In order to elucidate the past distribution and colonization routes of broad-leaved evergreen (lucidophyllous) forests, we investigated the intraspecific phylogeographic patterns of lucidophyllous forests in Japan and surrounding areas. We selected 6 component species with a similar geographic distributions growing in Castanopsis-dominant forests. We defined possible important refugia during the glacial periods as the regions rich in rare haplotypes (with a frequency of 5% or less), or as regions rich in the number of common haplotypes (with a frequency of more than 5%). We then located the sites of refuge by comparing the intraspecific phylogeographic patterns among 6 component species of lucidophyllous forests with respect to these two parameters (i.e., haplotype uniqueness and the number of haplotypes). The following results were obtained during the course of this study: (1) rare haplotypes were distributed among islands around the main islands of Japan; (2) rare subtypes and the greatest numbers of common haplotypes were observed in Kyushu, a finding which agreed with fossilized pollen data demonstrative of the past existence of refugia in southern Kyushu; and (3) rare haplotypes were found on the Muroto Peninsula, and the second greatest numbers of common haplotypes were observed on the Kii Peninsula, a finding which suggested the existence of additional important refugia along the Pacific coast of Japan during the glacial ages.

A phylogeographic study of four tree species (Padus grayana, Euonymus oxyphyllus, Magnolia hypoleuca, and Carpinus laxiflora) growing in Japanese deciduous broad-leaved forests was conducted based on chloroplast DNA (cpDNA) variations. Using nucleotide sequences of 702-1,059 bp of intergenic spacers of cpDNA, 20, 27, eight, and eight haplotypes were detected among 251, 251, 226, and 262 individuals sampled from 67, 79, 75, and 71 populations of the above species, respectively. The geographical pattern of the cpDNA variations was highly structured in each species, and the following three regional populations were genetically highly differentiated among all four species: (1) the Sea of Japan-side area, (2) the Kanto region, and (3) southwestern Japan. Based on some interspecific similarities among the phylogeographic patterns, the following migration scenario of Japanese deciduous broad-leaved forests was postulated. During the last glacial maximum (LGM), the forests were separately distributed in six regions. After LGM, as the climate warmed, the forests in eastern Japan separately expanded from each of the refugia along the Sea of Japan-side or along the Pacific Ocean-side. In contrast, those in southwestern Japan retreated and moved to high altitudes from each of the continuous forests.

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Climatic changes during glacial periods have had a major influence on the recent evolutionary history of living organisms, even in the warm temperate zone. We investigated phylogeographical patterns of a weevil Curculio hilgendorfi (Curculionidae), a host-specific seed predator of Castanopsis (Fagaceae) growing in the broadleaved evergreen forests in Japan. We examined 2709 bp of mitochondrial DNA for 204 individuals collected from 62 populations of the weevil. Four major haplogroups were detected, in southwestern and northeastern parts of the main islands and in central and southern parts of the Ryukyu Islands. The demographic population expansion was detected for the two groups in the main islands but not for the Ryukyu groups. The beginning time of the expansion was dated to 39,000-59,000 years ago, which is consistent with the end of the last glacial period. Our data also demonstrated that the southwestern population of the main islands has experienced a more severe bottleneck and more rapid population growth after glacial ages than the northeastern population. At least three refugial areas in the main islands were likely to have existed during the last glacial periods, one of which had not previously been recognized by analyses of intraspecific chloroplast DNA variation of several plant species growing in the broadleaved evergreen forests. Our results represent the first phylogeographical and population demographic analysis of an insect species associated with the broadleaved evergreen forests in Japan, and reveal more detailed postglacial history of the forests.

The broadleaved evergreen forests of the East Asian warm temperate zone are characterised by their high biodiversity and endemism, and there is therefore a need to extend our understanding of its genetic diversity and phylogeographic patterns. Castanopsis (Fagaceae) is one of the dominant tree species in the broadleaved evergreen forests of Japan. In this study we investigate the genetic diversity, genetic structure and leaf epidermal morphology of 63 natural populations of C. sieboldii and C. cuspidata, using 32 Expressed Sequence Tag associated microsatellites. The overall genetic differentiation between populations was low (G ST = 0.069 in C. sieboldii and G ST = 0.057 in C. cuspidata). Neighbor-joining tree and Bayesian clustering analyses revealed that the populations of C. sieboldii and C. cuspidata were genetically clearly differentiated, a result which is consistent with the morphology of their epidermal cell layers. This suggests that C. sieboldii and C. cuspidata should be treated as independent species, although intermediate morphologies are often observed, especially at sites where the two species coexist. The higher level of genetic diversity observed in the Kyushu region (for both species) and the Ryukyu Islands (for C. sieboldii) is consistent with the available fossil pollen data for Castanopsis-type broadleaved evergreen trees during the Last Glacial Maximum and suggests the existence of refugia for Castanopsis forests in southern Japan. Within the C. sieboldii populations, Bayesian clustering analyses detected three clusters, in the western and eastern parts of the main islands and in the Ryukyu Islands. The west-east genetic differentiation observed for this species in the main islands, a pattern which is also found in several plant and animal species inhabiting Castanopsis forests in Japan, suggests that they have been isolated from each other in the western and eastern populations for an extended period of time, and may imply the existence of eastern refugia.