Phylogeny of Kemenyan Toba (Styrax sumatrana) Inferred

from trnl-trnf Chloroplast DNA Sequence

Arida Susilowati

, Henti Hendalastuti Rachmat

Wiza Noni Fadilah

and Yosie Syadza Kusuma

Faculty of Forestry, Universitas Sumatera Utara. Jl. Tri Dharma Ujung No. 1, Kampus USU,

Medan 20155, North Sumatra, Indonesia

Forest Research, Development and Innovation Agency Ministry of Environment and Forestry. Jl. Gunung Batu No. 5. PO

Box 165, Bogor 16001, West Java, Indonesia. Tel.: +62-251-8633234; 7520067. Fax. +62-251-8638111

Keywords: DNA, Styrax sumatrana, trnL-trnF, Phylogeny.

Abstract: Styrax sumatrana is a member of genus styrax that cultivated by local comunities in North Sumatra due to its

higher rosin and cinnamic acid content compared to others. This species is widely distributed in Tapanuli

Utara, Pakpak Bharat, and Humbang Hasundutan District. The information on Styrax sumatrana molecular

phylogeny in North Sumatra has not determinet yet, whereas it is important for future breeding and

conservation efforts. Therefore, this research was conducted to determined the phylogenetic relationship

Styrax sumatrana in North Sumatra and other member of genus Styrax in the world. The material for genetic

analysis were leaves sample from 10 individuals and collected from Humbang Hasundutan, Tapanuli Utara,

and Pakpak Bharat. Samples then extracted by using CTAB (Cetyl Trimethyl Ammonium Bromide) method.

DNA amplification was perform using PCR with annealing temperature 50°C. Sequence data analysis was

conducted by using BioEdit software and phylogenetic tree construction was using Mega 5.05. The results

showed that 3 sampled populations of S. sumatrana were grouped into four haplotypes. Phylogenetic tree

analysis result showed that Styrax sumatrana has the closest relationship with Styrax suberifolius and Styrax

chinensis, both are Chinese kemenyan species, with 63% bootstrap value.

1 INTRODUCTION

Indonesia known as megabiodiversity country with

huge number of endemic flora and fauna. Information

on plant species diversity in Indonesia is needed for

future conservation strategy and loosen the rate of

diversity loss. Among those of important native and

multipurpose tree in North Sumatra comes from

Styrax Genus, and locally are known as kemenyan

species. Steenis (1953) mentions four species of

kemenyan which were found and cultivated by local

farmer in North Sumatra, those were: kemenyan toba

(Styrax sumatrana J.J.SM), kemeyan durame (Styrax

benzoin), kemenyan siam (Styrax tonkinennsis) and

kemenyan bulu (Styrax paralleloneurum). There are

several local names standing both for similar and

different species of the Styrax.

Styrax sumatrana known by local people as the

best rosin producer in North Sumatera compared to

other because of it whitish color (preffered by market)

and stronger odor in which they were assumed have

higher rosin and cinnamic acid content (Hidayat et al.

2018). Rosin from this family has long been known

for local medicinal, traditional event and

pharmaceutical purpose and globally named as

benzoin resin (Susilowati et al. 2017).

The general purpose of phylogenetic

reconstruction using molecular evidence is done on

the basis of a homology sequence by aligning DNA

sequences (Thomy et al. 2018). Variations in the

sequence of nucleotide caused by substitution of base

or the indel. Compared to another marker, the

secondary structure of the intron trnL is often

constructed to infer homology position, for example

in Annonaceae (Pirie et al. 2007). Among the plant

DNA regions, non-coding areas such as trnL-trnF and

trnH-psbA chloroplast markers usually exhibit high-

level variations, including indel polymorphism, and

for some cases can provide good capacity for species

identification (Rachmat et al. 2017)

The use of non-coding region sequences of

chloroplast genome also has potential in phylogenetic

research (Soltis and Soltis 1998). Non-coding region

trnL-trnF is region with the highest mutation

frequency so that in most varied trnL-trnF sequence

plants (Taberlet et al. 1991). Therefore, our research

Susilowati, A., Rachmat, H., Fadilah, W. and Kusuma, Y.

Phylogeny of Kemenyan Toba (Styrax sumatrana) Inferred from trnL-trnF Chloroplast DNA Sequence.

DOI: 10.5220/0008387300260029

In Proceedings of the International Conference on Natural Resources and Technology (ICONART 2019), pages 26-29

ISBN: 978-989-758-404-6

was conducted to determined phylogenetic

relationship of kemenyan toba (Styrax sumatrana) in

North Sumatra. An understanding of genetic identity

of the species and/or population is great practical

importance both to conservation biologists and

silviculturist.

2 MATERIAL AND METHODS

2.1 Plant Material

Kemenyan leaf sample collected from tree different

populations those were Humbang Hasundutan,

Pakpak Bharat and North Tapanuli (Figure 1). This

population were choosen based on their different of

altitude and also the presence of physical barrier of

Bukit Barisan mountain range. Ten individuals S.

Sumatrana was taken from healthy and mature trees

of each population.

Figure 1. The origin of kemenyan sample from tree

population.

The total of 30 samples were sampled originated

from 15 years or more mature trees with the average

of dbh was 21 cm. Leaf samples were cut to ± 2 cm x

2 cm size and then stored into an plastic clip filled

with silica gel at a ratio of 1:5, and kept until all leaf

samples ready for extracted. The total genomic DNA

was extracted by using a modified CTAB (Cetyl

Trimethyl Ammonium Bromide) method according

Murray and Thompson (1980).

2.2 DNA Sequencing of trnL-trnF

Chloroplast DNA (cpDNA)

The trnL-trnF cloroplast region was amplified by

PCR using the universal c and f primers described in

Taberlet et al. (1991). PCR process was performed

using 20 uL of a solution containing 10 ng of genomic

DNA, 5 pmol of each forward and backward primer,

and 10 uL of Go Taq® Hot Start Colourless Master

Mix (Promega, Wisconsin, 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 50°C and polymerization

at 72°C for 45 min, and final extension at 72°C for 7

min. Prior to sequencing, the PCR products were

purified and automatic sequenced by Genetic Science

(Singapore). DNA sequencing was performed for

both strands with the primers were used for the PCR

amplifications.

2.3 Data Analysis

The successful rate of amplification then assembled

using a nucleotide assembly software. In this study

the assembly of nucleotides more clearly using

BioEdit software (Hall, 1999). Sequences are aligned

using MEGA 5.05 software (Tamura et al. 2011) in

the ClustalW menu (Larkin et al. 2007) and then

manually adjusted.

Phylogenetic studies were analyzed using MEGA

5.05 software on the phylogeny menu using the

Neighbor-Joining (NJ) method. The consistency of

NJ phylogenetic trees was tested by the bootstrap

method (Felsenstein, 1985) of 1,000 repetitions.

Genetic distance between samples was analyzed

using Kimura 2-parameter method (K2P) (Kimura,

1980). Phylogenetic studies of another Styrax species

using trnL-trnF primers can be obtained from the

sequence databases of various deposited Styrax types

in NCBI (https://www.ncbi.nlm.nih.gov), the DNA

sequence is then aligned with Mega 5.05 using Align

by Muscle and then select the Phylogeny menu to

obtain the phylogenetic tree.

3 RESULT AND DISCUSSION

Not all of 30 samples produce clearly

chromatographic sequences and graph, at the end we

only used 26 individuals that yield clear and unbiased

sequence read. The four excluded individuals were

originated two from Humbang Hasundutan (SS13HB,

and SS20HB) and two more individuals from

Tapanuli Utara (SS23TU and SS24TU).

After alignment we obtained 941 bp of sequence

length from all individuals which were divided into 4

haplotypes (Rachmat et al. 2017). Phylogenetic

analysis was performed using Mega 5 software, using

Phylogeny of Kemenyan Toba (Styrax sumatrana) Inferred from trnL-trnF Chloroplast DNA Sequence

Neighbour Joining (NJ) method. The analysis of the

trnL-trnF gene involved 53 data, containing 26

Styrax sumatrana sequences and 27 other Styrax

species, including:. Others styrax sequences

references were downloaded from NCBI. Styrax

suberifolius, Styrax chinensis, Styrax gentryl, Styrax

pentlandianus, Styrax nunezii, Styrax latifolius,

Styrax peruvianus, Styraz camporum, Styrax

leprosus, Styrax pohlii, Styrax obtusifolius, Styrax

ferrugineus, Styrax rotundatus, Styrax acoustic,

Styrax tomentosus, Styrax lanceolatus, Styrax glaber,

Styrax portoricensis, Styrax martii, Styrax laberi,

Styrax ubargenteus, Styrax officinalis, Styrax

benzoin, Styrax aureus, Styrax japonicus, and Styrax

agrestis. The reference sequences were aligned using

the Align by Muscle menu. The phylogenetic tree is a

graph used to describe the interconnecting kinship

between species consisting of a number of nodes and

branches with only one branch connecting the two

closest nodes. Each node represents the taxonomic

units and each branch represents the relationships

between units that describe the hereditary relationship

with the ancestor.

The phylogenetic tree produced by the Neighbour

Joining (NJ) method produces a hypothesis of kinship

relationships between samples based on the genetic

distance in the trnL-trnF gene. In the present study,

phylogenetic trees were tested statistically using the

bootstrap method of 1000 replications presented in

Figure 2.

Reconstruction of phylogenetic trees based on

molecular markers trnL-trnF shows the separation of

several groups. The sample group of Styrax

sumatrana is supported with 83% bootstrap values

consisting of four sub-groups with bootstrap values

ranging from 56 – 94 % (See Figure 2). From the

phylogenetic tree, it can be seen that Styrax

sumatrana has the closest relationship with two China

species of Styrax suberifolius and Styrax chinensis

with a bootstrap value of 63%. Even though shared

similar habitat, the relationship among S. sumatrana

with that of S. benzoin seemed to be far enough.

Considering the phylogenetic relationship as

described in Figure 2, we can determine that S.

sumatrana had close ancestry with those of China

species of Styrax suberifolius and Styrax chinensis

rather than S. benzoin which grow and share similar

habitat type. Phylogenetic trees provide information

about the classification of populations based on their

evolutionary relationships. The roots of the tree

illustrate the first branching point or origin of each

population on the assumption that the rate of

evolution is running constant (Dharmayanti, 2011).

Figure 2: Phylogenetic tree of Styrax sumatrana and the

other Styrax from all over the world with the same marker.

Note : SS (Styrax sumatrana), PB (Pakpak Bharat), TU

(Tapanuli Utara), HB (Humbang Hasundutan).

Among their inter population differences, we

found that the genetic distance between Styrax

sumatrana from Tapanuli Utara and Pakpak Bharat is

0.003 or 99.7% similarity, Tapanuli Utara with

Humbang Hasundutan similarity is 99%, while

Humbang Hasundutan value with Pakpak Bharat is

99.7%.

The genetic distance of Styrax sumatrana with

Styrax chinensis and Styrax suberifolius is 0.005 or

with similarity of 99.5%. While Styrax sumatrana

with Styrax benzoin have similarity of 99,3%.

4 CONCLUSIONS

Our result on the phylogenetic tree of Styrax

sumatrana showed that this species has the same

monophyletic with Styrax suberifolius and Styrax

chinensis with a bootstrap value of 63%. Although S.

sumatrana and S. benzoin were planted together in

North Sumatera, both of species separated into

different group.

ICONART 2019 - International Conference on Natural Resources and Technology

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Phylogeny of Kemenyan Toba (Styrax sumatrana) Inferred from trnL-trnF Chloroplast DNA Sequence