Genetic Variation of Trigonobalanus Verticillata , a Primitive Species Of

Genes Genet. Syst. (2002) 77, p. 177–186 Genetic variation of Trigonobalanus verticillata, a primitive species of Fagaceae, in Malaysia revealed by chloroplast sequences and AFLP markers Koichi Kamiya1, Ko Harada1*, Mahani Mansor Clyde2, and Abdul Latiff Mohamed2 1Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan 2Faculty of Science and Technology, University Kebangsaan Malaysia, 43600 UKM, Bangi, Selangor, Malaysia (Received 15 January 2002, accepted 16 May 2002) The genetic variation of Trigonobalanus verticillata, the most recently described genus of Fagaceae, was studied using chloroplast DNA sequences and AFLP fingerprinting. This species has a restricted distribution that is known to include seven localities in tropical lower montane forests in Malaysia and Indonesia. A total of 75 individuals were collected from Bario, Kinabalu, and Fraser’s Hill in Malaysia. The sequences of rbcL, matK, and three non-coding regions (atpB-rbcL spacer, trnL intron, and trnL-trnF spacer) were determined for 19 individuals from these populations. We found a total of 30 nucleotide substitutions and four length variations, which allowed identification of three haplotypes characterizing each population. No substitutions were detected within populations, while the tandem repeats in the trnL-trnF spacer had a variable repeat number of a 20-bp motif only in Kinabalu. The differentiation of the populations inferred from the cpDNA molecular clock calibrated with paleontological data was estimated to be 8.3 MYA between Bario and Kinabalu, and 16.7 MYA between Fraser’s Hill and the other populations. In AFLP analysis, four selective primer pairs yielded a total of 431 loci, of which 340 (78.9%) were polymorphic. The results showed rel- atively high gene diversity (HS = 0.153 and HT = 0.198) and nucleotide diversity (πS = 0.0132 and πT = 0.0168) both within and among the populations. Although the cpDNA data suggest that little or no gene flow occurred between the populations via seeds, the fixation index estimated from AFLP data (FST = 0.153 and NST = 0.214) implies that some gene flow occurs between populations, possibly through pollen transfer. monotypic genera, Trigonobalanus verticillata, Formano- INTRODUCTION dendoron doichangensis, and Colombobalanus excelsa Trigonobalanus is the most recently described genus of (Nixon and Crepet, 1989). The collection of Trigonobal- Fagaceae, which was originally considered to contain two anus verticillata and establishing as a new genus has species, T. verticillata from Malaysia and Indonesia, and been one of the great botanical topics of the last century T. doichangensis from Thailand and South China (For- because the presence of some ambiguous characteristics man, 1964). Later, a third species, T. excelsa, was des- suggests this genus to be primitive in the Fagaceae (For- cribed from Colombia (Lozano et al., 1979). Although man, 1964; Corner, 1990). For example, the floral appa- these species were originally placed in a single genus, ratus indicates affinities to Quercus, and the wood Trigonobalanus, one or more unique morphological fea- structure indicates affinity to Quercus and Lithocarpus, tures suggest that the three species may have diverged in while fruit and seedling development resembles that seen ancient times and they are currently treated as three in Fagus (Forman, 1990). Molecular phylogenetic study of Fagaceae suggested that Trigonobalanus have diversi- Edited by Hidenori Tachida fied after Fagus and basal to Castanea and Quercus * Corresponding author. E-mail: [email protected] (Manos and Steele, 1997). 178 K. KAMIYA et al. Trigonobalanus verticillata has a restricted distribu- large numbers of samples (Travis et al., 1996). tion that is known in the seven localities in Peninsular In this study, we examined the genetic diversity Malaysia, Borneo, Sumatra, and Celebes (Forman, 1964; between and within populations of T. verticillata.We Forman and Cutler, 1967; Nixon and Crepet, 1989; Fig. used cpDNA sequences to analyze haplotypic diversity in 1). Although this species is commonly found in some T. verticillata, and especially to focus on the level of pop- areas in Peninsular Malaysia and Borneo, only a few ulation differentiation. In addition, we estimated the specimens have been collected in central Celebes and divergence time between the populations as inferred from Sumatra, suggesting that it is less common in Indonesia the cpDNA molecular clock, so as to shed light on the (K. C. Nixon and M. Hotta, personal communications). possible process of migration of this species. AFLP fin- This species occurs in tropical lower montane forests at gerprinting was used to evaluate the intra- and inter-pop- elevations of 800–1,500 m and coexists with other faga- ulational genetic variation on the basis of allelic ceous species, Lauraceae, and several gymnosperms frequency analysis. In a wide range of Fagaceae species, such as Agathis (Araucariaceae) and Podocarpus chloroplast genome have been demonstrated to be trans- (Podocarpaceae). The areas where T. verticillata is mited via seeds (Demesure et al., 1995). While, AFLP abundant contain a remarkably rich diversity of faga- markers reveal both plastid and nuclear DNA fragments. ceous species. For example, it is well known that there Application of both maternally and biparentally inherited are about 50 species of Fagaceae in Kinabalu, as well as markers allowed us to compare the levels of migration via other plant species (Corner, 1996). Eighteen fagaceous seed and via pollen based on the levels of population sub- species have been found in a one-hectare plot at Fraser’s division inferred from cpDNA and AFLP, respectively. Hill, but far fewer in the Cameron Highlands located only 120 km north of Fraser’s Hill in a mountain system of Banjaran Titiwangsa. Moreover, in South China, where MATERIALS AND METHODS Fagaceae has broadly diversified, one species related to Trigonobalanus, F. doichangensis, is found. These Study sites and sampling. Sampling was carried out observations may suggest that the area where T. verticil- on three populations in Malaysia, i.e., Bario, Kinabalu, lata occurs has not been seriously affected by environ- and Fraser’s Hill (Fig. 1 at positions 2, 1, and 5, mental changes during past geological ages. Thus, the respectively). In Bario, located in northeastern Sarawak area might have provided a suitable refugium for Trigo- about 1,100 m above sea level, the vegetation consists of nobalanus and other montane species. submontane forests. A wide area of the vegetation has Although chloroplast DNA (cpDNA) is not thought to be been affected by degradation of the natural forests and very useful for detecting intraspecific genetic variation, a transformed to wet and dry kerangas forests (Latiff et al., number of studies have successfully examined the varia- 1998b). A small population is found in Bario, where the tions in this plastid genome (Ferris et al., 1993; Ferris et habitat seems to have been largely disturbed by human al., 1995; Fujii et al., 1997a; Ferris et al., 1998). Fujii et activity. In Kinabalu, samples were collected from two al. (1997b) identified 17 haplotypes in Pedicularis cha- locations, Kundasang and Kinabalu National Park areas missonis based on the sequences of three cpDNA non-cod- (Table 1). In both areas, the species is predominantly ing regions. Ferris et al. (1998) demonstrated the routes distributed in the lower montane forest up to 1,500 m of postglacial migration of European oaks, Quersus robur above sea level (Soepadmo et al., 2000). The Fraser’s and Q. petraea, and identified three refugial sources for Hill population is found on both sides of Sungai Jeriau up these species using the trnL intron sequences. A similar to 1,100 m above sea level. In this area, the species coex- study has been conducted for Japanese beech, Fagus cre- ists with Lithocarpus and several conifers, and the distri- nata (Okaura and Harada, 2002). These studies have bution seems to be related to the allocation of some demonstrated that cpDNA is a useful tool for determining elements in the soil (Latiff et al., 1998a). the phylogeography, interspecific variation, and gene flow Leaf samples were collected from 12 mature trees in of plants (Newton et al., 1999). Bario, 30 in Kinabalu, and 33 in Fraser’s Hill (Table Amplified fragment length polymorphism (AFLP) is one 1). The trees often have multi-stems and shoots around of the most powerful methods recently developed (Vos et the bottom of the main trunk (so-called coppice shoots). al., 1995) for characterizing the genetic diversity within We treated a tree with many trunks and shoots as one and among populations (Travis et al., 1996; Anamthawat- individual, and collected leaf samples from individual Jónsson et al., 1999; Muluvi et al., 1999; Krauss, 2000; trees located at least 10 m apart from each other. Total Negi et al., 2000; Cresswell et al., 2001). This technique DNA was isolated following the CTAB method (Doyle and can generate more than 50 variable genetic markers Doyle, 1990). through a single iteration and can provide an efficient tool for studying the genetic diversity in rare and/or Sequence analysis. Nineteen samples, i.e., four from endangered species, in which it is often difficult to collect Bario, eight from Kinabalu, and seven from Fraser’s Hill, Genetic variation of Trigonobalanus 179 Fig. 1. Present distribution of Trigonobalanus verticillata (Nixon and Crepet, 1989). 1) Fraser’s Hill, 2) Bario, 3) Hose Mountains,

Load more