
), FST (Weir and Cockerham, 1984
), and RST (Slatkin, 1995
; Rousset, 1996
) were estimated using FSTAT. HT is the gene diversity in the total population, HS is the average gene diversity within populations, and FST is the coefficient of genetic differentiation among populations under an infinite allele model. RST is also a coefficient of genetic differentiation among populations, but it is defined under a stepwise mutation model. The significance of differentiation at each locus was tested by the log-likelihood (G)-based exact test (Goudet et al., 1996
), using a Markov chain method in GENEPOP.
Pairwise FST values between populations for nSSRs and cpSSRs were estimated using ARLEQUIN version 2.000 (Schneider et al., 2000
). To test the presence of isolation by distance, a Mantel test (Mantel, 1967
) between population-pairwise geographic distance and FST/(1 – FST) was applied (Rousset, 1997
). The geographic distances between populations were determined between the centroids of genets in populations. The population F, which had less than five genets, was excluded from this analysis.
A dendrogram was constructed to interpret relationships among populations by a neighbor-joining (NJ) method (Saitou and Nei, 1987
) based on Nei's genetic distances between populations (Nei, 1972
) using PHYLIP version 3.64 (Felsenstein, 1993
). The node significances of the dendrogram were evaluated using bootstrap probabilities based on 1000 replicates, and the population F (which had less than five genets) was excluded.
We analyzed the relationship between spatial distance and mating frequency between parents within the population Y. The frequencies of matings were calculated as the number of matings at each distance class divided by the total number of matings for each seed parent, then plotted as a function of the distance between parents. The frequencies of mating were averaged over all seed parents and fitted to an exponential function. The data for seed parent Y56 were excluded due to small sample size.
RESULTS
Genetic diversity
High levels of genetic diversity in nSSRs were consistently observed in all the populations, as indicated by gene diversity (HE) estimates ranging from 0.738 to 0.800 with an average of 0.773 (Table 1). All FIS values calculated at each nSSR locus in each population (80 values) had non-significant deviations from Hardy–Weinberg equilibrium. No significant linkage disequilibrium between loci was observed for any population, except at one pair of loci in one population (M6D8 and stm222 in population T), so all loci were used for further analyses. The exclusion probability for the second parent was 0.99992.
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Genetic differentiation among populations
There was significant genetic differentiation among populations in both nDNA and cpDNA. The FST value for cpSSRs (0.137) suggests that historical gene flow via seeds alone moderately reduced the differentiation for nDNA. The genetic differentiation among populations in cpDNA was more than twice that in nDNA, as indicated by the difference in FST values between cpSSRs and nSSRs (0.053). The likeliest reasons for this are (1) that gene flow is higher for nDNA, for which gene flow occurs via both seed and pollen, than in cpDNA, for which gene flow is solely via seed movements, and (2) the effective number of genes is lower in cpDNA than in nDNA, indicating that the effect of genetic drift is stronger on cpDNA than on nDNA.
The FST value (0.137) for cpSSRs in M. stellata in this study was fairy low compared with reported values (0.29–0.81) for other angiospermous tree species with bird-dispersed seeds (El Mousadik and Petit, 1996a
; Fineschi et al., 2000
; Rapse et al., 2000
; Mohanty et al., 2002
; Hampe et al., 2003
; Rendell and Ennos, 2003
), probably because the populations examined in the cited studies were distributed over large areas. In accordance with this hypothesis, Ueno et al. (2005)
estimated the value of 
st (an analogue to Fst) for cpSSRs in M. stellata at a geographical level (the entire distribution of the species) to be 0.62; within the reported range for other species. The low FST value for cpSSRs in this study suggests that seed dispersal is relatively frequent at the spatial scale represented here. Furthermore, the FST value for nSSRs (0.053) in M. stellata found in this study is also low compared to corresponding values (0.06–0.25) for other insect-pollinated angiospermous tree species with bird-dispersed seeds (El Mousadik and Petit, 1996b
; Rapse and Jacquemart, 1998
; Fineschi et al., 2000
). These differences could be explained by the same reasons as described for cpDNA, because Ueno et al. (2005)
estimated the st value for nSSRs in M. stellata at the geographical level to be 0.29.
No isolation-by-distance pattern was detected in the cpDNA differentiation. This absence could be due to random seed dispersal by birds attracted to the seeds by their fleshiness and red color (Callaway, 1994
). However, isolation-by-distance has been detected at the geographical level using the same cpSSR markers (Ueno et al., 2005
). This inconsistency might reflect the activity range of the birds, i.e., they may move randomly at a landscape level, while at the geographical level their movements are restricted by the geographical distances. Another possible explanation for the lack of isolation-by-distance patterns in cpDNA differentiation is that genetic drift has a stronger effect on cpDNA than on nDNA and thus may have disrupted such patterns in cpDNA. However, the moderate or high levels of cpDNA haplotype diversity throughout all populations suggest that genetic drift has not greatly affected the haplotype frequencies of the populations. The former explanation may therefore be more likely than the latter. An isolation-by-distance pattern was detected for nDNA, but not cpDNA, and the topology of the dendrogram based on nSSR frequencies clearly reflects the geographic relationships among populations. These results indicate that the level of pollen flow between populations is negatively correlated with the geographical distance between populations, which may have caused the isolation-by-distance pattern for nDNA. If the seeds had been randomly dispersed by birds, no isolation-by-distance pattern would have been detected for nDNA. However, at the within-population level, the seeds are probably dispersed not only by birds but also by gravity because there is spatial genetic structure over short distances within populations, probably due to spatially limited, barochorous seed dispersal (Setsuko et al., 2004
). An isolation-by-distance pattern has also been detected in nSSR at the geographical level (Ueno et al., 2005
), although the nSSR markers used in the cited study were different from those in this study.
Patterns of contemporary pollen flow
The shape of the pollen dispersal curve shows that there were high frequencies of short-distance events and low frequencies of long-distance events and that spatial distance greatly restricted pollen dispersal. These trends are consistent with those of other tree species, both wind pollinated and insect pollinated (Streiff et al., 1999
; Burczyk et al., 2002
; Nakanishi et al., 2004
; Oddou-Muratorio et al., 2005
).
In the study population, the maximum distance of pollen flow was 420 m, although the average distance was only 25.0 m, and the proportion of pollen flow from outside the population (9.3 ha) was only 2.5%. These values of pollen transfer are very low compared to reported distances for other insect-pollinated trees. For instance in studies of Magnolia obovata (Isagi et al., 2004
), Pithecellobium elegans, a large tropical tree pollinated by hawkmoths (Chase et al., 1996
), and Neobalanocarpus heimi, a tropical emergent tree pollinated by bees (Konuma et al., 2000
), the average distance of pollen flow within the respective research sites (69, 32, and 42 ha) was 157.1, 142, and 524 m, and the proportion of pollen flow from outside them was 18.3, 28.6, and 33.9%, respectively. The shorter average distance of pollen flow and lower frequency of pollen flow from outside the population for M. stellata in comparison with M. obovata may be due to a number of factors. First, M. stellata flowers in early spring from March to April, while M. obovata flowers in early summer from May to June (Satake et al., 1989
); hence the activities and varieties of pollinators are likely to be lower during the flowering season of M. stellata than that of M. obovata. Second, M. stellata is distributed in clumps along small rivers and valleys, while M. obovata is distributed sparsely, its density is usually very low (a few trees per hectare; Kikuzawa, 1987
), and pollen dispersal distances tend to be shorter in high-density populations than in low-density populations (Levin and Kerster, 1974
; Fenster, 1991
).
Small, flower-visiting insects were observed in the study population of M. stellata; most commonly members of the Coleoptera, Thysanoptera, and Diptera (S. Setsuko et al., unpublished manuscript). Small beetles belonging to the Staphylinidae (2–3 mm in length; Coleoptera) were the most frequently observed, and the high frequency of short-distance pollination most likely resulted from pollination by these small beetles. The low frequency of long-distance pollen flow suggests that pollinators capable of flying long distances rarely visited the flowers. Bees belonging to the Bombidae were observed to visit M. stellata flowers at the study site, and although they visited infrequently, they could act as long-distance pollen flow vectors. However, there are other candidates for long-distance pollen flow vectors. It is unlikely that small insects could fly more than 400 m over the ridges, but many studies of thrips as pests have reported that they are primarily dispersed by wind (Lewis, 1997
). Chan and Appanah (1980)
pointed out that the pollination of Shorea by thrips could be viewed as an intermediate strategy between wind pollination and insect pollination. Nason et al. (1998)
also reported that the pollen dispersal of figs (Ficus) in tropical forests was estimated to occur routinely over distances of 5.8–14.2 km, despite the minute size (1–2 mm) of their pollinator wasps (Aganoidae, Chalcidoidea), possibly because of passive wind-mediated transport. Therefore, small insects such as Staphylinidae and thrips may have been dispersed over long distances by wind while transporting M. stellata pollen on their bodies.
Comparison between indirect and direct estimates of pollen flow
Assuming the island model of migration with neither mutation nor selection, the level of genetic differentiation among populations for biparentally inherited markers, Fst(b), is given by Fst(b) = 1/(4Nmb + 1) (Wright, 1951
) and that for maternally inherited markers, Fst(m), is given by Fst(m) = 1/(2Nmm + 1) (Takahata and Palumbi, 1985
; Birky et al., 1989
), where N is the effective population size and mb and mm are migration rates for biparentally and maternally inherited markers, respectively. Using these formulas and the estimated FST values in this study, the estimated number of genes that migrate via pollen flow among populations per generation is 2.64 (2Nmb – 2Nmm = 8.94 – 6.30).
The number of gene that migrate via pollen flow among populations per generation can also be calculated from the directly estimated contemporary pollen flow in this study. Assuming that the size of population Y (84 adult genets) remains the same across generations and that there are no differences in mating patterns, fecundity, or the average survival rates of offspring among adult genets, fertilization by pollen flow from outside population Y will give rise to 2.5% of the adult genets. Thus, the estimated number of genes migrating among populations via pollen flow per generation is 2.10 (84 x 0.025).
The estimated number of genes that migrate via pollen flow based on FST values represents the effective number that migrated via historical pollen flow and is somewhat higher than the estimate based on contemporary pollen flow. However because the effective population size is usually smaller than the actual population size (the number of adults) (Frankham, 1995
), the effective number of genes that migrate via contemporary pollen flow would be lower than the above estimate (2.10). The high levels of genetic variation throughout all populations, irrespective of their size, suggest that a recent, rapid reduction in population size. Thus, the level of pollen flow among populations may have declined from historical levels. The estimated levels of gene flow based on FST values should be treated cautiously because this approach is based on unrealistic assumptions, such as equilibrium between genetic drift and gene flow in a large number of populations of constant size that never go extinct (Whitlock and McCauley, 1999
).
Importance of gene flow among populations
The results of this study indicate that seed dispersal among populations moderately reduced the extent of genetic differentiation among populations in both nDNA and cpDNA and that pollen flow among populations reduced the extent of genetic differentiation in nDNA. However, the extent of their differentiation was partially determined by the different magnitudes of genetic drift for nDNA and cpDNA. The gene flow patterns via seeds and pollen in M. stellata could be described as follows. At the among population level, gene flow by seeds occurs randomly, probably by birds, while at the within-population level, spatially distance-dependent gene dispersal by seeds occurs. In contrast, gene dispersal by pollen is restricted by spatial distance at both the within- and among-population levels.
We also indirectly evaluated the historical seed dispersal patterns within and among populations (see also Setsuko et al., 2004
). However, the contemporary seed dispersal pattern is still unresolved and needs to be evaluated in further studies by analyzing seeds dispersed by birds and dormant seeds in the soil (seed bank).
We have ascertained that the populations within the metapopulation are linked through gene flow. Gene flow among populations can compensate for losses of genetic variation by genetic drift, and it is important for the persistence of populations and ultimately, for conservation of the species, M. stellata. In attempts to conserve rare species, only specific, large populations are often designated as conservation targets. However, other unprotected areas are also important because nearby populations can provide sources of pollen and seeds with different genetic variations. To conserve M. stellata, it is therefore important to conserve the whole population by maintaining the metapopulation structure and gene flow between the local populations. The results of this study suggest that the level of pollen flow among populations has fallen recently, probably due to rapid reductions in population size, although our results are based on a single population in only 1 year. More detailed research is thus needed before definitive conclusions can be drawn. In some cases, artificial treatment to maintain the gene flow among populations may be necessary, e.g., thinning surrounding trees of other species may be beneficial if they are adversely affecting M. stellata habitats and thus reducing the frequency of gene flow among its populations.
FOOTNOTES
1 The authors are grateful to S. Yamamoto, A. Nakanishi, K. Hiraoka, and other members of the Laboratory of Forest Ecology and Physiology of Nagoya University for useful discussions and field assistance and to S. Hiroki, Y. Isagi, and A. Kanazashi for their support. They also thank the Aichi Prefectural Forest Office for permitting the study. This research was supported by Grants-in-Aid for Young Scientists (no. 17007746) and Scientific Research (nos. 14206017, 16380100) from the Japan Society for the Promotion of Science and a grant from the Ministry of the Environment of Japan. 
2 Author for correspondence (e-mail address: setsuko{at}agr.nagoya-u.ac.jp
)
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