Characterization and application of newly developed polymorphic microsatellite markers in the Ezo red fox (Vulpes vulpes schrencki).

The Ezo red fox (Vulpes vulpes schrencki), a subspecies endemic to Hokkaido island, Japan, is a known host species for the tapeworm Echinococcus multilocularis. To develop tools for molecular ecological studies, we isolated 28 microsatellite regions from the genome of Ezo red fox, and developed 18 polymorphic microsatellite markers. These markers were characterized using 7 individuals and 22 fecal samples of the Ezo red fox. The number of alleles for these markers ranged from 1 to 7, and the observed heterozygosity, estimated on the basis of the genotypes of 7 individuals, ranged from 0.29 to 1.00. All markers, except DvNok5, were in Hardy-Weinberg equilibrium (P > 0.05), and no linkage disequilibrium was detected among these loci, except between DvNok14 and DvNok28 (P = 0.01). Moreover, six microsatellite loci were successfully genotyped using feces-derived DNA from the Ezo red fox. The markers developed in our study might serve as a useful tool for molecular ecological studies of the Ezo red fox.

Male-biased hermaphrodites in a gynodioecious shrub, Daphne jezoensis.

Gynodioecy, a state where female and hermaphrodite plants coexist in populations, has been widely proposed an intermediate stage in the evolutionary pathway from hermaphroditism to dioecy. In the gynodioecy-dioecy pathway, hermaphrodites may gain most of their fitness through male function once females invade populations. To test this prediction, comprehensive studies on sex ratio variation across populations and reproductive characteristics of hermaphrodite and female phenotypes are necessary. This study examined the variation in sex ratio, sex expression, flower and fruit production and sexual dimorphism of morphological traits in a gynodioecious shrub, Daphne jezoensis, over multiple populations and years. Population sex ratio (hermaphrodite:female) was close to 1:1 or slightly hermaphrodite-biased. Sex type of individual plants was largely fixed, but 15% of plants changed their sex during a 6-year census. Hermaphrodite plants produced larger flowers and invested 2.5 times more resources in flower production than female plants, but they exhibited remarkably low fruit set (proportion of flowers setting fruits). Female plants produced six times more fruits than hermaphrodite plants. Low fruiting ability of hermaphrodite plants was retained even when hand-pollination was performed. Fruit production of female plants was restricted by pollen limitation under natural conditions, irrespective of high potential fecundity, and this minimised the difference in resources allocated to reproduction between the sexes. Negative effects of previous flower and fruit production on current reproduction were not apparent in both sexes. This study suggests that gynodioecy in this species is functionally close to a dioecious mating system: smaller flower production with larger fruiting ability in female plants, and larger flower production with little fruiting ability in hermaphrodite plants.

Development of microsatellite markers for Cinnamomum camphora (Lauraceae).

PREMISE OF THE STUDY: Cinnamomum camphora is an evergreen tree distributed in southern Japan, Taiwan, and southeastern China. Because of its vast utilization and cultivation, the natural distribution area of this species has been controversial. METHODS AND RESULTS: I isolated and characterized 22 microsatellite loci in C. camphora. Levels of polymorphism were evaluated in 104 adult trees from three populations in Japan: Meiji Jingu (Shinto Shrine), Kajiya Plantation, and Manazuru Peninsula. The mean number of alleles per locus ranged from 4.1 to 8.0 among populations. The mean observed and expected heterozygosities per population ranged from 0.53 to 0.60 and 0.55 to 0.68, respectively. CONCLUSIONS: All of 22 loci showed a clear and strong single band for each allele, and revealed a useful degree of polymorphism. The microsatellite markers described here will be useful to study the history, population dynamics, mating system, and genetic structure of C. camphora.

Heating applicator based on reentrant cavity with optimized local heating characteristics.

PURPOSE: A reentrant-cavity-based applicator can produce a concentrated electric field between reentrant electrodes for localized heating. However, this field is inadequate for treating early small tumors localized in the head and neck. In order to safely heat such well-localized lesions, the electric field distribution should be more localized. MATERIALS AND METHODS: In order to achieve localized heating, four parameters of the reentrant cavity (applicator height, outer diameter, reentrant diameter, and reentrant gap size), which influence the distribution of the electric field produced in the reentrant gap, are optimized using the Taguchi method. The variation in the heating characteristics affected by the size of the heating object is estimated using the signal-to-noise ratio (SNR) index. In this study, the electromagnetic field distributions in a cylindrical phantom and an oblate sphere phantom are analyzed by the three-dimensional finite element method, and the full width at half height (FWHH) of the specific absorption rate (SAR) distribution in the reentrant gap is evaluated. RESULTS: It is shown that the optimized applicator yields both the maximum SNR and minimum mean FWHH, and the sizes of the heating region in the phantom expressed using the averaged FWHH values of the SAR distribution are 60 and 80 mm along the radial and long-axis directions of the applicator, respectively. CONCLUSIONS: A heating region can be robustly and optimally localized by using the Taguchi method and considering the variation in the size of the heating object.

A hybrid zone dominated by fertile F1s of two alpine shrub species, Phyllodoce caerulea and Phyllodoce aleutica, along a snowmelt gradient.

In alpine ecosystems, the steep environmental gradients produced by the difference in snowmelt timing create a dynamic selective regime for alpine plants. As these gradients directly alter flowering phenology, they can affect pollen-mediated gene flow among populations of single and related species. In northern Japan, we found a hybrid zone dominated by fertile F(1)s of two alpine shrub species, Phyllodoce caerulea and P. aleutica, along a snowmelt gradient. Seed germination confirmed the fertility of F(1) hybrid, making the rarity and absence of backcross and F(2) plants puzzling. The long-term clonal perpetuation of F(1) hybrids (at least a few thousand years ago) contributes the maintenance of this unique hybrid zone. The distribution patterns of chloroplast DNA haplotypes suggest that F(1) formation might be caused by directional pollen flow between parental species along the snowmelt gradient. Based on these results, we discuss the ecological and evolutionary significance of this unique hybrid zone.

Effects of nifedipine and interleukin-1alpha on the expression of collagen, matrix metalloproteinase-1, and tissue inhibitor of metalloproteinase-1 in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Nifedipine-induced gingival overgrowth is known to be characterized by fibrosis and some degree of inflammation. However, the molecular mechanism of the fibrosis is not fully understood. The purpose of this study was to investigate in vitro the effects of nifedipine and interleukin-1alpha on the molecules involved in fibrosis, namely type I collagen, matrix metalloproteinase-1 (MMP-1), and tissue inhibitor of metalloproteinase-1 (TIMP-1). MATERIAL AND METHODS: Four human gingival fibroblast strains, derived from four healthy volunteers, were cultured in media containing nifedipine (1 microg/ml), with or without interleukin-1alpha (0.05 ng/ml). The mRNAs of type I collagen, MMP-1, and TIMP-1 were measured by reverse transcription-polymerase chain reaction (RT-PCR). The proteins of MMP-1 and TIMP-1 were examined by enzyme-linked immunosorbent assay (ELISA), and the ratios of MMP-1 to TIMP-1 proteins were calculated. RESULTS: The mRNA expression of type I collagen showed no significant change. Both mRNA expression and protein production of MMP-1 were up-regulated by interleukin-1alpha, either alone or in combination with nifedipine, whereas those of TIMP-1 were up-regulated by nifedipine alone or in combination with interleukin-1alpha. The ratio of MMP-1 to TIMP-1 was not changed by nifedipine alone, but it was increased by interleukin-1alpha alone or in combination with nifedipine. However, in two of the four cell strains tested, nifedipine reduced the ratio of MMP-1 to TIMP-1 compared with that for interleukin-1alpha alone. CONCLUSION: These results suggest that nifedipine may predispose to fibrosis in some individuals in situations where interleukin-1 levels are raised.

Seasonal changes in pollinator activity influence pollen dispersal and seed production of the alpine shrub Rhododendron aureum (Ericaceae).

In alpine ecosystems, microscale variation in snowmelt timing often causes different flowering phenology of the same plant species and seasonal changes in pollinator activity. We compared the variations in insect visitation, pollen dispersal, mating patterns, and sexual reproduction of Rhododendron aureum early and late in the flowering season using five microsatellites. Insects visiting the flowers were rare early in the flowering season (mid-June), when major pollinators were bumblebee queens and flies. In contrast, frequent visitations by bumblebee workers were observed late in the season (late July). Two-generation analysis of pollen pool structure demonstrated that quality of pollen-mediated gene flow was more diverse late in the season in parallel with the high pollinator activity. The effective number of pollen donors per fruit (N(ep)) increased late in the season (N(ep) = 2.2-2.7 early, 3.4-4.4 late). However, both the outcrossing rate (t(m)) and seed-set ratio per fruit were smaller late in the season (t(m) = 0.89 and 0.71, seed-set ratio = 0.52 and 0.18, early and late in the season, respectively). In addition, biparental inbreeding occurred only late in the season. We conclude that R. aureum shows contrasting patterns of pollen movement and seed production between early and late season: in early season, seed production can be high but genetically less diverse and, during late season, be reduced, possibly due to higher inbreeding and inbreeding depression, but have greater genetic diversity. Thus, more pollinator activity does not always mean more pollen movement.

DNA vaccine against hamster oral papillomavirus-associated oral cancer.

Previously we developed a carcinogenesis model involving the combination of 9,10-dimethyl-1,2-benzanthracene (DMBA) application with physical wounding of hamster lingual mucosa. The presence of a novel hamster oral papillomavirus (HOPV) was demonstrated and its genome sequenced. In the present study, this HOPV hamster model was used to test whether vaccination with the L1 gene could prevent the development of oral carcinoma. DNA plasmids encoding the L1 gene or the vector alone were injected intramuscularly into 20 vaccinated and 20 control hamsters, respectively. The lingual tips of the hamsters were painted with DMBA for 8 weeks. A portion of the lingual tips was excised, and the tips were then painted daily with DMBA until the animals were killed 13 days later. All control hamsters developed lingual carcinoma, whereas 12 of the L1-vaccinated hamsters showed no lesions. These results suggest that immunization with L1 DNA vaccines may prevent the development of papillomavirus-associated oral cancer.

Enhanced anti-tumour effect of cisplatin with low-voltage electrochemotherapy in hamster oral fibrosarcoma.

The aim of this study was to determine the effects of low-voltage electrochemotherapy with intraperitoneal cisplatin on hamster oral fibrosarcoma. Oral fibrosarcoma was transplanted submucosally into the cheek pouch mucosa of 100 hamsters. After transplantation, the hamsters were randomly divided into four equal groups. These groups received no treatment (D-E-); 2 mg/kg body weight cisplatin treatment without electroporation (D+E-); electroporation without cisplatin treatment (D-E+); or 2 mg/kg body weight cisplatin treatment followed by electroporation (D+E+). Electrical pulse treatment together with cisplatin injection markedly reduced the size of the tumour, whereas cisplatin injection or electrical pulse treatment alone did not. These results clearly indicate that the anti-tumour effect of cisplatin on hamster oral fibrosarcoma was considerably potentiated or enhanced by the administration of local electrical pulses at low voltages.

Enhancement of crude bone morphogenetic protein-induced new bone formation and normalization of endochondral ossification by bisphosphonate treatment in osteoprotegerin-deficient mice.

Osteoprotegerin (OPG) is a novel secreted member of the tumor necrosis factor receptor family which plays a crucial role in negative regulation of osteoclastic bone resorption. We investigated both the quantity and quality of heterotopic new bone induced by crude bone morphogenetic protein (BMP) as a means of examining bone metabolism by bisphosphonate administration in OPG-/- mice. Four weeks after implantation of crude BMP, the volume of heterotopic new bone in OPG-/- mice without alendronate was significantly less than in wild-type (WT) mice. Alendronate treatment of OPG-/- mice resulted in enhancement of the volume of heterotopic new bone. Histological findings revealed that WT mice showed normal bone formation with persistent cartilage that was interspersed with islands of bone. In contrast, the cartilage was replaced by trabecular bone and bone marrow adipocytes in OPG-/- mice without alendronate. However, some cartilage was still present in OPG-/- mice with alendronate compared to those without alendronate. All bone formation-related parameters and bone resorption-related parameters were significantly lower in OPG-/- mice with alendronate than in those without alendronate. These findings suggest that in stimulated osteoclastogenesis without OPG, osteoinductive activity induced by crude BMP is inhibited and endochondral ossification induced by crude BMP is accelerated. On the other hand, alendronate treatment of OPG-/- mice caused osteoinductive activity induced by crude BMP to increase and endochondral ossification induced by crude BMP to be decelerated. In conclusion, inhibition of stimulated osteoclastogenesis results in the enhancement of new bone formation and normalization of endochondral ossification.

Effect of a selective agonist for prostaglandin E receptor subtype EP4 (ONO-4819) on the cortical bone response to mechanical loading.

The influence of a selective agonist for prostaglandin E receptor subtype EP4 (ONO-4819) on the bone response to mechanical loading was evaluated. Six-month-old female Wistar rats were used and assigned to three groups (n = 12/group): Vehicle administration (EP4-V), low-dose ONO-4819 administration (EP4-L, 3 microg/kg BW), and high-dose ONO-4819 administration (EP4-H, 30 microg/kg BW). ONO-4819 was subcutaneously injected in the back twice a day for 3 weeks. Loads on the right tibia at 39.4 N for 36 cycles at 2 Hz were applied in vivo by 4-point bending every other day for 3 weeks. Whole-body bone mineral content showed a significant difference between EP4-V and EP4-H (P < 0.05). Bone mineral density (BMD) of the total and regional tibia (the region with maximal bending at the central diaphysis) was higher in EP4-H than EP4-V, showing a significant effect of loading (P < 0.001) and ONO-4819 (P < 0.05). BMD of the total femur was higher in EP4-H than EP4-V (P < 0.01) and that of the distal femur was higher in EP4-H than EP4-V (P < 0.001). Histomorphometry of the cortical bone showed that loading increased formation surface (FS/BS), mineral appositional rate (MAR), and bone formation rate (BFR/BS) significantly at the lateral periosteal surface (P < 0.001); however, the effect of ONO-4819 was not significant. At the medial periosteal surface, loading increased the three parameters (P < 0.001) and ONO-4819 increased FS/BS (P < 0.001) and MAR (P < 0.05) significantly. At the endocortical surface, the effects of both loading and ONO-4819 were significant on all three parameters (for loading; FS/BS P < 0.01, MAR P < 0.05, BFR/BS P < 0.03, for ONO-4819 all P < 0.001). It was concluded that ONO-4819 increased cortical bone formation in rats and there was an additive effect on the bone response to external loading by 4-point bending.

Human papillomaviruses in the normal oral cavity of children in Japan.

The purpose of this study was to determine the frequency of human papillomavirus (HPV) infections in the normal oral cavity of children in Japan. Oral squamous cell specimens were collected from 77 children (44 boys and 33 girls), aged 3 and 5 years. Extracted DNA was evaluated for HPV infections by polymerase chain reaction (PCR) methods, using consensus primers for the L1 region, specific primers, and direct DNA sequencing analysis. Thirty-seven of 77 specimens (48.1%) were positive for HPV DNA. Positive rates of boys and girls in all specimens were 28.3 (22/77) and 19.5 (15/77)%, respectively. The positive rate in 3-year-old children was 45.2 (14/31)%, and positive rates in boys and girls were 52.6 (10/19) and 33.3 (4/12)%, respectively. The positive rate in 5-year-old children was 50.0 (23/46)%, and positive rates in boys and girls were 48.0 (12/25) and 52.4 (11/21)%, respectively. HPV types were determined by specific PCR and direct DNA sequencing analysis. Frequent HPV types in the specimens of all children were HPV-16 (11/37; 29.7%),-1 (6/37; 16.2%),-2 (6/37; 16.2%),-75 (6/37; 16.2%). The results of the present investigation indicate that many HPVs, including HPV-16 (a high-risk type for cancer), are present in the oral cavity of 3- and 5-year-old children. It is suggested, therefore, that the oral cavity is already a reservoir of HPVs in childhood where later HPV-associated diseases, such as oral cancer and other oral lesions, may develop.

Hemichrome formation observed in human haemoglobin A under various buffer conditions.

AIM: To observe hemichrome formation in human haemoglobin A under various buffer conditions. METHOD: Hemichrome formation of human oxyhaemoglobin A (HbO2) was studied spectrophotometrically in 0.1 m buffer at various temperatures and pH values. RESULTS: Following autoxidation in ferrous HbO2, it was evident that formation of hemichrome, which tends to precipitate, occurred at various stages during the course of the autoxidation reaction namely at initial, intermediate or final stages, depending on temperature and pH of the solution. By varying temperature of the solution from 35 to 55 degrees C and pH from 4.5 to 10.5, it is shown here that HbO2 exhibits high susceptibility for hemichrome formation and its occurrence is a function of pH, temperature and progress of autoxidation of HbO2. Unlike HbO2 and its separated haemoglobin chains, monomeric bovine heart myoglobin (MbO2) did not easily form hemichrome. CONCLUSION: These findings provide a clue on the crucial role of haemoglobin molecule for senescent cell recognition or homeostasis in the blood circulation.

The effect of high sugar intake on the development of periradicular lesions in rats with type 2 diabetes.

Diabetes mellitus is associated with depression of natural defenses against infection and increases the risk of periodontal disease. However, the effects of diabetes on periradicular tissue, which differs structurally from periodontal tissue, are not known. In this study, we evaluated the effects of type 2 diabetes on the development of periradicular lesions after exposure of the pulp in the left mandibular first molar through the occlusal surface in rats. GK rats with spontaneous non-insulin-dependent diabetes mellitus and Wistar rats (controls) received a normal laboratory diet and either water or a 30% sucrose solution. At both 2 and 4 weeks after pulp exposure, histologic analysis showed that alveolar bone resorption was most severe and the periradicular lesions were largest in diabetic rats given the sucrose solution. These results suggest that the metabolic conditions produced by type 2 diabetes enhance the development of periradicular lesions in rats.

Human papillomavirus type 38 infection in oral squamous cell carcinomas.

In this study, 53 paraffin-embedded oral squamous cell carcinoma (OSCC) biopsy specimens were used. Human papillomavirus type 38 (HPV-38) infection was demonstrated in OSCCs using the PCR technique, DNA sequencing analysis, in situ hybridization, and immunohistochemical techniques. Additionally, the correlation between HPV-38 infection and expressions of proliferating cell nuclear antigens (PCNA) or p53 protein was analyzed immunohistochemically. Using consensus primers for the L1 region (L1-PCR), we identified 35 of 53 specimens (66%) as positive for HPV-38 DNA. Furthermore, specimens from patients over 60 years of age revealed a lower prevalence for the HPV-38 (56.7%) than did those below that age (78.3%). Immunohistochemically, positive stainings for PCNA and p53 protein were more frequently detected in HPV-38 positive OSCCs than HPV negative ones. These results indicate that HPV-38 positive OSCCs were higher in proliferative cellular activity than HPV negative ones. Moreover, the findings suggest that HPV-38 infection may cause malignant transformation of the oral mucosal epithelium.

Epithelial cell proliferation in oral lichen planus.

Although the pathogenesis of oral lichen planus (OLP) is not clear, a small proportion of cases with OLP are reported to transform to cancer. We examined the epithelial cell proliferation status of OLP to relate the labelling index to microscopic features surveyed routinely in pathology. Mucosal biopsies obtained from 44 cases diagnosed with OLP with an intact oral epithelium and 10 normal control specimens from Japanese subjects were immunohistochemically stained with MIB and p53 antibodies. The Ki67 labelling index (LI) was significantly higher in OLP compared with normal controls. A particularly large number of OLP lesions (64%) were p53 positive. No association was, however, found with p53 expression and the Ki67 LI. Atrophic and flat epithelia had a quantitatively higher LI, which did not significantly differ from acanthotic biopsies. Increased cell proliferation in OLP is likely to be a secondary phenomenon due to the damage inflicted on keratinocytes by infiltrating mononuclear cells in the submucosa.

Cytochrome oxidase activity and confocal laser scanning microscopic analysis of the hamster submandibular gland using microwave irradiated fixation.

Submandibular glands of the hamster were irradiated in 2% paraformaldehyde (pFA)-0.5% pure glutaraldehyde (PGA) with a microwave (MW) processor at temperatures of 10 degrees and 37 degrees C. Electron microscopy showed that cytochrome oxidase activity was taking place in the mitochondrial intermembrane-intracristal space of the granular duct cell when the temperature of the MW-irradiated fixatives was at 10 degrees C. However, a decrease of this activity was observed when we took care to keep the temperature of the MW-irradiated fixatives at 37 degrees C. The distinct reduction of cytochrome oxidase activity allowed by MW irradiation seems to be due the thermal affects of fixatives. Of course, the possibility cannot be excluded that MW irradiation caused other undetectable membrane damage. Then, we used confocal laser scanning microscopy for the preservation check of the mitochondrial membrane for cytochemistry with MW-irradiated fixation. The fluorescence of rhodamine 123 was observed in the inner spaces of the mitochondria at temperatures of 10 degrees and 37 degrees C. When the same tissues were fixed with 2% pFA using an MW processor as the sole fixative at 10 degrees C, no mitochondrial fluorescence was observed. Cytochrome oxidase activity, by contrast, could be seen in the mitochondrial intermembrane-intracristal spaces in the same condition. Formaldehyde is not the best aldehyde for the purpose of ultrastructural preservation. On the other hand, light and electron microscopy showed that the endogenous peroxidase activity was localized in the nuclear envelope, endoplasmic reticulum, secretory granules, and Golgi apparatus of the hamster submandibular gland using 2% pFA-0.5% PGA fixative with and without MW irradiations at temperatures of 10 degrees and 37 degrees C. Some of the same cells were fixed with only 2% pFA under MW irradiation at 10 degrees C; however, marked diffuseness of the peroxidase activity was observed. Therefore, these results indicated that cytochrome oxidase activity was sensitive to heat with MW-irradiated fixation. Peroxidase activity was very resistant to heat with MW-irradiated fixation but not with pFA solo fixation, therefore, PGA had to be used.

Release of mercury from dental amalgam fillings in pregnant rats and distribution of mercury in maternal and fetal tissues.

Mercury vapor released from a single amalgam restoration in pregnant rats and mercury concentrations in maternal and fetal rat tissues were studied. Dental treatment was given on day 2 of pregnancy. Mercury concentration in air sample drawn from the metabolism chamber with the rat was measured serially for 24 h on days 2, 8 and 15 of pregnancy. An average mercury concentration in the air samples from the rats given amalgam restorations was 678.6+/-167.5 ng/day on day 2. The average mercury concentration in the air samples tended to decline as time elapsed but a marked amount (423.2+/-121.5 ng/day) was observed even on day 15. The amount of mercury in the air samples increased 7--20-fold after chewing. The placement of the single amalgam restoration (3.8--5.5 mg in weight) increased the levels of mercury approximately three to 6 times in the maternal brain, liver, lung, placenta and 20 times in the kidneys. The highest mercury concentration among fetal organs was found in the liver, followed by the kidneys and brain. Mercury concentrations in maternal organs and fetal liver were significantly higher than those of the controls, and concentrations in maternal whole blood, erythrocytes and plasma, and in fetal whole blood were also significantly higher. Mercury concentrations in the fetal brain, liver, kidneys and whole blood were lower than those of the maternal tissues.

Ca2+-independent phospholipase A2 activity in apical plasma membranes from the rat parotid gland.

An apical-enriched plasma membrane fraction (A-PM) was prepared from rat parotid gland by Mn2+ precipitation. In this fraction, phosphatidylcholine (PC) labelled at the sn-2 position was mainly decomposed into two labelled compounds (free fatty acid and 1,2-diacylglycerol) under Ca2+-free conditions. Studies using double-labelled PC and 2,3-diphosphoglycerate (as a phospholipase D inhibitor) showed that they were produced through different pathways: free fatty acid was released by phospholipase A2 (PLA2) while 1,2-diacylglycerol may be produced by sequential action of phospholipase D and phosphatidate phosphatase. The PLA2 in A-PM did not require Ca2+ for its activity and was highly activated by Triton X-100 and ATP. The inhibitor of the well-documented Ca2+-independent PLA2, bromoenol lactone, did not inhibit the PLA2 activity in A-PM. Although PLA2 activity was detected in other subcellular fractions, the highest specific activity was in A-PM. Its distribution among various fractions was roughly similar to that of the marker enzyme of apical plasma membranes. These findings suggested that Ca2+-independent PLA2 activity is present in apical plasma membranes from rat parotid gland. In addition, to clarify the involvement of the PLA2 in exocytosis, the fusion of exogenous PLA2-treated membranes with secretory granules was examined by fluorescence dequenching assay. This study clearly demonstrated the facilitation of fusion by PLA2 treatment, which suggests some involvement of apical PLA2 in saliva secretion.

Patterns and levels of gene flow in Rhododendron metternichii var. hondoense revealed by microsatellite analysis.

Parentage analysis was conducted to elucidate the patterns and levels of gene flow in Rhododendron metternichii Sieb. et Zucc. var. hondoense Nakai in a 150 x 70 m quadrant in Hiroshima Prefecture, western Japan. The population of R. metternichii occurred as three subpopulations at the study site. Seventy seedlings were randomly collected from each of three 10 x 10 m plots (S1, S2, and S3) on the forest floor of each subpopulation (A1, A2, and A3). Almost all parents (93.8%) of the 70 seedlings were unambiguously identified by using 12 pairs of microsatellite markers. Within the quadrant, adult trees less than 5 m from the centre of the seedling bank (plots S1, S2, and S3) produced large numbers of seedlings. The effects of tree height and distance from the seedling bank on the relative fertilities of adult trees were highly variable among subpopulations because of the differences in population structure near the seedling bank: neither distance nor tree height had any significant effect in subpopulation A1; distance from the seedling bank had a significant effect in subpopulation A2; and tree height had a significant effect in subpopulation A3. Although gene flow within each subpopulation was highly restricted to less than 25 m and gene flow among the three subpopulations was extremely small (0-2%), long-distance gene flow from outside the quadrant reached 50%. This long-distance gene flow may be caused by a combination of topographical and vegetational heterogeneity, differences in flowering phenology, and genetic substructuring within subpopulations.