Intraspecific Phylogenetic Relationships of Caryopteris incana in the Tsushima Islands, Japan, Using DNA Sequence Analysis


Caryopteris incana is a perennial shrub distributed in the temperate zone of the East Asia. It is found in West Kyushu in Japan, where it is designated as an endangered species. Tsushima, Nagasaki, which experienced repeated connection and fragmentation between the Korean Peninsula and Japan, is an island on the route along which C. incana moved to Japan from continental Asia. We conducted field work and confirmed the genetic structure of populations using DNA sequence analysis to construct a detailed distribution map and clarify the intraspecific phylogenetic relationships of C. incana in Tsushima Island. We confirmed 72 populations in Tsushima. Using the leaves of individuals cultivated from seeds collected from each natural population, we analyzed the chloroplast and nuclear DNA sequence variations. Among the populations, sequence variations were confirmed in six regions of chloroplast DNA, and six haplotypes, including base substitutions, were distinguished. Two haplotypes were mainly divided at the border of the northern part of the southern island in Tsushima. One population in the northwestern part of the north island showed a haplotype derived from the southern part. This finding revealed that the distribution of C. incana had been artificially influenced. Several haplotypes were confirmed by sequence variations in the northern populations, but only one haplotype in the southern populations, suggesting that C. incana on the north island had separated early from the south island in Tsushima.

Share and Cite:

Ando, M. , Watanabe, H. , Matsubara, K. and Taniguchi, A. (2015) Intraspecific Phylogenetic Relationships of Caryopteris incana in the Tsushima Islands, Japan, Using DNA Sequence Analysis. American Journal of Plant Sciences, 6, 2361-2373. doi: 10.4236/ajps.2015.614239.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Darwin, C. (1859) On the Origins of Species by Means of Natural Selection. John Murray, London.
[2] Avise, J.C., Arnold J., Ball, R.M., Bermingham, E., Lamb, T., Neigel, J.E., Reeb, C.A. and Saunders, N.C. (1987) Intraspecific Phylogeography: The Mitochondrial DNA BRIDGe between Population Genetics and Systematics. Annual Reviews of Ecology and Systematics, 18, 489-522.
[3] Hewitt, G.M. (1996) Some Genetic Consequences of Ice Ages, and Their Role in Divergence and Speciation. Biological Journal of the Linnean Society, 58, 247-276.
[4] Medail, F. and Diadema, K. (2009) Glacial Refugia Influence Plant Diversity Patterns in the Mediterranean Basin. Journal of Biogeography, 36, 1333-1345.
[5] Comes, H.P. and Kadereit, J.W. (1998) The Effect of Quaternary Climatic Changes on Plant Distribution and Evolution. Trends in Plant Science, 3, 1360-1385.
[6] Qiu, Y.X., Fu, C.X. and Comes, H.P. (2011) Plant Molecular Phylogeography in China and Adjacent Regions: Tracing the Genetic Imprints of Quaternary Climate and Environmental Change in the World’s Most Diverse Temperate Flora. Molecular Phylogenetics and Evolution, 59, 225-244.
[7] Bai, W.N., Liao, W.J. and Zhang, D.Y. (2010) Nuclear and Chloroplast DNA Phylogeography Reveal Two Refuge Areas with Asymmetrical Gene Flow in a Temperate Walnut Tree from East Asia. New Phytologist, 188, 892-901.
[8] Aoki, K., Suzuki, T., Hsu, T.W. and Murakami, N. (2004) Phylogeography of the Component Species of Broad-Leaved Evergreen Forests in Japan, Based on Chloroplast DNA Variation. Journal of Plant Research, 117, 77-94.
[9] Nakanishi, H. (2010) Distribution and Ecology of Islet Biased Plants in northern Kyushu, Japan. Vegetation Science, 27, 1-9.
[10] Itow, S. (1997) A Review of Phyto- and Vegetation Geography in the Japan-Korea Strait Region. Bulletin of the Faculty of Liberal Arts, Nagasaki University, Natural Science, 38, 25-51.
[11] Nakanishi, H. (1996) Plant Species with Northbound Distribution in Western-Kyushu, Japan: Definition, Composition and Origin. Acta phytotaxonomica et Geobotanica, 47, 113-124.
[12] Abu-Asab, M.S., Cantino, P.D., Nowicke, J.W. and Sang, T. (1993) Systematic Implications of Pollen Morphology in Caryopteris (Labiatae). Systematic Botany, 18, 502-515.
[13] Cantino, P.D., Wagstaff, S.J. and Olmstead, R.G. (1999) Caryopteris (Lamiaceae) and the Conflict between Phylogenetic and Pragmatic Considerations in Botanical Nomenclature. Systematic Botany, 23, 369-386.
[14] Gao, J. and Han, G. (1997) Cytotoxic Abietane Diterpenoids from Caryopteris incana. Phytochemistry, 44, 759-561.
[15] Gao, J., Igalashi, K. and Nukina, M. (1999) Radical Scavenging Activity of Phenylpropanoid Glycosides in Caryopteris incana. Bioscience, Biotechnology, and Biochemistry, 63, 983-988.
[16] Gao, J., Igalashi, K. and Nukina, M. (2000) Three New Phenylethanoid Glycosides from Caryopteris incana and Their Antioxidative Activity. Chemical and Pharmaceutical Bulletin, 48, 1075-1078.
[17] Li, J. and Wang, Y. (2004) Synthesis of Trisaccharide of Incanoside from Caryopteris incana. Synthetic Communications, 34, 515-522.
[18] Zhao, D.P., Matsunami, K. and Otsuka, H. (2009) Iridoid Glucoside, (3R)-oct-1-en-3-ol Glycosides, and Phenylethanoid from the Aerial Parts of Caryopteris incana. Journal of Natural Medicines, 63, 241-247.
[19] Chu, S.S., Liu, Q.Z., Zhou, L., Du, S.S. and Liu, Z.L. (2011) Chemical Composition and Toxic Activity of Essential Oil of Caryopteris incana against Sitophilus zeamais. African Journal of Biotechnology, 10, 8476-8480.
[20] Itow, S. and Kawasato, H. (1988) The Distribution and Ecology of Caryopteris incana Maxim. (Verbenaceae) in Western Kyushu, Japan. Hikobia, 10, 135-143.
[21] Itow, S., Kim, C.S., Kim, M.H., Kawasato, H. and Oh, J.G. (1993) Biogeography and Ecology of Caryopteris incana Maxim. (Verbenaceae) in Regions of Japan-Korea Strait. Bulletin of the Faculty of Liberal Arts, Nagasaki University, Natural Science, 34, 45-50.
[22] Environment Agency of Japan (2000) Threatened Wildlife of Japan, Red Data Book 2nd Edition. Japan Wildlife Research Center, 8, 521.
[23] Doyle, J.J. and Doyle, J.L. (1987) A Rapid DNA Isolation Procedure for Small Quantities of Fresh Leaf Tissue. Phytochemical Bulletin, 19, 11-15.
[24] Lassner, M.W., Peterson, P. and Yoder, J.I. (1989) Simultaneous Amplification of Multiple DNA Fragments by Polymerase Chain Reaction in the Analysis of Transgenic Plants and Their Progeny. Plant Molecular Biology Reporter, 7, 116-128.
[25] Hilu, K.W. and Liang, H.P. (1997) The matK Gene: Sequence Variation and Application in Plant Systematics. American Journal of Botany, 84, 830-839.
[26] Shi, S., Du Y., Boufford, D.E., Gong, X., Huang, Y., He, H. and Zhong, Y. (2003) Phylogenetic Position of Schnabelia, a Genus Endemic to China: Evidence from Sequences of cpDNA matK Gene and nrDNA ITS Regions. Chinese Sci- ence Bulletin, 48, 1576-1580.
[27] Taberlet, P., Gielly, L., Pautou, G. and Bouvet, J. (1991) Universal Primers for Amplification of Three Non-Coding Regions of Chloroplast DNA. Plant Molecular Biology, 17, 1105-1109.
[28] Drew, B.T. and Sytsma, K.J. (2011) Testing the Monophyly and Placement of Lepechinia in the Tribe Mentheae (Lamiaceae). Systematic Botany, 34, 1038-1049.
[29] Shaw, J., Lickey, E.B., Schilling, E.E. and Small, R.L. (2007) Comparison of Whole Chloroplast Genome Sequences to Choose Noncoding Regions for Phylogenetic Studies in Angiosperms: The Tortoise and the Hare III. American Journal of Botany, 94, 275-288.
[30] Drew, B.T. and Sytsma, K.J. (2012) Phylogenetics, Biogeography, and Staminal Evolution in the Tribe Mentheae (Lamiaceae). American Journal of Botany, 99, 933-953.
[31] White, T.J., Bruns, T., Lee, S. and Taylor, J. (1990) Amplification and Direct Sequencing of Fungal Ribosomal RNA Genes for Phylogenetics. In: Innis, M.A., Gelfand, D.H., Shinsky, J.J. and White, T.J., Eds., PCR Protocols: A Guide to Methods and Applications, Academic Press, Inc., New York, 315-322.
[32] Steane, D.A., Scotland, R.W., Mabberley, D.J. and Olmstead, R.G. (1999) Molecular Systematics of Clerodendrum (Lamiaceae): ITS Sequences and Total Evidence. American Journal of Botany, 86, 98-107.
[33] Huang, M., Crawford, D.J., Freudenstein, J.V. and Cantino, P.D. (2008) Systematics of Trichostema (Lamiaceae): Evidence from ITS, ndhF, and Morphology. Systematic Botany, 33, 437-446.
[34] Hall, T.A. (1999) BioEdit: A User-Friendly Biological Sequence Alignment Editor and Analysis Program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41, 95-98.
[35] Tamura, K., Stecher, G., Peterson, D., Filipski, A. and Kumar, S. (2013) MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Molecular Biology and Evolution, 30, 2725-2729.
[36] Huson, D.H. (1998) Splitstree: Analyzing and Visualizing Evolutionary Data. Bioinformatics, 14, 68-73.
[37] Charlesworth, D. and Charlesworth, B. (1987) Inbreeding Depression and Its Evolutionary Consequences. Annual Review of Ecology and Systematics, 18, 237-268.
[38] Keller, L.F. and Waller, D.M. (2002) Inbreeding Effects in Wild Populations. Trends in Ecology and Evolution, 17, 230-241.
[39] Stephens, P.A., Sutherland, W.J. and Freckleton, R.P. (1999) What Is the Allee Effect? Oikos, 87, 185-190.

Copyright © 2022 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.