Further analysis of intraspecific sequence variation of chloroplast DNA in Primula cuneifolia Ledeb. (Primulaceae): Implications for biogeography of the Japanese alpine flora

In order to clarify evolutionary patterns and processes of intraspecific diversification of Primula cuneifolia Ledeb. (Primulaceae), we analyzed intraspecific variation of the nucleotide sequence of non-coding regions of chloroplast DNA: the intergenic spacers between trnT (UGU) and the trnL (UAA) 5'exon, the trnL (UAA) 3'exon and trnF (GAA), and atpB and rbcL. In 20 populations of P. cuneifolia, 22 nucleotide substitutions and five insertions/deletions were inferred, and their genetic distances ranged from 0.001 to 0.008. Eight distinct haplotypes could be recognized and each haplotype was found to be geographically structured. Three major clades (the Northern, Hokkaido and Southern clades) were revealed in phylogenetic analyses of the haplotypes. The haplotypes of the Northern clade had a wider distribution area in the populations of Mt. Rausu and Rishiri island of eastern and northern Hokkaido in Japan, northward to Unalaska island in the Aleutians, and those of the Hokkaido clade were distributed in the populations of central Hokkaido and Mt. Iwaki of the northern Honshu in Japan in addition, those of the southern clade were observed only in the populations of the central Honshu. It was shown that the genetic diversifications of the Southern clade were higher than those of the Northern and Hokkaido clades. Furthermore, it was shown that the topology within the Southern clade was hierarchical, and the haplotypes of the Southern populations in the clade were derivative. From these results, we concluded that the cpDNA haplotypes of the three clades in P. cuneifolia arose and assumed the present distribution areas through several cycles of glacial advance and retreat in the Pleistocene