The Phylogenetic Relationship Among Varieties of Lansium domesticum Correa Based on ITS rDNA Sequences

Lansium domesticum Corr. with vernacular name in Indonesian duku has been reported containing therapeutic bioactive compounds, and some of these compounds shown to be potent antitumor, anticancer, antimalaria, antimelanogenesis, antibacteria, and antimutagenic activities. This plant is commonly known as duku, kokosan and langsat by the local community in Indonesia. The morphological appearance of all varieties is nearly the same, and identifi cation of the varieties is very diffi cult for growers. Variation of DNA sequences of the ITS (Internal transcribed spacer) region can be used as a molecular character to determine the phylogenetic relationship of different varieties of L. domesticum. The aims of this study were to determine taxonomy status of duku, kokosan, and langsat, also phylogenetic relationship among varieties of L. domesticum based on ITS rDNA sequencing. DNA was isolated from leaves of plant and then amplifi ed using F1 and R1 primers. Nucleotide sequences were identifi ed using Sequence Scanner Software Programm version 1.0, nucleotide sequences from 18S, ITS1, 5.8S, ITS2 and 26S region, that has been mergered using EditSeq and SegMan in software Suite for Sequence Analysis DNASTAR Lasergene DM version 3.0.25. The results of study showed that DNA fragments ranging in size from 782-810 bp. Different pattern of DNA fragments indicated polymorphism among duku, kokosan, and langsat. Based on the results of the ITS rDNA sequencing and phylogenetic tree analysis. It was determined that Lansium and Aglaia are a separated genus with the similarity index value of 0.98. Duku, kokosan and langsat were divided into two cluster, namely cluster kokosan-langsat and cluster duku with the similarity index value of 0.996.


Introduction
Lansium domesticum is an important fruit tree and a highly variable species, with different forms that have been classifi ed by some taxonomists as distinct species.In Southeast Asia, the plant has numerous common names, that is known as duku, kokosan and langsat (Indonesia); duku, langsak (Burmese); buahan, lansone, lansones, lanzon, lanzone (Philippine); langseh, langsep, lansa (Malay); duku, langsat, longkong (Thai) and Bòn-bon (Vietnamese).It still occurs wild or naturalized in these area and is one of the major cultivated fruits.The greatest producers of L. domesticum are Malaysia, Thailand, the Philippines and Indonesia.On a small scale, this plant is also cultivated in Vietnam, Burma, India, Sri Lanka, Hawaii, Australia, Surinam and Puerto Rico (Yaacob and Bamroongrugsa, 1991;Lim, 2012).There are numerous varieties of L. domesticum, both the plants and the fruit.Overall, there are three main varieties of these fruits, in Indonesia i.e duku, kokosan and langsat (Yulita, 2011;Hanum et al., 2012).
Several parts of the plant have medicinal uses.The fruit peel is dried and burned to repel mosquitoes, it is also used to treat intestinal parasites and diarrhea.Powdered seeds are used to reduce fever, and the bark is used to treat malaria and scorpion stings (Naito, 1995;Loekitowati and Hermansjah, 2000;Saewan et al., 2006).Skin and leaf extracts of fruit of L. domesticum interrupt the lifecycle of Plasmodium falciparum, and are active towards a chloroquine-resistant strain of the parasite (T9) in vitro.Study indicates extracts of L. domesticum are potential source for compounds with activity against chloroquine-resistant strains of P. falcifarum (Yapp and Yap, 2003) Cosmeceutical value from its antioxidant, moisturizing, whitening and lightening effects.Dry extract of fruit is used for skin depigmentation and as a moisturizer (Tilaar et al., 2008;Manosroi et al., 2012).Extracts of this plant showed that strong inhibition of melanin production of B16 melanoma cells without signifi cant cytotoxicity, presenting as a potential ingredient for skin-whitening cosmetics (Arung et al., 2009).
The air-dried fruit peel of L. domesticum yielded fi ve onoceroid triterpenes; the airdried seeds yielded one onoceroid triterpene (lansionic acid) and germacrene D. Studies of the compounds showed various degrees of activity against P. aeruginosa, B subtilis, C albicans, A niger among others (Ragasa et al., 2006).
Phylogenetic relationship among duku, kokosan, and langsat in infraspecies level are still not clear.At the level of infraspecies, there are two different taxonomic status of duku, kokosan and langsat.Ridley (Lim, 2012) suggested that duku, kokosan and langsat (pisitan) belonging to two varieties which were L. dommesticum var.typica (duku) and L. dosmesticum var.pubescens (pisitan and kokosan), and Morton (1987) suggested that L. domesticum Corr.divided into two varieties, namely L. domesticum var.domesticum (duku) and L. domesticum var.pubescent (pisitan/ langsat).Another study by Yee et al. (1993), based on the observation of the anatomy of leaves, fl owers, and fruits of duku separated them as varieties within the same species, namely L. domesticum.Morton (1987) and Yee et al. (1993) did not mentioned kokosan taxonomic status at the level of varieties.Lim (2012) stated that duku, kokosan and pisitan were grouped into two groups namely L. domesticum'duku group' and L. domesticum'langsat-longkong group'.According to Brandenburg (1986) and Hettercheid et al. (1996), the term group on crop can be equated varieties with the formal classifi cation.
Molecular approach is an effective technique in genetic analysis, that can be used to determine genetic relatedness among duku, kokosan, and langsat in Indonesia.DNA sequences of the internal transcribed spacer (ITS) of the ribosomal RNA transcription unit have proven useful in resolving phylogenetic relationship of closely related taxa and in distinguishing species in plant (Hershkovitz and Zimmer, 1996;Muellner et al., 2008;Pandey and Ali, 2012).
Ribosomal DNA (rDNA) genes are organized in clusters of tandemly repeated units, each of which consists of coding regions (18S, 5.8S, and 28S) and 2 internal transcribed spacers (ITS) and 1 non-transcribed spacer (NTS) region.ITS sequences have been used successfully in studying phylogenetic and genomic relationships of plants at lower taxonomic levels and the ITS regions are therefore valuable in more discrete phylogenetic separation of closely related species, recognition of new species, determination of conspecificity between isolates, discrimination within a species, and differentiation between species and subspecies (Samigullina et al., 1998;Aktas et al., 2007;Song et al., 2012).I T S a n i n t e r e s t i n g s u b j e c t f o r evolutionary/phylogenetic investigations, that which are found on either side of 5.8S rRNA gene and are described as ITS1 and ITS2.The length and sequences of ITS regions of rDNA repeats are believed to be fast evolving and therefore may vary.The Hanum et al. nucleotide sequence variation found in both of ITS-1 and ITS-2 is used extensively for the systematic analysis of closely related taxa, at least in part due to the speedy rate of evolutionary change characterizing these DNA regions.Universal PCR primers designed from highly conserved regions fl anking the ITS and its relatively small size (600-700 bp) enable easy amplifi cation of ITS region due to high copy number (Baldwin et al., 1995;Hershkovitz and Zimmer, 1996).The aims of this study to determine taxonomy status of duku, kokosan, and langsat, also phylogenetic relationship within different varieties of L. domesticum based on squencing of ITS rDNA.

Plant materials
Samples used in this study were 10 samples of duku, kokosan, and langsat leaves from eight province in Indonesia with all of their vernacular names, summarized in Table 1.

DNA isolation
Total DNA was isolated from leaves using the Nucleon Phytopure Plant and Fungal DNA kit exraction RPN-8511/GE (Healthcare, U.K.) following the procedure described by Daryono and Natsuaki (2002).

Amplification condition and agarose gel electrophoresis
DNA amplification was conducted based on Kasiamdari et al. (2002) with s l i g h t m o d i f i c a t i o n s .D N A w e r e amplified by polymerase chain reaction ( P C R ) u s i n g f o r w a r d p r i m e r F 1 5'GATCGCGGCGGCGACTTGGGCGGTT C3' and reverse primer R1 5'GGTAG TCCCGCCTGACCTGGG3' (Muellner et al., 2008)

Data Analysis
Analysis of nucleotide sequences was Similiarities sequence analysis between samples were carried out using BioEdit programs (Hall, 1999) followed by construction of phylogenetic tree.Phylogenetic analysis was performed by Neighbor-Joining (NJ) methods using ClustalIX2 and MEGA5 programs, whereas genetic distance analysis relied on parameter Kimura-2 model.Grouping stability was calculated using 1000 Bootstrap value (analysis formation of branch of phylogenetic tree) (Tamura et al., 2011)

Amplification of Duku, Kokosan, and Langsat ITS region
PCR amplifi cation using primers (F1 and R1) specifi cally recognized ITS region.The PCR products were visualized by agarose gel electrophoresis under UV light to chek the presence of amplifi ed bands.Figure 1 showed clearly amplified bands (single band ) of ~800 bp that was generated from DNA sequencing of ITS region using forward primer (F1) and reverse primer (R1) produced nucleotide sequences with size ranged from 782-810 bp which consisted of 18S; ITS 1; 5.8S; ITS 2; and 28S region (Table 2).
The results in Table 2 also showed that ten samples of plant has produced size of varied fragment ITS region, ITS 1 ranged from 346-367 bp and ITS 2 ranged from 223 -228 bp while 5.8S region was more stable at the size of 164 bp.The results obtained in this studies, the same the report Muellner et al. (2005) on Aglaia is for ITS 1 ranging from 263-274 bp and ITS 2 ranged from 221-227 bp, while 5.8S was stable at the size of 164 bp.
Variation of fragment size resulted from amplifi cation of ITS region indicated that there was variation on the length of ITS region.Aktas et al. (2007) reported that the ITS1 length varied more than the ITS2, ITS1 varied in length from 482 to 1634 bp and was longer than the ITS2 region (268-525 bp) in all Theileria isolates.Some rRNA genes were organized in clusters of tandemly repeated units, each of which consisted of coding regions (18S, 5.8S, and 28S) and 2 internal transcribed spacers (ITS) and 1 nontranscribed spacer (NTS) region.While the coding regions were evolutionarily conserved and had been utilized for phylogenetic inferences for major phyla (Hills and Dixon, 1991).
In this experiment, region of 5.8S rRNA had similar fragment size of 164 bp in all samples.Accoding to Hidayat and Pancoro (2001), region of 5.8S rRNA was more constant because of these gene encode rRNA which part constitute of ribosom small subunit to be a benefi t to synthesis of protein.Ritland et al. (1993) reported that 5.8S region was relatively unvaried and ITS region do not encode an rRNA subunit and showed the expected greater sequence variation than that in the 5.8S.
Fragment sizes of ITS region (ITS 1, 5.8S, and ITS2) in this study with size ranged from 733-761 bp were different with fragment sizes that had been reported by Muellner et al. (2005), in species of Aglaia (Meliaceae) including L. domesticum with size ranging between 627-664 bp in size.The fragment sizes of ITS regions resulted from this research were similar to fragment sizes that commonly found in Angiospermae.Baldwin et al. (1995) reported that generally fragment sizes of ITS region of Angiospermae approximately 700 bp with the sizes of ± 300 bp, ± 165 bp, and ± 300 bp, respectively.
N u c l e o t i d e s e q u e n c e a n a l y s i s for comparing genetic information of L. domesticum and some species from Aglaia genus on ITS region was done compaty with foreknown sequences of DNA in GenBank databases.The homologous nucleotide sequences of each samples were analysed using BLAST (Basic local alignment search tool) software versi 2.2.24 on server of DDBJ (DNA Data Bank of Japan) as mention in Table 3.The result showed that all samples produced 99% similarity with L. domesticum voucher MWC2113 (AY695586.1)and L. domesticum voucher Muellner 130 (AY695587.1)but had no similiarity to another species (Table 3).
Similarity index values of DKom, DSle, DDre, LOKI, LSle, LHat, LMat, LTan, LPung, KKal, L. domesticum voucher MWC2113, L. domesticum vouchers Muellner130, and A. rugulosa, A. coriacea, A. spectabilis, A. korthalsii, and A. teysmanniana as outgroup were given on Table 4.The highest and lowest sequence similarity index values were 0.999 and 0.987, respectively.Highest sequence similarity index value was found on DKom and LOKI meanwhile the lowest sequence similarity index values was found in all types of Aglaia was used as an out group with of duku, kokosan, langsat, L. domesticum voucher MWC2113 and L.domesticum voucher Muellner130.
Grouping pattern and relatedness among duku, kokosan, and langsat with A. rugulosa, A. coriacea, A. spectabilis, A. korthalsii, and A. teysmanniana as outgroup based on sequence similarity index value were analyzed with MEGA5 resulted as phylogenetic tree (Figure 2).
Figure 2 showed that the phylogenetic tree had two main clusters namely cluster I, which consisted of duku, kokosan, and langsat and cluster II consisted of several species of Aglaia.Based on the clustering pattern suggested that Lansium and Aglaia is a monophyletic group that had a high similarity among the members.According to Hidayat and Pancoro (2008) in a phylogenetic approach, a group of organisms whose members have a lot of similarities of character is considered to have a very close relationship and estimated descended from a common ancestor.
Our studies supported studies that had been reported by Pennington & Style (1975)   on our reseach of nucleotide sequences of the ITS region further strengthens position of DDre in the group of kokosan.The second cluster consisted of langsat namely LMat, LSle, LTan, and LPung with the similarity index value of 0.998.
Subcluster B consisted of two sub-clusters, the first cluster consisted of L. domesticum voucher Muellner130 and L. domesticum voucher MWC2113 to the value of similarity index of 0.998.The second cluster consists of DSle, LHat, DKom, and LOKI.Similarity index values between DKom with LOKI are 0.999, while the value of similarity index between DSle with LHat are 0998.The inclusion of LOKI and LHat in group of duku is possibly occurred by mistake in vernacular name and genetic variation occurred on LOKI and LHat.According to Suryanto (2003), genetic variation can occur because of alteration in nucleotides constituent of DNA.Genetic variation of duku, kokosan, and langsat likely occurred because there has been a cross-pollination and vegetative propagation.
Dispersal by humans can indirectly cause genetic variation.Genetic variations may occur due to natural mutations due to the influence of environmental stress the place of origin of the plant.Plants survive by adapting to their environment, breed and pass on their genes to the next generation.This process has been going on for decades causing genetic changes in LOKI dan LHat.Pandin (2010) stated that alteration occur due to the changes in reimbursement mechanisms and alteration in DNA nucleotide bases does not necessarily change the morphological characters, so that the use of markers that directly integrates with the genetics system will be better able to describe the actual state of the genome.Hanum et al. (2012) reported based on the RAPD approach of duku, kokosan, and langsat was known that LOKI and LHat were included in the group of duku.
Based on the results of the ITS rDNA sequencing and phylogenetic tree analysis, it can be determined that Lansium and Aglaia are separate genera with the similarity index value of 0.98, and duku, kokosan and langsat were divided into two clusters, namely cluster kokosan-langsat and cluster duku with the similarity index value of 0.996.
which stated Lansium and Aglaia were different genus.Muellner et al. (2005) also suggested that Lansium as a separate genus from Aglaia based on 16rps intron regions and secondary metabolites.This statement was reinforced by the results obtained byMuellner et al. (2008) based on the ITS region of Meliaceae plants, also got the same grouping pattern, that was Lansium and Aglaia separated into different groups and put Lansium and Aglaia on tribus Aglaieae.Cluster I was divided into two subclusters, namely subcluster A and subcluster B. Sub cluster A consisted of two clusters, the fi rst cluster consisted of KKal and DDre with the similarity index value of 0.998.The inclusion of DDre in group kokosan is possibly occured by mistake in vernacular name by local communities.DDre was collected from Bengkalis Island, Pekan Baru.Recently, it is known that kokosan has only been recognized and is found in Java Island.Although based on fruit morphology DDre has similarities with kokosan but DDre has sweet fruit flavors like duku, unlike the kokosan that taste is very sour.Hanum et al (2012) reported based on RAPD approach stated DDre into the group kokosan.Based

Figure 2 .
Figure 2. Phylogenetic tree of duku, kokosan, dan langsat based on ITS rDNA.The values on each of branch refers to values of bootstrap.Scala underneath of tree refers to genetic distance among samples.