Genetic structure and gene flow in a metapopulation of an endangered plant species,Silene tatarica

We investigated the distribution of genetic variation within and between seven subpopulations in a riparian population of Silene tatarica in northern Finland by using amplified fragment length polymorphism (AFLP) markers. A Bayesian approach-based clustering program indicated that the marker data contained not only one panmictic population, but consisted of seven clusters, and that each original sample site seems to consist of a distinct subpopulation. A coalescent-based simulation approach shows recurrent gene flow between subpopulations. Relative high FST values indicated a clear subpopulation differentiation. However, amova analysis and UPGMA-dendrogram did not suggest any hierarchical regional structuring among the subpopulations. There was no correlation between geographical and genetic distances among the subpopulations, nor any correlation between the subpopulation census size and amount of genetic variation. Estimates of gene flow suggested a low level of gene flow between the subpopulations, and the assignment tests proposed a few long-distance bidirectional dispersal events between the subpopulations. No apparent difference was found in within-subpopulation genetic diversity among upper, middle and lower regions along the river. Relative high amounts of linkage disequilibrium at subpopulation level indicated recent population bottlenecks or admixture, and at metapopulation levels a high subpopulation turnover rate. The overall pattern of genetic variation within and between subpopulations also suggested a 'classical' metapopulation structure of the species suggested by the ecological surveys.     

THE RELEVANCE OF GENE FLOW IN METAPOPULATION DYNAMICS OF AN OCEANIC ISLAND ENDEMIC,OLEA EUROPAEA SUBSP. GUANCHICA

Theoretical and empirical studies suggest that geographical isolation and extinction–recolonization dynamics are two factors causing strong genetic structure in metapopulations, but their consequences in species with high dispersal abilities have not been tested at large scales. Here, we investigated the effect of population age structure and isolation by distance in the patterns of genetic diversity in a wind‐pollinated, zoochorous tree (Olea europaea subsp. guanchica) sporadically affected by volcanic events across the Canarian archipelago. Genetic variation was assessed at six nuclear microsatellites (nDNA) and six chloroplast fragments (cpDNA) in nine subpopulations sampled on four oceanic islands. Subpopulations occurring on more recent substrates were more differentiated than those on older substrates, but within‐subpopulation genetic diversity was not significantly different between age groups for any type of marker. Isolation‐by‐distance differentiation was observed for nDNA but not for cpDNA, in agreement with other metapopulation studies. Contrary to the general trend for island systems, between‐island differentiation was extremely low, and lower than differentiation between subpopulations on the same island. The pollen‐to‐seed ratio was close to one, two orders of magnitude lower than the average estimated for other wind‐pollinated, animal‐dispersed plants. Our results showed that population turnover and geographical isolation increased genetic differentiation relative to an island model at equilibrium, but overall genetic structure was unexpectedly weak for a species distributed among islands. This empirical study shows that extensive gene flow, particularly mediated by seeds, can ameliorate population subdivision resulting from extinction–recolonization dynamics and isolation by distance.     

Comparative phylogeography of mainland and insular species of Neotropical molossid bats (Molossus)

Historical events, habitat preferences, and geographic barriers might result in distinct genetic patterns in insular versus mainland populations. Comparison between these two biogeographic systems provides an opportunity to investigate the relative role of isolation in phylogeographic patterns and to elucidate the importance of evolution and demographic history in population structure. Herein, we use a genotype‐by‐sequencing approach (GBS) to explore population structure within three species of mastiff bats (Molossus molossus, M. coibensis, and M. milleri), which represent different ecological histories and geographical distributions in the genus. We tested the hypotheses that oceanic straits serve as barriers to dispersal in Caribbean bats and that isolated island populations are more likely to experience genetic drift and bottlenecks in comparison with highly connected ones, thus leading to different phylogeographic patterns. We show that population structures vary according to general habitat preferences, levels of population isolation, and historical fluctuations in climate. In our dataset, mainland geographic barriers played only a small role in isolation of lineages. However, oceanic straits posed a partial barrier to the dispersal for some populations within some species (M. milleri), but do not seem to disrupt gene flow in others (M. molossus). Lineages on distant islands undergo genetic bottlenecks more frequently than island lineages closer to the mainland, which have a greater exchange of haplotypes.     

Contrasting demographic and genetic estimates of dispersal in the endangered Coahuilan box turtle: a contemporary approach to conservation

The evolutionary viability of an endangered species depends upon gene flow among subpopulations and the degree of habitat patch connectivity. Contrasting population connectivity over ecological and evolutionary timescales may provide novel insight into what maintains genetic diversity within threatened species. We employed this integrative approach to evaluating dispersal in the critically endangered Coahuilan box turtle (Terrapene coahuila) that inhabits isolated wetlands in the desert‐spring ecosystem of Cuatro Ciénegas, Mexico. Recent wetland habitat loss has altered the spatial distribution and connectivity of habitat patches; and we therefore predicted that T. coahuila would exhibit limited movement relative to estimates of historic gene flow. To evaluate contemporary dispersal patterns, we employed mark–recapture techniques at both local (wetland complex) and regional (intercomplex) spatial scales. Gene flow estimates were obtained by surveying genetic variation at nine microsatellite loci in seven subpopulations located across the species’ geographical range. The mark–recapture results at the local spatial scale reveal frequent movement among wetlands that was unaffected by interwetland distance. At the regional spatial scale, dispersal events were relatively less frequent between wetland complexes. The complementary analysis of population genetic substructure indicates strong historic gene flow (global F_ST = 0.01). However, a relationship of genetic isolation by distance across the geographical range suggests that dispersal limitation exists at the regional scale. Our approach of contrasting direct and indirect estimates of dispersal at multiple spatial scales in T. coahuila conveys a sustainable evolutionary trajectory of the species pending preservation of threatened wetland habitats and a range‐wide network of corridors.     

Isolated in an ocean of grass: low levels of gene flow between termite subpopulations

Habitat fragmentation is one of the most important causes of biodiversity loss, but many species are distributed in naturally patchy habitats. Such species are often organized in highly dynamic metapopulations or in patchy populations with high gene flow between subpopulations. Yet, there are also species that exist in stable patchy habitats with small subpopulations and presumably low dispersal rates. Here, we present population genetic data for the ‘magnetic’ termite Amitermes meridionalis, which show that short distances between subpopulations do not hinder exceptionally strong genetic differentiation (F_ST: 0.339; R_ST: 0.636). Despite the strong genetic differentiation between subpopulations, we did not find evidence for genetic impoverishment. We propose that loss of genetic diversity might be counteracted by a long colony life with low colony turnover. Indeed, we found evidence for the inheritance of colonies by so‐called ‘replacement reproductives’. Inhabiting a mound for several generations might result in loss of gene diversity within a colony but maintenance of gene diversity at the subpopulation level.     

Fine scale population genetic structure of pumas in the Intermountain West

In this study, I examined the population genetic structure of subpopulations of pumas (Puma concolor) in Idaho and surrounding states. Patterns of genetic diversity, population structure, levels of inbreeding, and the relationship between genetic differentiation and dispersal distance within and between 15 subpopulations of pumas were compared. Spatial analyses revealed that the Snake River plain was an important barrier to movement between northern and southern regions of Idaho. In addition, subpopulations south of the Snake River plain exhibited lower levels of genetic diversity, higher levels of inbreeding, and a stronger pattern of isolation by distance relative to subpopulations north of the Snake River plain. Lower levels of diversity and restricted gene flow are likely the result of historically lower population sizes in conjunction with more recent changes in habitat use and available dispersal corridors for movement. The subdivision of puma populations north and south of the Snake River plain, along with the patterns of genetic diversity within regions, indicate that landscape features are affecting the population genetic structure of pumas in Idaho. These results indicate that information about the effects of landscape features on the distribution of genetic diversity should be considered when designing plans for the management and conservation of pumas.     

Genetic variation,population structure and identification of yellow catfish, <Emphasis Type="Italic">Mystus nemurus</Emphasis> (C&;V) in Thailand using RAPD,ISSR and SCAR marker

Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to investigate the genetic structure of four subpopulations of Mystus nemurus in Thailand. The 7 RAPD and 7 ISSR primers were selected. Of 83 total RAPD fragments, 80 (96.39%) were polymorphic loci, and of 81 total ISSR fragments, 75 (92.59%) were polymorphic loci. Genetic variation and genetic differentiation obtained from RAPD fragments or ISSR fragments showed similar results. Percentage of polymorphic loci (%P), observed number of alleles, effective number of alleles, Nei’s gene diversity (H) and Shannon’s information index revealed moderate to high level of genetic variations within each M. nemurus subpopulation and overall population. High levels of genetic differentiations were received from pairwise unbiased genetic distance (D) and coefficient of differentiation. Mantel test between D or gene flow and geographical distance showed a low to moderate correlation. Analysis of molecular variance indicated that variations among subpopulations were higher than those within subpopulations. The UPGMA dendrograms, based on RAPD and ISSR, showing the genetic relationship among subpopulations are grouped into three clusters; Songkhla (SK) subpopulation was separated from the other subpopulations. The candidate species-specific and subpopulation-specific RAPD fragments were sequenced and used to design sequence-characterized amplified region primers which distinguished M. nemurus from other species and divided SK subpopulation from the other subpopulations. The markers used in this study should be useful for breeding programs and future aquacultural development of this species in Thailand.     

Population structure and dispersal patterns in Scottish Golden Eagles Aquila chrysaetos revealed by molecular genetic analysis of territorial birds

Conservation management of species distributed across fragmented habitats requires consideration of population genetic structure and relative levels of genetic diversity throughout the relevant geographical range. The Golden Eagle Aquila chrysaetos is monitored within Scotland to ensure its survival in the face of land‐use pressure, persecution and future climate change. In this study we constructed DNA profiles for 271 individual birds using a collection of over 1600 moulted feathers collected from 148 territories, representing 34% of known Scottish territories in the largest population genetic study of Golden Eagles undertaken to date. The results, based on data from 10 nuclear microsatellite loci, revealed previously unreported genetic structure between the islands of the Outer Hebrides and the rest of Scotland (F_ST = 0.03), together with evidence of reduced genetic diversity in the Outer Hebridean population compared with mainland Scotland. Analysis of gene flow supports a hypothesis of limited, predominantly male‐mediated, dispersal from the Outer Hebrides to mainland Scotland. The persistence of this pattern is discussed with respect to variation in population density and persecution pressure across Scotland. A finding of non‐random mating within the Outer Hebrides is interpreted as evidence of natal philopatry that was revealed by more intensive sampling in these islands, and is likely to be accentuated by the apparent degree of isolation of the islands from the rest of Scotland.     

Temporal change in the spatial genetic structure of a sika deer population with an expanding distribution range over a 15-year period

Since the 1980s, the sika deer (Cervus nippon Temminck, 1838) population of Hokkaido, Japan, has grown, resulting in range expansion. To assess the effects of this range expansion on the spatial genetic structure of the population, we compared subpopulation structures during 2 different periods (168 samples for 1991–1996, and 169 samples for 2008–2010), using mitochondrial DNA (mtDNA; D-loop) and microsatellites (9 loci). The number of gene-based subpopulations decreased across the 15-year period; specifically from four to three subpopulations based on mtDNA, and from two to one subpopulation based on microsatellite DNA. The fusion of the two northern subpopulations caused the change to the mtDNA-based structure, which might be explained by the dispersal of females from higher to lower density subpopulations. In comparison, the reason for the change in the microsatellite DNA-based structure was unclear, because no significant genetic differentiation was observed between the two study periods. A stable mtDNA-based structure was maintained in the north and central population separated by a west-to-east boundary, while a north-to-south boundary in eastern Hokkaido maintained stability in the eastern subpopulation versus all other subpopulations. The findings of this study demonstrate the importance of understanding gene flow within a structured population to implement effective management efforts; for instance, the culling of one subpopulation might not affect an adjacent subpopulation, because deer movement is limited between the subpopulations.     

Local genetic population structure in an endangered plant species, Silene tatarica (Caryophyllaceae)

Genetic substructuring in plant populations may evolve as a consequence of sampling events that occur when the population is founded or regenerated, or if gene dispersal by pollen and seeds is restricted within a population. Silene tatarica is an endangered, perennial plant species growing along periodically disturbed riverbanks in northern Finland. We investigated the mechanism behind the microspatial genetic structure of S. tatarica in four subpopulations using amplified fragment length polymorphism markers. Spatial autocorrelation revealed clear spatial genetic structure in each subpopulation, even though the pattern diminished in older subpopulations. Parentage analysis in an isolated island subpopulation indicated a very low level of selfing and avoidance of breeding between close relatives. The mean estimated pollen dispersal distance (24.10 m; SD = 10.5) was significantly longer and the mean seed dispersal distance (9.07 m; SD = 9.23) was considerably shorter than the mean distance between the individuals (19.20 m; SD = 13.80). The estimated indirect and direct estimates of neighbourhood sizes in this subpopulation were very similar, 32.1 and 37.6, respectively. Our results suggested that the local spatial genetic structure in S. tatarica was attributed merely to the isolation-by-distance process rather than founder effect, and despite free pollen movement across population, restricted seed dispersal maintains local genetic structure in this species.     

Relatedness structure in Rhododendron metternichii var. hondoense revealed by microsatellite analysis

The relatedness structure of Rhododendron metternichii Sieb. et Zucc. var. hondoense Nakai was analysed in a 150 x 70-m quadrat in Hiroshima Prefecture, western Japan. The population of R. metternichii occurred as three subpopulations at the study site (A1-A3). Pairwise relatedness based on microsatellite genotypes at eight loci and Mantel tests revealed a hierarchical structure of relatedness within and among subpopulations: (i) relatedness between individuals within 10 m of one another was significantly positive; (ii) relatedness between individuals in the same subpopulation was significantly positive, but negative between individuals in distant subpopulations; and (iii) relatedness was not significantly different from zero among neighbouring subpopulations. In detail, however, relatedness within each subpopulation was significantly positive in subpopulation A1, relatively weak but significantly positive in subpopulation A2, and not significantly different from zero in subpopulation A3. Relatedness within each subpopulation was inversely related to correlations between interindividual distance and relatedness. The aggregation of related individuals at short distances from one another may lead to decreasing relatedness within subpopulations as a whole. Moreover, negative correlations between interindividual distance and relatedness corresponded to high flowering densities at less than 10-m distance, implying that high flowering densities reduce pollinator foraging distance and lead to stronger genetic structure within subpopulations. Small individuals (< 2.0 m in height) showed stronger genetic structure compared with that of large individuals (> or = 2.0 m in height). The different relatedness structure within and among subpopulations may be caused by various degrees of gene flow affected by distribution patterns of individuals and population density.     

Effect of the landscape matrix on gene flow in a coastal amphibian metapopulation

Functional connectivity is crucial for the persistence of a metapopulation, because migration among subpopulations enables recolonization and counteracts genetic drift, which is especially important in small subpopulations. We studied the degree and drivers of connectivity among occupied patches of a coastal dune metapopulation of the Natterjack Toad (Epidalea calamita Laurenti), on the basis of microsatellite variation. As spatial landscape heterogeneity is expected to influence dispersal and genetic structure, we analyzed which landscape features affect functional connectivity and to what extent. Sixty different landscape resistance scenarios as well as the isolation-by-distance model were compared using two landscape genetics approaches. We identified three subpopulations with unidirectional levels of gene flow among the two most geographically separated subpopulations, while inferred gene flow into the geographically intermediate subpopulation was limited. Urbanization and vegetation height negatively affected connectivity. Low estimates of genetic diversity and effective population size indicate that conservation measures in the smallest and most isolated subpopulation are required.     

Temporal genetic dynamics among mosquitofish (<Emphasis Type="Italic">Gambusia holbrooki</Emphasis>) populations in invaded watersheds

The temporal components of genetic diversity and geographical structure of invasive mosquitofish populations are poorly known. Through the genetic monitoring of four consecutive cohorts of Gambusia holbrooki from three different river basins we aimed to determine temporal patterns of regional genetic variation and dispersal rates within invasive populations. Despite showing evidence of strong population size fluctuations, genetic diversity levels were maintained among local cohorts. We only detected temporal allele frequency changes associated with seasonal flooding that did not modify major trends on population structure among cohorts. Downstream gene flow coupled with increased connectivity at lowland locations to increase genetic diversity levels in these areas. A large proportion of local fish (up to 50 %) were dispersers, often originated from locations within the same river basin. High dispersal capability, ecological tolerance, and reproductive traits likely promote river colonization. Finally, our results also confirmed that human-assisted translocations promote within and among basin gene flow and maintained levels of genetic diversity, particularly in upstream locations.     

Large‐scale population genetic structure in Bonelli's Eagle Aquila fasciata

In bird species that have a high movement capacity, dispersal may connect subpopulations over vast geographical regions, with important consequences for the design of conservation management strategies. Here we used a molecular approach to infer the patterns and rates of dispersal among eight Mediterranean subpopulations of the endangered Bonelli's Eagle, based on 245 individuals screened at 17 microsatellite loci. There was moderate genetic differentiation between subpopulations sampled in the western (Iberia and Morocco) and eastern (Cyprus) Mediterranean, whereas differentiation among subpopulations in the former region was weak to moderate and followed a pattern of isolation by distance. Within the western Mediterranean, the small, peripheral and ecologically unique population of southwest Portugal had the lowest genetic diversity and the highest differentiation. The remaining subpopulations formed two loose clusters, one including Morocco and southwest and eastern Spain, and the other northeast Portugal and western and central Spain. Few recent migrants were detected, and they originated primarily from adjacent subpopulations. Our findings suggest that western Mediterranean Bonelli's Eagles may have a large‐scale metapopulation structure, with subpopulations connected to some extent by distance‐dependent dispersal, probably influenced by natal philopatry and the geographical configuration of subpopulations. The combination of marked ecological and genetic divergence suggests that the peripheral subpopulation of southwest Portugal may be regarded as a distinct management unit.     

SPECIES-WIDE POPULATION STRUCTURE IN A SOUTHEASTERN U.S. FRESHWATER FISH,HETERANDRIA FORMOSA: GENE FLOW AND BIOGEOGRAPHY

The phylogeography of the freshwater fish fauna of the southeastern United States has almost achieved paradigm status in evolutionary biology (Avise 1992), and the major geographic features responsible for shaping species distributions are well-characterized. Nevertheless, variation among species in distributions of allele or haplotype frequencies suggests that species-specific processes (e.g., migration) may also play a role in establishing those distributions. There has also been relatively little investigation into how population structure may differ among subregions in the Southeast, for example, on the Florida peninsula versus the U.S. mainland to the northwest and/or northeast. The geology of the peninsula is such that both physical and biotic fluctuations may have been (and still be) particularly important in establishing the population structure of freshwater taxa. This possibility leads to two interesting questions in population genetics. (1) Does gene flow in freshwater species of the region better approximate a one- or two-dimensional pattern? (2) Are populations on the peninsula farther from migration-genetic drift equilibrium than their counterparts on the mainland? These questions were addressed by examining the population strucuture of a livebearing fish, Heterandria formosa; several features of the biology of the species make it particularly likely that recent gene flow has been important in its evolution. I surveyed electrophoretic variation in 34 populations distributed throughout the species range. The phylogeographic patterns observed are in general concordance with those found in other species, although with some differences. A two-dimensional hypothesis of gene flow on the Florida peninsula better explains the data than does a one-dimensional one. There is no evidence that populations on the peninsula are farther from migration-drift equilibrium than those to the northwest. Populations in the northeast have lower genetic diversity than those to the south and west and show no isolation by distance; those results are consistent with a recent range expansion into the northeast, although smaller historical effective population sizes could also explain the pattern.     

Genetic structure of introduced populations: 120‐year‐old DNA footprint of historic introduction in an insular small mammal population

Wildlife populations have been introduced to new areas by people for centuries, but this human‐mediated movement can disrupt natural patterns of genetic structure by altering patterns of gene flow. Insular populations are particularly prone to these influences due to limited opportunities for natural dispersal onto islands. Consequently, understanding how genetic patterns develop in island populations is important, particularly given that islands are frequently havens for protected wildlife. We examined the evolutionary origins and extent of genetic structure within the introduced island population of red squirrels (Sciurus vulgaris) on the Channel Island of Jersey using mitochondrial DNA (mtDNA) control region sequence and nuclear microsatellite genotypes. Our findings reveal two different genetic origins and a genetic architecture reflective of the introductions 120 years ago. Genetic structure is marked within the maternally inherited mtDNA, indicating slow dispersal of female squirrels. However, nuclear markers detected only weak genetic structure, indicating substantially greater male dispersal. Data from both mitochondrial and nuclear markers support historic records that squirrels from England were introduced to the west of the island and those from mainland Europe to the east. Although some level of dispersal and introgression across the island between the two introductions is evident, there has not yet been sufficient gene flow to erase this historic genetic “footprint.” We also investigated if inbreeding has contributed to high observed levels of disease, but found no association. Genetic footprints of introductions can persist for considerable periods of time and beyond traditional timeframes of wildlife management.     

Scale‐dependent post‐establishment spread and genetic diversity in an invading mollusc in South America

Aims Our study aimed to characterize the dispersal dynamics and population genetic structure of the introduced golden mussel Limnoperna fortunei throughout its invaded range in South America and to determine how different dispersal methods, that is, human‐mediated dispersal and downstream natural dispersal, contribute to genetic variation among populations. Location Paraná–Uruguay–Río de la Plata watershed in Argentina, Brazil, Paraguay and Uruguay. Methods We performed genetic analyses based on a comprehensive sampling strategy encompassing 22 populations (N = 712) throughout the invaded range in South America, using the mitochondrial cytochrome c oxidase subunit I (COI) gene and eight polymorphic nuclear microsatellites. We employed both population genetics and phylogenetic analyses to clarify the dispersal dynamics and population genetic structure. Results We detected relatively high genetic differentiation between populations (F_ST = ?0.041 to 0.111 for COI, ?0.060 to 0.108 for microsatellites) at both fine and large geographical scales. Bayesian clustering and three‐dimensional factorial correspondence analyses consistently revealed two genetically distinct clusters, highlighting genetic discontinuities in the invaded range. Results of all genetic analyses suggest ship‐mediated ‘jump’ dispersal as the dominant mode of spread of golden mussels in South America, while downstream natural dispersal has had limited effects on contemporary genetic patterns. Main conclusions Our study provides new evidence that post‐establishment dispersal dynamics and genetic patterns vary across geographical scales. While ship‐mediated ‘jump’ dispersal dominates post‐establishment spread of golden mussels in South America, once colonies become established in upstream locations, larvae produced may be advected downstream to infill patchy distributions. Moreover, genetic structuring at fine geographical scales, especially within the same drainages, suggests a further detailed understanding of dynamics of larval dispersal and settlement in different water systems. Knowledge of the mechanisms by which post‐establishment spread occurs can, in some cases, be used to limit dispersal of golden mussels and other introduced species.     

Spatial genetic structure of a tropical understory shrub,PSYCHOTRIA OFFICINALIS (RuBIACEAE)

Analyses of fine-scale and macrogeographic genetic structure in plant populations provide an initial indication of how gene flow, natural selection, and genetic drift may collectively influence the distribution of genetic variation. The objective of our study is to evaluate the spatial dispersion of alleles within and among subpopulations of a tropical shrub, Psychotria officinalis (Rubiaceae), in a lowland wet forest in Costa Rica. This insect-pollinated, self-incompatible understory plant is dispersed primarily by birds, some species of which drop the seeds immediately while others transport seeds away from the parent plant. Thus, pollination should promote gene flow while at least one type of seed dispersal agent might restrict gene flow. Sampling from five subpopulations in undisturbed wet forest at Estación Biologíca La Selva, Costa Rica, we used electrophoretically detected isozyme markers to examine the spatial scale of genetic structure. Our goals are: 1) describe genetic diversity of each of the five subpopulations of Psychotria officinalis sampled within a contiguous wet tropical forest; 2) evaluate fine-scale genetic structure of adults of P. officinalis within a single 2.25-ha mapped plot; and 3) estimate genetic structure of P. officinalis using data from five subpopulations located up to 2 km apart. Using estimates of coancestry, statistical analyses reveal significant positive genetic correlations between individuals on a scale of 5 m but no significant genetic relatedness beyond that interplant distance within the studied subpopulation. Multilocus estimates of genetic differentiation among subpopulations were low, but significant (F_st = 0.095). Significant F_st estimates were largely attributable to a single locus (Lap-2). Thus, multilocus estimates of F_st may be influenced by microgeographic selection. If true, then the observed levels of IBD may be overestimates.     

Patterns and levels of gene flow in Rhododendron metternichii var. hondoense revealed by microsatellite analysis

Parentage analysis was conducted to elucidate the patterns and levels of gene flow in Rhododendron metternichii Sieb. et Zucc. var. hondoense Nakai in a 150 x 70 m quadrant in Hiroshima Prefecture, western Japan. The population of R. metternichii occurred as three subpopulations at the study site. Seventy seedlings were randomly collected from each of three 10 x 10 m plots (S1, S2, and S3) on the forest floor of each subpopulation (A1, A2, and A3). Almost all parents (93.8%) of the 70 seedlings were unambiguously identified by using 12 pairs of microsatellite markers. Within the quadrant, adult trees less than 5 m from the centre of the seedling bank (plots S1, S2, and S3) produced large numbers of seedlings. The effects of tree height and distance from the seedling bank on the relative fertilities of adult trees were highly variable among subpopulations because of the differences in population structure near the seedling bank: neither distance nor tree height had any significant effect in subpopulation A1; distance from the seedling bank had a significant effect in subpopulation A2; and tree height had a significant effect in subpopulation A3. Although gene flow within each subpopulation was highly restricted to less than 25 m and gene flow among the three subpopulations was extremely small (0-2%), long-distance gene flow from outside the quadrant reached 50%. This long-distance gene flow may be caused by a combination of topographical and vegetational heterogeneity, differences in flowering phenology, and genetic substructuring within subpopulations.