PHYLOGENETIC RELATIONSHIPS WITHIN FAMILIES MYRTACEAE IN TRIPA PEAT SWAMP FOREST USING INTERNAL TRANSCRIBED SPACER (ITS)

The Tripa peat swamp forest is located on the west coast of Aceh province and falls administratively under the two districts of Nagan Raya and Aceh Barat Daya. Tripa peat swamp forest has a high diversity of plants. The previous research discovered that Myrtaceae is one of the dominant families in the forest. This study aimed to predict phylogenetic relationships among trees species within families Myrtaceae that live in the Tripa peat swamp forest using Internal Transcribed Spacer (ITS). The research was conducted at Laboratory of Forest Genetics and Molecular Forestry, Faculty of Forestry, IPB from June 2015 to December 2016. The methods were conducted with an experimental laboratory of molecular identification consisting of DNA extraction, amplification, and sequencing. Data was analyze using BioEdit, Clustal X, Mega 6, and BLAST. The morphology identification reported that there were four trees species within families Myrtaceae, i.e., Syzygium sp 1, Syzygium garciniifolium, Syzygium leptostemon and Tristaniopsis whiteana. Reconstruction of phylogenetic tree using the Neighbor-Joining (NJ) method 1000x showed that the ITS region was successfully to predict phylogenetic relationships within genus Syzygium and Tristaniopsis.


I INTRODUCTION
Tropical peat swamp forest is a unique ecosystem that is most extensive in the Southeast Asia, where it is under enormous threat from logging, fire, and land conversion.Recent research has shown this ecosystem's significance as a global carbon store, but its value for biodiversity remains poorly understood [1].The Tripa peat swamp forest is located on the west coast of Aceh province and falls administratively under the two districts of Nagan Raya and Aceh Barat Daya.The Tripa peat swamp forest received special designation as part of the Leuser Ecosystem Zone (Kawasan Ekosistem Leuser/KEL) based on Presidential Decree No. 33/1998 on Leuser Ecosystem Management [2].Degradation of peat swamp forest in Tripa affects the ecosystem's function such as tree species diversity and tree density.However, complete conversion of forest into a non-natural vegetation cover is a much more significant concern [2].The impact of degradation has been a renewal of the concern over the decline of biodiversity in this forest.Ref. [3] reported there were 17 families of trees species that can be found in Tripa peat swamp forest i.e.Myrtaceae, Apocynaceae, Sapotaceae, Anacardiaceae, Sterculiaceae, Moraceae, Euphorbiaceae, Rubiaceae, Stemonuraceae, Thymelaeaceae, Ochnaceae, Rhizophoraceae, Annonaceae, Dipterocarpaceae, Myristicaceae, Elaeocarpaceae, and Arecaceae.Ref. [3] also explained that among all the families, Myrtaceae was the dominant families in the forest.Inline with Ref. [4] also reported that the most species in peat swamp forests belong to the Myrtaceae and Dipterocarpaceae working on timber species of Pondok Tanjong peat swamp forest has reported that Syzygium grandis, is the most dominant species recorded in two 100 m 2 study plots.
The main goal of this study is to predict phylogenetic relationships among species within families Myrtaceae.Analysis of the relationships of trees species is using Internal Transcribed Spacer (ITS).The nuclear Internal Transcribed Spacer (ITS) regions have been used as molecular markers because of their relative variability and ease of PCR amplification [5].The ITS consists of the entire ITS1, 5.8S and ITS2 regions of the nuclear rDNA cistron.It is a multigene family with the potential for variation among tandem repeats.ITS sequences are typically found to be more similar within species and more divergent between species [6].Prior to, the ITS was used for phylogenetic analysis of the genus Hexachlamys within families Myrtaceae [7].

Study area
The research was conducted in Tripa peat swamp forest, Darul Makmur District, Nagan Raya (Figure 1) and Forest Genetics and Molecular Forestry Laboratory, Faculty of Forestry, Bogor Agricultural University.The research was begun from September 2015 to August 2016.

Sample Collection
The sampling method in the field using purposive sampling and taken three different locations in Tripa peat swamp forest: primary, secondary and tertiary.The study samples were collected as leaf material from field samples.As many as fifteen trees species were collected in the field.Twelve species included in families Myrtaceae and three species included in families Arecaceae were used as an out group.Every leaf consisted of three replications (one sample for DNA extraction, two samples for herbaria).Samples were collected as silica gel dried leaves from natural populations and as leaves from herbaria.Voucher specimens were collected and deposited in Indonesian Institute of Sciences (LIPI).Plot design for collecting the samples is shown in Figure 2.

DNA
Extraction, Amplification and Sequencing Total genomic DNA was isolated using DNeasy® Plant Mini Kit according to the manufacturer's instructions (QIAGEN, Hilden, Germany).initial denaturation at 94˚C for 3 min, 30 cycles consists of the 3 stages: denaturation (at 94˚C for 30s), annealing (at 58˚C for 30s), and extension (at 72˚C for 1 min), and ends with an elongation stage at 72°C for 10 min.The primers were used in this study in Table 1.
Table 1 Primers used in the present study

DNA region
Primer name Sequence (5'-3') Ref. [8] All PCR products were visualized by electrophoresis on 2% agarose gels stained with red gel.Amplified PCR products were sequenced based on the selective incorporation of chain-terminating dideoxynucleotides method using DNA polymerase during in vitro DNA replication according to the manufacturer's instructions and run on an ABI-3100 automatic sequencer (Applied Biosystems) [9,10].Both DNA strands were fully sequenced.

Editing and Sequence Alignment
Data were analyzed using BioEdit, BLAST and MEGA 6. Manual data review was performed by using BioEdit version 7.0.5.2 [11].The Basic Local Alignment Search Tool (BLAST) from the NCBI homepage (http://www.ncbi.nlm.nih.gov/blast/blast.ci)was then used to compare these sequences with inhouse sequences and GenBank database sequences.The software of Molecular Evolutionary Genetics Analysis (MEGA) version 6.0 [12] was used to predict phylogenetic relationships among trees species in Tripa peat swamp forest.The reconstructing of the phylogenetic tree using Neighbor Joining method (NJ) bootstrap 1000x [13].

III RESULT AND DISCUSSION
The morphological identifications showed that there were four trees species from families Myrtaceae, i.e.Syzygium sp., Syzygium garciniifolium, Syzygium leptostemon, and Tristaniopsis whiteana.More details about all species are presented in Table 2.The most dominant species of the tree in Tripa peat swamp forest was Tristaniopsis whiteana.This species was discovered almost in every location included three areas, i.e. primary, secondary, and tertiary.The morphology characters of T. whiteana that were discovered in Tripa peat swamp forest, i.e. tree with height>20 meters, the diameter of tree around 20-30 cm, terms of its peeling barks, has small flowers, and small leaves.Inline with [14] was reported that Tristaniopsis is distinctive in terms of its peeling barks with mixed reddish brown to gray-white.According to Ref. [6] morphological characteristics of T. whiteana, i.e. has a canopy, occasionally shortly emergent tree to 45 m high, to 1.5 m with concavely rounded buttresses.Bark at first was white to light greenish grey with the occasional hint of very pale orange, smooth, later peeling in scroll-like strips; the older unpeeled bark was evenly pale grey-dull light olive; peeled bark scrolls were pale to dark grey with mauve-brown patches; the inner bark was whitish.The species of T. whiteana at Tripa peat swamp forest is shown in Figure 3.

Basic Local Allignment Search Tool (BLAST)
The BLAST result analysis of 4 trees species within families Myrtaceae from Tripa peat swamp forest is presented in Table 3.The table shows that all species of trees within families Myrtaceae from Tripa peat swamp porest had a high level of similarity with the sequences in GenBank DNA database at the genus levels.The Query Cover for four trees species has a values in the range of 91-100%.A Query Cover is a number that describes how much of the query sequence is covered by the target sequence.If the target sequence in the database spans the whole query sequence, then the query cover is 100%.The E-value for all species of trees is 0.0.
Based on E-value=0.0,we can assume that the sequence is identical to the BLAST sequence result.The lower the E-value is, the more significant the match.According to Ref [16] explained the E-value in BLAST takes into account both the length and composition of the alignment along with the percentage identity found.A number close to 0.0 means that the hit has to be significant.

Phylogenetic relationships Based on Internal Transcribed Spacer (ITS) region
The phylogenetic tree in Figure 4. explained about phylogenetic relationships among species within families Myrtaceae.Based on the result, ITS region was successful in grouping the species originated from Tripa peat swamp forest (TPSF) with species from GenBank (GB) into the same clade.It can be presumed that Syzygium sp. 1 (TPSF) together with Syzygium australe and Syzygium samarangense were from a single common ancestor.
Inline with Syzygium garciniifolium (TPSF), this species also formed the monophyletic group together with four species from GenBank.According to [17] clade is a piece of a phylogeny that includes an ancestral lineage and all the descendants of that ancestor.This group of organisms has the property of monophyly, so it may also be referred to as a monophyletic group.
Based on morphological Syzygium sp. 1 and S. australe are two different species.Syzygium sp. 1 is species originated growing in peat swamp forest.On the other hand, S. australe is a common plant of coastal and highland rainforests from Southern New South Wales to central Queensland.In nature, it can reach over 25 m in height, but it is usually smaller in cultivation.The leaves are opposite (each pair emerges from the stem at the same location but on opposing sides), rounded and bright, glossy green in color [18].Conversely, refer to-molecular identifications S. australe is suggested as the sister group to Syzygium sp. 1 for the future studies.In addition, S. samarangense also recommended as the sister groups to S. garciniifolium, together their formed the same branches with 66% bootstrap support.According to Ref. [19] sister group is a phylogenetic term denoting the closest relatives of another given unit in an evolutionary tree.

CONCLUSION
Internal Transcribed Spacer (ITS) region was successful to predict the phylogenetic relationships among four species within families Myrtaceae.Also, the ITS region is recommended as a candidate barcode for the tree species from peat swamp forest.

Figure 1
Figure 1 Location of sample collected in Tripa peat swamp forest, Aceh, Indonesia

Figure 2
Figure 2 Sampling plot design for tree species Amplification segments of DNA were conducted using 20 µL Polymerase Chain Reaction (PCR) reactions (Kapa Taq PCR MasterMix).All of the components consists of 10 µL (1X Kapa Taq), 1 µL forward primer, 1 µL reverse primer, 3 µL DNA template, and 5 µL nuclease-free water.The temperature for PCR condition were start to

Figure 3
Figure 3 The population of Tristaniopsis whiteana in Tripa peat swamp forest

Table 2
The species of trees that exist in Tripa peat swamp forest, all Familes belong to Myrtaceae.
Phylogenetic Relationships Within Families Myrtaceae In Tripa Peat Swamp Forest … (Ardhana Yulisma, Zairin Thomy and Essy Harnelly) Due to DNA GenBank database does not provide the same sequence for each species.In the future, it is very important to develop of studies about forest genetic in the tropical rainforest, especially species of tree from peat swamp forest.Here, Internal Transcribed Spacer (ITS) is recommended as candidate barcode for the tree species from peat swamp forest.According to Ref.[22]among six tested loci i.e. matK, rbcL, rpoB, rpoC1, trnH-psbA, and ITS.The ITS is recommended as a possible barcode for plants, providing 100% species identification.In line with Ref.[23], the Internal Transcribed Spacer (ITS) of nuclear ribosomal DNA is one of the most commonly used DNA markers in plant phylogenetic and DNA barcoding analyses and it has been recommended as a core plant DNA barcode.