Contrasting cpDNA Variation in Two Indonesian Endemic Lowland Dipterocarp Species and Implications for their Conservation

MATERIALS AND METHODS

Plant materials: Leaf samples of S. javanica were collected from five Sumatran populations and one pooled Java population to represent the current geographical distribution of this species. In Sumatra, 15 adult trees (for each individual sampled minimum diameter at breast height (dbh) was 30 cm and minimum distance between individuals sampled was 50 m) were sampled from each population, originating from four repong dammar populations and one remnant natural population in Kubu Perahu, Bukit Barisan Selatan National Park. Because only one remaining tree was found in Sancang Nature Reserve, samples were also taken from a tree in Bogor Botanical Garden in Java, (exact sampling site is not known; personal communication). Therefore, these samples were pooled together as the Java population. A total of 77 adult trees were sampled from seven different geographic locations for S. javanica. For S. selanica, leaf samples were collected from three populations in the Moluccas, eastern Indonesia. Leaf samples were taken from adult trees at minimum dbh of 30 cm and from individuals spaced at a minimum distance of at least 50 m. Nine individuals were sampled from the Sula Islands, 10 individuals from Seram Island and eight individuals from Buru Island. In total, 27 individuals of S. selanica were analyzed in this study. Sampling location and collected individuals are described in Fig. 1 and Table 1. Sampled leaves were dried with silica gel and subsequently used for DNA extraction.

Loci studied: Eight chloroplast DNA regions (cpDNA) were tested from S. javanica (trnL-trnF, trnH-trnK, psbC-trnS, petB, trnS-trnT, petL-psbE, trnS-trnfM and trnT-trnL) and five cpDNA from S. selanica (trnL-trnF, trnH-trnK, psbC-trnS, trnS-trnfM and trnT-trnL).

Table 1:	Genetic variation and population differentiation of the two Indonesian endemic lowland dipterocarp species, S. javanica and S. selanica, as inferred by non-coding chloroplast DNA region sequences
N: No. of individuals analyzed per population, S: No. of segregating sites in nucleotide sequences, Hd: Haplotype diversity, π: Nucleotide diversity, na: Not available, FST: Population differentiation, ***p<0.0001

Fig. 1:	Sampling location for S. javanica and S. selanica, (a, b, c, d, e, f, g): Sampling sites for S. javanica; (h, i, j): Sampling sites for S. selanica

For S. javanica, three cpDNA regions could be amplified successfully by PCR using the universal primers described by Taberlet et al. (1991) for trnL-trnF and those described by Demesure et al. (1995) for psbC-trnS and trnS-trnfM. For S. selanica two cpDNA intergenic spacers, trnT-trnL and trnL-trnF (Taberlet et al., 1991) were amplified.

DNA isolation, amplification and sequencing: Genomic DNA was isolated from adult leaves using a modified CTAB method (Murray and Thompson, 1980). Polymerase Chain Reaction (PCR) amplifications for S. javanica samples were performed in a volume of 20 μL containing 10 ng of genomic DNA, 5 pmol of each forward and backward primer and 10 μL of Go Taq^® Hot Start Colourless Master Mix (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Initial denaturation was performed at 95°C for 2 min, followed by 30-35 cycles of denaturation at 95°C for 1 min, annealing at 52-62°C and polymerization at 72°C for 2 min and final extension at 72°C for 7 min. PCR amplifications for S.selanica samples were performed in a volume of 25 μL containing 10 ng of genomic DNA, 5 pmol of each forward and backward primer, 10x buffer KOD-Plus, 2 mM dNTPs, 25 mM MgSO₄, KOD Plus DNA polymerase and nuclease free water following the manufacturer’s instruction (TOYOBO Co., Osaka, Japan). A touchdown PCR program was applied at 94°C for 2 min; 4 cycles at 94°C for 45 sec, 59°C for 45 sec (decreasing 1 degree each cycle) and at 68°C for 2.5 min; then 26 cycles at 94°C for 45 sec, 55°C for 45 sec and 68°C for 2.5 min and holding at 68°C for 30 min. Prior to sequencing, the PCR products were purified using rAPid Alkaline Phosphatase^TM (Roche, Germany) and Exonuclease I (New England Biolabs, Ipswich, MA, USA). Purified products were directly sequenced on both strands using an ABI Prism 3100 automatic sequencer (Applied Biosystems, Foster City, CA, USA).

Data analyses: DNA sequences were checked visually and then forward and reverse traces were assembled using the ATGC program (Genetyx Corporation, Japan). To assess levels of nucleotide polymorphism, nucleotide diversity (π) (Nei, 1987) and Haplotype Diversity (Hd) (Nei, 1987) for each of the investigated loci were estimated. To test for deviations from selective neutrality and other assumptions (e.g., constant population size with no migration), Tajima’s D (Tajima, 1989) test was performed for individual loci. Population structure was examined using ARLEQUIN ver. 3.5 (Excoffier and Schneider, 2005). Genetic differentiation, F_ST of nuclear loci was estimated through analysis of molecular variance (AMOVA). Pairwise F_ST was also calculated to determine population structure between pairs of populations (Gaggiotti and Excoffier, 2000).

RESULTS AND DISCUSSION

Genetic diversity and population structuring: In S. javanica, sequences of 953, 1510 and 1475 bp (3938 bp in total) were determined, excluding alignment gaps, for three non-coding regions of cpDNA, trnL-trnF, psbC-trnS and trnS-trnfM, respectively. There were no variants except for single occurrence of a 6-bp indel (TTTTTA) at the psbC-trnS region in four of the 15 individuals from the Gk population. Considering the highly specific habitat and very restricted distribution of S. javanica, it is expected that populations would show low levels of genetic variation as compared to other dipterocarp species with wider distributions. This is indeed the case for S. javanica, both at the population and species level. There were no variants in three cpDNA regions among the 77 individuals analyzed in this study. It could be that the low level of genetic variation is a specific characteristic of the cpDNA regions examined in this study. However, considerable numbers of substitutions were previously observed within and between Shorea species in the trnL-trnF and psbC-trnS regions (Tsumura et al., 2011).

In S. selanica, the alignment lengths for non-coding chloroplast DNA regions trnT-trnL and trnL-trnF were 1010 and 951 bp, respectively (1961 bp in total). Haplotype diversity for the pooled population (Hd) was 0.798 and population nucleotide diversities ranged from 0 (Seram) to 0.00044 (Buru), with 0.00041 for pooled populations (Table 1). This value is lower than that reported for cpDNA nucleotide diversity in the common species Shorea curtisii (π_sil = 0.00155; Kamiya et al., 2012) from the Malay Peninsula and Borneo.

The study result suggests that the extremely low level of genetic variation in the cpDNA regions examined here is not specific to the studied loci, but specific to S. javanica as other Shorea species maintain some degree of intrapopulation genetic variation. Three nucleotide substitutions were also found at the same loci (trnL-trnF) in lowland endemic S. selanica in this study. The low level of genetic variation in two endemic lowland dipterocarps in this study is congruent with that of Hamrick and Godt (1996) which stating that endemic species tend to have low levels of genetic diversity due to their small population sizes. Overall, the result of this study also supports the general expectation of reduced genetic diversity in rare endemic species and showed that genetic diversity in rare endemic species and showed that species with a narrow geographic distribution harbor less genetic variation than do widespread species (Hamrick et al., 1992).

Table 2:	Pairwise FST among populations of S. selanica as inferred by non-coding chloroplast DNA region sequences
FST, population differentiation; ***p<0.0001

While nucleotide substitution was only detected in S. selanica, the level of genetic differentiation between populations (F_ST) could only be estimated for this species. Significant differentiation was found among populations (F_ST = 0.702) (Table 1). Pairwise F_ST values between sampled populations indicated significant population differentiation for all pairs of populations, with values ranging from 0.39 (Seram-Sula) to 0.90 (Seram-Buru) (Table 2). The chloroplast genome is primarily maternally inherited in angiosperms and is considered a single, non-recombining unit of inheritance which is structurally stable (Olmstead and Palmer, 1994; Birky Jr, 1995). The level of differentiation observed in the chloroplast genome of S. selanica is most probably due to the separation and isolation of populations over an extended period of time during the evolutionary history of this species. The genetic structuring of populations of organism and ultimately the establishment of independent evolutionary lineages, is strongly influenced by patterns of genetic exchange (gene flow) within and between populations. Seed dispersal for Shorea species is facilitated by wind or gravity. Distances for seed dispersal can be up to 500 m, but under forest conditions, more than half of the mature seeds will land within 50 m of the parent trees (Chan and Appanah, 1980; Takeuchi et al., 2004). Furthermore, seed dispersal is limited even for species with comparatively light seeds and long-winged fruits (Fukue et al., 2007). Disjunction of the distribution of the species would lead to the evolution of different lineages among separated islands through the effects mutation and drift. The absence of permanent land bridges among islands (De Jong, 1998) strengthens the isolation process and limits gene flow between populations.

The pairwise F_ST values describe the occurrence of population structuring in S. selanica, in which populations have become differentiated from each other. The Moluccas which are the islands where S. selanica grows naturally, have a distinctive geological history. It should be noted that the absence of permanent land bridges among these islands since the land emerged (De Jong, 1998) could have restricted historical gene flow and increased genetic differentiation among populations. For example, the pairwise F_ST value between the extant adjacent islands Buru and Seram was quite high (F_ST = 0.90 for cpDNA). While there is close stratigraphic similarity between Seram and Buru, the two islands show very different structural styles (Charlton, 2000). At present, although Buru and Seram are located close to each other; however, 5 mya, Buru was located farther to the north of Seram island (Hall, 1996), too distant to permit intensive gene flow.

Implications for conservation: An understanding of the partitioning the genetic diversity within and between populations (known as genetic structure) is important for developing a conservation strategy for endangered species, especially if not all populations can be protected. Species with low levels of population structure could be simplified, as the loss of single population may have little impact on the species-wide genetic diversity. In contrast, for species with a high level of population structure, the loss of a single population might significantly reduce overall genetic diversity. The uniformity of nucleotide sequences across individuals and populations of S. javanica in this study revealed that no population differentiation could be detected within this species based on cpDNA variation, suggested that each population (from either repong dammar or natural stands) harbored similar genetic characteristics. However, modern selection pressure from farmers preferentially collecting and planting seeds from high-dammar-producing trees, together with the declining population sizes, could reduce inter and intra-population gene flow and may generate systematic genetic erosion. In contrast to S. javanica, S. selanica exhibits distinct genetic structure among populations, indicating that they are genetically differentiated. Different pattern of population structuring between the two endemic lowland dipterocarps in this study suggested the need for different management and conservation strategies for each species.

For S. javanica, connectivity within and among populations, including augmentation of population sizes across the species range should be promoted. Even though repong dammar have been established primarily for dammar production, they have also been managed for adaption to the local environmental conditions. Maintenance of these plantations can preserve both biological and genetic diversity and enable them to serve as refugia for biodiversity. Thus, maintenance of repong dammar should be a primary focus and conversion to other land uses, especially monoculture-based systems, should be avoided. Conserving S. javanica populations in native habitat is also essential.

The appropriate conservation strategy for S. selanica would require a combination of different approaches. The first would consist of an in situ conservation plan that would define core areas completely free from perturbation in each population. The second strategy would employ ex situ conservation to maintain genetic variation by pooling populations from various islands. In the short term, recovery of as many seeds as possible spanning their entire distribution will be critical to obtain a good representation of each specie’s genetic diversity.

CONCLUSION

This study was carried out to determine genetic variation of two lowland dipterocarp species endemic to Indonesia, S. javanica (Sumatran and Java endemic) and S. selanica (Moluccan endemic) inferred from non-coding chloroplast DNA regions. The result showed extremely no variation for S. javanica while low level of genetic variation was attributed to S. selanica. In contrast with no population structuring of S. javanica, S. selanica exhibited high levels of genetic differentiation among populations. According to different pattern of population structuring for the two species determined in this study, distinct management and conservation strategies are required for each species. Connectivity within and among populations, including augmentation of population size across the species range should be promoted for S. javanica while an in situ conservation plan that defines core areas completely free from perturbation within each population is necessary for S. selanica.