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There have been many studies estimating genetic variation of dipterocarps using different markers, such as allozyme, and microsatellite e.g. Ujino et al. 1998, Konuma et al. 2000, Lim et al. 2002, Takeuchi et al. 2004, Lee et al. 2004, Ng et al. 2004, Lee et al. 2009, Pandek and Geburek 2009, Abasolo et al. 2009, Senakun et al. 2009, Pandek and Geburek 2010, Estoque 2011. Apparently, the present study is the first investigation to genetic diversity of Shorea johorensis using microsatellite markers.
The present study utilized microsatellite markers which had been reported successfully amplified in different dipterocarp species. Ujino et al. (1998) reported that Shc01, Shc07, and Shc09 were reported to be highly polymorphic (He > 0.8) and the loci were well conserved within dipterocarps family. These loci were found strongly amplified in many species of dipterocarps.
According to Ng et al. (2009), significant heterozygote deficiencies were observed for loci SleE14 (p-value < 0.05). This is confirmed by the present study that SleE14 had extremely high Fis (0.74). Ng et al (2009) examined the transferability of SleE05, SleE07, and SleE14 to thirty-six species from 10 genera of dipterocarps, which resulting in successful amplification of SleE05 mostly in Shorea species, while SleE07 and SleE14 showed successful amplification in all the 36 species.
In studies by Lee et al. (2004) and Pandey and Geburek (2009), it is revealed that the heterozygosity of Sle566 and Sle562 was relatively low. In contrast, the expected heterozygosity of these two loci was estimated high (0.69 and 0.87, respectively). The reason of this could be the limited number of samples in those previous studies (24 and 30 samples, respectively). Further, Lee et al. (2004) reported that Sle267 was not amplified in Shorea parvifolia. Otherwise, Pandek and Geburek (2009) found Sle267 was amplified in S. robusta.
Abasolo et al. (2009) revealed the monomorphism of Sle111a in Parashorea malaanonan while in the present study the respective locus revealed to be polymorphic in S. johorensis. The controversy is also happened to Sle118, which the heterzygosity was very high in S. parvifolia (Lee et al. 2004) while in S. johorensis the heterozygosity was extremely lower than the former.
In the present study, six loci were excluded (SleE01, SleE02, SleE08, SleE10, SleE17, and Sle280). SleE01 failed to amplify in Shorea johorensis, although it is succeeded almost in all species of Shorea tested by Ng et al. (2009). The same thing happened to SleE08 and SleE10 during PCR amplification which in Ng et al.' case it could not amplify in many species of Shorea. Furthermore, SleE02 and SleE10 were found to be monomorphic in a study by Villarin et al. (in preparation) confirming the monomorphism of those loci in the present study. While in a study by Abasolo et al. (2009), Sle280 was observed to be successfully amplified in Parashorea malaanonan but it was monomorphic as happened in S. johorensis.
Thereby, each locus has been observed differently in different studies. Concern may be expressed that the number of samples and populations included in studies might affect the genetic diversity revealed from utilized loci. However, microsatellite markers are proven to be applicable in Shorea johorensis.
IV.2. Genetic diversity of microsatellite markers on Shorea johorensis
To date, only few studies about genetic pattern of Shorea johorensis are available. A study by Siregar et al. (2008) using RAPD resulted in a relatively low genetic diversity of S. johorensis compared to other dipterocarps. Meanwhile, a study by Cao et al. (2009) reported S. johorensis had moderate level of genetic diversity (He) as revealed by AFLPs. The mean number of alleles (Na) revealed by these two studies were relatively low compared to Na in the present study. Dominant markers, such as RAPD and AFLP, are expected to be less heterozygous than codominant marker such as SSR.
The mean number of alleles (Na) in the present study is considerably high. The mean number of alleles per locus is affected greatly by sample size (Leberg 2002). Therefore, the high Na could possibly due to the large number of sample size of the present study. A study by Pandek and Geburek (2010) showed lower Na although sampling size was much higher. However, it could also be due to the the heterozygosity of the species which is relatively high as shown above.
The mean expected heterozygosity is the most widely used measure of the genetic variation employing genetic markers (White et al. 2007). The mean of expected heterozygosity in the present study (0.69) are comparable to similar studies using microsatellite markers in different dipterocarp species, e.g. the estimation of expected heterozygosity (He) in Shorea leprosula by Ng et al. (2004) resulted in average to 0.70 based on seven loci and 178 samples from one population, He of S. curtisii studied by Ujino et al. (1998) based on eight loci and 40 samples from one population was averaged to 0.64, He of S. robusta studied by Pandek and Geburek (2010) based on four loci and 450 samples from 15 populations was averaged to 0.69, He of S. obtusa studied by Senakun et al. (2011) based on five loci and 146 samples from 5 populations was averaged to 0.66, He of S. guiso studied by Estoque (2011) based on six loci and 136 samples from 8 populations was averaged to 0.76, He of Dryobalanops aromatica studied by Lim et al. (2002) based on seven loci and 5 populations each with about 18 samples was averaged to 0.71, He of Neobalanocarpus heimii studied by Konuma et al. (2000) based on four loci and 30 samples from one population was averaged to 0.78.
The mean proportion of observed heterozygosity (Ho) also could be used to measure genetic variation, but the magnitude of Ho depends on the level of inbreeding (White 2007). In the present study, Ho was found higher than He in 2 loci (Sle392 and SleE07), showing excess of heterozygosity. However, the overall He was averaged higher than Ho as the mean of inbreeding coefficient (Fis) was positive. According to Balloux (2004), the Fis is reliable only if it is estimated using at least 10 markers, but the validity of this assumption is unclear.
According to Hamrick et al. (1992), long-lived woody species have a higher proportion of polymorphic loci, more alleles per locus, higher effective number of alleles per locus and more genetic diversity than other life forms. Many study about genetic variation of short-lived woody species using SSR markers (e.g. Ribeiro 2010, Marulanda 2012) revealed that most of them has relatively low heterozygosity compared to Shorea in general.
The characteristics such as geographical range, outcrossing, long life span are probably responsible for the high level of genetic diversity in S. johorensis.
IV.3. Genetic variation within and among populations
One of the main objectives of this study was the comparison of variation levels in natural and planted populations. In the present study, natural population in SJM and SBK site was found to have higher expected heterozygosity (He) than the planted population, while in BFI the He in natural population was observed lower than in planted population. A study by Siregar et al. (2008) revealed that natural population of S. johorensis sampled from SBK has slightly higher He than planted population from the same study site. Furthermore, AMOVA revealed the p-value of genetic differentiation between natural populations and planted populations in the present study was less than 0.05. Therefore, hypothesis of lower genetic variation in plantations than in natural populations are rejected as the heterozygosity of both population groups are insignificantly different. Estoque (2011) observed no significant different between natural and planted populations of S. guiso. Moreover, a study by Gauli (2009) revealed similarity between natural and planted populations of Pinus roxburghii. This indicates the plantation of S. johorensis in Borneo has no significant impact to the genetic variation of the species. However, the source of planting materials should be considered in further analysis. In this case, the planting materials for the plantation was sourced from natural stands. Therefore, there are close relatedness between these two population groups.
Furthermore, the present study revealed that nursery population has the lowest heterozygosity. Previous study by Siregar et al. (2008) also revealed the low heterozygosity of reproductive materials of S. johorensis compared to adult trees. The reason of this could be due to the young age of the regeneration which means the individual has not been subjected long enough to evolutionary force such as mutation as happened to adult trees. However, the genetic differentiation between regeneration plant and adult plant covered in the present study was insignificant. This is most likely because all sampled regeneration were sexual offspring collected from natural stands.
The present study was aimed to proof the hypothesis of higher genetic differentiation within populations than among populations due to the characteristic of the genus which is outcrossing with relatively long distance of pollen and seed movement and wide distributed in the region. According to Hamrick et al. (1992), long-lived woody species have high genetic diversity within their populations and low genetic diversity among its populations. AMOVA revealed approximately >90% of the total variation in the present study resided within all three population groups. Accordingly, genetic diï¬€erentiation among population groups is considerably low. Therefore, the hypothesis is necessarily accepted. White (2007) suggested that the small values of genetic diversity among populations observed in most tree species indicate that past gene flow was occurred extensively.
Genetic structures of plantations are very similar to those of natural populations, and genetic distances between natural populations and neighbouring plantations are as low as those between natural stands and more distant plantations. Thus, the hypothesis of genetic similarities decrease with geographical distance are rejected.
Mantel matrix-correspondence test revealed no significant correlation between genetic distance (D) and geographic distance. Even though Ulu Sedili was nearest to Lenggor in terms of geographic distance, genetic distance between these two places was not the lowest. The same applied for the furthest genetic distance, between Lesong and Ulu Sedili, which was not reflected geographically, with the furthest distance being between Ulu Sedili and Kanching.
In general, it is expected that fragmentation of habitat constricts the genetic neighborhood of species by reducing the population sizes and increasing inter-population distances (Templeton et al. 1990), which increases the likelihood of breeding among relatives.
IV.4. Implication to the genetic conservation of Shorea johorensis
An effective strategy for conserving its genetic resources has to consider the amount and extent of genetic diversity within and between populations. Conserving 'within population diversity' should involve preserving large core populations that will not lose diversity due to drift, whilst conserving 'among populations diversities' should focus on preserving the most genetically distinctive populations (peripheral populations) (Millar and Libby 1991).
Conservation and management of plant species, in addition to ecological information, requires a sound understanding of underlying genetic processes as well as variation within and among populations (CHANGTRAGOON 2001; LEE et al. 2006).
The genetic information generated could be utilized in conservation and management of the species in future.
Genetic diversity is essential to the long-term survival of species; without it, species cannot adapt to environmental changes and are more susceptible to extinction (White et al. 2007).
Natural stands are main sources for forest reproductive material in the absence of an advanced tree improvement program for a plantation species in a particular region.
As shown above, a large number of genetically highly variable Shorea johorensis exist in Borneo. The phenotypically best plantations should be developed into seed production areas as a ï¬rst step towards an operational tree improvement programme and as ex situ conservation stands.
Ex situ and in situ conservation activities for the purpose of conserving genetic resources are considered to have an important role in the forestry program. Species that are not available in the planting region are introduced by ex situ conservation in the plantation area, while the conservation of the original forests within a plantation area represents in situ conservation.
High level of outcrossing is particularly important to maintaining genetic diversity within populations. Outcrossing promotes heterozygosity and maintenance of recessive allele that might otherwise be exposed to selection. Furthermore, mating between heterozygous individuals leads to genetic recombination and creation of vast arrays of different genotypes in offspring. On the other hand, inbreeding results in less recombination and increased homozygosity, leading not only to loss of variation, but to exposure of deleterious, recessive alleles, reducing average viability of offspring (inbreeding depression) (White 2007).
In situ conservation of D. aronlatica only requires a few populations. This is due to the higher diversity within populations compared to among populations. For ex-situ conservation, it is suggested that focus is given to variation among individuals within populations so that all the variations are sampled. Population selection should be done based on the highest diversity in terms of alelle or genotype.
To carry out a selection program effectively, selection of more individuals within population needs to be done so that high genetic diversity can be maintained.
The establishment of the plantations had no noticeable effects on the genetic structures of the established man-made forests in comparison to natural stands. In particular, genetic diversity was not or only slightly reduced. This result is mainly due to the low diï¬€erentiation observed among natural stands as potential origins, and due to the mode of harvesting and production of seeds and seedlings for plantation establishment.
Concern may be expressed that the number of loci and populations included in studies might affect estimates of genetic diversity.
To assess the effect of self-incompatibility on the genetic structures in Shorea johorensis, it will be necessary to investigate its breeding systems.