「森林総合研究所研究報告」(Bulletin of FFPRI), Vol.5, No.2 (No.399), 175-181, June 2006 論 文（Original article） Preliminary results for genetic variation and populational differentiation of Japanese alder (Alnus japonica), a pioneering tree species in the Kushiro Marshland, Hokkaido KONDO Kei 1), KITAMURA Keiko 2)* and IRIE Kiyoshi 3) Abstract In recent years, an Alnus japonica (Japanese alder) forest has been expanding into the Kushiro Marshland, bringing conspicuous changes to its ecosystem and landscape. To prevent these rapid changes in wetland ecosystems, we sought to clarify the genetic variation and structure of 23 populations for A. japonica using isozyme analysis, which might provide basic information for estimating the distribution expansion mechanism of A. japonica in the Kushiro Marshland. Our results show significantly high expected heterozygosity (He) for populations in upstream regions than in downstream regions. Other genetic parameters show no significant differences, such as the mean number of alleles per locus (Na), effective number of alleles per locus (Ne), observed heterozygosity (Ho), and the coefficient that measures excesses of homozygotes relative to panmictic expectations within respective populations (FIS). The FST value is high (0.183), indicating differences in allele frequencies among populations. Significant clinal differences between populations in the upstream and downstream regions might be partly attributable to (i) the founder effect, a fluctuation of genetic diversity during foundation, or (ii) natural selection to certain alleles to/against different environments. The high FST value among populations might be partly attributable to the founder effect as a pioneer species and one with rapid expansion during establishment. This result might relate to the characteristic to the pioneering tree species, which establishes suitable sites by chance. Key words : founder effect, isozyme, Japanese alder (Alnus japonica), the Kushiro Marshland Introduction of A. japonica forest. Edaphic dryness of the marshland is The Japanese alder (Alnus japonica) is a deciduous broad- inferred to be a major factor for the expanded distribution of A. leaved tree species that grows in wet lowlands such as open japonica forest. Nakamura et al. (2002) reported that housing swamps and fallow fields with wet and open environments. The and farmland development causes soil and sand inflow to the tree reaches a maximum diameter of 60 cm and 20 m height. marshland, which engenders edaphic dryness. This species is distributed widely from Ussuri in China, to Genetic variation and structure of populations might Japan, Korea, and Taiwan. The growth form of the A. japonica provide basic information for clarifying the distribution tree changes according to local environmental conditions: it has expansion process of present A. japonica establishment sites. a tall single trunk in fertile lands, but it is shrubby with high For that reason, we studied genetic variation and structure of levels of groundwater (Fujita, 2002; Nakamura et al., 2002). A. japonica populations over the Kushiro Marshland using to Alnus japonica is wind pollinated and has wind-dispersal seeds obtain basic information for controlling the rapid expansion of A. (Suzuki, 1975); it is an intolerant tree species (Koike, 1991). japonica. Therefore, A. japonica might be a pioneer tree species. In the last 50 years, the expanded distribution of A. Materials and Methods japonica has made them increasingly common in the Kushiro Study site Marshland. Fens that had been covered with Japanese reeds We sampled 23 populations of A. japonica in the Kushiro (Phragmites communis) are changing to swamp forests of A. Marshland, eastern Hokkaido (Fig. 1; Table 2). All populations japonica. Moreover, A. japonica is invading into the core of are located in wetlands of spring-water swamps in foot hills, the marshland, where it had only grown at the edges before. creek floodplains, and marshland edges. The populations are Rapid change of the Kushiro Marshland landscape might affect often mixed with ash (Fraxinus mandshurica) and elm (Ulmus its ecosystem. It is necessary to prevent the rapid expansion japonica). 原稿受付：平成 17 年 7 月 6 日 Received July. 6, 2005 原稿受理：平成 18 年 1 月 24 日 Accepted Jan. 24, 2006 * Hokkaido Research Center, Forestry and Forest Products Research Institute (FFPRI), Hitsujigaoka 7, Toyohira, Sapporo 062-8516, Japan 1) Ce� plan Co. Ltd., 4-9-5-27 Tsukisamuhigashi Toyohira-ku, Sapporo, Hokkaido, 062-0054 2) Hokkaido Research Center, Forestry and Forest Products Research Institute (FFPRI) 3) Docon Co. Ltd., 1-5-4-1 Atsubetsuchuo Atsubetsu-ku, Sapporo, Hokkaido, 004-8585 176 KONDO K. et al. (PGM; EC 2.7.5.1), and shikimate dehydrogenase (ShDH; EC 1.1.1.25) (Table 1). Data analysis Phenotypes were interpreted into genotypes, and allele frequencies of respective loci were calculated. The following genetic parameters were calculated for all populations, the mean number of alleles per locus (Na), the effective number of alleles per locus (Ne), observed heterozygosity (Ho), and expected heterozygosity (He) using the POPGENE computer program (Yeh et al., 1997) for loci with at least two alleles. The genetic structure within and among populations was Fig. 1. Study sites for Alnus japonica in the Kushiro Marshland. estimated using Wright’s (1965) F-statistics: a coefficient Numbers indicate respective populations. See Table 2. represents excesses of homozygotes relative to panmictic Enzyme electrophoresis expectations within respective populations (FIS); another Ten plants were collected randomly from each population. coefficient measures excesses of homozygotes relative to Leaves were collected from each tree during June–September panmictic expectations over all populations (FIT); and another 2003 and were stored at -30°C until enzymes were extracted. coefficient estimates relative population differentiation (FST). Isozyme analyses were carried out according to Shiraishi (1988). We compared genetic parameters (Na, Ne, Ho, He, FIS) for First, 100 µg of leaf tissue was homogenized with liquid populations according to the stream length from the river mouth nitrogen and 1.0 mL of extract buffer with 80 mg polyvinylpoly- to respective populations (parametric test – Pearson correlation pyrrolidone. Of the resulting supernatant, 17 µL was loaded on coefficient; non-parametric test – Kendall’s rank correlation a polyacrylamide vertical slab gel after centrifugation (20000 coefficient); tests of significance were also conducted (Student’s g, 20 min). Electrophoresis was conducted at 2°C, 10 mA/cm2 t-test). for 150 min. Gels were stained for the following 12 enzyme systems: alcohol dehydrogenase (ADH; EC 1.1.1.1), amylase Results (AMY; EC 3.2.1.1), diaphorase (DIA; EC 1.6.4.3), glutamate Genetic diversity dehydrogenase (GDH; EC 1.4.1.2), glutamate oxaloacetate This study scored 16 loci from 12 enzyme systems. All transaminase (GOT; EC 2.6.1.1), glucose-6-phosphate 16 studied loci showed detectable polymorphisms. No locus dehydrogenase (G6PD; EC 1.1.1.49), leucine aminopeptidase showed monomorphism. Among these loci, we used 10 loci (LAP; EC 3.4.11.1), malate dehydrogenase (MDH; EC 1.1.1.37), (Amy-1, Amy-2, Dia, Gdh-1, Gdh-2, G6p, Lap, Mdh, Pgi, Shdh) 6-phosphogluconate dehydrogenase (6 PGD; EC 1.1.1.44), with a clear banding pattern for further analyses (Table 1). phosphoglucoisomerase (PGI; EC 5.3.1.9), phosphoglucomutase The mean number of alleles per locus (Na) was 2.81 within populations, ranging 2.00–3.20. The effective number Table 1. List of enzyme systems employed. of alleles per locus (Ne) was 2.13 within populations, ranging Enzyme E.C. No. No. of loci scored 1.65–2.53. The observed heterozygosity (Ho) was 0.421 within ADH 1.1.1.1 - populations, ranging 0.233–0.567. The expected heterozygosity AMY 3.2.1.1 2 (He) was 0.513 within populations, ranging 0.285–0.588 (Table DIA 1.6.4.3 1 GDH 1.4.1.2 2 2). Most populations show high genetic variations (e.g. Shdh GOT 2.6.1.1 - locus (Fig. 3)). G6P 1.1.1.49 1 Most populations show that the expected heterozygosity LAP 3.4.11.1 1 was higher than the observed heterozygosity, suggesting MDH 1.1.1.37 1 an excess of homozygotes. The homozygote excess is also 6PGD 1.1.1.44 - reflected in a population mean of 0.106 for Wright's F (Table 3); PGI 5.3.1.9 1 IS PGM 2.7.5.1 - Wright's FIT value was 0.270 (Table 3). ShDH 1.1.1.25 1 We also analyzed relationships between respective genetic - indicates that the locus was not included for scoring because parameters for each population and the stream length from the of the lack of enzyme activity in certain individuals tested or river mouth to each population. Significant correlation was because of smeared banding patterns. found between the expected heterozygosity (He) and the stream length from the river mouth (parametric test – r =0.439, p < 0.05; 森林総合研究所研究報告 第 5 巻 2 号 , 2006 Preliminary results for genetic variation and populational differentiation of Japanese alder (Alnus japonica), 177 a pioneering tree species in the Kushiro Marshland, Hokkaido Table 2. Calculated genetic parameters for 23 populations for A. japonica. Standard deviations are shown in parentheses. Sample size (N), mean number of alleles per locus (Na), effective number of alleles per locus (Ne), observed heterozygosity (Ho), expected heterozygosity (He) and a coefficient measures excesses of homozygotes relative to panmictic expectations within respective populations (FIS). No. River Population Stream length(km)* N Na Ne Ho He FIS 1 Kushiro Toya1 9.50