Interspecific and annual variation in pre-dispersal seed predation by a granivorous bird in two East Asian hackberries, Celtis biondii and Celtis sinensis.

Pre-dispersal seed predation by granivorous birds has potential to limit fruit removal and subsequent seed dispersal by legitimate avian seed dispersers in bird-dispersed plants, especially when the birds form flocks. We monitored pre-dispersal seed predation by the Japanese grosbeak, Eophona personata, of two bird-dispersed hackberry species (Cannabaceae), Celtis biondii (four trees) and Celtis sinensis (10 trees), for 3 years (2005, 2007 and 2008) in a fragmented forest in temperate Japan. Throughout the 3 years, predation was more intense on C. biondii, which, as a consequence, lost a larger part of its fruit crop. Grosbeaks preferred C. biondii seeds that had a comparatively lower energy content and lower hardness than C. sinensis, suggesting an association between seed hardness and selective foraging by grosbeaks. In C. biondii, intensive predation markedly reduced fruit duration and strongly limited fruit removal by seed dispersers, especially in 2007 and 2008. In C. sinensis, seed dispersers consumed fruits throughout the fruiting seasons in all 3 years. In C. biondii, variation in the timing of grosbeak migration among years was associated with annual variation in this bird's effects on fruit removal. Our results demonstrate that seed predation by flocks of granivorous birds can dramatically disrupt seed dispersal in fleshy-fruited plants and suggest the importance of understanding their flocking behaviour.

Lineage admixture during postglacial range expansion is responsible for the increased gene diversity of Kalopanax septemlobus in a recently colonised territory.

We aimed to reveal the effects of range expansion and subsequent lineage admixture from separated glacial refugia on genetic diversity of Kalopanax septemlobus in Japan, by combining nuclear microsatellite data and ecological niche modelling. Allelic richness and gene diversity were compared at the population and regional level. We also statistically examined these indices as a function of population accessibility to the last glacial maximum (LGM) palaeodistribution reconstructed by ecological niche modelling to test a simple range expansion scenario from glacial refugia. Genetic diversity was highest in the populations of southern Japan and gradually decreased towards the north. However, an additional centre of genetic diversity, when measured as gene diversity, was found in northern Honshu Island, where distinct lineages were shown to be in contact. Positive effects of population accessibility to the LGM range were detected in both diversity indices at different spatial scales. The combined data support independent postglacial range expansions towards the north from the edge populations on the exposed coastal shelf of Pacific and Sea of Japan in northern Honshu during the LGM, which subsequently resulted in markedly low genetic diversity in the northernmost extant range, Hokkaido. The regional increase in gene diversity in northern Honshu is likely to be the result of postglacial lineage admixture. Relative difference in the spatial scales best relating population genetic diversity with the LGM distribution can be explained by a higher rate of allelic richness diversity loss during range expansions and stronger effects of lineage admixture on gene diversity.

Isolation and characterization of microsatellite loci in Ainsliaea faurieana (Asteraceae), an endemic plant species on Yakushima Island, Japan, and cross-species amplification in closely related taxa.

Fourteen microsatellite loci were isolated and characterized for Ainsliaea faurieana, an endemic perennial plant species on Yakushima Island. In an analysis of 37 individuals from Yakushima Island, the observed number of alleles ranged from two to six. The expected and observed heterozygosities were 0.109-0.811 and 0.000-0.27, respectively. The inbreeding coefficient was 0.489-1.000. The interspecific applicability of these microsatellites was evaluated by analysing three closely related taxa from the Ryukyu Islands. All primer pairs for the 14 loci tested successfully amplified in all taxa.

Development of 17 microsatellite markers for Ceratosolen constrictus, the pollinating fig wasp of Ficus fistulosa.

The obligate mutualism between figs (Ficus) and fig pollinating wasps (Agaonidae) is regarded as a classic example of mutualism. Seventeen polymorphic microsatellite loci were developed for Ceratosolen constrictus, the pollinating wasp of the dioecious fig Ficus fistulosa. The number of alleles per locus ranged from two to 15 and the observed and expected heterozygosities ranged from 0.040 to 0.846 and from 0.040 to 0.916, respectively. These microsatellite loci offer a powerful tool for evolutionary and population genetic studies in C. constrictus, and gene flow of F. fistulosa.

Isolation and characterization of 10 microsatellite loci in Callicarpa subpubescens (Verbenaceae), an endemic species of the Bonin Islands.

Ten microsatellite loci were isolated and characterized for Callicarpa subpubescens (Verbenaceae), an endemic tree species of the Bonin Islands. The observed number of alleles at each locus ranged from two to eight with an average of 4.9, and the expected heterozygosity ranged from 0.238 to 0.690 with an average of 0.483. All 10 loci were screened in cross-amplification tests for two other endemic Callicarpa species that also inhabit the Bonin Islands. All loci were successfully amplified in these species.

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.

Effect of gene flow on spatial genetic structure in the riparian canopy tree Cercidiphyllum japonicum revealed by microsatellite analysis.

Few studies have analyzed pollen and seed movements at local scale, and genetic differentiation among populations covering the geographic distribution range of a species. We carried out such a study on Cercidiphyllum japonicum; a dioecious broad-leaved tree of cool-temperate riparian forest in Japan. We made direct measurement of pollen and seed movements in a site, genetic structure at the local scale, and genetic differentiation between populations covering the Japanese Archipelago. Parentage analysis of seedlings within a 20-ha study site indicated that at least 28.8% of seedlings were fertilized by pollen from trees outside the study site. The average pollination distance within the study site was 129 m, with a maximum of 666 m. The genotypes of 30% of seedlings were incompatible with those of the nearest female tree, and the maximum seed dispersal distance within the study site was over 300 m. Thus, long-distance gene dispersal is common in this species. The correlation between genetic relatedness and spatial distance among adult trees within the population was not significant, indicating an absence of fine-scale genetic structure perhaps caused by high levels of pollen flow and overlapping seed shadows. Six populations sampled throughout the distribution of C. japonicum in Japan showed significant isolation-by-distance but low levels of genetic differentiation (F(ST) = 0.043), also indicating long-distance gene flow in C. japonicum. Long-distance gene flow had a strong influence on the genetic structure at different spatial scales, and contributes to the maintenance of genetic diversity in C. japonicum.

Variation in pollen dispersal between years with different pollination conditions in a tropical emergent tree.

We examined differences in pollen dispersal efficiency between 2 years in terms of both spatial dispersal range and genetic relatedness of pollen in a tropical emergent tree, Dipterocarpus tempehes. The species was pollinated by the giant honeybee (Apis dorsata) in a year of intensive community-level mass-flowering or general flowering (1996), but by several species of moths in a year of less-intensive general flowering (1998). We carried out paternity analysis based on six DNA microsatellite markers on a total of 277 mature trees forming four spatially distinct subpopulations in a 70 ha area, and 147 and 188 2-year-old seedlings originating from seeds produced in 1996 and 1998 (cohorts 96 and 98, respectively). Outcrossing rates (0.93 and 0.96 for cohorts 96 and 98, respectively) did not differ between years. Mean dispersal distances (222 and 192 m) were not significantly different between the 2 years but marginally more biased to long distance in 1996. The mean relatedness among cross-pollinated seedlings sharing the same mothers in cohort 96 was lower than that in cohort 98. This can be attributed to the two facts that the proportion of intersubpopulations pollen flow among cross-pollination events was marginally higher in cohort 96 (44%) than in cohort 98 (33%), and that mature trees within the same subpopulations are genetically more related to each other than those between different subpopulations. We conclude that D. tempehes maintained effective pollen dispersal in terms of outcrossing rate and pollen dispersal distance in spite of the large difference in foraging characteristics between two types of pollinators. In terms of pollen relatedness, however, a slight difference was suggested between years in the level of biparental inbreeding.

Clonal structure and flowering traits of a bamboo [Phyllostachys pubescens (Mazel) Ohwi] stand grown from a simultaneous flowering as revealed by AFLP analysis.

Abstract Although many bamboo species are characterized by simultaneous flowering at long intervals of up to 120 years, few studies have revealed the length of the flowering interval for very-long-lived bamboo species by observing the whole life cycle of a single clone. The flowering interval of Phyllostachys pubescens had been determined to be 67 years by means of observation through the entire life cycle from 1912 to 1979. We observed and analysed the clonal structure and flowering traits of a P. pubescens community which had regenerated from seed in 1930 and began to flower in 1997. Although this interval was again 67 years, flowering and nonflowering culms were mixed, and the flowering event lasted three years in the community. AFLP analysis of DNA samples showed distinct genets that originated from the previous flowering event and that each genet had its own flowering time. This is the first report to show that (i) different genets are mixed in a community of a large bamboo species with a leptomorphic rhizome system, and (ii) a community that originated from a single flowering event can have a range of flowering years.

Microsatellite genetic variation in small and isolated populations of Magnolia sieboldii ssp. japonica.

Magnolia sieboldii ssp. japonica, distributed mainly in western Japan, is restricted to high elevation areas (1000-2000 m above sea level) and usually forms small isolated populations. Four microsatellite loci were assayed for 19 populations from six regions spanning the range of distribution, and the levels and distribution of genetic variation were estimated. All four loci were variable, with a total of 39 alleles, but the overall level of microsatellite genetic variation was low, especially compared with a related species, M. obovata. Genetic structure in M. sieboldii was characterised by low intrapopulational genetic variation (A = 3.74 and H(o) = 0.366 on average) and high genetic differentiation even among regional populations. Highly significant isolation-by-distance (IBD) models at the short distance were detected. Genetic drift and limited gene flow was considered to be important in determining the genetic structure within regions. Total genetic differentiation was remarkably high (F(ST) = 0.488 and R(ST) = 0.538), suggesting genetic barriers among regions. Neighbour-joining dendrograms relating the 19 populations, and further analysis on the IBD models, revealed that a stepwise mutation model was more suited than an infinite allele model to explain the genetic differentiation among regions. It is suggested that mutation at microsatellite loci might be influential in generating the genetic differentiation among regions. These results showed the potential of hypervariable microsatellite loci to evaluate the effects of genetic drift and population isolation within regions, and to detect genetic distinctiveness, in spite of the loss of overall genetic variation in M. sieboldii.

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.

Microsatellite analysis of the regeneration process of Magnolia obovata Thunb.

We analysed the regeneration process of Magnolia obovata using polymorphic microsatellite markers. Eighty-three adult trees standing in a watershed covering an area of 69 ha, and saplings collected from a smaller research plot (6 ha) located at the centre of the watershed were genotyped using microsatellite markers. Among 91 saplings analysed, 24 (26%) had both parents, 31 (34%) had one parent and 36 (40%) had no parent within the watershed. The proportion of genes in saplings inherited from the adults within the watershed was 43%, and therefore 57% were from outside the site, indicating active gene exchange across the watershed area. Average distance between parents and saplings (264.6 +/- 135.3 (SD) m) was significantly smaller than that of pairs randomly chosen between adults and saplings (436.7 +/- 203.0 (SD) m). The distance of pollen movement inferred from the distance between the two parents of each sapling ranged from 3.2 m to 540 m with an average of 131.1 m +/- 121.1 m (SD). Because 34% ( = 31/91) of saplings had only one parent within the watershed, the estimate of average pollen movement must be smaller than the actual one. Long-distance seed dispersal by birds, inbreeding depression and limitation in acceptance of pollen because of the difference of phenology in each individual flower were considered to be the probable causes of large gene exchange across the watershed.