Makoto Miwa

Outcrossing and paternity analysis of Pinus densiflora (Japanese red pine) by microsatellite polymorphism.

This study employed microsatellite loci to analyse outcrossing rate and pollen dispersal in Japanese red pine (Pinus densiflora) in an isolated stand. The average offspring outcrossing rate for 29 cones was 0.955. Significant differences in outcrossing rates between offspring groups on individual branches that extended in different directions at different heights were not detected. Male parents of 874 offspring collected from the maternal tree were assessed by exclusion using polymorphisms at three microsatellite loci. Paternity analysis indicated that at least 31% of the offspring were fertilized by pollen from trees outside the stand. The average distance of pollen migration within the study stand was 68 m, with a maximum value of 325 m. There was excess mating with nearby P. densiflora trees, of which only a few were predominant pollen donors. In addition, a weakly directional bias in P. densiflora pollination was also detected in the study stand, suggesting that female strobili on a branch of the maternal tree were more easily fertilized by pollen from trees in that direction.

Spatial distribution of the subterranean mycelia and ectomycorrhizae of Suillus grevillei genets

Suillus grevillei, and found that one, SG-5, is species-specific to S. grevillei and polymorphic. Using the SG-5 marker, we analyzed for the first time the horizontal and vertical distribution of the subterranean parts of several S. grevillei genets. The spatial distribution of S. grevillei genets in the soil demonstrated that the development of S. grevillei sporocarps is correlated with that of extra-radical mycelia and ectomycorrhizae of the same genet, which are distributed in a narrow area. However, sporocarps are not always centered over the subterranean parts and the amount of subterranean mycelia and mycorrhizae is not always correlated to the number of sporocarps formed on them. No S. grevillei mycelia and mycorrhizae were detected beneath the positions where S. grevillei sporocarps emerged in the previous year. The observation indicates the rapid alteration of the subterranean parts of S. grevillei genets, and suggests that S. grevillei genets change location rather than merely extend their habitat year after year.

Patch establishment and development of a clonal plant, Polygonum cuspidatum, on Mount Fuji

Microsatellite analysis was used to investigate the patch establishment and development of Polygonum cuspidatum Sieb. et Zucc, a clonal herbaceous plant that dominates the primary succession on the southeast slope of Mount Fuji. Genotypes of P. cuspidatum in 155 patches at the study site differed from each other. This indicates that P. cuspidatum patches are initially established by seed dispersed on the bare scoria field, and not by clonal rhizome extension. Genetic differentiation was estimated using the FST values between subpopulations at the study site. There was almost no genetic differentiation between subpopulations, indicating the presence of massive gene flow. The pollen fathers of seeds and maternal genets of current‐year seedlings were inferred from the microsatellite allele composition by a simple exclusion method. The wide, random distribution of pollen fathers suggests that pollen dispersal occurs over a broad area. Maternal analysis showed a tendency for seed dispersal to be biased to the area nearby and down slope from the mother plants. Patch establishment under massive gene flow may result from such pollen and seed dispersal. To understand the process of patch development, aerial photographs taken from 1962 to 1999 were compared, and then genets in each of 36 patches were identified from the microsatellite genotypes of P. cuspidatum shoots. The comparison of aerial photographs showed that most of the patches enlarged each year and that some neighbouring patches combined during growth. Genet analysis demonstrated a high correlation between patch area and the area of the largest genet within it, and that new genets were recruited at the patch periphery. These findings indicate that both vegetative and sexual reproduction, i.e. rhizome extension and the establishment of new seedlings, contribute to the development of P. cuspidatum patches.

Genet Distribution of Ectomycorrhizal Fungus Suillus grevillei Populations in Two Larix kaempferi Stands over Two Years

Suillus grevillei in two Larix kaempferi stands was determined over two years by inter-simple sequence repeat (ISSR) polymorphism analysis using primers, (GTG)5, (GCC)5 and (GACA)4. Thirty-five genets were identified from 67 sporocarps at the older stand (stand A in which the distribution of S. grevillei genet in 1997 was analyzed previously) in 1998, and 14 genets from 52 sporocarps at the younger stand (stand B) in 1997 and 1998. The characteristics of S. grevillei genets in stand A in 1998 were similar to those in 1997. A single genet was represented by 1.8 and 3.7 sporocarps on average in stands A and B, respectively. In stand A, 42 out of 61 genets, i.e., about 70% were represented by individual sporocarps compared to five out of 14 genets, i.e., about 35% in stand B. The largest and the average genet sizes was 6.8 m and less than 1 m in stand A, and 11 m and 2.3 m in stand B, respectively. A t-test showed the genet size in stand A to be significantly smaller than that in stand B. The above results indicate that the smaller genets of the S. grevillei population in stand A might be due to environmental conditions not genetic traits specific to this species. Observations over two years showed that although some genets formed sporocarps in both 1997 and 1998, many formed sporocarps only in one of the two years. Emerging positions of sporocarps in 1997 and 1998, which belonged to the same genet, were similar but not identical, about 2 m apart, suggesting mobility in the subterranean parts of ECM fungal genets.

Analysis of clonal structure of Melaleuca cajuputi (Myrtaceae) at a barren sandy site in Thailand using microsatellite polymorphism

Abstract. The clonal structure of Melaleuca cajuputi growing in Thailand was investigated using ten polymorphic microsatellite markers and excavation of root connections. A large-scale test plot (25×45xa0m) and a small-scale one (10×12xa0m) within it were established in an M. cajuputi population at a barren sandy location in Narathiwat, southern Thailand (6° 30′ N, 101° 45′ E). Eleven clonal groups were identified from 31 ramets in the small-scale test plot based on root connections between ramets. To evaluate the effectiveness of microsatellite markers for genet analysis of M. cajuputi, clonal groups in the small-scale test plot identified by root connection were compared with genets determined by analysis of ten microsatellite markers. Most of the clonal groups identified by root connection completely matched the genets determined by the microsatellite markers. This indicates that microsatellite markers are very useful for genet analysis of M. cajuputi. Eleven genets were identified from 53 ramets in the large- and small-scale test plots using the ten microsatellite markers; the largest genet was about 23xa0m wide. This suggests that vegetative reproduction, i.e., shoot sprouting from roots, plays an important role in the establishment of M. cajuputi populations at this barren sandy site.

Indirect prediction of surface ozone concentration by plant growth responses in East Asia using mini-open top chambers

We developed small and mobile open top chambers (mini-OTC) measuring 0.6xa0m (W)u2009×u20090.6xa0m (D)u2009×u20091.2xa0m (H) with an air duct of 0.6xa0m (W)u2009×u20090.23xa0m (D)u2009×u20091.2xa0m (H). The air duct can be filled with activated charcoal to blow charcoal filtered air (CF) into the chamber, as opposed to non-filtered ambient air (NF). Ozone sensitive radish Raphanus sativus cv. Red Chime and rosette pakchoi Brassica campestris var. rosularis cv. ATU171 were exposed to NF and CF in mini-OTCs at different locations in East Asia. A total of 29 exposure experiments were conducted at nine locations, Shanghai, China, Ha Noi, Vietnam, Lampang, Phitsanulok and Pathumtani, Thailand, and Hiratsuka, Kisai, Abiko and Akagi, Japan. Although no significant relationships between the mean concentrations of ambient O3 during the experimental period and the growth responses were observed for either species, multiple linear regression analysis suggested a good relationship between the biomass responses in each species and the O3 concentration, temperature, and relative humidity. The cumulative daily mean O3 (ppb/day) could be indirectly predicted by NF/CF based on the dry weight ratio of biomass, mean air temperature, and relative air humidity.