Mineaki Aizawa

Genetic structure of Sakhalin spruce (Picea glehnii) in northern Japan and adjacent regions revealed by nuclear microsatellites and mitochondrial gene sequences

The genetic structure of Sakhalin spruce (Picea glehnii) was studied across the natural range of the species, including two small isolated populations in south Sakhalin and Hayachine, by using six microsatellite loci and maternally inherited mitochondrial gene sequences. We also analyzed P. jezoensis, a sympatric spruce in the range. Genetic diversity of P. glehnii was higher in central Hokkaido and the lowest in the Hayachine. Bayesian clustering and principal coordinate analysis by using the microsatellites indicated that the Hayachine was clearly distinct from other populations, implying that it had undergone strong genetic drift since the last glacial period. P. glehnii harbored four mitochondrial haplotypes, two of which were shared with P. jezoensis. One of the two was observed without geographical concentration, suggesting its derivation from ancestral polymorphism. Another was observed in south Sakhalin and in P. jezoensis across Sakhalin. The Bayesian clustering—by using four microsatellite loci, including P. jezoensis populations—indicated unambiguous species delimitation, but with possible admixture of P. jezoensis genes into P. glehnii in south Sakhalin, where P. glehnii is abundantly overwhelmed by P. jezoensis; this might explain the occurrence of introgression of the haplotype of P. jezoensis into P. glehnii.

Comparison of fine-scale spatial genetic structure of two sympatric Rhododendron shrub species in forest habitat having different seed weights: A case study

Abstract Restricted seed dispersal is one of the most prevalent determinants of spatial genetic structure (SGS) at a fine spatial scale within a plant population. Rhododendron kaempferi and R. semibarbatum are common and coexistent Ericaceous species in the shrub layer of secondary deciduous broad-leaved forests in the northern Kanto District, central Japan. The two species have entomophilous flowers and are thought to have similar pollination styles. However, R. kaempferi produces threefold heavier seeds than R. semibarbatum. Therefore, we tested the hypothesis that the intensity of SGS was stronger in R. kaempferi than in R. semibarbatum in a forest stand. We comparatively examined the SGS for 73 individuals of R. kaempferi and 36 individuals of R. semibarbatum by using highly variable nuclear microsatellite loci. The analysis revealed significant SGS in both species at the shortest distance (<3 m); a measure to quantify SGS showed a counterintuitive result: R. semibarbatum exhibited stronger SGS than R. kaempferi. This result might be explained by the ecological consequences of R. semibarbatum producing lighter seeds, which might have greater dispersal efficacy, but its safe sites could be more restricted than those of R. kaempferi; in contrast, R. kaempferi producing heavier seeds might have more limited seed dispersal, but its safe sites for seedling establishment could be more prevalent than those for R. semibarbatum. The different strategies for the trade-off between seed weight and site selection of the two Rhododendron species might be reflected in the difference in the intensity of SGS in this study plot.

Natural hybridization of Yezo and Sakhalin spruce in central Hokkaido, revealed by DNA markers with contrasting modes of inheritance

Yezo spruce (Picea jezoensis var. jezoensis) and Sakhalin spruce (Picea glehnii) occur across Hokkaido and co-occur in some forest habitats. This leads to the potential for natural hybridization between these two species, which has been shown to occur at low frequencies. The purpose of this study was to identify these hybrids and their possible mating patterns, using various Pinaceae DNA markers with different modes of inheritance. The markers used were maternally inherited mitochondrial DNA (mtDNA), paternally inherited chloroplast DNA (cpDNA) and biparentally inherited nuclear microsatellites (nSSRs). Seven putative natural hybrids, four artificially-crossed F1 hybrids, four parent plants from each species, and two artificially-backcrossed hybrids of putative natural hybrids and their parents were analyzed using the diagnostic DNA markers developed in this study. We found Yezo spruce and Sakhalin spruce to be distinct (J and G types, respectively), and the modes of inheritance held true for the two species, as was previously reported to be the case in Pinaceae. Four of the seven putative natural hybrids harbored J-type cpDNA, G-type mtDNA and J/G-type nSSRs, indicating that natural F1 hybrids are likely to arise from a G (female) × J (male) crossing. One natural hybrid harbored G-type cpDNA, J-type mtDNA and J/G-type nSSRs, which implies that hybrids produced by J (female) × G (male) crossings occur at low frequencies. The two remaining hybrids harbored J-type cpDNA and mtDNA with either J/G or J/J-type nSSRs, suggesting that they may be F2 hybrids resulting from backcrossing between an F1 hybrid and a Yezo spruce.

Characterization of 13 polymorphic microsatellite loci in the Japanese land leech

We developed 13 polymorphic microsatellite loci of the Japanese land leech (Haemadipsa japonica; Haemadipsidea) using an Illumina MiSeq sequencing approach. A total of 42,064 nuclear DNA contigs were filtered for microsatellite motifs, among which 30,873 simple sequence repeat loci were identified. From these sequences, we selected 30 primer sets, and 13 of these loci were successfully amplified. Polymorphism of the 13 loci was tested using 16 individuals sampled from sixteen populations across Japan. The number of alleles and polymorphism information content varied from 5 to 17 and 0.335 to 0.883, respectively, and observed and expected heterozygosity values ranged from 0.143 to 0.875 and 0.349 to 0.893, respectively, indicating that these loci are polymorphic. Furthermore, we established useful multiplex PCR using these loci. The 13 microsatellite loci described in this paper are the first nuclear microsatellite markers for a land leech species.

Relationships between tree size and reaction wood formation in 23 Japanese angiosperms

The objective of this study is to clarify the relationships between tree sizes and the anatomical, chemical, and physical characteristics of reaction wood. Naturally inclined stems or branches in 23 Japanese angiosperms, including 3 tree sizes at adult trees, 7 arbor, 7 subarbor, and 9 shrub species, were used. The presence of a gelatinous (G-) layer, basic density, vessel frequency, and lignin content were examined on the upper and lower sides of each sample. Almost all the species showed a decrease in vessel frequency and lignin content on the upper side, which are typical characteristics of reaction wood in angiosperms. A G-layer was formed in four arbor, one subarbor, and two shrub species. The ratio of the upper and lower sides in vessel frequency and lignin content gradually decreased with an increase in tree size. Based on the results of the principal component and cluster analyses, shrub species were classified into the different groups: from arbor and subarbor groups. These results indicate that the magnitude of alteration in the characteristics due to reaction wood formation of arbor and subarbor might differ from that of shrub, although the characteristics changed by reaction wood formation might be the same in the three tree sizes at adult trees.

Highly polymorphic nuclear microsatellite markers reveal detailed patterns of genetic variation in natural populations of Yezo spruce in Hokkaido

The Yezo spruce (Picea jezoensis) has attracted attention as a species commonly used for afforestation in Hokkaido. However, there is no information available for assessing appropriate seed and planting zones for this species. We developed 21 nuclear microsatellite markers for P. jezoensis and used them to examine the genetic structure of nine natural populations (288 trees). Based on the 31 samples, the 21 markers were highly polymorphic, with an average expected heterozygosity greater than 0.84. When we applied ten effective markers to nine populations in Hokkaido, no clear regional differences in genetic diversity were detected. STRUCTURE analysis revealed that, aside from certain geographically isolated or peripheral populations, the populations within planting zones belonged to a single cluster. Thus, based on our marker data, with a few exceptions, there appears to be a single seed zone within the planting zones in Hokkaido.