Generally, oaks dominate the broadleaf deciduous forests in Japan. The genetic variation in 6 cpDNA regions (trnT-trnL, trnL-trnF, atpB-rbcL, and trnHpsbA speacers, trnL intron, and matK gene) with regard to the Japanese oak (Quercus mongolica var. crispula) and 3 related species in the section Prinus (Q. serrata, Q. dentata and Q. aliena) was investigated in 598 trees belonging to 44 populations distributed throughout the Japanese archipelago. Additional samples were collected from Korea, China, and Russia (Sakhalin). Thirteen haplotypes (I to XIII) were identified on the bases of 15 nucleotide substitutions and 3 indels. Haplotypes I and II were discovered in northeastern Japan, whereas haplotypes III to IX were distributed in southwestern Japan. The boundary distinguishing these 2 groups was located in central Japan coincident with the Itoigawa-Shzuoka tectonic line. Haplotype I was also found in Sakhalin, whereas haplotypes VI, VII, VIII, X, XI, XII, and XIII were found in Korea and China. Four oak species in the same location shared identical haplotypes, suggesting cpDNA introgression by occasional hybridization. Both the values of total haplotype diversity (H T ) and haplotype diversity within populations (H S ) in Q. mongolica var. crispula were higher in the southwestern populations than in the northeastern populations. A haplotype network indicated that haplotype VI is the ancestral haplotype. The presence of identical haplotypes in Korea, China, and Japan suggested that the haplotypes diversified on the Eurasian continent before the last glacial period. The difference in genetic structure between the northeastern and southwestern regions indicates a difference in the history of migration and recolonization in Japan during the last glacial period.

When considering the genetic implications of immigrant gene flow, it is important to evaluate both the proportions of immigrant gametes and their genetic composition. We simultaneously investigated paternal and maternal gene flow in dispersed seeds in a natural population of Pinus densiflora located along a ridge. The paternity and maternity of a total of 454 dispersed seeds (in 2004 and 2005) were accurately and separately assigned to 454 candidate adult trees, by analyzing the nuclear DNA of both diploid biparentally derived embryos and haploid maternally derived megagametophytes of the seeds. The relative genetic diversities and differences between within-population and immigrant groups of both paternally and maternally derived gametes (4 groups) that formed the genotypes of the seeds were evaluated. Using 8 microsatellite markers, we found that 64.0-72.6% of paternally derived gametes, and 17.8-20.2% of maternally derived gametes, were from other populations. Principal coordinate analysis showed that the 4 gamete groups tended to be plotted at different locations on the scattergram, indicating that they each have different genetic compositions. Substantial paternal and maternal immigrant gene flow occurred in this population, and therefore, the overall genetic variation of dispersed seeds is enhanced by both paternally and maternally derived immigrant gametes.

To investigate how accurately biparentage assignments for coniferous seeds can be improved by using different kinds of seed tissues, we assigned biparentage to dispersed seeds in a natural stand of Pinus densiflora Siebold & Zucc. (288 mature trees) using two procedures: with or without megagametophyte haplotype data (“MH” and “ordinary” procedures, respectively). Using archived clones, we confirmed the conifer-specific modes of inheritance of three kinds of seed tissues from certain maternal trees using six microsatellite markers. In the natural stand, under the MH procedure, 39.2% of male parents and 77.0% of female parents for a total of 204 seeds analyzed were assigned to at least one mature tree within the study area. At that time, the proportion of exactly matching seeds out of seeds with one matching parent under the MH procedure was significantly larger than that of the seeds under the ordinary procedure. The biparentage assignments under the ordinary procedure corresponded to only 53.7% of the accurate separate assignments under the MH procedure. It is suggested that analyzing different seed tissues is effective for exact and accurate biparentage assignments in investigations of biparentally mediated gene flow in coniferous populations, particularly at the seed-dispersal stage.

Although the variations in within-crown cone production have mainly been associated with resource availability, trees with relatively fewer cones allocate more cones to the optimal vertical layer for cone production than their expected resource availability suggests. We investigated the number of cones (NCone) per branch basal area (BBA) and the proportion of cones (PCone) in the three crown layers (upper, middle, and lower) for 72 Pinus densiflora Sieb. et Zucc. clones in 2004 and 2005. We also measured cone characteristics in each layer to infer their resource or pollen availability. Further, in 2006 we conducted pollination experiments for another 19 P. densiflora ramets, manipulating pollen quality (open-pollinated, self-pollinated (selfed), and polycrossed) in their two crown layers (upper and lower), and examined how pollen quality could affect the among-layer differences. Among 72 clones investigated, PCone in the upper crown layer was significantly greater, with a decrease of the total NCone per BBA; at that time, the seed/ovule ratio (S/O) in the upper crown layer cones was generally greater than that found in the middle and lower crown layers. In the pollination experiments, self-pollination resulted in a decreased S/O in the cones, regardless of the crown layer: S/O in the selfed cones, which were pollinated with controlled-quality pollen, did not differ between the upper and lower layers. Clones with relatively fewer cones allocated more resources to producing cones in the optimal cone-producing layer (upper crown layer) than would be expected from their biomass allocation, suggesting that optimal cone production is the result of a pollination-related factor.

Microsatellites were isolated and characterized for the Japanese oak species, Quercus mongolica var. crispula , distributed in temperate deciduous forests of Japan. Eleven of the 48 primer pairs designed successfully amplified unambiguous and polymorphic single loci among 67 Q. mongolica var. crispula individuals within a plot in southwestern Japan. The observed and expected heterozygosities of the 11 microsatellite markers ranged from 0.522 to 0.896 and from 0.536 to 0.882, respectively. These polymorphic microsatellite markers are useful for estimating pollen-mediated gene flow in Q. mongolica var. crispula .