Genes Genet. Syst. (2002) 77, p. 177–186 Genetic variation of Trigonobalanus verticillata, a primitive species of , 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 popula-

tions 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 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 , 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 , 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 in of (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 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, 4) Lawas, 5) Mt. Kinabalu, 6) Central Celebes, and 7) Northern Sumatra. Collection sites in this study are shown by open circle.

Table 1. Samples of Trigonobalanus verticillata. Population No. of Samples Locations Accession Numbersa Bario 12 Pa’Umor, Bario, Sarawak AB084766/ AB084769/ AB084772/ AB084775/ AB084778 Kinabalu 30 A) Kinasaraban villege, Kundasang, AB084767/ AB084770/ AB084773/ AB084776/ AB084779 Ranau, Kota Kinabalu B) Liwagu trail, Kinabalu National Park, Ranau, Kota Kinabalu Fraser’s Hill 33 Jeriau Valley, Fraser’s Hill, AB084768/ AB084771/ AB084774/ AB084777/ AB084780 Pahang a Accession numbers are shown in the order rbcL, matK, atpB-rbcL spacer, trnL intron, and trnL-trnF spacer, respectively. were randomly selected for sequence analysis. The rbcL matK-390R, 5’-GCATCTTTCACCCGGTAACG-3’; matK- gene, matK gene, atpB-rbcL spacer, trnL intron, and 444F, 5’-ACGAATACCCTACCCCATCCATCTG-3’; matK- trnL-trnF spacer were amplified by PCR using the uni- 919R, 5’-GACATTGCCATAAACTGACAAGGT-3’; matK- versal primers described by Franscaria et al. (1993) for 789F, 5’-CATCCCATGCTTACTCAAGGAT-3’; and matK- rbcL, Demesure et al. (1995) for matK, Terachi (1993) for 1022F, 5’-CAATGGTGCGGAGTCAAATGCTA-3’, and the atpB-rbcL spacer, and Taberlet et al. (1991) for the matK-8R, 5’-AAAGTTCTAGCACAAGAAAGTCGA-3’ (Ooi trnL intron and trnL-trnF spacer. PCR products were et al., 1995). directly sequenced after purification using a GENE- The sequences were aligned visually. The average CLEAN KIT III (BIO101). DNA sequencing was per- number of nucleotide substitutions was calculated based formed using an ABI 310 Genetic Analyzer (Perkin on the method of Jukes and Cantor (1969). A neighbor- Elmer) with an ABI BigDye Terminator Cycle Reaction joining tree (NJ; Saitou and Nei, 1987) with bootstrap Kit following the manufacturer’s instructions. The analysis (Felsenstein, 1985) was constructed using sequencing matK was performed using the following MEGA var. 2 (Kumar et al., 2001). The substitution rate internal primers originally designed by us except the last per site per year (r) can be estimated by dividing the num- one: matK-86F, 5’-ATGCACTTGCTCATGATCATGGT- ber of nucleotide substitutions per site (K) by 2T, where 3’; matK-152R, 5’-AATGCAAATTCTTGTTGTACCC-3’; T is the divergence time between two nucleotide sequ-

180 K. KAMIYA et al. ences (Li, 1997). In order to estimate r, the molecular substitutions per site among individuals was estimated, clock was calibrated by dating the unequivocal trigo- following the method of Innan et al. (1999). The average nobalanoid fossil to 32 million years ago (MYA) (middle values of pairwise distances (nucleotide diversity) within

Oligocene; Nixon and Crepet, 1989). The mean diver- each population (πS) and for the entire population (πT) gence time and the 95% confidence intervals between the were calculated. The inter-populational nucleotide diver- populations were estimated using synonymous sites of sity was compared to total nucleotide diversity to give NST rbcL and matK following the method of Haubold and = πT – πS / πT (Nei and Kumar, 2000). The UPGMA tree Wiehe (2001). was constructed based on the pairwise distance using NEIGHBOR program in PHYLIP 3.57c (Felsenstein, AFLP analysis. AFLP analysis was performed accord- 1993). ing to Vos et al. (1995) using an AFLP™ Core Reagent Kit following the instruction manual of the manufacturer (Life Technology, Inc.) with minor modifications. RESULTS Approximately 100 ng of the genomic DNA cleaved by EcoRI and MseI was ligated to EcoRI- and MseI-specific CpDNA analysis. CpDNA analysis was performed adaptors. Pre-amplification was performed with the using 19 individuals randomly selected from the three Pre-amplification Core Mix I (Life Technology, Inc.). populations. Among the three populations, a total of 30 PCR was performed with 20 cycles of 94°C for 30 sec, nucleotide substitutions and four indels were found, but 56°C for1 min, and 72°C for 1 min. Selective amplifica- no variations were found within a given population (Table tion was carried out using four selective primer pairs: 2 and Fig. 2). EcoRI-AAC/MseI-CTC, EcoRI-AAC/MseI-CAA, EcoRI- In complete rbcL sequences (1,446 bp, Fig. 2A), four ACG/MseI-CAA, and EcoRI-ACG/MseI-CAG. The ampli- synonymous and one nonsynonymous substitutions were fication products were subjected to PCR by incubation detected, while no substitutions were found in the 3’ under the following conditions: 94°C for 2 min; 10 cycles flanking region of this gene (64 bp). In complete matK of 94°C for 1 min, 65°C in the first cycle, and then reduced sequences (1,512 bp, Fig. 2B), seven synonymous and five the annealing temperature by 1°C each cycle for the next nonsynonymous substitutions were found, while there nine cycles for 30 sec, and 72°C for 2 min; and 20 cycles were no substitutions in both flanking regions (277 bp of of 94°C for 1 min, 56°C for 30 sec, and 72°C for 2 5’ and 249 bp of 3’ flanking regions). In atpB-rbcL spacer min. The respective amplification products were sepa- (Fig. 2A), nine substitutions and two insertion-deletions rated using an ABI 310 Genetic Analyzer (Perkin (indels) of mononucleotides were found. In the trnL Elmer). The data were collected using Genetic Analyzer intron (Fig. 2C), two substitutions were found in the Collection Software, and analyzed using GeneScan® 3.1 Fraser’s Hill population. In the trnL-trnF spacer (Fig. software. The fragment size was determined using 2C), two substitutions and two indels were found. A sin- GeneScan-500 [ROX] size standards (Perkin Elmer) that gle 22-bp insertion sequence (5’-CAATCATTCTACT- were loaded with the amplification products. We CTTTTACAA-3’) at position 114 was found in Fraser’s detected fragments ranging in size from 60 to 500 bp, and Hill, but not found in the other populations. Moreover, subject them to further analysis. a tandem repeat of a 20-bp-long motif (5’- CAATATTTGT- The amplification fragments were manually scored as GATATATATG-3’) at position 162 was found only in present (1) or absent (0). Some unclear fragments were Kinabalu. Since length variation was detected on the gel ignored. Genetic diversity was assessed within each (Fig. 3), the sequence of this region was determined for all population and in the total population based on the pro- 30 samples from Kinabalu. Eleven types of length vari- portion of polymorphic bands and average heterozygosity. ants containing four to 21 repeats of the motif were found, The proportion of polymorphic bands (P) was estimated with 17 repeats of the motif being the most frequent by dividing the number of loci having a band frequency of (43%). more than 0.05 and less than 0.95 by the total number of The nucleotide diversity among the populations ranged loci. Considering the AFLP marker to be dominant, the from 0.0006 for rbcL nonsynonymous sites to 0.0115 for average heterozygosity (H) was estimated under the matK synonymous sites (Table 2). The phylogenetic Hardy-Weinberg equilibrium; the frequency of a recessive analysis based on each region as well as the combined allele (q) for the null band at each locus was estimated as data clearly demonstrated the topology: (Fraser’s Hill the square root of x, which is the fraction of individuals (Bario, Kinabalu)) when the sequences of Fagus crenata in a population without the marker, and then the het- were used as an outgroup taxon. This indicates the erozygosity for each locus was estimated as 2pq (p = 1 monophyly of T. verticillata and that the Fraser’s Hill –q). The relative magnitude of genetic differentiation population is distantly related to the populations Bario among populations (FST) was calculated for each locus and and Kinabalu (data not shown). This topology is then averaged over all loci. The number of nucleotide strongly supported by 100% bootstrap probability and is Genetic variation of Trigonobalanus 181

Table 2. Polymorphism of rbcL, matK, atpB-rbcL spacer, trnL intron, and the trnL-trnF spacer among popula- tions of Trigonobalanus verticillata. Locus No. of sites analyzed No. of substitution Nucleotide diversitya rbcL overall 1,446 5 0.0023 ± 0.0010 synonymous 351 4 0.0077 ± 0.0037 nonsynonymous 1,095 1 0.0006 ± 0.0006 matK overall 1,512 12 0.0053 ± 0.0016 synonymous 351 5 0.0115 ± 0.0045 nonsynonymous 1,161 7 0.0035 ± 0.0014 atpB-rbcL 751 9 0.0084 ± 0.0026 trnL intron 523 2 0.0028 ± 0.0015 trnL-trnF 438-460 2 0.0034 ± 0.0015 a The standard error of the nucleotide diversity was estimated by the bootstrap method (Nei and Kumar, 2000; Kumar et al., 2001).

Fig. 2. The nucleotide substitutions and indels of A) the rbcL gene and the 5’ flanking region (atpB-rbcL spacer), B) the matK gene, and C) the trnL intron and trnL-trnF spacer in T. verticillata. Solid boxes indicate coding regions, and solid lines indicate non-coding regions. Asterisks indicate the nonsynonymous substitutions. Gaps are indicated by -. The sequences of the insertions in the trnL- trnF spacer are as follows: 114: 5’-CAATCATTCTACTCTTTTACAA-3’, and 162: 5’-CAATATTTGTGATATATATG-3’. The latter inser- tion occurs as a tandem repeat of varying copy numbers in the Kinabalu population. consistent with the geographical distribution of the pop- ibrate the molecular clock. The nucleotide divergence ulations. based on rbcL and matK synonymous sites was calculated By the molecular phylogenetic analysis, Trigonobala- to be 0.0115 ± 0.0039 between Fraser’s Hill and Bario- nus was affiliated at a sister position of the allied genera Kinabalu, and 0.0057 ± 0.0028 between Bario and Kina- of Castanea, Castanopsis, Lithocarpus, and Quercus, but balu. Using these values, the age of the population dif- not with Fagus (Manos and Steele, 1997; Kamiya and ferentiation was estimated to be 16.7 MYA with 95% Harada, unpublished results). The nucleotide diver- confidence intervals of 6.7 MYA and 32.9 MYA between gence between Trigonobalanus and above mentioned Fraser’s Hill and Bario-Kinabalu, and 8.3 MYA with con- allied genera was estimated to be 0.02293 ± 0.0039 based fidence intervals of 2.1 MYA and 19.4 MYA between on rbcL and matK synonymous sites and was used to cal- Bario and Kinabalu. 182 K. KAMIYA et al.

Fig. 3. PCR products of the trnL-trnF spacer in T. verticillata. Length polymorphism among the three populations and within the Kinabalu population were observed on an ethidium bromide-stained agarose gel. A negative image is shown. The size markers in the outermost lanes are the fragments of the lambda DNA (Takara) digested with HindIII. Lanes 1 and 2, Fraser’s Hill; lanes 3 and 4, Bario; lanes A1-A15, Kundasang, Kinabalu; B1-B15, Kinabalu National Park, Kinabalu.

Table 3. Diversity statistics for the three populations of Trigonobalanus verticillata by AFLP analysis. Population Pa Hb Average no. of different bands πc Bario 0.482 0.134 ± 0.008 71.1 ± 10.5 (16.5%) 0.01259 ± 0.00002 Kinabalu 0.632 0.170 ± 0.009 85.1 ± 14.2 (19.7%) 0.01390 ± 0.00012 Fraser’s Hill 0.625 0.154 ± 0.009 76.3 ± 10.4 (17.7%) 0.01321 ± 0.00008 All 0.789 0.198 ± 0.009 95.5 ± 16.7 (22.2%) 0.01683 ± 0.00006

FST = 0.153 NST = 0.214 a P = proportion of polymorphic loci (95% criteria). b H = average heterozygosity with standard error estimated on the assumption of Hardy-Weinberg equilibrium. c π = Average nucleotide diversity estimated following the method of Innan et al. (1999) with standard error.

Fig. 4. UPGMA tree based on the pairwise distance estimated by the method of Innan et al. (1999) among 75 individuals of T. verticillata. The number in parenthesis indicates the number of tandem repeats in the trnL-trnF spacer. Genetic variation of Trigonobalanus 183

AFLP analysis. A total of 75 samples were used for the Although Trigonobalanus is thought to be derived taxa AFLP analysis. Of the 431 bands scored, 206 (47.8%), from the northern temperate region (Crepet and Nixon, 270 (62.6%), and 267 (61.9%) were polymorphic in the 1989), the current distribution is somewhat puzzling Bario, Kinabalu, and the Fraser’s Hill populations, because the genus has largely disappeared from continen- respectively. The total number of polymorphic bands tal Asia (Corner, 1990). Our estimate of the divergence was 340 (78.9%). The average heterozygosity within a time of the populations using the cpDNA molecular clock population (HS) ranged from 0.134 in Bario to 0.170 in may provide a clue about the routes of migration. The Kinabalu, with a mean value of 0.153. The total het- divergence of the Fraser’s Hill population from the others erozygosity (HT) was 0.198, and the coefficient of genetic was estimated to be 16.7 MYA. The 95% upper confi- differentiation among populations (FST) turned out to be dence limit (32.9 MYA) indicates that T. verticillata 0.153. reached the Malay peninsula no earlier than the The average number of bands that differed between Oligocene. Paleobotanical evidence has shown that the individuals in a given population was lowest in Bario Oligocene and early Miocene (about 35 to 21 MYA) were (71.1; 16.5%) and highest in Kinabalu (85.1; 19.7%). The periods of relatively dry and cool climates in Southeast nucleotide diversity within a population ranged from Asia (Morley, 1998; Whitmore, 1998). The cooler cli- 0.01259 to 0.01321, and 0.01683 for entire population mates could have allowed the montane species to expand (Table 3). The proportion of inter-populational nucle- to lower altitudes and migrate to lower latitudes. This otide differentiation (NST) turned out to be 0.214. suggests that T. verticillata could have expanded to the Figure 4 shows the UPGMA tree on the basis of pair- south and subsequently migrated as far as Borneo during wise distance estimated by the method of Innan et al, this period. Early Miocene paleogeographical recon- (1999) . The individuals from each population formed a struction in the Sunda-Sahul region suggests that one monophyletic group. The Fraser’s Hill population was a possible migration route would have been the corridor sister clade to that of Bario and Kinabalu, consistent with across the Malay peninsula and Borneo (Morley and Flen- the result of cpDNA analysis. Samples of the Kinabalu ley, 1987). Morley (1998) also suggested that warm and population were collected from two locations (Table 1), moist climatic conditions prevailed during the initial but no difference between these locations was seen in the parts of the middle Miocene (c.a. 15 MYA) throughout tree. large parts of East and Southeast Asia. Thus, a large area of the regions was covered by tropical lowland ever- green forest species. Species like Trigonobalanus, which DISCUSSION were adapted to a cooler climate would then have with- In this study, we used cpDNA sequences and AFLP to drawn to a higher altitude in the montane area and could study genetic diversity within and between populations of have been isolated. On the other hand, the divergence T. verticillata. Both analyses showed a significant level time of the Bario and Kinabalu populations was esti- of population differentiation. The cpDNA data showed a mated to be 8.3 MYA. The lower 95% confidence limit large number of nucleotide substitutions between popula- (2.1 MYA) indicates that the differentiation occurred no tions, but no substitutions were found within a given later than the Pliocene. The idea that a cooler and sea- population. This implies very limited gene flow via sonal climate started during the Pleistocene and Quater- seeds between populations of T. verticillata. The level of nary is supported by the fact that pollen assemblies found intraspecific variation shown here is higher than the at the Subang airport in the Malay peninsula were dom- interspecific variation within genera in Fagaceae; for inated by Pinus and grass pollens (Whitmore, example, in rbcL no substitutions were found within Jap- 1998). Furthermore, marks of Quaternary glaciers were anese species and Malaysian species of Quercus subgenus found in Mt. Kinabalu below about 3,000 m (Whitmore, Cyclobalanopsis (Kamiya and Harada, unpublished 1998). Morley and Flenley (1987) also suggested that results), while four substitutions were found among T. there was a seasonal climate within the Malay peninsula verticillata populations. In matK, only two substitutions during the middle Pleistocene. This could have permit- were found in the various species of Quercus (Kamiya and ted montane species to expand to lowland areas and Harada, unpublished results), while 12 substitutions allowed frequent migration between populations. were found in T. verticillata. This suggests that the A 20-bp-long repeat motif, 5’-CAATATTTGTGATAT- Trigonobalanus populations have been isolated for a long ATATG-3’, was found in the trnL-trnF spacer specifically time and are therefore evolutionarily more divergent. in the Kinabalu population. Simple sequence repeats Moreover, Forman and Cutler (1967) observed that mor- (so-called SSRs) such as mono- or di-nucleotide repeats phology of cupule-valves of specimens from Kinabalu have been reported in cpDNA (Powell et al., 1995a; Powell appeared distinctive from that of specimens from Fraser’s et al., 1995b; Palmer, 1987). However, to the best of our Hill; i.e., those of the former are narrower and more knowledge, tandem repeats that consist of such a long tomentous externally than those of the latter. motif (so-called VNTR or minisatellite in Hartl, 2000) 184 K. KAMIYA et al. have not been detected previously in cpDNA. Three species is certainly a subject for future study. monophyletic groups were identified in the Kinabalu pop- The AFLP analysis indicates that T. verticillata shows ulation based on the AFLP-based UPGMA tree (Fig. 4); a relatively large genetic differentiation (FST = 0.153 and however, neither the sampling sites nor the distribution NST = 0.214), concurring with the results of cpDNA of repeat type was related to the phylogenic relationship analysis. The UPGMA tree (Fig. 4) based on nucleotide in Kinabalu. This may suggest that multiple changes of diversity also indicated significant population differenti- the repeat number have occurred within the Kinabalu ation. The longer branch leading to each individual indi- population. cates that the individual is genetically differentiated from The absence of nucleotide substitutions in cpDNA each other. Although it has been suggested that there is sequences within populations suggests that the effective vegetative reproduction of T. verticillata, in which epicor- size of the cpDNA has been kept very small or that severe mic (coppice) shoots are produced at the base when the bottlenecks occurred in the past. Nevertheless, the main trunk died or is injured (Forman, 1964; Corner, AFLP analysis showed relatively large genetic variation 1990), no evidence for a clonal state was detected in this within populations of T. verticillata. The average het- study. In our field observation, many seedling could be erozygosity was high even in the Bario population (H = found, suggesting possible sexual reproduction of this 0.134) (which contains only a small number of individu- species. Thus, for T. verticillata, asexual reproduction als) as compared to the values obtained by AFLP analysis does not play a major role for extending the habitat. of the Japanese beech, Fagus crenata (H = 0.102) and Richards (1996) suggested that the formation of ‘clumps’ Japanese , Quercus mongolica var. grosserata (H = 0. by Trigonobalanus is a method of crown enlargement 133) (Okaura and Harada, 1998). These two fagaceous rather than reproduction, as the secondly trunks do not species are the main components of the cool deciduous for- usually become independent trees. ests and widely distributed throughout Japan. Travis et The values for genetic differentiation observed here al. (1996) studied the genetic variation of the endangered also suggest that some gene flow occurs between popula- species, Astragalus cremnophylax var. cremnophylax, tions, possibly through pollen transfer. The ratio of seed using AFLP markers. Their results clearly indicate that to pollen flow has been calculated for some European the smallest population had lost genetic diversity (H = Fagaceae species using the formula of Ennos (1994). The 0.018 in South Rim Site 2). Furthermore, they found results showed that the pollen/seed flow ratio is generally unexpectedly lower diversity in larger populations con- high for the anemophilous species such as Quercus and sisting of about 500 individuals (H = 0.037 in South Rim Fagus (< 100), while the value for entomophilous species Site 1). According to Hamrick et al. (1991), endemic spe- such as Castanea is close to 1 (Fineschi et al., 2000). The cies in restricted areas might have been maintained in ratio for T. verticillata must be high (this is evident even small populations for long periods of time. Such popula- though the formula of Ennos is not applicable because of tions often show rather low genetic variation due to forced complete fixation of cpDNA). The haploid nature of inbreeding within populations. On the other hand, cpDNA in comparison with the diploid nature of AFLP larger genetic variations is often associated with wide might also explain the restricted gene flow via seeds vs. geographic ranges, out-crossing, and wind-pollination possible wind pollination in T. verticillata. In addition, (Hamrick et al., 1991). In this study, the Kinabalu pop- due to the essentially complete linkage of cpDNA, adap- ulation showed the highest average heterozygosity (H = tive mutations of some genes may have been fixed easily 0.170) and the Bario population showed the lowest (H = through selective sweep (Hartl, 2000). 0.134). In Kinabalu and Fraser’s Hill, relatively large numbers of adult trees were found; for example, the We thank A. Zainudin Ibrahim, M. Nazre Saleh, B. Parumal, Fraser’s Hill population consists of about 700 mature and L. F. S. Julia for assistance during fieldwork, and T. Okaura trees in the confined area of Jeriau Valley (L. F. S. Julia, for kindly providing his unpublished results. This work was supported by the New Energy and Industrial Technology Devel- personal communication). In contrast, the Bario popula- opment Organization (NEDO) under the Malaysia-Japan tion consists of very few individuals, probably due to the Research Corporation on Conservation and Sustainable Use of expansion of rice fields (Latiff et al., 1998b). This result Tropical Bioresources, and IRPA Grant 08-02-02-0022 from the suggests that the genetic diversity of the populations in Malaysian Government. T. verticillata is not correlated with the current popula- tion size and the populations have kept fairly large size REFERENCES for a long period of time. It should be noted that we are assuming random mating, and the heterozygosity would Anamthawat-Jónsson, A., Bragason, B. T. H., Bödvarsdóttir, S. be overestimated if there is some fraction of inbreeding. K., and Koebners, R. M. D. (1999) Molecular variation in Leymus species and populations Mol. Ecol. 8, 309–315. Although all the samples were collected from mature Corner, E. J. H. (1990) On Trigonobalanus (Fagaceae). Bot. J. trees and the deviation from random mating is expected Linn. Soc. 102, 219–223. to be small, investigation of the breeding system in this Corner, E. J. H. (1996) The plant life of Kinabalu - An introduc- Genetic variation of Trigonobalanus 185

tion. In: Kinabalu Summit of Borneo (eds.: K. M. Wong, and Hartl, D. L. (2000) A Primer of Population Genetics, 3rd Edition. A. Philipps), pp. 101–150. The Sabah Society in associa- Sinauer Associates, Massachusetts, USA. tion with Sabah Park, Malaysia. Haubold, B., and Wiehe, T. (2001) Statistics of divergence time. Crepet, W. L, and Nixon, K. C. (1989) Earliest megafossil Mol. Biol. Evol. 18, 1157–1160. evidence of Fagaceae: phylogentic and biogeographic Innan, H., Terauchi, R., Kahl, G., and Tajima, F. (1999) A implications. Amer. J. Bot. 76, 842–845. method for estimating nucleotide diversity from AFLP data. Cresswell, A., Sackville Hamilton, N. R., Roy, A. K., and Viegas Genetics 151, 1157–1164. B. M. (2001) Use of amplified fragment length polymor- Jukes, T. H., and Cantor C. R. (1969) Evolution of protein mole- phism markers to assess genetic diversity of Lolium species cules. In: Mammalian Protein Metabolism (ed.: H. N. from Portugal. Mol. Ecol. 10, 229–241. Munro), pp. 21–132. Academic Press, New York. Demesure, B., Sodzi, N., and Petit, R. J. (1995) A set of univer- Krauss, S. L. (2000) Accurate gene diversity estimates from sal primers for amplification of polymorphic non-coding amplified fragment length polymorphism (AFLP) markers. regions of mitochondrial and chloroplast DNA in plants. Mol. Ecol. 9, 1241–1245. Mol. Ecol. 4, 129–131. Kumar, S., Tamura, K., Jakobsen I. B., and Nei, M. (2001) Doyle, J., and Doyle, L. (1990) Isolation of plant DNA from fresh MEGA2: Molecular Evolutionary Genetics Analysis tissue. Focus 12, 13–15. software. Bioinformatics (in press). Ennos, R. A. (1994) Estimating the relative rates of pollen and Latiff, A., Mat-Salleh, K., Faridah, H. I., Zakri, A. H., and seed migration among plant populations. Heredity 80, Ogino, K. (1998a) Ecosystem conservation in Malaysia. In: 584–593. Proceedings of the Tokyo International Forum on Conserva- Felsenstein, J. (1993) PHYLIP (phylogeny inference package) tion and Sustainable Use of Tropical Bioresources, pp. 26– version 3.5,c. Distributed by author. Department of Genet- 39. Japan Bioindustry Association, Tokyo, Japan. ics, University of Washington, Seattle. Latiff, A., Zainudin, A. I., and Mat-Salleh, K. (1998b) An account Felsenstein, J. (1985) Confidence limits on phylogenies: An and checklist of the flowering plants at Kelabit Highlands, approach using the bootstrap. Evolution 39, 783–791. Sarawak. In: A Scientific Journey through Borneo: Bario: Ferris, C., King, R. A., Vainola, R., and Hewitt, G. M. (1998) The Kelabit Highlands of Sarawak (eds.: G. Ismail and L. B. Chloroplast DNA recognizes three refugial sources of Euro- Din), pp. 29–50. Pelanduk Publications (M) Sdn. Bhd., pean oaks and suggests independent eastern and western Malaysia. immigrants to Finland. Heredity 80, 584–593. Li, W. -H. (1997) Molecular Evolution. Sinauer Associates, Mas- Ferris, C., Oliver, R. P., Davy, A. J., and Hewitt, G. M. (1993) sachusetts, USA. Native oak chloroplasts reveal an ancient divide across Lozano-C., G., Harnandez-C., J., and Henao-S., J. E. (1979) Hal- Europe. Mol. Ecol. 2, 337–344. lazgo del genero Trigonobalanus Forman, 1962 (Fagaceae), Ferris, C., Oliver, R. P., Davy, A. J., and Hewitt, G. M. (1995) en el Neotropico-I. Caldasia 12, 517–537. Using chloroplast DNA to trace postglacial migration routes Morley, R. J. (1998) Palynological evidence for Tertiary plant of oak into Britain. Mol. Ecol. 4, 731–738. dispersal in the SE Asian region in relation to plate tecton- Fineschi, S., Taurchini, D., Villani, F., and Vendramin, G. G. ics and climate. In: Biogeography and Geological Evolution (2000) Chloroplast DNA polymorphism reveals little geo- of SE Asia (eds.: R. Hall and D. Holloway), pp. 211–234. graphical structure in Castanea sativa Mill. (Fagaceae) Backhuys Publishers Leiden, the Netherlands. throughout southern European countries. Mol. Ecol. 9, Morley, R. J., and Flenley, J. R. (1987) Late Cainozoic vegeta- 1495–1503. tional and environmental changes in the Malay archipelago. Forman, L. L. (1964) Trigonobalanus, a new genus of Fagaceae, In: Biogeographical Evolution of the Malay Archipelago (ed.: with notes on the classification of the family. Kew. Bull. T. C. Whitmore), pp. 50–59. Oxford Monograph on Biogeog- 17, 381–396. raphy 4, Oxford Scientific Publications. Forman, L. L., and Cutler, D. F. (1967) Additional notes on Trig- Muluvi, G. M., Sprent, J. I., Soranzo, N., Provan, J., Odee, D., onobalanus Forman (Fagaceae). 1, Trigonobalanus in Folkard, G., McNicol, J. W., and Powell, W. (1999) Amplified Malaya and Sarawak. Kew. Bull. 21, 331–334. fragment length polymorphism (AFLP) analysis of genetic Frascaria, N., Maggia, L., Michaud, M., and Bousquest, J. (1993) variation in Moringa oleifera Lam. Mol. Ecol. 8, 463–470. The rbcL gene sequence from indicates a slow rate Negi, M. S., Singh, A., and Lakshmikumaran, M. (2000) Genetic of evolution in the Fagaceae. Genome 36, 668–671. variation and relationship among and within Withania spe- Fujii, N., Ueda, K., Watano, Y., and Shimizu, T. (1997a) cies as revealed by AFLP markers. Genome 43, 975–980. Intraspecific sequence variation in chloroplast DNA of Nei, M. (1987) Molecular Evolutionary Genetics. Colombia Primula cuneifolia Ledeb. (Primulaceae). J. Phytogeogr. University Press, New York. Taxon. 43, 15–24. Nei, M., and Kumar, S. (2000) Molecular Evolution and Phylo- Fujii, N., Ueda, K., Watano, Y., and Shimizu, T. (1997b) Intra- genetics. Oxford University Press, New York. specific sequence variation of chloroplast DNA of Pedicu- Nei, M., and Gojobori T. (1986) Simple methods for estimating laris chamissonis Steven (Scrophulariaceae) and geographic the numbers of synonymous and nonsynonymous nucleotide structuring of Japananese “Alpine” plants. J. Plant Res. substitutions. Mol. Biol. Evol. 3, 418–426. 110, 195–207. Newton, A. C., Allnutt, T. R., Gillies, A. C. M., Lowe A. J., and Hamrick, J. L., Godt, M. J. W., Murawski, D. A., and Loveless, Ennos R. A. (1999) Molecular phylogeography, intraspecific M. D. (1991) Correlations between species traits and alloz- variation and the conservation of tree species. TREE 14, yme diversity: Implications for conservation biology. In: 140–145. Genetics and Conservation Biology (eds.: D. A. Falk, and K. Nixon, K. C., and Crepet, W. L. (1989) Trigonobalanus (Faga- E. Holsinger), pp. 75–86. Oxford Univ. Press, New York. ceae): taxonomic status and phylogenetic relationships. Hamrick, J. L., Godt, M. J., and Sherman-Broylers, S. L. (1992) Amer. J. Bot. 76, 828–841. Factors influencing levels of genetic diversity in woody Okaura, T., and Harada, K. (1998) Genetic structure of Japa- plant species. New Forests 6, 95–124. nese beech and a kind of oak in the population in western 186 K. KAMIYA et al.

part of Japan. Genes Genet. Syst. 73, 451 (abstract only). new method for reconstructing phylogenetic trees. Mol. Okaura, T., and Harada, K. (2002) Phylogeographical structure Biol. Evol. 4, 406–425. revealed by chloroplast DNA variation in Japanese beech Soepadmo, E. (1972) Fagaceae. Flora Malesiana 7 (2), 266–403. (Fagus crenata Blume). Heredity 88, 322–329. Soepadmo, E., Julia, S., and Go, R. (2000) Fagaceae. In: Tree Ooi, K., Endo, Y., Yokoyama, J., and Murakami, N. (1995) Use- Flora of Sabah and Sarawak (eds.: E. Soepadmo and L. G. ful primer designs to amplify DNA fragments of the plastid Saw), pp. 1–117. Ampang Press Sdn. Bhd., Kuala Lumpur, gene matK from Angiosperm plants. J. Jpn. Bot. 70, 328– Malaysia. 331. Taberlet, P., Gielly, L., Pautou, G., and Bouvet, J. (1991) Univer- Palmer, J. D. (1987) Chloroplast DNA evolution and biosystem- sal primers for amplification of three non-coding regions of atic uses of chloroplast DNA variation. Am. Nat. 130, S6– chloroplast DNA. Plant Mol. Biol. 17, 1105–1109. S29. Terachi, T. (1993) Structural alterations of chloroplast genome Powell, W., Morgante, M., Andre, C., McNicol, J. W., Machray, and their significance to the higher plant evolution. Bull. G. C., Doyle, J. J., Tingey, S. V., and Rafalski, J. A. (1995a) Inst. Natl. Land Util. Developm. Kyoto Sangyo Univ. 14, Hypervariable microsatellites provide a general source of 138–148. polymorphic DNA markers for the chloroplast genome. Travis, S. E., Maschinski, J., and Keim, P. (1996) An analysis of Curr. Biol. 5, 1023–1029. genetic variation in Astragalus cremnophylax var. cremno- Powell, W., Morgante, M., McDevitt, R., Vendramin, G. G., and phylax, a critically endangered plant, using AFLP markers. Rafalski, J. A. (1995b) Polymorphic simple sequence repeat Mol. Ecol. 5, 735–745. regions in chloroplast genomes: applications to the popula- Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., tion genetics of plants. Proc. Natl. Acad. Sci. USA. 92, Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M., 7759–7763. and Zabaeu, M. (1995) AFLP: a new technique for DNA Richards, P. W. (1996) The Tropical Rain Forest 2nd Edition. fingerprinting. Nucleic Acids Res. 23, 4407–4414. Cambridge University Press, Cambridge. Whitmore, T. C. (1998) An Introduction to Tropical Rain Forests Saitou, N., and Nei, M. (1987) The neighbor-joining method: a 2nd Edition. Oxford University Press, New York.