A single application of fertilizer can affect semi-natural grassland vegetation over half a century.

Restoration of species-rich semi-natural grassland requires not only a seed source but also appropriate soil properties. In Europe, approximately 10 years are required for the properties of fertilized soils to reach suitable conditions and be considered successfully restored. However, restoration may require additional time in Japan because heavier precipitation causes leaching of basic cations from soils, resulting in soil acidification; volcanic ejecta also forms active Al and Fe hydroxides with high phosphate sorption. Within this context, we aimed to answer the following questions: i) whether and how the impacts of fertilization remain in the soil properties after half a century in Japan; and ii) how fertilization affects the restoration of semi-natural grasslands in Japan. We investigated the vegetation and soil properties of a Zoysia japonica pasture improved half a century ago with a single application of fertilizer and an adjacent semi-natural grassland (native pasture) in Japan, and found the following: (1) the two pastures had similar dominance of Z. japonica, but differed in the species composition; (2) the improved pasture exhibited lower species richness than the native pasture; (3) soil nutrients, including N, P, K, Mg, and Ca, were higher in the improved pasture than in the native pasture; and (4) many chemical properties of the soils were associated with species composition; namely, the vegetation on nutrient-rich soil had more alien species and fewer native species. We conclude that a single dose of fertilization can affect soil properties in semi-natural grasslands over half a century in Japan, leading to species loss and changing the species composition. We suggest that fertilized soils under grazing in Japan may require more than half a century to restore the nutrients to suitable levels for the establishment of a species-diverse grassland.

Genetic diversity and population structure of Nuphar submersa (Nymphaeaceae), a critically endangered aquatic plant endemic to Japan, and implications for its conservation.

Nuphar submersa (Nymphaeaceae) is a critically endangered freshwater macrophyte indigenous to central Japan, with only four small extant populations represented across its entire range. We investigated the genotypic and genetic diversity as well as the genetic structure of all extant individuals of N. submersa based on analysis of 15 microsatellite loci. Among 278 individual ramets, 52 multilocus genotypes were detected: 30 genotypes in Nikko City (NIK), 18 in Nasukarasuyama City (NAS), 3 in Mooka City (MOK), and 1 in Sakura City (SAK). The average number of alleles per locus ranged from 1.20 to 1.93, whereas the observed and expected heterozygosities ranged from 0.11 to 0.33 and from 0.10 to 0.24, respectively. With the exception of SAK, all populations contained multiple clones, but our results indicated low levels of within-population genetic diversity. The populations NIK and NAS comprised few large or middle-sized genets and many small genets. The populations NIK and NAS were suggested to comprise large old, old fragmented, and/or young small genets resulting from seedling establishment. All four populations were differentiated, and gene flow between the populations was restricted (average level of gene flow (Nm) = 0.122, G' ST  = 0.639). Of the total genetic diversity, 67.20 and 9.13% were attributable to inter- and intra-population diversity, respectively. STRUCTURE analysis revealed two or three well-differentiated groups of populations. Cluster I comprised one population (NIK) and cluster II comprised the remaining populations at K = 2. The populations NIK, NAS, and the remaining populations were assigned to clusters I, II, and III, respectively, at K = 3. For conservation practices, we recommend that each cluster be regarded as a different management unit. We further suggest that artificial gene flow among MOK and SAK populations is an appropriate option, whereas NIK should not be reinforced with genotypes from the remaining populations.

Predominance of a single clone of the most widely distributed bamboo species Phyllostachys edulis in East Asia.

Phyllostachys edulis, one of the most dominant bamboo species with the leptomorph rhizome system, has been asexually expanding its range into adjacent natural forest sites by shooting new culms. The resulting ecological problems include simplification of stand structure and decline in the species diversity of local flora. In this study, the genetic diversity of P. edulis for the entire distribution range from Japan to China was analyzed using 16 microsatellite markers. Among these, 12 loci were fixed by a single allele, whereas only two alleles were detected for each of the remaining 4 loci; all adult samples shared the same genotype at all loci including the four heterozygous loci. These observations indicate that all current samples from Japan and China comprise an identical clone. The clone is distributed over more than 2,800 km with an estimated biomass of approximately 6.6 × 10(11) kg, which is exceptionally large. Among seedlings from flowering events in 2005 and 2006, 20 different genets were generated by recombination through selfing of a single flowering genet. Predominance of a single clone in the wild and a diverse composition of genets among seedlings suggest that the intermittent flowering of P. edulis in the wild has produced a variety of clones through recombination. However, the resulting seedlings cannot compete with other tree species or adult P. edulis, and almost all adult P. edulis growing in Japan and China likely propagated through vegetative reproduction of a single clone by human transplantation, and subsequently expanded into adjacent forest sites by shooting young sprouts. The relatively small size of the flowering area and rapid culm reproduction has led to the stability of P. edulis communities. However, the low genetic diversity is an important consideration for the long-term management of this prevailing bamboo species.

Outcrossing rates and organelle inheritance estimated from two natural populations of the Japanese endemic conifer Sciadopitys verticillata.

The Japanese endemic conifer Sciadopitys verticillata is one of the most phylogenetically isolated species of all plants. Occurring in small and scattered populations, the species is currently classified as Near Threatened by the International Union for Conservation of Nature and Natural Resources (IUCN) and as Vulnerable in three prefectures of Japan. This study investigated two major factors that should impact the genetic structure of the species at both the nuclear and organelle DNA level, the mating system and the inheritance of both the chloroplast and mitochondrial genomes. The mating system is crucial to determining the degree of outcrossing of plant species and thus should have a key role in shaping the species' population level genetic diversity and gene flow between populations but as yet has not been studied in S. verticillata. Nine mother trees and their seedling progeny from two natural populations were genotyped using genetic markers from three plant genomes (eight nuclear microsatellites and DNA sequence for the chloroplast and mitochondria). Using a maximum likelihood method implemented in the software MLTR, the study found an outcrossing rate in the seedling stage of 0.49 and 0.79 for Aburazaka and Mount Shirotori populations, respectively, and an average of 0.66 at the species level. These outcrossing rates were low for conifers and therefore may have potential deleterious implications for the conservation of the species. The test of organelle inheritance supported paternal transmission of both the chloroplast and mitochondria consistent with previous microscopic evidence.