Genetic Diversity of Enhalus acoroides (L.) Royle from Coastal Waters of Pramuka Island, Lembongan Island, and Waigeo Island, Indonesia, Based on Microsatellite DNA.

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Genetic Diversity of Enhalus acoroides (L.)
Royle from Coastal Waters of Pramuka Island,
Lembongan Island, and Waigeo Island,
Indonesia, Based on Microsatellite DNA
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RESEARCH ARTICLE
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Advanced Science Letters
Vol. 21, 199–202, 2015

Genetic Diversity of Enhalus acoroides (L.) Royle
from Coastal Waters of Pramuka Island,
Lembongan Island, and Waigeo Island,
Indonesia, Based on Microsatellite DNA
Made Pharmawati1 3 ∗ , I Nyoman Giri Putra2 3 , Yuliana Fitri Syamsuni3 , and
I Gusti Ngurah Kade Mahardika3
1

Biology Departement, Faculty of Mathematics and Natural Sciences, Udayana University,
Kampus Bukit Jimbaran, Bali, 80361, Indonesia
2
Department of Marine Science and Technology, Faculty of Fisheries and Marine Sciences,
Bogor Agricultural University, Jalan Rasamala, Kampus IPB Darmaga, Bogor, 16680, Indonesia
3
Indonesian Biodiversity Research Center, Udayana University, Jalan Sesetan Gang Markisa No. 6,
Denpasar, Bali, 80223, Indonesia
Enhalus acoroides (L.) Royle is one of sea grass species that is found in most Indonesian marine waters.
A study was conducted to analyze genetic diversity of E. acoroides collected from waters of Pramuka Island,
Lembongan Island and Waigeo Island, Indonesia using microsatellite DNA markers. Allele frequencies, observed
and expected heterozigosities were calculated using GenAlEx 6.501. The average of observed heterozigosity

was higher at Pramuka Island compared to the other two sites. In general, analysis of the genetic relationships
revealed that E. acoroides collected from three sites in Indonesia were grouped based on their habitats although
there are some exceptions. There is a possibility of gene flow between the three sampling areas. More samples
and microsatellite primers have to be included to further confirm E. acoroides connectivity.

Keywords: Enhalus acoroides, Genetic Diversity, Indonesia, Microsatellite DNA.

1. INTRODUCTION
Sea grass bed is one of coastal ecosystem that is dominated by
sea grass vegetation. Sea grass bed can be formed by single sea
grass species or by mix of two to 12 sea grass species on a
substrate.1 Sea grass bed has important ecological functions, for
example, as nursery grown, spawning and protection areas for
marine biota such as fishes, mollusks, shrimps, sea urchin, and
starfish2 . Besides that, sea grass is also food for dugong (Dugong
dugon) and green turtle (Chelonia mydas).3
Sea grass grows in shallow coastal areas from intertidal zone to
sub littoral zone and around rocky islands of Indonesian water.4
There are 60 sea grass species identified in the world5 and among
them, there are 13 species found in Indonesia.6

One of sea grass species is Enhalus acoroides which has bigger
size with wider and longer leaf as compared to other species.
Its leaves can reach 100 cm long. Enhalus acoroides has high
productivity in intertidal zone as food source for marine biota and
serves as carbon sequestration. Therefore, it can reduce impact
∗

Author to whom correspondence should be addressed.

Adv. Sci. Lett. Vol. 21, No. 2, 2015

of climate change.7 Enhalus acoroides is sea grass species that is
mostly found in almost all types of marine water and grow well
in shallow sea water and in open area during low tide.8
In Indonesia, E. acoroides is distributed throughout Indonesia,
such as Java, Bali, Kalimantan, Sumatra, Papua, Nusa Tenggara,
Sulawesi, Ambon and Papua.6 9 Seribu archipelago is located
off Jakarta’s Northern Coast and consists of 110 islands.10 Here,
E. acoroides is reported as one of common sea grass species.11 12
One of the islands in Seribu archipelago is Pramuka Island. This

island was chosen as one study area, representing west of Indonesia. In central Indonesia, Lembongan Island which is a small
island in east of Bali Island is famous for its marine biota including E. acoroides. Lembongan Island water was the second sampling location for this study. In Papua, Raja Ampat archipelago
is place where E. acoroides is also found.13 Weigo Island which
is located in Raja Ampat area was the third sampling location
that represents eastern part of Indonesia.
The genetic diversity of E. acoroides from Indonesia has not
been reported. Therefore, the aims of this study were to analyze the genetic diversity and relationships of E. acoroides from

1936-6612/2015/21/199/004

doi:10.1166/asl.2015.5861

199

RESEARCH ARTICLE
Table I.

Location of sample collection and number of sample.

Location (ISLAND)

Pramuka
Lembongan
Waigeo

Adv. Sci. Lett. 21, 199–202, 2015

GPS position

No. of sample

S 05 45’06”
E 106 36’44”
S 08 39’978”
E 115 28’116”
S 00 26’11.05”
E 130 46’37.45”

12
28
11

waters of Pramuka Island, Nusa Lembongan and Waigo Island
by using microsatellite data. Microsatellite DNA markers have
been widely used in genetic diversity and molecular ecology
studies both in plants and animals. Microsatellite marker has several advantages as compared to other DNA markers. Microsatellites have high mutation rate, hence, high polymorphism can
be detected, becoming high informative molecular markers.14
Information of E. acoroides genetic diversity is important for
ecosystem conservation, population management and biodiversity
maintenance.

2. MATERIALS AND METHODS
2.1. Sample Collection
Samples were collected during low tide in three areas of Indonesia (Table I, Fig. 1). Samples were taken from the shoreline up to
a perpendicular distance of 100 m–200 m. Young leaf blades of
E. acoroides were taken from individuals that separated at least
5 m apart. Leaves were cleaned, dried, cut about 1.5 cm long and
kept in silica gel in plastic bag. The samples were then transferred to laboratory for further analysis.
2.2. DNA Extraction and PCR
DNA was extracted from 0.1 g leaf using Qiagen Plant Mini
kit according to manufacturer instruction. DNA was quantified

using comparison with known concentration of lambda DNA in
1% agarose gel electrophoresis. PCR was conducted using five
primer pairs (Eaco1, Eaco9, Eaco19, Eaco51, Eaco55).15
The PCR reaction consisted of 1 × PCR buffer, 2.5 mm MgCl,
200 m dNTP, 1U taq polymerase (Amplitaq Gold-Life Technologies), 0.75 m each forward and reverse primers, 50 ng
template DNA and sterile water to make up 20 l PCR reaction.
The PCR cycles were as follows: pre denaturation at 95  C for
15 min, followed by 32 cycles of denaturation at 94 C for 30 s,
annealing at 58  C for 1.5 min and extension at 72  C for 1 min.

Final extension was done at 60  C for 30 min. The PCR products
were sent to UC Berkeley, Department of Molecular and Cell
Biology Sequencing Facility, USA for fragment identification.
2.3. Data Analyses
Data were analyzed using GenAlEx 6.501 software and presented
as allele frequencies, expected heterozigosity (He) and observed
heterozigosity (Ho). A dendrogram showing genetic relationships
of samples from three regions was developed by MVSP Software
using UPGMA based on Euclidean distance matrix.

3. EXPERIMENTAL RESULTS
The number of alleles from five loci used in this study varied
from 6 to 18 (data not shown). The highest number of allele
was at locus Eaco51 which had 18 alleles while locus Eaco1 and
locus Eaco19 had the lowest number of allele (6 alleles). Locus
Eaco9 had 10 alleles and there were 15 alleles at locus Eaco55.
The number of total alleles at Pramuka was 26 while there were
40 alleles from Lembongan and 27 alleles from Waigeo. Lembongan had the highest number of alleles with low frequency of
each allele.
Allele 254 bp at locus Eaco51 was a common allele at Pramuka Island and Nusa Lembongan while allele 205 bp had the
highest frequency at Waigeo Island.
Analysis of heterozigosity showed that E. acoroides from Pramuka Island had the highest value of observed heterozigosity
which was 0.767. Observed heterozigosity at population at Nusa
Lembongan was 0.436 which was slightly lower than that in
Waigeo ie. 0.582 (Table II). The high levels of observed heterozigosity at water of Pramuka Island may be due to scattered
individual distribution of E. acoroides. At study site, E. acoroides
do not form continuous sea grass bed.
A dendrogram using UPGMA based on Eucliden distance
showing genetic relationships of a total of 51 E. acoroides samples from waters of Pramuka, Lembongan and Waigeo Islands is
presented in Figure 2.

The dendrogram divided all samples into four groups which
were group A, B, C and D. There is no previous study on genetic
relationship of E acoroides from Indoneian waters, therefore, this
is the first report on its genetic relationships from three locations in Indonesia. In general, the samples were grouped into
their growing locations, although there are some exceptions. Several individuals from Lembongan Island water were grouped with
E. acoroides from Waigeo water (Group A). Group C consisted
of individuals from waters of Pramuka, Lembongan and Waigeo
Islands.
Table II. Heterozigosity of E. acoroides in Pramuka Island, Nusa Lembongan and Waigeo Island.
Location

Locus

Fig. 1. Map of location of samples collection, Pramuka Island (•), Nusa
Lembongan (), Weigeo Island (⋆).

200

Eaco1
Eaco9

Eaco19
Eaco51
Eaco55
Average

Pramuka Island

Lembongan Island

Waigeo Island

He

Ho

He

Ho

He

Ho

0.152
0.761
0.577
0.667
0.712
0.574

0167
1
1
0833
0833
0767

0.438
0.676
0.406
0.850
0.867
0.647

0.429
0.463
0.143
0.964
0.909
0.436

0.454
0.849
0.298
0.835
0.806
0.648

0636
1
0364
0909
0909
0582

RESEARCH ARTICLE

Adv. Sci. Lett. 21, 199–202, 2015

WO
WO
WO
WO
WO
WO
WO
WO
LB
WO
LB
LB
LB
PR
LB
LB
LB
LB
LB
LB
LB
LB
LB
LB
LB
LB
LB
LB
LB
LB
LB
LB
LB
LB
LB
WO
WO
LB
LB
LB
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR
PR

A

B

C

D

Euclidean distance
Fig. 2.

Dendrogram showing genetic relationships of E. acoroides collected from waters of Pramuka, Lembongan and Waigeo Islands.

Genetic structure of marine species in Indonesia is also
affected by oceanic factors such as ocean current of Indonesian
through flow current that convey water from Pacific to Indian
Oceans.18 This may facilitate gene flow of E. acoroides between
locations. There is also a dynamic seasonally reversing current
in Java sea18 which may contribute to the mixed position of
E. acoroides from Pramuka Island water, Lembongan and Waigeo
waters (Group C). More samples and microsatellite primers are
needed to further elucidate the grouping and connectivity of
E. acoroides from these three areas.
It is believed that there is genetic divergence between populations of Indian and Pacific Oceans.16 However, several studies showed that there was genetic exchange between oceans.17
This may explain the grouping of some E. acoroides individuals from Waigeo and Lembongan waters. Similar situation was
reported for pearl oyster species (Pinctada maxima) from West
Papua and Bali where there was genetic exchange between the
two populations.18

4. CONCLUSIONS
Enhalus acoroides from coastal waters of Pramuka Island, Lembongan Island and Waigeo Island have high heterozigosity. The
samples were clustered into four groups and in general they were
grouped based on their growing location although in group A and
group C, there were some individuals from different locations.

Acknowledgments: This work was supported by USAID
under NAS Sub-Grant No. PGA-2000003438. We would like to

thank Andri Kuncoro from Papua University for helping us with
sample collection at Waigeo Island.

References and Notes
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Received: 29 September 2014. Accepted: 25 October 2014.

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