Comparison of Spatial Genetic Structures in Fagus crenata and F. japonica by the Use of Microsatellite Markers By T. TAKAHASHI1, A. KONUMA1,4, T. OHKUBO2, H. TAIRA1 and Y. TSUMURA3 (Received 10th November 2003) Abstract combination of limited gene dispersal and mating of individu- The spatial genetic structures of the tree species Fagus cre- als with their ancestors (DOLIGEZ et al., 1998). Furthermore, a nata and F. japonica were investigated by using 4 microsatel- long life span should enhance the effect of generation overlap lite markers. The study site was a 2-ha plot within a mixed and promote the development of spatial genetic structure. population of these species. We used 2 different statistics, However, whether differences in the number of generations per genetic relatedness, and the number of alleles in common lifetime affect population genetic structure has not yet been (NAC), to study the extent of spatial genetic structure. Signifi- reported. cant negative correlation between genetic relatedness and spa- Fagus crenata and F. japonica are monoecious, long-lived, tial distance was detected among all individuals in each woody angiosperms (beech species) with outcrossing breeding species. However, this correlation was weak and likely resulted from extensive pollen dispersion caused by wind pollination. systems based on wind pollination. The seeds are dispersed Spatial genetic clustering in F. japonica was stronger than in F. mainly by gravity but also secondary by animals (WATANABE, crenata over short distance classes. This result may be due to 1990; MIGUCHI, 1994). In a natural forest, F. crenata regener- the different reproductive and breeding systems, which were ates only by seedlings, and F. japonica regenerates from seeds the overlapping of individuals of kinship. and by production of spouts from the root collar. In this study, we investigated the genetic structures of F. crenata and F. Key words: Fagus crenata, F. japonica, genetic structure, Japanese japonica. The seed- and pollen-dispersal systems of both beech, NAC, Relatedness. species are similar (KITAMURA and MURATA, 1980), but the life- Introduction time of a generation is longer in F. japonica than in F. crenata (HARA, 1996). We studied individuals in a sympatric habitat of Spatial genetic structure within forest tree populations has these species by using microsatellite markers to investigate been investigated in many species and interpreted in terms of how differences in ecological characters such as regeneration population history, level extent of gene flow, and selection system and life span may affect their spatial genetic struc- (PERRY and KNOWLES, 1991; MERZEAU et al., 1994; BREG and tures. HAMRICK, 1995; SHAPCOTT, 1995; SHNABEL and HAMRICK, 1995; TANI et al., 1998; UENO et al., 2000). Knowledge of the genetic Materials and Methods structure of a population is important for in situ conservation of natural populations (RITLAND, 1985; WILLIAMS and HAMRICK, Materials 1996). Poor understanding of the spatial genetic structure Fagus crenata and F. japonica are distributed in the cool could lead to misinterpretation of the importance of mating temperate zone of the Japanese archipelago. Fagus crenata is systems and gene flow mechanisms and result in implementing found mainly in the heavy snowfall zone adjacent to the Sea of the wrong conservation program for the natural population. Japan and in scattered areas of the zone with little snow on the The spatial genetic structure within a population is influ- Pacific Ocean side. Fagus japonica is found only on the Pacific enced by several factors, such as seed dispersal and mating Ocean side, reaching less far north than F. crenata (SASAKI, system. Simulation studies based on isolation-by-distance mod- 1970; NOZAKI and OKUTOMI, 1990). Both are allogamous, wind- els have demonstrated that strong spatial genetic structure pollinated species with gravity-dispersed seeds. Fagus crenata tends to arise quickly and to persist across generations when regenerates by seedlings that establish during recovery of gene dispersal is limited with respect to distance (TURNER et canopy gaps in natural forests (NAKASHIZUKA and NUMATA, al., 1982; SOKAL and WARTENBERG, 1983; BREG and HAMRICK, 1982; NAKASHIZUKA, 1983; YAMAMOTO, 1989). Fagus japonica 1995). Recently, a simulation study by DOLIGEZ et al. (1998) regenerates both from seeds and by production of sprouts, showed that not only limited gene dispersal but also generation characteristically forming stools arising from vigorous sprout- overlapping and self-incompatibility can influence spatial ing from the root collar (TOHYAMA, 1965; OHKUBO et al., 1988; genetic structuring. In their study, generation overlapping PETERS and OHKUBO, 1990; OHKUBO, 1992). Within a F. japonica resulted in strong spatial genetic structure (DOLIGEZ et al., stool, one or more of the sprouts may replace a canopy trunk 1998). This effect of overlapping generations is presumably due that has died (OHKUBO, 1992), and the stools can reach sub- to the increased kinship between mates that results from the stantial ages. Since new sprouts are formed on the outside of each stool, the stool gradually increases in size (OHKUBO et al., 1996). The genetic homogeneity of such clusters of trunks has 1 Graduate School of Science and Technology, Niigata University, Niiga- ta, Niigata 950-2181, Japan been demonstrated by isozyme analysis (KITAMURA et al., 1992). 2 Department of Forest Science, Utsunomiya University, Utsunomiya Study site and sampling methods 321-8505, Japan 3 Corresponding author: YOSHIHIKO TSUMURA, Department of Forest The study site was located in the Forest Reserve near Mount Genetics, Forestry and Forest Products Research Institute, Tsukuba, Takahara (36° 53’ N) at an altitude of ca. 900 m, on a south- Ibaraki 305-8687, Japan. Phone +81-29-873-3211, Fax +81-29-874- east-facing slope ranging from 13° to 36° on the Pacific Ocean 3720. E-mail address: [email protected] side of Japan (Figure 1). The 4-ha plot (100×400 m) was estab- 4 Present address: Department of Biological Safety, National Institute for Agro-Environmental Sciences, Kannondai, Tsukuba, Ibaraki, 350- lished for a long-term ecological survey (PETERS and OHKUBO, 8604, Japan 1990). The 2-ha subplot in this 4-ha plot that was chosen for Silvae Genetica 52, 5-6 (2003) 287 Figure 1. – Location of the 2-ha study site and distribution map of 147 Fagus crenata (DBH > 5 cm) and 127 F. japonica (DBH > 5 cm) individuals. Closed circles: Fagus crenata individuals; open triangles: F. japonica individuals. observations on the genetic structure within F. crenata and F. ABI 310 Genetic Analyzer and GeneScan software version 2.0.1 japonica populations was located in an area containing a rela- (PE Applied Biosystems). tively high density of individuals of both species (Figure 1). Leaf samples were collected from 147 F. crenata (diameter at Data analysis breast height, DBH > 5 cm) and 127 F. japonica (DBH > 5 cm) For each locus, the allele frequencies and observed heterozy- individuals and were stored at –30°C prior to DNA extraction. gosity (HO) were calculated with genotype data. The expected Even large stools of F. japonica usually consist of single indi- heterozygosity, HE, and fixation index, F = 1 – (HE /HO), were viduals, so we collected leaf samples from only one sprout of calculated according to WRIGHT (1965). Null allele frequencies each stool to avoid resampling the same individuals. were estimated from the deviation from the Hardy-Weinberg equilibrium, with the Cervus 1.0 program (MARSHALL et al., DNA extraction and microsatellite genotyping 1998). Total DNA was extracted from leaves of each F. crenata and Measurements of genetic relatedness, as defined by QUELLER F. japonica individual by a modified CTAB method (TSUMURA et and GOODNIGHT (1989), were used to detect genetic structure in al., 1996). The genotypes of 4 microsatellite loci (TANAKA et al., F. crenata and F. japonica. In general, genetic relatedness is 1999) for each individual of each species were determined. the probability of 2 individuals sharing a gene that is identical Microsatellite primer pairs from F. crenata could be used for F. by descent. The genetic relatedness between all possible pairs japonica because they are closely related (TANAKA et al., 1999). of the 147 F. crenata individuals and 127 F. japonica individu- PCR amplification was performed in 10-µl reaction volumes als was calculated with the Relatedness program, version 5.0.6. containing 5 ng of genomic DNA, 1× PCR buffer, 0.2 mM of (GOODNIGHT, 1999), according to QUELLER and GOODNIGHT each dNTP, 1.5 mM MgCl2, 0.2 µM of each primer (the forward (1989). The spatial distance between all individual pairs was primer labeled with dye in each case), and 0.25 units of Taq also calculated for each species. The correlation between genet- polymerase, with the GeneAmp PCR System Model 9700 (PE ic relatedness and spatial distances among all individuals in Applied Biosystems). The annealing temperature and number each species was tested by Mantel tests with the R Package of PCR cycles for the primer pairs (mfc2, mfc5, mfc11, and program, version 4.0 (CASGRAIN and LEGENDRE, 2000). We used mfc12) were 64 °C, 58 °C, 56 °C, and 56 °C for 30 s; and 30, 35, 5000 times randomization in the Mantel test, which is a realis- 35, and 30 cycles, respectively. The denaturation and extension tic minimum for estimating a significance level of about 0.01 conditions were the same for all primer pairs: 94°C and 72 °C (MANLY, 1997). The spatial distances between individuals were for 30 s, respectively. The genotype was determined with an divided into 12 distance classes, at 5-m intervals from 0 to 60 288 m. The mean genetic relatedness was calculated for each of the the infinite allele model; NEI, 1973) and RST (based on the step- 12 distance classes by averaging the pairwise genetic related- wise mutation model; SLATKIN, 1995) values of 0.061 and 0.672, ness values.