Habitat fragmentation influences genetic diversity and differentiation: Fine‐scale population structure of Cercis canadensis (eastern redbud)

Abstract Forest fragmentation may negatively affect plants through reduced genetic diversity and increased population structure due to habitat isolation, decreased population size, and disturbance of pollen‐seed dispersal mechanisms. However, in the case of tree species, effective pollen‐seed dispersal, mating system, and ecological dynamics may help the species overcome the negative effect of forest fragmentation. A fine‐scale population genetics study can shed light on the postfragmentation genetic diversity and structure of a species. Here, we present the genetic diversity and population structure of Cercis canadensis L. (eastern redbud) wild populations on a fine scale within fragmented areas centered around the borders of Georgia–Tennessee, USA. We hypothesized high genetic diversity among the collections of C. canadensis distributed across smaller geographical ranges. Fifteen microsatellite loci were used to genotype 172 individuals from 18 unmanaged and naturally occurring collection sites. Our results indicated presence of population structure, overall high genetic diversity (H E = 0.63, H O = 0.34), and moderate genetic differentiation (F ST = 0.14) among the collection sites. Two major genetic clusters within the smaller geographical distribution were revealed by STRUCTURE. Our data suggest that native C. canadensis populations in the fragmented area around the Georgia–Tennessee border were able to maintain high levels of genetic diversity, despite the presence of considerable spatial genetic structure. As habitat isolation may negatively affect gene flow of outcrossing species across time, consequences of habitat fragmentation should be regularly monitored for this and other forest species. This study also has important implications for habitat management efforts and future breeding programs.


| INTRODUC TI ON
Habitat fragmentation involves discontinuities in the distribution of an organism due to geographical and/or geological barriers, and/or human activities (Kolb & Diekmann, 2005;Kwak, Velterop, & Andel, 1998). For example, roadways have transected forests and imposed long, linear artificial structure to forested patches that, in turn, may be invaded by a mix of ornamental plants, ornamental plant cultivars, and their wild-type progeny (Hamberg, Lehvävirta, & Kotze, 2009;Hardiman & Culley, 2010;Kwak et al., 1998). Such fragmentation impacts the genetic diversity and population structure of a species in various ways depending on the ecology and biology of the species (Cuartas-Hernández & Núñez-Farfán, 2006;Suárez-Montes, Chávez-Pesqueira, & Núñez-Farfán, 2016). In a constantly changing natural setting, the dynamics of a plant population and ecological succession of plant species are shaped by the following three major challenges: species dispersal, establishment, and persistence in a specific environment (Weiher et al., 1999).
Habitat fragmentation may affect these processes through a number of ecological and biological modifications imposed on the fragmented area (Haila, 1999;Kolb & Diekmann, 2005;Suárez-Montes et al., 2016). For instance, pollination-and animal-based seed dispersal mechanisms are negatively impacted by forest fragmentation, especially at local levels (Dickson, 1990;Santos, Tellería, & Virgós, 1999;Sato & Kudoh, 2014). Disruptions in dispersal processes can reduce gene flow and increase inbreeding within these spatially isolated populations (Kearns, Inouye, & Waser, 1998;Van Geert, Rossum, & Triest, 2007). Loss of habitat, or even its degradation caused by fragmentation, can reduce the availability of suitable habitat and, therefore, negatively influence species establishment (Haila, 1999;Kolb & Diekmann, 2005). Finally, habitat fragmentation can lead to reduction in population size that results in reduced genetic variations, adaptive potential, and survival of the members in the smaller, isolated population (Sherwin & Moritz, 2000;Van Geert et al., 2007). Additionally, fragmentation reduces the natural habitat of a species, but also creates artificial edges that differ in plant composition from the rest of the forested area (Hamberg et al., 2009).
The presence of forest edges may increase the habitat area for different ecological niches typically found in higher numbers in these types of ecosystems (Hamberg et al., 2009).
In many cases, tree species in temperate forests are less likely to be impacted by genetic consequences of forest fragmentation compared to tropical tree species (Kramer, Ison, Ashley, & Howe, 2008). This outcome is partly explained by the higher tree density and undisturbed pollen-seed dispersal. This can, in turn, ensure sufficient gene flow across these isolated populations, thus reducing the potential threat of genetic declines to temperate tree species in North America (Byrne, Elliott, Yates, & Coates, 2008;Kramer et al., 2008;Nason, Herre, & Hamrick, 1998). Several economically and socially important tree species that have been extensively exploited as a result of logging and forest fragmentation have shown little to no genetic consequences of these demographic events challenging their sustainability (Marquardt, Echt, Epperson, & Pubanz, 2007;Victory, Glaubitz, Rhodes, & Woeste, 2006). In contrast, Taxus baccata L., a forest tree in Spain, was negatively affected by chronic fragmentation and revealed strong spatial structure with a recent bottleneck history in spite of abundant seed dispersal mechanisms (Dubreuil et al., 2010). Fragmented systems can result in a reduction in overall species health that can in turn restructure forest compositions in impacted communities and disrupt the ecosystem processes of tree species (Hall, Motzkin, Foster, Syfert, & Burk, 2002). Since the European settlement of the United States, more than 220 plant species have become extinct in North America and Hawaii (Noss, LaRoe, & Scott, 1995;Russell & Morse, 1992). Moreover, in the list of threatened and endangered species in the United States, 81% were affected by anthropogenic activities (Cook & Dixon, 1989;Noss et al., 1995).
In geographically reduced or fragmented populations, genetic diversity level can be affected through genetic drift and inbreeding, which can additionally reduce the ability of the affected individuals to regenerate and respond to changes of selection pressures (Hadziabdic et al., 2010;Suárez-Montes et al., 2016). Genetic drift and inbreeding in a plant species can erode the overall population fitness and the prospects for adaptive change, thus increasing the possibility of species decline, mortality, or extinction (Fischer & Matthies, 1998;Severns, 2003;Young, Boyle, & Brown, 1996). Due to constantly changing forest structures as a result of fragmentation, urbanization, and environmental conditions, knowledge of current genetic diversity and spatial structure of economically and/or environmentally important species is often unknown or limited. Here, we focused on the U.S. native Cercis canadensis L. (eastern redbud) wild or naturally occurring populations distributed across a smaller, fragmented geographical area around the Georgia and Tennessee border, USA.
To address the effect of fragmentation, in this study we used a native, understory tree Cercis canadensis (eastern redbud; Figure 1).
The tree is widely distributed across the eastern United States and extends into the northern part of central Mexico (Figure 2; Couvillon, 2002;Davis, Fritsch, Li, & Donoghue, 2002;Dirr, 1990). Cercis canadensis is a small-to-medium-size ornamental tree that presents an umbrella-shaped crown and foliage that exhibits varying colors across the season that ranges from deep purple, green to yellow (Pooler, Jacobs, & Kramer, 2002;Trigiano, Beaty, & Graham, 1988;Wadl, Trigiano, Werner, Pooler, & Rinehart, 2012). Characteristic heart-shaped leaves, wide range of foliage and floral colors, and early spring blooms make C. canadensis a popular ornamental landscape tree in the temperate North America (Figure 1; Dickson, 1990;Wadl et al., 2012). There are more than three dozen commercial cultivars of C. canadensis and other Cercis species currently available in retail and wholesale trade (Thammina, Kidwell-Slak, Lura, & Pooler, 2017;Wadl et al., 2012). Consequently, Cercis spp. cultivars have achieved an annual U.S. market value of 27 million USD (USDA, 2014).
In addition, C. canadensis is well adapted to mesic, semiarid to sometimes in xeric environments. This self-incompatible tree can grow as a shade-tolerant understory tree in closed forests (the mid-to deep southern United States), open woodlands, and forest borders (northern region of the species distribution) in full sunlight (Abram, 1986;Griffin, Ranney, & Pharr, 2004;Norcini, Knox, & Andersen, 1991;Pooler et al., 2002). The tree is commonly found on southward slopes with full sunlight and open forest edges where there are low levels of competition with other tree species. Also, C.
canadensis tolerates a wide range of soil types and pH throughout the species' wide geographical distribution (Dickson, 1990). Cercis canadensis is self-incompatible; therefore, it relies on pollen and potentially animal-mediated seed dispersal to enable reproduction (Roberts, Werner, Wadl, & Trigiano, 2015). Currently, our knowledge regarding the effects of forest fragmentation at local levels on the genetic diversity and spatial distribution of wild populations of C. canadensis is limited.
Given the popularity of this species as a landscape specimen, we investigated genetic diversity and spatial structure of wild C.
canadensis across a small geographical area in eastern Tennessee and around the Tennessee-Georgia border in the eastern United States. Over the last century, forest fragmentation has increased in this area because of regional urbanization and human infrastructure development (Lloyd, 2012;Zhang et al., 2012). Our hypothesis was that despite increased habitat fragmentation, wild populations of C. canadensis at local levels would have high genetic diversity with the presence of population structure and moderate-to-high gene flow. To test this hypothesis, previously developed microsatellite loci  were used to achieve the following objec-

| Sample collection
Samples of C. canadensis were collected from eastern Tennessee and around the Georgia-Tennessee border, USA, which is located near the center of the current native distribution range of the species F I G U R E 1 Flowers of Cercis canadensis in full bloom. Ramiflorous flowers of C. canadensis emerge in clusters directly from beneath bark on bare branches with flowering occurring prior to expansion of juvenile leaves F I G U R E 2 Geographical distribution of Cercis canadensis in the eastern United States (insert) and map of 18 collection sites used in this study ( Figure 2) and not far from The University of Tennessee, Knoxville laboratory (35°56′53.8″N; −83°56′28.9″W). Young and newly expanded leaves from 10 to 12 noncultivated (wild) trees per collection site were collected at 18 locations (Table S1; n = 180 trees).
From each tree, five to seven leaves were randomly collected from branches that were at cardinal directions around each tree. Leaves were placed in paper envelopes to dry and geographical coordinates were recorded for each sampled tree. Samples collected from each location were considered a single collection site.

| DNA extraction
DNA was isolated from approximately 100 mg of dried leaf tissue.
Samples were homogenized four times using a Bead Mill 24 (Fisher Scientific) with the settings of S (speed) = 6.00 m/s, T (time) = 30 s.
Between each homogenization step, samples were frozen in liquid nitrogen for 5 min to improve the tissue homogenization. Genomic DNA (gDNA) from each sample was isolated using the Qiagen DNeasy Plant Mini Kit (Qiagen) using the manufacturer's protocol with several modifications, which included the following: 2% w/v polyvinylpyrrolidone (PVP) was mixed into lysis buffer (AP1); 8 µl of RNase was added into each sample tube; the time period of incubation at 65°C was increased to 45 min and inverted gently to mix the sample every two minutes; incubation step at −20°C was followed with increased time of incubation to one hour. Additionally, before adding the elution buffer, ethanol was used to wash the spin columns if there was any debris left from the sample tissue. Finally, 50 μl of elution buffer preheated at 65°C was added, and the step was repeated twice, for a total elution of 100 μl. The quality and concentration of the isolated gDNA was assessed using ND1000 Visible (UV-Vis) Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA).

| Microsatellite primers and genotyping conditions
Primers for 68 genomic microsatellite loci  were synthesized by Integrated DNA Technologies (IDT). Because the 68 primers were developed for a cross-species amplification study among eight species representing the genus Cercis and another closely related species Bauhinia faberi Oliver , further screening was needed to optimize these primers for the wild C. canadensis samples used in this study. We used gDNA samples of five C. canadensis individuals from the University of Tennessee (UTK) Gardens to screen all of the primers used in this study. From our initial screening of the 68 microsatellite loci, 15 primer pairs were selected based on polymorphic loci, as well as a successful amplification rate across the five tested samples (Table 1).
Polymerase chain reaction (PCR) amplifications were completed in 10 µl reaction mixture consisting of the following: 1 µl undiluted gDNA, 1 µl of 10 µM of each forward and reverse primer, 0.5 µl of dimethyl sulfide, 4 µl of GoTaq G2 Hot Start Master Mix (Promega Corp), and 2.5 µl water. Both positive control (a DNA sample that amplified across all microsatellite loci) and negative control (control reaction without any DNA sample) samples were used for every primer tested to ensure validity of the data. Amplification of reactions was completed in 96 well plates using an Eppendorf thermocycler (Eppendorf AG) with the following thermal profile: initial denaturation at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s. and an extension at 72°C for 30 s, with a final extension of 72°C for 4 min. Amplified PCR products were visualized using a QIAxcel Capillary Electrophoresis System (Qiagen) and sized with a 15/600 bp internal alignment marker and a 25 to 500 bp DNA size marker. All 180 C. canadensis gDNA samples were tested against each of the 15 microsatellite loci using the procedure described above.
Failed reactions were repeated twice before considering them as missing data in the dataset.

| Genetic diversity
The FLEXBIN Excel macro version 2 (Amos et al., 2007) was used to bin raw alleles into statistically similar allelic classes. The binned allelic data were used for all statistical analyses. Samples were divided into the following two groups based on geographical distance from each other: the 10 collection sites from eastern Tennessee were combined in one group (north group) and the eight collection sites from the Georgia-Tennessee border into the second group (south group; Table   S1). To avoid overrepresentation of possible clonal samples in the dataset (e.g., originating from a planted cultivar, rather than a pollinated, wild-type tree), clone correction was completed using POPPR version 2.8.2 (Kamvar, Tabima, & Grünwald, 2014) in R (R Core Team, 2019) version 3.5.3. Only unique multilocus genotypes (MLGs) per collection site were used for further analyses so that unbiased allele frequency estimates could be obtained (Tsui et al., 2012). Eight samples were discarded due to missing data in more than 25% of loci resulting in 172 samples for all subsequent analyses.
Genetic diversity indices across 15 microsatellite loci and 18 collection sites of C. canadensis were calculated using package POPPR. For each microsatellite locus, the number of alleles, observed heterozygosity (H o ; calculated as the number of the individual heterozygotes present at a locus divided by sample size), and expected heterozygosity (H e ; expected heterozygosity per tested locus (Nei, 1978)) were estimated. Allelic richness (Ar; a measure of rarefied allelic counts per locus) is an estimation of the long-term potential of a population to adapt and persist in a given population, was calculated using package hierfstat (El Mousadik & Petit, 1996;Hurlbert, 1971;Petit, Mousadik, & Pons, 1998   the probability that two alleles at a random locus are from the same ancestor (Wright, 1990)) were also calculated using package hierfstat. In addition, the Shannon-Wiener diversity index (H) was estimated which combines both allele richness and evenness of the allele distribution (Shannon, 1948). H index value increases with increased richness and evenness. Moreover, as H is a logarithmic function, exp(H) will provide the number of expected alleles that are evenly distributed in the studied dataset (Grünwald, Goodwin, Milgroom, & Fry, 2003). Gene flow (Nm) among collection sites was estimated using GenAlEx 6.5 software (Peakall & Smouse, 2006 and was calculated as number of effective migrants per locus on the basis of F-statistics. Also, pairwise F ST among 18 collection sites were estimated using the R package adegenet version 2.1.1 (Jombart & Ahmed, 2011).  (Gao, Bryc, & Bustamante, 2011;Gao, Williamson, & Bustamante, 2007). This program considers the presence of clonal populations (K = 1) and disregards the assumption of Hardy-Weinberg equilibrium within groups of populations.

| Population structure
The following parameters were used in this analysis: 20 independent MCMC chains for each K value (1-18) with a burn-in period of 500,000 and 500,000 repetitions of the thinning interval using the admixture model. An admixture model was implemented to evaluate the proportion of mixed ancestry in an individual and improve the clustering (Lind and Gailing, 2013). Deviance information criterion (DIC) values were estimated to obtain an optimum K value (Gao et al., 2011).
Two model-free clustering approaches were implemented to delineate the population clusters. Nei's genetic distance was used to construct a neighbor-joining dendrogram. Discriminant analysis of principal components (DAPC), a model-free multivariate analysis approach, is a useful tool to investigate and visualize the presence of genetic clusters (Jombart, Devillard, & Balloux, 2010

| Demography
The program BOTTLENECK version 1.2.02 (Cornuet & Luikart, 1996) was implemented to investigate the evidence of a recent bottleneck.
To test whether a recent bottleneck or expansion of C. canadensis Significance of this test under either of these models was evaluated by the Wilcoxon sign-rank test with 10,000 simulations, as the number of loci for this study was under 20 (Cornuet & Luikart, 1996;Piry, Luikart, & Cornuet, 1999).   Private alleles (n = 15) were detected in 10 out of 18 collection sites (Table 2). When data were partitioned into two collection sites, 33 private alleles were detected (16 and 17 across the north and south groups, respectively;  Table S2). All of these five collection sites belonged to the south group.

| Population structure
Using the Bayesian clustering analysis, STRUCTURE results indi-  Table 3). When partitioned by two clusters according to STRUCTURE results, only 27.85% (p < .001) of the variation was present between the clusters, whereas 7% (p < .001) of the variation was attributed among collection sites within these two clusters (Table 3). The greatest level of variance was partitioned among two clusters-65.14% (p < .001). When the data were divided into two geographical groups (north and south), 14.88% (p < .001) of observed variation occurred between the groups, whereas only 14.33% (p < .001) of the variation was attributed among collection sites within these two groups (Table 3). The vast majority of the variance was present among individuals within collection sites (70.79%, p < .001; Table 3).
Isolation-by-distance analysis suggested that the geographical distance was linearly correlated (r = .39, p < .001) with genetic distance of the sampled data ( Figure S3). However, when analyzed separately by geographical groups, there was no evidence of correlation between genetic and geographical distance among C.
F I G U R E 3 STRUCTURE bar graph representing two genetic clusters (ΔK = 2) among 18 collection sites of Cercis canadensis. Each vertical bar represents an individual sample, and the color of the bar indicates the assignment probability of that individual to belong to one of the two identified clusters (designated by different colors). The geographical groups are designated as 1 for the north group and 2 for the south group in collection sites label in X-axis

| Population demography
Using the BOTTLENECK software with S.M.M., I.A.M., and T.P.M., data were partitioned and analyzed based on STRUCTURE results.
Sign tests revealed no significant excess of heterozygosity among C. canadensis collection sites for the analyzed loci. Also, the results indicated a normal L-shaped allele frequency distribution observed in both clusters, indicating that C. canadensis collection sites from eastern Tennessee and along the Georgia-Tennessee border have not been subjected to any recent bottleneck events (Table S3).

Long-term evolutionary success and ultimately species survival is in-
fluenced by population size, genetic diversity, allelic richness, fitness, and substantial gene flow, which are of fundamental importance in plant ecology, evolution, and conservation (Leimu, Mutikainen, Koricheva, & Fischer, 2006). Our study, which is part of a larger population assessment effort for C. canadensis in the United States, has revealed high levels of genetic diversity and allelic richness, moderate genetic differentiation, presence of genetic structure, and high gene flow between C. canadensis wild populations distributed within fragmented forest patches in eastern Tennessee and along the Georgia-Tennessee border. These results are congruent with other studies of temperate tree species, suggesting that populations of woody forest trees maintain high levels of genetic diversity at the population level compared to herbaceous plants (Chang, Bongarten, & Hamrick, 1998;Hamrick & Godt, 1996;Marquardt & Epperson, 2004;Victory et al., 2006).

F I G U R E 4
Neighbor-joining tree of 18 collection sites of Cercis canadensis (constructed and visualized using Nei's genetic distance). The geographical groups are designated as "1" for the north group and "2" for the south group in labels. Numbers indicate the percentage of bootstrap support using 1,000 replications | 3665 ONY et al.
kentukea, C. canadensis is widely distributed across the United States and found in a wide range of ecological habitats. Therefore, high genetic differentiation is probably related to tree reproductive biology, as well as a combination of other factors, including dispersal method and local isolations.
Based on our study results, we reject the hypothesis of limited gene flow as a result of increased distance between the examined groups of C. canadensis. Limited gene flow typically leads to reduced genetic diversity, increased population structure, and inbreeding within populations (Byrne et al., 2008;Sherwin & Moritz, 2000;Young et al., 1996), which were not consistent with our findings.
Other studies focused on forest trees in fragmented landscapes found that regardless of moderate-to-high habitat fragmentation, many insect-pollinated tree species were able to maintain high levels of gene flow across isolated patches through increased long distance pollen dispersal (Bacles, Burczyk, Lowe, & Ennos, 2005;Colabella, Gallo, Moreno, & Marchelli, 2014;Nason & Hamrick, 1997;Wang, Stephen, & Xiao-Yong, 2011). Our study findings are consistent with these studies as shown by the high level of gene flow present across fragmented C. canadensis populations. Therefore, forest fragmentation did not negatively influence the gene flow across isolated populations of C. canadensis in the eastern Tennessee and Georgia-Tennessee border.
Eastern redbud is similar to many other self-incompatible species (Roberts et al., 2015), wherein C. canadensis depends on insect-, mammal-, and bird-mediated pollination for dispersal. In its natural distribution pollinators of C. canadensis include honeybees, megachilid bees, small sweat bees, butterflies, and beetles (Dickson, 1990;Kraemer & Favi, 2010;Tucker, 2002). These insects are usually capable of flying one to several kilometers, depending on the insect species and environmental conditions, so pollen-based gene flow was expected to be high across fine-scale geographical ranges (Hagler, Mueller, Teuber, Machtley, & Deynze, 2011;Kramer et al., 2008;Pasquet et al., 2008). In a large, continuous forest area, the majority of the pollination that occurs by insects is among neighboring trees.
Although pollen dispersal by insects could be a primary medium of gene flow (Vekemans & Hardy, 2004), a substantial amount of C. canadensis seed movement depends on birds and F I G U R E 5 Discriminant analysis of principal components (DAPC) plots of Cercis canadensis individuals with 18 collection sites (a) and 12 collection sites (b). In plot A, the first 35 principal components explained 97.3% of the variation in C. canadensis individuals from all 18 collection sites. In plot A, allele 149 at locus 680a explained 9.55% of the variance and allele 95 at locus 220a explained 7.03% of the variance on the first axis (threshold = 0.07). In plot B, the first 24 principal components explained 87.8% of the variation in C. canadensis individuals from the 12 collection sites. In plot B, allele 108 at locus 220a explained 24.36% of the variance and allele 95 at locus 220a explained 11.51% of the variance on the first axis (threshold = 0.07). Datasets were cross-checked using 1,000 permutations. Discriminant Analysis (DA) eigenvalues are also presented in the plots TA B L E 3 Analysis of molecular variance (AMOVA) of Cercis canadensis across 15 microsatellite loci for (a) 18 collection sites into two groups according to two clusters of STRUCTURE and (b) 18 collection sites as two groups (north and south groups) other small mammals. The gene flow introduced via animal-mediated seed dispersal is therefore dependent upon the dispersal mode and behavior patterns of the animals that forage on the fruits and eat the seeds. Heavy C. canadensis fruits (pods) harbor seeds with a hard testa that usually fall in close proximity to the parent tree and then germinate within a year or two (Dickson, 1990;Hayden, 2013). Hence, some of the seedlings could grow in close proximity to the mother tree, which could potentially create half-sib neighborhoods on a small spatial scale (Gonzales, Hamrick, Smouse, Trapnell, & Peakall, 2009;Nakanishi, Tomaru, Yoshimaru, Manabe, & Yamamoto, 2008;Schnabel, Laushman, & Hamrick, 1991;Vekemans & Hardy, 2004). Assessments of pollen or seed dispersal patterns in C. canadensis were beyond the scope of this study; therefore, we can only provide this as a plausible explanation for our findings. An alternative explanation for presence of the half-sib neighbors is that fruits are also ingested by small rodents (e.g., gray squirrel, eastern woodrat), white-tailed deer, quail, pheasants, and several other bird species which are then dispersed in scat (Dickson, 1990;Post, 1992;Wakeland & Swihart, 2009;Wright, Fleming, & Post, 1990). Related individuals (from seeds that originated with the same mother tree) can be carried to a nearby location by an individual animal feeding on the fruits from the same mother tree. For example, the hoarding behavior of the rodents helps related propagules to be dispersed to a close-proximity destination (Post, 1992;Setoguchi, 1990). On the other hand, birds occasionally ingest C. canadensis seeds and can dispose of them in new localities across longer distances, resulting in an increased gene flow among the populations (Hadziabdic et al., 2012;Sullivan, 1994). The direction and rate of dispersal of such C. canadensis seeds would depend on the foraging behavior of the seed carrier and the ecological conditions of their habitat.

Source of variations
Although animals may occasionally choose C. canadensis seed pods as a food source during summer and winter months when other food resources are scarce (Post, 1992;Short & Epps, 1976), this is less common scenario for seed dispersal (Dickson, 1990;Halls & Crawford, 1960;Wakeland & Swihart, 2009). Moreover, C. canadensis seed dispersal that is mostly animal-dependent can become more limiting within isolated areas, further increasing half-sibling mating and structuring among C. canadensis populations (Koprowski, 2005). Therefore, our hypothesis was that spatial genetic structure of C. canadensis could be influenced by the forest fragmentation despite the presence of pollen-mediated gene flow in the small geographical location (Chung, Nason, Epperson, & Chung, 2003;Wang et al., 2011). Furthermore, our study revealed significant isolation by distance in local populations of C. canadensis and indicated that genetic distance between C. canadensis populations was significantly correlated with the geographical distance between them. Therefore, geographical distance or barriers may influence spatial genetic structure in C.
canadensis populations. Additional research undertaken to evaluate pollen and seed dispersal methods and distances covered by seed carriers would help articulate the gene flow mechanisms for this native tree species.
Our results indicated high genetic diversity and high gene flow, which suggests reproductive isolation of C. canadensis caused by fragmentation may not be of great concern for current populations. These findings are congruent with other population genetics studies of temperate, self-incompatible tree species. Also, current isolated patches and remnant populations are genetically stable allowing for maintenance of viable populations at a geographically fine-scale level. Although natural stands of C. canadensis seemingly maintain genetically fit populations, we cannot rule out the possible negative effect of forest fragmentation on C. canadensis population viability. As this is an outcrossing, self-incompatible, and animal-dispersed tree species, limitation or reduction in the number of seed dispersal agents (e.g., small rodents) in these fragmented populations can affect the fitness of C. canadensis populations in the future. Therefore, it is important to ensure that healthy and diverse habitats are present for animals responsible for seed and pollen dispersal of C. canadensis. Also, to better understand the effect of habitat fragmentation on C. canadensis, we suggest further studies be conducted on its sexual reproduction and life-history traits.
Over the past few decades, a number of studies have been conducted to better understand the genetic consequence of habitat fragmentation and anthropogenic disturbances on rare forest species. However, very few studies have been conducted to evaluate the effects of these disturbances on common forest species (Aguilar, Ashworth, Galetto, & Aizen, 2006). Our study showed the consequences of recent habitat fragmentations on an economically important and widely distributed native species C. canadensis. We suggest that despite the current resilient genetic diversity and high evolutionary potential, C. canadensis populations and other species in the fragmented habitats may suffer severe consequences following further environmental changes and climate threats. Therefore, to better understand the potential consequences of these threats, research on the population genetics of other concurring species is necessary for conservation efforts and habitat management of forest ecosystems. Our results also suggested that wild populations of C. canadensis with high genetic variations can be used as reservoirs of desired genetic variations and can be utilized in breeding programs to improve phenotypic traits in the nursery stocks.

CO N FLI C T O F I NTE R E S T
None declared.

AUTH O R S ' CO NTR I B UTI O N
DH, WK, and RT conceived and planned the experiments including the main conceptual ideas and proof outline. MO and SB carried out the experiments, MO, MN, SB, and DH troubleshot technical details.
All authors contributed to sample collection and preparation. MO, MN, JZ, and DH contributed to data analyses. All authors contributed to the interpretation of the results, manuscript writing, and editing. All authors provided critical feedback and helped shape the research, analysis, and manuscript.