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The study area is located in the Provincial Nature Reserve of V. mangachapoi, Wanning City, Hainan Province (China). Two V. mangachapoi populations (SM and RY) in the coastal forest separated by villages, roads and human facilities, and one population (TT) in the lowland rainforest near the coast were selected (Table 1, Fig. 2). In total, 188 V. mangachapoi trees individually spaced out more than 25 m apart and with DBH > 5 cm were sampled. Mature leaves lacking disease spots were selected, dried by silica gel and then were stored in a −20 °C refrigerator. The voucher specimens of V. mangachapoi were kept in Hainan University (Hainan, China).
Table 1. Genetic diversity indices of the three V. mangachapoi populations based on 12 SSR markers.
Population Location N Na Ne Ho He Fis SM 110.26691° E, 18.66671° N 91 8 3.647 0.547 0.690 0.207 RY 110.17952° E, 18.59768° N 39 7 3.704 0.605 0.700 0.142 TT 110.24941° E, 18.67744° N 58 7.5 3.694 0.566 0.692 0.167 Average 7.5 3.682 0.572 0.694 0.172 Figure 2.
Geographic distribution of the coastal V. mangachapoi-dominated forest and the locations of the three sampled V. mangachapoi populations. Gene flow among them was estimated, with the width of lines being proportional to the intensity of gene flow.
Genomic DNA extraction, PCR amplification and SSR genotyping
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A modified CTAB method[27] was used to extract the genomic DNA of V. mangachapoi. Twelve pairs of polymorphic SSR primers developed by Guo et al.[28] were used in this study. PCR amplification were performed in a total volume of 10 μL, containing 1.0 μL of genomic DNA (around 50 ng), 5.0 μL of Taq PCR Master Mix (GeneTech), 0.5 μL of forward and reverse primers, and 3.0 μL of ddH2O. Amplification was carried out as follows: pre-denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 20 s, annealing at 52−62 °C for 15 s, extension at 72 °C for 30 s, and finally extension at 72 °C for 7 min. PCR products were separated by capillary electrophoresis using ABI3730xl (Applied Biosystem) and SSR genotypes were analyzed by the GeneMarker software.
Data analysis
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Population genetic parameters, including number of alleles (Na), effective number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), inbreeding coefficient (Fis) and genetic differentiation among populations (
) were estimated using GenAlex 6.51[29]. Polymorphism information content (PIC) and Nei & Chessers[30] genetic distances were calculated by PowerMarker 3.25[31]. Analysis of molecular variance (AMOVA) of the V. mangachapoi population was performed using Arlequin 3.5[32]. Potential population bottleneck was examined by Wilcoxon sign-rank test and model shift using the Stepwise Mutation Model and Two-phased Mutation Model implemented in Bottleneck 1.3.2[33].$ {F}_{\mathrm{s}\mathrm{t}} $ Bayesian clustering analysis was performed using Structure 2.3.4[34]. The values of K were set from 1 to 10, and for each value of K, 10 independent replicates were run with 100,000 burn-in iterations followed by 200,000 MCMC (Markov chain Monte Carlo) iterations. The best K was determined according to the delta K of STRUCTURE Harvester[35]. The results of the 10 replicates were combined by the Greedy algorithm implemented in Clumpp1.1.2[36], and the result of individual clustering were drawn using Distruct1.1[37]. NJ trees were constructed using MEGA 11.0[38] based on Nei & Chessers[30] genetic distances. Principal co-ordinate analysis (PCoA) was performed with GenAlex 6.51.
The effective population size (θ) of the three V. mangachapoi populations and the migration rate (M) between them were calculated using MIGRATE[39], a software based on the coalescent theory and Bayesian inference to estimate values of parameters of a user-specified population model. MIGRATE analyses were run under a Brownian motion model, and four heat chains with different temperatures of 1.0, 1.5, 3.0 and 1.0 × 105 were simulated. Gene flow was calculated according to the equation Nm = θ*M/x. For SSR markers, x was set to 4. Three independent replications were run to ensure the convergence of the Markov chain Monte Carlo methods implemented in MIGRATE.
The fine-scale spatial genetic structure (FSGS) within-population was assessed using SPAGeDi 1.5[40]. The kinship coefficients (Fij, kinship coefficients) between any two individuals were calculated and regressed against the natural logarithm of the spatial distance to obtain the regression slope bF[41]. We divided the distance between any pair of individuals sampled from the SM population into 10 distance classes (35, 50, 75, 100, 150, 300, 500, 700, 850, 1,100 m), with at least 30 pairs of individuals per distance class[42, 43].
The 95% confidence intervals of Fij were calculated from 9,999 permutations of spatial distance among pairs of adults for 10 distance classes. If the Fij was higher than the upper bound of the 95% confidence interval, there is significant spatial genetic structure in population and a high level of genetic similarity among individuals; if the Fij fell within the 95% confidence interval, there is no spatial genetic structure in population and individuals were considered to be spatially randomly distributed; if the Fij was less than the lower bound of the 95% confidence interval, individuals were considered to be uniformly distributed in space without spatial genetic structure. The value of the Sp statistic reflects the strength of FSGS and is defined as Sp = -bF / (1-F(1) ), where bF is the regression slope, and F(1) is the mean pairwise kinship coefficient of the first distance class.
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There is no significant difference in the level of genetic diversity between the coastal (SM and RY) and the rainforest (TT) populations (Table 1). The inbreeding coefficient was greater than 0, indicating inbreeding and an excess of homozygotes in the three V. mangachapoi populations. Totally 90 alleles were detected from the 12 SSR loci, and the number of alleles at a single locus ranged from 4.667 to 11.000, with an average of 7.500 alleles per locus. The observed and expected heterozygosity ranged from 0.303 to 0.769 and from 0.415 to 0.808, respectively. The primer sequences, range of allele sizes and genetic diversity indices of the 12 SSR loci are shown in Table 2.
Table 2. Primer sequences, allele size and genetic diversity indices of the 12 SSR markers.
Loci Primer sequences (5’-3’) Repeat
motifAllele size GenAlex PowerMarker Na Ne Ho He PIC VM1 F:GAACCCTTATTGGCCTGCCTAC (AT)11 166−184 7.333 4.231 0.740 0.763 0.7430 R:GGGACCAAATGACTTGAGTAATCT VM2 F:ACCCTAACAATTCTCTTTGTTTCCT (TAA)11 152−195 9.667 4.120 0.513 0.755 0.7364 R:CCCCAATCTCAGTAAGGACTCA VM3 F:CTTGTGTCGAGCATGCATGTAT (AT)11 175−191 8.333 4.857 0.761 0.793 0.7659 R:TGCTGGCCTTTTATGTTAGGGT VM4 F:ATAGCAGGCACTTCGGAAGTAC (TA)8 261−277 8.667 4.613 0.370 0.781 0.7533 R:CCTGAGAAACAAAGCAACGCAT VM5 F:GCACTAGCACTAGCACTAGCTT (CT)11 218−226 4.667 2.908 0.629 0.651 0.6026 R:GGCTTTTCCAATTTCCATGGCT VM6 F:AGTTAAGGGACCAAATTTAGCGT (TA)7 259−269 5.000 2.794 0.593 0.636 0.5902 R:GTGTTTGTCAACTGGGCTTCAA VM7 F:CCCATGTGCTAGGCTAATGCTA (AT)6 229−239 5.000 2.394 0.303 0.582 0.5409 R:AAATCAGCATGAAACTTCTCCATT VM8 F:CACCACCACAGGCTTGAGTATA (TA)7 168−182 5.667 1.722 0.374 0.415 0.4044 R:GAAGGCCAACTAATCAAGCTGC VM9 F:TCATTTCTGTCTCACTCGACCC (TTC)10 148−168 5.667 3.010 0.639 0.666 0.6097 R:TCATCGACGAATCACTGTTCGA VM10 F:ACGGATAAGTTAACGGACTAGACA (TA)10 215−227 9.333 4.713 0.568 0.776 0.7997 R:AGATTTTCCCCCAGTCATCGAC VM11 F:GCTGGCACTTAGGATGCCTTAA (ATT)11 138−150 11.000 3.564 0.610 0.702 0.6657 R:AGCAACCAATTAGCTCAAATCAA VM12 F:GGGCAGCCTCGTAAATCAATTAC (ATT)13 225−249 9.667 5.253 0.769 0.808 0.7958 R:ATTACCTGGCACAACCTTAGCC Genetic differentiation was weak among the three V. mangachapoi populations (Fst = 0.008~0.013). The result of AMOVA showed that 99% of genetic variation was partitioned within population, in line with little divergence among populations (Supplemental Table S1).
The Wilcoxon sign rank test found that the p-values were not significant under either the S.M.M or the T.P.M model for the three V. mangachapoi populations, and their allele frequency distributions were generally L-shaped (Fig. 3), indicating that the three populations have not experienced genetic bottlenecks recently.
Population genetic structure
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STRUCTURE analysis found that delta K was maximized at K = 2, indicating two genetic clusters of the studied V. mangachapoi populations. The distribution of the two clusters did not differ significantly between the three populations, and this is also true for K = 3 or 5 (Fig. 4). Consistent with the results of STRUCTURE analyses, NJ tree (Supplemental Fig. S1) and PCoA analysis (Fig. 5) also suggest a homogeneous genetic structure of the three V. mangachapoi populations.
Figure 4.
Results of STRUCTURE analysis. (a) Best K determined using the delta K method. (b) Log probabilities and delta K values for K from two to ten. (c) The results of individual assignment at K = 2, 3 and 5. Each vertical bar represents an individual, and the proportion of the colors corresponds to the posterior probability of genetic clusters assigned to each individual.
Figure 5.
Principal co-ordinate analysis (PCoA) based on Nei & Chessers[30] genetic distance among individual samples of V. mangachapoi.
Gene flow and effective population size
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The effective population sizes of the three V. mangachapoi populations estimated by MIGRATE were similar, but the intensity of gene flow varied among them (Fig. 2, Table 3). The gene flows from RY to the other two populations were less than their reverse gene flows, however, the gene flows from SM to the other two populations were greater than their reverse gene flows. These results suggested that gene flows between the V. mangachapoi populations were asymmetric.
Table 3. Mutation-scaled migration rate, effective population size and gene flow estimated by program MIGRATE.
Direction of
gene flowMigration
rate (M)Effective population
size (θ)Gene flow (Nm) SM→RY 129.615 θSM = 0.09790 3.172327 SM→TT 179.839 4.401560 RY→SM 63.241 θRY = 0.09686 1.531380 RY→TT 117.490 2.845020 TT→SM 115.412 θTT = 0.09746 2.812013 TT→RY 134.062 3.266421 Fine-scale spatial genetic structure
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No spatial genetic structure was detected at any of the 10 distance classes in the SM population (Fig. 6). The values of Fij were less than zero over multiple distance classes, indicating that individual trees of V. mangachapoi were spatially uniformly distributed. Based on the mean affinity (F(1)) for the first distance class (0.0151) and the regression slope bF (−0.004605), the strength of FSGS (Sp) was derived as 0.004675 for the V. mangachapoi population in Shimei Bay.
Figure 6.
Fine-scale genetic structure of V. mangachapoi in Shimei Bay. The solid line represents the mean Kinship coefficient F (Loiselle et al.[41]), and the dashed lines represent the 95% confidence intervals of the mean Kinship coefficient F.
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The fragmented coastal V. mangachapoi-dominated forest in Shimei Bay have not yet exhibited significant genetic differentiation and diversity loss. The winged fruits of V. mangachapoi may promote seed dispersal and maintain gene flow between populations, which could mitigate genetic drift and lead to random distribution of genotypes within population. In addition, comparing with the generation time of V. mangachapoi, the time of fragmentation of the coastal V. mangachapoi forest is relatively short, so there is not enough time to accumulate differentiation for populations from the fragmented forest. Based on the above findings, we suggest to strengthen the protection of the coastal V. mangachapoi forest to prevent further deforestation. Besides, saplings of V. mangachapoi should be planted to connect isolated populations and facilitate the restoration of the unique coastal V. mangachapoi forest.
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About this article
Cite this article
Duan J, Wang H, Tang L. 2023. Effects of habitat fragmentation on the coastal Vatica mangachapoi forest (Dipterocarpaceae) in Shimei Bay, Hainan Island, China. Tropical Plants 2:8 doi: 10.48130/TP-2023-0008
Effects of habitat fragmentation on the coastal Vatica mangachapoi forest (Dipterocarpaceae) in Shimei Bay, Hainan Island, China
- Received: 06 May 2023
- Accepted: 14 June 2023
- Published online: 05 July 2023
Abstract: Habitat fragmentation can cause isolation and decline of a formerly continuously distributed population, which leads to loss of genetic variation and increased risk of extinction. Vatica mangachapoi Blanco is a dominant tree species growing in the lowland rainforests of Hainan Island, China. Remarkably, this species dominates a coastal forest in Shimei Bay, Wanning City of Hainan Province (China). Due to logging, expansion of farmland and villages, and construction of tourism facilities, the coastal V. mangachapoi-dominated forest has become fragmented, threatening its future. To evaluate the effects of habitat fragmentation on this unique coastal forest, two V. mangachapoi populations (SM and RY) along the coast and one population in the lowland rainforest near the coast were selected, and their genetic diversity was assessed based on 12 SSR markers. In addition, the genetic structure of the three populations and gene flow among them, and the fine-scale spatial genetic structure (FSGS) of the SM population were also studied. The results show that the three V. mangachapoi populations had comparable levels of genetic variation, and differentiation among them is negligible ($ {F}_{\mathrm{s}\mathrm{t}} $ = 0.008 ~ 0.013). Model-based clustering, Principal co-ordinate analysis and the Neighbor-joining (NJ) methods consistently support a homogeneous genetic structure of the three populations, and strong gene flow was detected among them by MIGRATE analyses. Moreover, there is no significant FSGS in the SM population. A relatively short time since habitat fragmentation and gene flow mediated by seed dispersal might be the likely reasons for the high levels of genetic variation and an absence of genetic structure of the coastal V. mangachapoi populations. In conclusion, even though there are no significant effects of fragmentation on the coastal V. mangachapoi forest, strict protection is required to prevent further deforestation and fragmentation. Besides, saplings of V. mangachapoi should be planted in forest gaps to reconnect fragments of the coastal forest, which would be of benefit for the long-term survival of the tropical coastal V. mangachapoi-dominated forest.