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

Abstract

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.

Distribution and Phylogeography of <i>Caryopteris incana</i> (Lamiaceae) Based on Chloroplast DNA Sequences in West Kyushu, Japan

“…Like the sequence variation of the populations on the Tsushima Islands [18], several substitution sites between Genbank registration sequences and those of the study populations were confirmed in the matK, trnL-trnF and rpl32-trnL regions( Table 2). However, there were no indications of substitution among the study populations in these sites.…”

“…We have confirmed 72 natural populations by conducting fieldwork in the Tsushima Islands, Nagasaki [18]. In this study, we investigated the Nagasaki Mainland, Goto Islands, some remote islands, and the western parts of Saga adjacent to the Nagasaki and Koshikijima Islands, based on the report by Itow and Kawasato [16] (Figure 1).…”

“…The management of plant samples from seeds collected from each natural population for the investigations, and the extracting method from the leaves of individuals cultivated followed the previous report [18].…”

“…Each primer was newly prepared from the original paper or sequence information in Genbank registration regions, and universal primers were used for the remaining regions. The PCR condition, the sequences of primers used, and the equipment used were similar to the former report [18]. …”

“…The geographical distribution for each haplotype of chloroplast DNA sequences in the study areas are shown in Figure 3. In each area, five haplotypes were located on the Tsushima Islands [18] (Figure 3(a)). Two haplotypes were mainly separated between the southern part and the northern part of the Tsushima Islands, and several haplotypes were located in the center part and the north island.…”