**5. Conclusions**

Based on high-resolution SNPs, we compared the genetic response of *H. chrysotricha* to short-term and long-term habitat fragmentation. The levels of genetic diversity and the population substructure of *H. chrysotricha* populations were both higher in the TIL region than in the ZA region, while gene flow was higher and less asymmetric among ZA populations. The differences in genetic diversity between island and mainland populations in both regions were not significant, suggesting that genetic erosion of island populations does not increase as the spatial and temporal scales of fragmentation increase. Population connectivity in *H. chrysotricha* has also not been greatly modified by either short-term or long-term habitat fragmentation. The strong fruit (seed) dispersal capacity of *H. chrysotricha*, facilitated by the matrix of water in the island system, could buffer against the negative effects of habitat fragmentation and maintain the population connectivity over thousands of years.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants11141800/s1. Table S1: Raw statistical data for *Hedyotis chrysotricha* samples from the Thousand-Island Lake (TIL) and Zhoushan Archipelago (ZA) regions, generated by Illumina sequencing; Table S2: Genotype information for the 185 filtered SNPs from the TIL dataset; Table S3: Genotype information for the 188 filtered SNPs from the ZA dataset; Table S4: Migration rates (the fraction of individuals in population i (pop i) from population j (pop j)) and confidence intervals between populations of *H. chrysotricha* from the TIL region. Bold values represent the proportion of non-migrants within a population, and italic values indicate asymmetrical migration rates between populations; Table S5: Migration rates (the fraction of individuals in population i (pop i) from population j (pop j)) and confidence intervals between populations of *H. chrysotricha* from the ZA region. Bold values represent the proportion of non-migrants within a population, and values in italics indicate asymmetrical migration rates between populations; Figure S1: Analysis of isolation by distance based on *F*ST/(1 − *F*ST) and the geographic distance (log10 transformed) in (A) the Thousand-Island Lake (TIL) region and (B) the Zhoushan Archipelago (ZA) region; Figure S2: Histogram of the STRUCTURE analysis in the TIL region for the model with *K* = 2, 4, and 5. A vertical bar represents a single individual, and each color corresponds to a suggested cluster; Figure S3: Histogram of the STRUCTURE analysis in the ZA region for the model with *K* = 3–5. A vertical bar represents a single individual, and each color corresponds to a suggested cluster.

**Author Contributions:** N.Y. and Y.Q. conceived the study; N.Y., R.L. and S.L. collected the samples. N.Y. extracted the DNA samples. N.Y., S.W. and R.L. analyzed the genetic data and wrote the manuscript. H.P.C., R.L. and Y.Q. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Chinese National Natural Science Foundation (No. 31800312), the Jiangsu Innovative and Enterpreneurial Talent Programme (JSSCBS20211311), the Natural Science Foundation of Guangdong Province, China (No. 2021A1515010946), and the Forestry Science and Technology Innovation Project of Guangdong Province, China (No. 2020KJCX003).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** SNPs in VCF files across the Thousand-Island Lake (TIL) and the Zhoushan Archipelago (ZA) populations were deposited in Mendeley Data, V1, https://doi.org/10.1 7632/wsx8swxd27.1 (accessed on 22 May 2022).

**Acknowledgments:** Special thanks go to the anonymous referees for providing valuable and constructive comments to improve the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.
