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Genetic Variation and Population Structure in Korean Populations of Eurya japonica (Theaceae)
by Myong Gi Chung, Soon Suk Kang
Genetic Variation and Population Structure in Korean Populations of Eurya japonica (Theaceae)
Myong Gi Chung, Soon Suk Kang
American Journal of Botany
Updated: December 11th, 2012
Department of Biology, Gyeongsang National University,
Chinju, 660-701, The Republic of Korea
We investigated levels of genetic diversity, population genetic structure, and gene flow in Eurya japonica, a widespread and broad-leaved evergreen dioecious tree native to Japan, China, Taiwan, and the southern and southwestern coast of the Korean Peninsula. Starch-gel electrophoresis was conducted on leaves collected from 1,000 plants in 20 Korean populations. All 12 loci examined were polymorphic in at least one population, and the mean number of alleles per locus was 3.79. In addition, mean observed population heterozygosity (Hop = 0.425), expected heterozygosity (He, = 0.462), and total genetic diversity (H, = 0.496) were substantially higher than average values for species with similar life history traits. Although significant differences in allele frequency were detected between populations at all loci (P < 0.001), <7O/o of the genetic variation was found among populations (F,, = 0.069). There was a significant negative correlation between genetic identity and distance between populations (r = -0.341; P < 0.05), but this explained only a small amount of the diversity among populations. Indirect estimates of the number of migrants per generation (Nm)(3.37, calculated from F,,; 3.74, calculated from the mean frequency of eight private alleles) indicate that gene flow is extensive among Korean populations of E. japonica. Factors contributing to the high levels of genetic diversity found within populations of E. japonica include large and contiguous populations, obligating outcrossing (dioecious plant), high fecundity, and long generation time. Occasional seed dispersal by humans and pollen movement by domesticated honey bees may further enhance gene flow within the species.
Enzyme electrophoresis has been used to describe the ture provide a basis on which to build sound programs levels and distribution of genetic variation and the pop- for the conservation of genetic diversity of rare and en- ulation genetic structure of a variety of groups of plants dangered species (Soul&, 1986; Hamrick et al., 199 1). In (Stoneburner, Wyatt, and Odrzykoski, 199 1). The accu- addition, allozyme diversity can be used as a yardstick mulation of this information has provided insights into to measure the effectiveness of in situ and ex situ con- the relationships between allozyme diversity and life his- servation programs (Hamrick et al., 199 1). tory traits (Brown, 1979; Gottlieb, 1981; Loveless and Despite the importance of knowledge concerning ge- Hamrick, 1984; Hamrick and Godt, 1989). Until the mid- netic variation for providing information for conservation 1980s, most studies of genetic variation in trees focused purposes, detailed studies of the levels and distribution on conifers. Like conifers, long-lived, widespread, wind- of genetic variation are not available for most native taxa pollinated, outcrossing species should maintain more ge- in Korea, particularly woody plants. Eurya japonica netic variation within populations and show less genetic Thunb. (Theaceae), a broad-leaved evergreen woody pe- differentiation among populations than do species with rennial, is widely distributed in Taiwan, China, Japan other combinations of life history and ecological traits (Honshu, Shikoku, Kushu, and Ryu Kyu Islands), and (Hamrick and Godt, 1989; Hamrick, Godt, and Sherman- the southern and southwestern coastal parts of the Korean Broyles, 1992). Recently, attention has focused on allo- Peninsula. Eurya japonica grows on hillsides with Pinus zyme studies of woody angiosperms that are comparable densifora, deciduous Quercus spp., Rhododendron spp., to conifers with respect to their ecological and/or life and broad-leaved evergreen woody species such as Litsea history traits (e.g., Schnabel and Hamrick, 1990a, b; Je- spp., Neolitsea spp., Persea spp., Quercus spp., and Calinski and Cheliak, 1992; Sherman-Broyles, Broyles, and mellia japonica (mainly in island populations in Korea). Hamrick, 1992). These studies have shown that such an- Eurya japonica is an abundant plant over its range in giosperms do have high levels of genetic variation and Korea (Chung and Kang, personal observation). The spe- low proportion of their genetic diversity among popula- cies is dioecious, but rarely individuals with a mixture of tions. Generalizations derived from the allozyme litera- male, female, and hermaphroditic flowers are found in
western Honshu, Japan (Murata et al., 1991). These in- constant plants have not been encountered during our
Manuscript received 31 August 1993; revision accepted 10 March
field observations in Korea. Eurya japonica is also an
The authors thank A. Schnabel, J. L. Hamrick, J. B. Mitton, C. R. economically important species in Korea, as the branches Parks, R. Wyatt, and an anonymous reviewer for comments on the have been used for making floral tributes and wreaths. manuscript; J. L. Hamrick, M. J. W. Godt, and S. Sherman-Broyles for These activities have disturbed the natural habitats of helping to set up the senior author's laboratory; A. Schnabel for providing
Korean euryas. In addition, until the past several years,
a program used to calculate the statistics of genetic diversity and struc-
most parts ofKorean forests had been disturbed by cutting
ture; and D. S. Ku for laboratory assistance. This research was supported
trees and shrubs for firewood in rural areas. Today most
by a nondirected research fund, Korea Research Foundation, 1992 to
MGC. Korean farests are revegetated naturally and artificially. Author for correspondence (FAX: 0591-54-0086). Based on recent reviews (Hamrick and Godt, 1989;
Fig. 1. The geographical distribution (dashed line) of Euryajaponica and the location of the 20 sampled populations (numerical codes as in Table 1) in Korea.
Hamrick, Godt, and Sherman-Broyles, 1992), the eco- logical and life history traits ofEurya japonica (i.e., mostly dioecious, large and contiguous populations, long-lived woody perennial, high fecundity) allow us to predict high genetic diversity within populations but low genetic dif- ferentiation between populations in this species. Here we report levels and partitioning of allozyme diversity within and among populations of Eurya japonica. The purpose of this study was: 1) to estimate how much total genetic diversity is maintained in the species; 2) to describe how genetic variation is distributed within and among pop- ulations; and 3) to compare the level of genetic diversity in populations of E. japonica with plant species having similar life history traits.
MATERIALS AND METHODS
Population samples-Twenty populations were sam- pled from throughout the geographical range of the species in Korea (Fig. 1). Eurya japonicagrows on coastal hillsides and reaches its greatest abundance in forests of Pinus densiflora. In all population samples, individuals tended to be roughly of equal size. The individuals collected were ca. 2-4 m high and 10-20 years old depending on pop- ulations. For all populations, leaves were collected from 50 individuals. Leaf samples were put individually in plastic bags and placed on ice, transported to the labo- ratory, and stored in the refrigerator for 4-5 days. Voucher specimens for each population were deposited in the Gyeongsang National University Herbarium (GNUC).
Enzyme extraction and electrophoresis -Enzyme extraction was done by grinding four finely cut leaves under liquid nitrogen with a mortar and pestle and mixing the resulting powder with a phosphate-polyvinylpyrrolidone extraction buffer (Mitton et al., 1979). The crushed extract
was absorbed onto 4 x 6-mm wicks cut from Whatmann 3 MM chromatography paper. Sample wicks were stored at -60 C until needed for analysis,Electrophoresis was performed using 10.5% starch gels. Putative gene loci from nine enzyme systems were resolved using five electro- phoretic buffer systems. Two discontinuous histidine ci- trate buffer systems were used: a modification of Soltis et al. (1983) system 11 (pH 6.8, 0.456 M anhydrous citric acid trisodium salt for only electrode buffer) was used to resolve isocitrate dehydrogenase (IDH) and phosphoglu- comutase (PGM), and buffer system 1 (Soltis et al., 1983) resolved 6-phosphogluconate dehydrogenase (PGD) and diaphorase (DIA). A Poulik buffer system, a modification (Haufler, 1985) of Soltis et al. (1983) system 8, resolved triosphosphate isomerase (TPI) and leucine aminopep- tidase (LAP). Two morpholine citrate buffer systems were used: a buffer system (pH 6.1) by Clayton and Tretiak (1 972) was used to resolve phosphoglucose isomerase (PGI) and peroxidase (PER); and a modification of Clayton and Tretiak (1972), pH 6.3, gel vs. electrode buffer (l:9, v/v), resolved malate dehydrogenase (MDH). The staining pro- cedures for DIA followed the method described by Che- liak and Pitel (1 984). All other stain recipes were identical to those described by Soltis et al. (1983). Putative loci were designated sequentially, with the most anodally mi- grating isozyme designated l, the next 2, and so on. Like- wise, alleles were designated sequentially with the most anodally migrating alleles designated a. All eurya iso- zymes, with the exception of Tpi-1, expressed phenotypes that were consistent in subunit structure and genetic in- terpretation with most isozyme studies in plants as doc- umented by Weeden and Wendel (1 989). The genetic basis ofthe Tpi-1 isozymes is at present unknown. In this region individuals contain highly variable and complicated two to four bands of approximately equal spacing. Similar results were observed for Camellia japonica (Theaceae) (Wendel and Parks, 1982). Lap-1 was being expressed, but it was not scored because of poor activity and/or resolution, and Pgi-1 could not be resolved using the methods.
Data analyses-A locus was considered polymorphic if two or more alleles were observed, regardless of their frequencies. Four genetic parameters were estimated using a computer program developed by M. D. Loveless and
A. Schnabel: percent polymorphic loci (P), mean number of alleles per locus (A), effective number of alleles per locus (Ae), and gene diversity (He) (Hamrick, Godt, and Sherman-Broyles, 1992). Subscripts refer to species (s) or population (p) level parameters.
Observed heterozygosity was compared to Hardy-Weinberg expected values using Wright's (1922) fixation indices (fl.These indices were tested for deviations from 0 by a x2-statistic following Li and Horvitz (1953).
Because He, and He, are equivalent to HT and Hs (Nei, 1973, 1977), respectively, and GsT is also equivalent to FsTas calculated in this paper, we evaluated the distri- bution of genetic diversity within and among populations using only Wright's (1965) F-statistics: FIT, FIs, and &,. The FITand FI,coefficients measure relative excesses of homozygotes or heterozygotes compared with panmictic expectations within the entire samples and within pop- ulations, respectively. The FsTcoefficient estimates rel-
TABLE1. Estimates of genetic variation within 20 populations of E.
L" Ha,,(SD) He, (SD) Po Ae, A,
Mean 0.425 (0.014) 0.462 (0.013) 94.17 2.09 3.79
Abbreviations (subscripts p refers to population level parameter): P, percentage of polymorphic loci; A, mean number of alleles per locus; Ae, effective number of alleles per locus; Ho, observed heterozygosity; He, Hardy-Weinberg expected heterozygosity or genetic diversity.
Numerical codes as in Fig. 1.
ative population differentiation. Deviations ofFIT and FIs from zero were also tested using the x2-statistic (Li and Horvitz, 1953). A x2-statistic was used to detect significant differences in allele frequencies among populations for each locus (Workman and Niswander, 1 970). Nei's (1 972) genetic identity (I) was calculated for each painvise com- bination of populations. A correlation between genetic identity and geographical distance was calculated using PC-SAS (SAS Institute, Inc., 1982). In addition, we used NTSYS (Rohlf, 1988) to conduct a cluster analysis on genetic identities via the unweighted painvise groups method using arithmetic average (UPGMA).
Two indirect estimates of gene flow were calculated. One estimate of Nm (the number of migrants per gen- eration) was obtained based on FsT(Wright, 195 1). The second estimate was based on the average frequency of private alleles (Slatkin, 1985; Barton and Slatkin, 1986).
Genetic diversity-Eurya japonica maintains high lev- els of genetic diversity both at the population (Table 1) and species level. All of the 12 putative isozyme loci surveyed were polymorphic in at least one population (P, = 100%). Seventy-one alleles were scored across all loci, indicating that, on average, slightly more than six alleles were found at each locus (Table 2). The effective number of alleles per locus (Ae,), however, was only 2.22, which highlights the fact that many of the alleles were present at very low frequencies. Total gene diversity (He,) was
0.496 (Table 2).
Allozyme diversity was also very high within popula- tion. In no population was the number of polymorphic loci fewer than 1 1, and in six populations polymorphism
TABLE2. Genetic structure for 12 polymorphic loci in E. jap~nica.~
Dia- 1 8 Idh- 1 2 Lap-2 5 Mdh- 1 6 Mdh-2 6 Per- 1 8 Pgd- 1 2 Pgd-2 8 Pgi-2 9 Pgm- 1 6 Pgm-2 6 Tpi-2 5
Abbreviations: He,, total genetic diversity; FITand F,,, deviations of genotype frequencies from Hardy-Weinberg expectations over all populations and within each population, respectively; F,,, proportion of the total genetic diversity partitioned among populations. Asterisks indicate F-coefficients significantly different from zero (** = P < 0.01;*** = P < 0.001).
was 100% (2, 8, 9, 16, 17, and 19) (Table I). The average number of alleles per locus was greater than 3.0 for all populations, and six populations had averages of 4.0 or greater (Table 1). Again, the mean effective number of alleles was considerably lower, averaging 2.09 for all pop- ulations (Table 1). Populations 3, 13, and 15 had the highest expected diversity (0.49-0.51), while 2, 19, and 20 had the lowest (0.40-0.42).
Genetic structure- Analysis of fixation indices, calcu- lated for all polymorphic loci in each population, showed an overall slight deficiency of heterozygotes relative to H-W expectations. Sixty-two percent of fixation indices were positive (141/226), and 29 of those departed sig- nificantly from zero (P < 0.05). In contrast, of 85 negative fixation indices, only two were significantly different from zero (P < 0.01), indicating an excess of heterozygosity at those loci and in those populations (Mdh-I in 12, Pgm-2 in 13).
Wright's F-coefficients showed, first of all, that signif- icant deficiencies of heterozygotes exist for eight and nine of the 12 loci at the level of population and the sample as a whole, respectively (Table 2). Only a single locus (Mdh-2) showed significant excess heterozygosity, and the two remaining loci (Per and Pgd-2) did not deviate from H-W expectations at either level. The values of FIs(Table 2) varied from 0.377 to -0.2 18. This range of values of the inbreeding coefficient were substantially greater than those expected, suggesting that the acting unknown evo- lutionary forces differ in their impacts upon 12 loci. Sig- nificant differences in allele frequencies among popula- tions were found for all 12 loci (P < 0.00 1 in each case). The FsTvalues ranged from 0.026 for Pgd-2 to 0.13 1 for Pgm-I (Table 2), and overall, slightly <94O/o of the total variation in the species is common to all populations. Eight private alleles were found in six populations: 14 (Mdh-la), 15 (Per-lb), 19 (Pgi-29, 20 (Dia-Ic and Per-lh), 12 (Pgi:da and Lap-2'), and 10 (Tpi-2'). The indirect estimate of gene flow based on the mean FsTwas high
NEI'S GENETIC IDENTITY
Fig. 2. Phenogram from UPGMA cluster analysis based on Nei's (1972) genetic identities between the 20 populations of Eurya japonica.
(Nm = 3.39) and very similar to the estimate based on private alleles (Nm = 3.74). Average genetic identity for all pairs of populations was
0.920 (SD = 0.024), well within the range of values ex- pected for conspecific populations (Crawford, 1989). The UPGMA dendrogram gave few insights into the genetic structuring of the 20 populations (Fig. 2). Although the tightest clusters (i.e., the closely spaced populations 3-6 and the two disjunct populations [19 and 201 from Cheju Island) were as expected, most other groupings show little relationship with the UPGMA dendrogram; the corre- lation between genetic identity and geographic distance was low (r = -0.341, df = 188, P < 0.05) and indicated that greater than 88% of the variation in genetic identities was due to unknown factors other than distance. Data on allele frequencies are too lengthy to include here, but these data are available upon request from the senior author.
Usually, species with large and contiguous populations, high fecundities, high rates of outcrossing, wind polli- nation, long generation times, and occurrence in late- successional phases maintain high genetic diversity (Loveless and Hamrick, 1984). Except for wind pollina- tion, E. japonica exhibits all of these traits. Populations
of E. japonica in Korea are large and contiguous (Chung and Kang, personal observation). Each female plant has hundreds of fruits (ca. 4-6 mm in diameter), and each fruit has ten to 20 small seeds. Although the seed dispersal mechanisms are unknown, numerous fruits have been observed near female plants, indicating that gravity may be a main seed dispersal mechanism. As E. japonica is dioecious, outcrossing rates should be high. The flowers are ca. 5 mm long and are visited by honey bees (Chung and Kang, personal observation). The observation of the annual rings in E. japonica examined revealed that the stems were at least 10-20 years old. However, it is highly probable that the ages of individuals are much older since 10-20-year-old new shoots possibly had been harvested for firewood for at least several hundred years. As indi- viduals of E. japonica are long-lived, opportunities for the accumulation of mutations should be high (Ledig, 1986). The initially raised genetic diversity through the process of recombination following mutation has been augmented by the ability to regenerate by stump-sprouting when harvested for firewood (S. Kim and G. Oh, Gyeong- sang National University, Korea, personal communica- tion). Eurya japonica is one of the important elements of the coastal forest vegetation that is in the late successional phases in Korea.
As expected, Eurya japonica maintains higher levels of allozyme variation than most other long-lived, woody perennials, averaging 65.0% polymorphic loci, 2.22 alleles per locus, 1.24 effective alleles per locus (Hamrick, Godt, and Sherman-Broyles, 1992). For E. japonica, P, is loo%, As is 6.17, and Ae, is 2.22. Genetic diversity at the species level (He,) in E. japonica (0.496) is also higher than the mean values for long-lived woody perennials (0.177), gymnosperms (0.169), long-lived woody widespread spe- cies (0.257), and long-lived woody outcrossing-animal
(0.21 1) and outcrossing-wind-pollinated species (0.173). The same trend is observed at the population level. Mean percent polymorphic loci (P,) for long-lived, woody pe- rennials is 49.39'0, mean number of alleles per locus (A,) is 1.76, and mean effective number of alleles per locus (Ae,) is 1.20 (Hamrick, Godt, and Sherman-Broyles, 1992). Within E. japonica populations, P, is 94.2%; A,, 3.79; and Ae,, 2.09. Eurya japonica also maintains higher amounts of genetic diversity (He, = 0.462) than most gymnosperms (mean He, = 0.15 1) and exceeds that of long-lived, woody angiosperms (mean He, = 0.143). In addition, the species with very similar life history char- acteristics (i.e., dioecy, insect pollination, and high fe- cundities) such as Gleditsia triacanthos (0.255, recalcu- lated only for polymorphic loci: Schnabel and Hamrick, 1990b) is less genetically diverse than E. japonica. It is of interest to note that Camellia japonica (0.3 13, recal- culated only for polymorphic loci: Wendel and Parks, 1985), almost the only well-studied Asian tree and an important element of coastal vegetations with E. japonica, was also found to have lower amounts of genetic diversity than that of E. japonica.
The present distribution pattern ofE. japonica in Korea could be explained by the Holocene paleoclimatic history of the Korean Peninsula. The supposed refugium for broad- leaved evergreen woody species such as E. japonica, C. japonica, and Quercus spp. originated presumably from South China in the Tertiary during the Riss-Wiirm in- terglacial stage was at least the central Korean Peninsula (Kim and Hong, 199 1). It is highly probable that the glacial remnants of these subtropical trees after the glacial Wiirm adapted to the warm southern coastal Korean Peninsula (the average annual temperature of this region ranges from 12 C to 15 C). Although this study was only conducted on the Korean populations, one edge of the distribution of a very wide-ranging E. japonica, the levels of genetic variation were high and comparable with those for several conifers. With relatively high evolutionary potential, pop- ulations of E. japonica, like Pinus spp. in Korea, are contiguous and individuals are abundant in this region. In this regard, an investigation into the biology of the whole species of E.japonica and other very wide-ranging and abundant, broad-leaved evergreen woody species in eastern Asia, a group we know very little about, would be a significant contribution of our knowledge of woody angiosperms (C. R. Parks, University of North Carolina, personal communication). This study is now in progress.
In general, many outcrossing species show heterozy- gosities that are lower than expected, despite theoretical predictions that heterozygosity should be favored in out- crossing plants (Brown, 1979). An overall slight deficiency of heterozygotes was observed in E. japonica (Table 2). Although E. japonica is dioecious, the heterozygote de- ficiencies in several populations and at several loci (see Results) indicate that consanguineous matings might oc- cur within E.japonica populations. Numerous fruit have been observed near female plants, indicating the lack of specialized seed dispersal mechanisms in the species. This may favor the establishment of clusters of related indi- viduals which could lead to partial inbreeding (Hamrick, 1982) and/or create a Wahlund effect (Hart1 and Clark, 1989) causing heterozygote deficiencies.
Genetic differentiation among populations is princi- pally a function of gene flow among populations via pollen and seeds dispersal (Loveless and Hamrick, 1984; Ells- trand and Marshall, 1985). Species with more continu- ously distributed or contiguous populations should ex- perience more gene flow than species with discrete, isolated populations and therefore have relatively higher levels of variation within populations and lower variation among populations (Gibson and Hamrick, 1991). Of the total variation observed in E. japonica, <7% is due to differ- ences among populations (FsT = 0.069). This low level ofgenetic differentiation also suggests that gene flow among populations is high. Indirect gene flow estimates of Nm based on FsT-values and private alleles (3.37 and 3.74, respectively) were high. Hamrick (1987) reported an av- erage estimate of 1.15 and 0.80, respectively, for the num- ber of migrants per generation for 16 outcrossing, animal- pollinated species. Nm values >1 can be considered high, and, as a result, genetic drift should not be a major factor in such populations (Slatkin, 1987). Thus, the levels of gene flow we have calculated are of sufficient magnitude to counterbalance genetic drift and may play a major role in shaping the genetic structure of the populations. The high level of gene flow may be explained in part by the natural history information about seed and pollen dis- persal. For example, the periods of fruit maturation of E.
japonica is from October to December, and the matured
fruits are easily detached by slight hand-touch. Although
the branches and stems have been cut, sites may be re- populated by numerous fruits within stands (Chung and Kang, personal observation). It was found that numerous seedlings of E.japonica grow within its stands. These cut branches and stems have often been carried from hillside to nearby farmhouse during the past several hundred years
(G. Oh, Gyeongsang National University, personal com- munication). This occasional cutting and carrying of fruit- bearing branches and stems may have resulted in sec- ondary seed dispersal into adjacent areas. In addition, apiculture on hillsides has been common in Korea since earlier this century, and Korean apiculturists have moved honey bees from place to place to forage on early spring flowers, including E. japonica (D. S. Ku, Gyeongsang National University, personal communication). It is high- ly probable that gene dispersal, mediated by pollen trans- fer on bees, has also been promoted in this way.
It is of interest to note that genetic structure in E. japonica is more similar to that of gymnosperms (mean GsT= 0.073) with characteristics that promote very high gene flow (Hamrick, Godt, and Sherman-Broyles, 1992). Similar results were observed for Gleditsia triacanthos (FsT=0.059;Nm = 3.987: Schnabel and Hamrick, 1990b). Although the geographical range of Camellia japonica is similar to that of E. japonica (the former species is not native to China), populations of C. japonica showed sub- stantially higher levels of genetic differentiation (FsT = 0.144; Nm = 1.486: Wendel and Parks, 1985). Camellia japonica, like E.japonica, is a long-lived, perennial, wide- spread, insect-pollinated tree species. Nevertheless, two life history traits differ strikingly between two species. First, the fecundity of C.japonica is substantially lower than that ofE. japonica. For C. japonica, each adult plant bears at best less than 100 fruits, and each fruit usually contains one to three seeds (Chung and Kang, personal observation). It was often a task to obtain seeds from 20 fruiting trees even in a very large population in Japan (C.
R. Parks, personal communication). Second, Eurya ja- ponica is dioecious, whereas flowers of C. japonica are perfect. These two factors may account for the substan- tially lower levels of genetic differentiation observed in E.japonica.
The UPGMA and correlation analysis show very weak correspondence between genetic distance and geograph- ical distance. Only three most isolated populations (1, 19, and 20) give any hint of a relationship between geographic and genetic distance. Mean genetic identity between all populations was relatively low (I = 0.920; SD = 0.024). With the exception of the three isolated populations, pop- ulations of E.japonica are contiguous, which might result from life history traits and historical events (e.g., the Ho- locene paleoclimatic history) of the species, and appear to be genetically linked by periodic gene flow, as in a "stepping stone" model, resulting in higher levels of ge- netic diversity within populations and lower levels of genetic differentiation among populations.
In summary, E. japonica maintains high levels of ge- netic diversity and exhibits lower levels of population differentiation than expected on the basis of its life history traits. Higher levels of gene flow in the past may be one of the dominant effects shaping the current genetic struc- ture of the species. Factors affecting the high levels of genetic diversity found within E. japonica populations include large and contiguous populations, dioecy, high fecundity, long generation time, and occurrence in late- successional forests. In addition, occasional seed dispersal by humans and pollen transfer by domesticated honey bees may enhance gene flow within the species.
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