International Journal Sustainable Future for Human Security FORESTRY AGRICULTURE J-SustaiN Vol. 3, No. 1 (2015) 12-20 http://www.j-sustain.com effective conservation schemes as well as for providing Population Genetic basic data for future provenance trials of Dryobalanops Diversity in The Genus species. Keywords: Dryobalanops spp.; genetic diversity; genetic Dryobalanops Gaertn. f. structure; microsatellite. (Dipterocarpaceae) Based on Nuclear Microsatellite 1. Introduction Markers The genus Dryobalanops (Dipterocarpaceae), locally known as “Kapur”, is a prominent genus with few species (a total of a b seven species). Three species, i.e. D. aromatica Gaertn. f., D. Fifi Gus Dwiyanti , Lucy Chong , Bibian beccarii Dyer and D. oblongifolia Dyer have a broad Diwayb, Lee Ying Fahc, Iskandar distribution range over Borneo, Sumatra and the Malay Zulkarnaen Siregard, Atok Subiaktoe, Peninsula. D. aromatica occurs naturally in Sumatra f f (Angkola, Sibolga, Kelasan, Upper Singkil, Bengkalis, Siak Koichi Kamiya , Ikuo Ninomiya , Ko and Mursala Island), the Malay Peninsula (Johor, Pahang, Haradaf* Selangor and Terengganu), Lingga Archipelago (Lingga and Singkep Island) and Borneo (Sabah, Sawarak and Brunei). a United Graduated School of Agricultural Sciences, Ehime Two subspecies are recognized for D. oblongifolia, i.e., University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan subsp. oblongifolia Dyer and subsp. occidentalis P.S. Ashton, b Botanical Research Centre, Km 20 Jalan Puncak Borneo, with the former occurring in Borneo (Sarawak, West Semenggoh Kuching, Sarawak, 93250, Malaysia Kalimantan, Central Kalimantan and East Kalimantan) and c Forest Research Centre, Forest Department Sabah, KM 23, the latter in Sumatra (Bengkalis, Riau, Jambi and Labuk Road, Sepilok, PO Box 1407, Sandakan, Sabah, 90715, Malaysia. Palembang) and Malay Peninsula (Johor, Kelantan, Pahang, d Faculty of Forestry, Bogor Agricultural University, Darmaga Perak, Selangor, and Terengganu). D. beccarii occurs in the Campus, PO Box 168, Bogor 16680, Indonesia Malay Peninsula (Johor) and Borneo (Sabah and Sarawak) e Forest and Nature Conservation Research and Development [1,2]. The other four species, i.e. D. lanceolata Burck, D. Centre, Jl. Gunung Batu No. 5, Bogor 16001, Indonesia rappa Becc., D. keithii Symington and D. fusca Slooten are f Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, endemic to Borneo. The timber of Dryobalanops, which Matsuyama, Ehime 790-8566, Japan differs in density and strength between species, is an important, moderately heavy and durable construction Received: January 27, 2015/ Accepted: April 24, 2015 timber. This genus has excellent traits that allow rapid Abstract growth in plantations or through silvicultural manipulations [3]. The species also yields particular commodities The genus Dryobalanops Gaertn. f. is a small but important categorized as non-wood forest products, such as oil and genus that contains seven species and is found in the camphor crystals, which are obtained from the inner Southeast Asian tropical rain forests. Seven polymorphic portion of the stems. Camphor especially obtained from D. nuclear microsatellite markers were analyzed in 46 natural aromatica attracted early Arab traders, and was worth more populations of six extant Dryobalanops species (N = 700 than its weight in gold [4]. individuals) covering the distribution range in Western Similar to other dipterocarps, the distribution area Malesia. The mean gene diversity at the species level where Dryobalanops species once abundant has been ranged from 0.392 in D. rappa to 0.635 in D. aromatica. shrinking since the last half century due to over- The F value ranged from 0.156 in D. keithii to 0.283 in D. ST exploitation and changes in land use systems to agriculture beccarii and all F values were highly significantly greater ST and oil palm cultivation [5]. Increased use of forest than zero. These results suggest that gene flow between resources and a shrinking forestland base threaten the populations has been limited and intensive genetic drift sustainability of forest genetic resources and highlight the has occurred in all of the species. Plants with narrowly distributed species such as D. keithii and D. rappa, which are endemic to Borneo, tend to have lower levels of genetic * diversity compared with widespread species such as D. Corresponding Author aromatica. STRUCTURE analysis revealed that at least two Tel.: +8189-946-9870 ; Fax.: +8189-946-9870 ; E-mail: of the six species could be effectively discriminated each [email protected]. other. The results of this study will be helpful for building Dwiyanti, Fifi Gus; Chong, Lucy; Diway, Bibian; Fah, Lee Ying; Siregar, Iskandar Zulkarnaen; Subiakto, Atok; Kamiya, Koichi; Ninomiya, Ikuo; Harada, Ko/ J-SustaiN Vol. 3, No.1 (2015) 12-20 importance of conservation and sustainable management of II (QIAGEN Japan, Tokyo). Total genomic DNA was these resources [6]. In fact, the lowland dipterocarp forests extracted from each sampled leaf using the modified CTAB where these species often predominate, are ecologically method [12]. important for nursing the rich biological diversity and for controlling the atmosphere-water conditions at a global 2.2. Microsatellite genotyping level. Protecting these rain forests is one of the biggest environmental issues in this century. Obtaining information Seven nuclear microsatellite loci (Dra187, Dra428, Dra426, on population genetic parameters such as genetic diversity Dra519, Dra266, Dra471 and Dra569), developed for D. and structure of these tree species is a fundamental aromatica [13], were used in this study. The forward primer requirement for devising conservation schemes and future of each marker was labeled with 6-FAM, VIC, NED, or PET provenance trials. phosphoramidite (Applied Bio systems Japan, Tokyo). A Genetic diversity is essential to the long-term survival Type-it Microsatellite PCR kit (QIAGEN Japan, Tokyo) was and evolution of tree species. It is the foundation of used to amplify microsatellite loci. Multiplex PCR sustainability because it provides raw material for amplification was performed in a volume of 5 µl, containing adaptation, evolution, and survival of species and 1 × Type-it Multiplex PCR Master mix, 0.2 µM of forward individuals, especially under changed environmental and and reverse primers and about 40 ng of genomic DNA. An disease conditions [7]. Loss of diversity may cause a risk of Applied Bio systems 2720 thermal cycler was used for the extinction because of a lack of adaptive ability to a changing PCR amplification under the following conditions: initial environment [8]. Genetic diversity is also the basis for tree denaturation at 95°C for 5 min, then 31 cycles of improvement through selective breeding. Therefore, denaturation at 95°C for 30 s, annealing for 1 min 30 s and maintaining genetic diversity and conserving forest genetic extension at 72°C for 30 s, followed by a final incubation at resources are critical to forest sustainability, ecosystem 60°C for 30 min. The annealing temperatures depended on stability and species’ continued adaptation and survival [6]. the primer pairs and species used. Details of the markers, In recent years, a variety of DNA-based techniques have including the annealing temperatures used, are shown in Table 2. Fragment sizes were determined using an ABI been employed to study genetic diversity and structure in TM tree populations (e.g., Random Amplified Polymorphic DNA PRISM 310 Genetic Analyzer and visualized using Gene (RAPD), Amplified Fragment Length Polymorphisms Mapper 3.0 software (Applied Bio systems Japan, Tokyo). (AFLPs), Simple Sequence Repeats (SSRs) and Cleaved 2.3. Data analysis Amplified Polymorphic Sequences (CAPS)). Among the classes of molecular markers, microsatellites, or simple Basic statistics of genetic diversity, including number of sequence repeats (SSRs), are considered to be ideal markers alleles per locus (Na), observed heterozygosity (HO), for genetic studies because they combine several suitable expected heterozygosity (HE) and fixation index (F), were features for evaluating genetic variation such as co- calculated using Gen AlEx software version 6.41 [14]. FST was dominance, multiallelism and high polymorphism. This calculated to determine the level of population allows precise discrimination, even among closely related differentiation using ARLEQUIN Version 3.5.1.2 [15]. An individuals, and the possibility of efficient analysis by a analysis of molecular variance (AMOVA) was also carried rapid and simple polymerase chain reaction (PCR) assay [9, out using ARLEQUIN version 3.5.1.2 to apportion the 10, 11]. Thus, the aims of our study are 1) to estimate the variance among species [15]. Genetic distances between amount of genetic variation, 2) to examine the genetic populations were estimated using the Nei genetic distance, structure and 3) to evaluate the discrimination abilities of D [16], as implemented in Gen Alex version 6.41 [14]. The microsatellite markers for the six Dryobalanops species. Bayesian model-based clustering method implemented in STRUCTURE software version 2.3.4 [17] using admixture 2. Material and methods model with LOCPRIOR option and allele frequency correlated model was further used to estimate the number of genetically homogeneous populations (K) and determine 2.1. Plant materials the genetic structure of the sampled populations. A burn-in A total of 700 individual leaf samples were collected from of 20,000 steps followed by 100,000