Analisis Filogenetik Beberapa Kerapu Subfamili Epinephelinae (Serranidae) Indo-Pasifik Barat Dari Perairan Indonesia.

PHYLOGENETIC ANALYSIS OF SOME INDO WESTPACIFIC GROUPER SUBFAMILY EPINEPHELINAE
(SERRANIDAE) FROM INDONESIAN WATERS

YANTI ARIYANTI

GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2015

STATEMENT LETTER
I hereby declare that thesis entitled “Phylogenetic Analysis of Some IndoWest Pacific Grouper Subfamily Epinephelinae (Serranidae) from Indonesian
Waters” is original result of my own research supervised under advisory
committee and has never been submitted in any form at any institution before. All
information from other authors cited here are mentioned in the text and listed in
the reference at the end part of the thesis.

Bogor, October 2015
Yanti Ariyanti
G352130011

SUMMARY
YANTI ARIYANTI. Pylogenetic Analysis of Some Indo West-Pacific Grouper
Subfamily Epinephelinae (Serranidae) from Indonesian Waters. Supervised by
ACHMAD FARAJALLAH and IRMA SHITA ARLYZA
The serranid Subfamily Epinephelinae comprises about 159 species of marine
fishes in 15 genera, commonly known as groupers, rockcods, hinds, and
seabasses. These commercially important fishes are bottom-associated which is
found in tropical and subtropical waters. Most species occupies coral reefs, but
some inhabit estuaries or on rocky reefs. Grouper has potential economic value in
fisheries, however, the classification and their evolutionary relationship was often
constrained by the incredible number of species, wide distribution, and lack of
morphological key that it’s used in classification. It causes any kind fishes of this
subfamily members were caught in the field are often summed up as groupers.
The Indo-Pacific region has the most diverse types of grouper than the other areas
such as Western Atlantic, Eastern Atlantic, and Eastern Pacific. Therefore, it is
becoming a very interesting to understanding genetic relationship of groupers in
this region particularly in Indonesian water.
In this present work, we have used cytochrome oxidase c subunit 1 (CO1)
gene as a molekular marker in order to investigate the molecular relationship
among some Indo West-Pacific grouper. The specimens were obtained from

various sources including fishing rod, seafood market, and marine fishery station
in several sampling point as follow Aceh (Sumatera), Luwuk (Sulawesi), Kupang
(East Nusa Tenggara), Pangandaran (West Java), Raja Ampat (Papua), Sinjai, and
Selayar Island (South Sulawesi). Tissue samples were used as the source of DNA
is part of the dorsal muscle, gill and fins tissue. All samples for molecular analysis
were stored in 95% alcohol.
The 26 sequences belonging to 5 generas (Anyperodon, Cephalopholis,
Epinephelus, Plectropomus, and Variola) and 14 species of some Indo-West
Pacific grouper from several places in Indonesia were obtained and studied herein.
Several sequences have been submitted in to GenBank. Based on the partial
Cytochorome oxidase subunit 1 and using Haemulon scuderii as outgroup, a
molecular phylogenetic tree was constructed by Neighbor-Joining (NJ) method
(Kimura 2-parameter). Appearance of the Anyperodon within group of
Epinephelus causes this cluster become not monopyletic. Cephalopholis is more
primitive than genus Epinephelus. Plectropomus and Variola stand on the other
clade basal position and it is seem like the primitive group among the subfamily
Epinephelinae. Hitherto, phenotypic identification in serranid fish is based on
color pattern differences and some morphological characters such as number of
spines, number of rays and caudal fin shape. Yet, that identification method has
often constrained by intraspesipic variation and also by extremely different

phenotypic traits between juvenile and adult individual in same species. So that,
based on the previous statement, one of the case study that will be revealed in this
present study is the case of species determination on juvenile individual of

Epinephelus (Epinephelus erythrurus). Analysis were conducted by combining
morphological and molecular identification (CO1) that showed also within this
report.
This result will be helpful in taxonomy and to understanding phylogenetic
relationship analysis among some West Indo-Pacific grouper in Indonesian
waters.
Keywords: CO1, Epinephelinae, Grouper, Indo West-Pacific, Phylogenetic

RINGKASAN
YANTI ARIYANTI. Analisis Filogenetik Beberapa Kerapu Subfamili
Epinephelinae (Serranidae) Indo-Pasifik Barat dari Perairan Indonesia. Dibimbing
oleh ACHMAD FARAJALLAH dan IRMA SHITA ARLYZA
Kelompok ikan serranid dari subfamili Epinephelinae terdiri atas sekitar 159
spesies yang termasuk dalam 15 genera, umumnya dikenal sebagai ikan kerapu,
rockcods, hinds, dan seabasses. Ikan komersial penting ini merupakan ikan
penghuni dasar perairan yang dapat ditemukan di daerah perairan tropis maupun

subtropis. Sebagian besar spesies ikan ini menghuni habitat terumbu karang,
sedangkan sebagian lainnya hidup di muara atau karang berbatu. Kerapu memiliki
nilai ekonomi tinggi, namun kalsifikasi dan hubungan evolusi mereka seringkali
terkendala oleh jumlah spesies yang luar biasa banyak, distribusi yang luas, serta
kurangnya keahlian dalam identifikasi morfologi. Hal ini menyebabkan apapun
jenis ikan yang tertangkap di lapangan dari anggota subfamili Epinephelinae
seluruhnya disebut sebagai kerapu. Wilayah Indo-Pasifik memiliki jenis ikan
kerapu yang paling beragam daripada daerah lain seperti di Atlantik Barat,
Atlantik Timur, dan Pasifik Timur. Oleh karena itu perairan Indo-Pasifik menjadi
wilayah yang sangat menarik untuk memahami hubungan filogenetik ikan kerapu
khususnya di wilayah perairan Indonesia.
Dalam penelitian ini, digunakan ruas gen sitokrom oksidase c subunit 1 (CO1)
sebagai penanda molekuler untuk menganalisis hubungan antara beberapa jenis
kerapu dari wilayah perairan Indonesia. Spesimen diperoleh dari berbagai macam
sumber termasuk hasil pancing tradisional, pasar ikan, dan tempat pelelangan ikan
dari beberapa titik sampling yaitu Banda Aceh (Sumatera), Luwuk (Sulawesi),
Kupang (East Nusa Tenggara), Pangandaran (West Java), Raja Ampat (Papua),
Sinjai, and Selayar Island (South Sulawesi). Jaringan yang digunakan sebagai
sumber DNA berasal dari otot dorsal, insang, serta sirip. Seluruh sampel jaringan
untuk keperluan analisis molekuler disimpan dalam alkohol 95%.

Sebanyak 26 urutan sekuen DNA dari 14 spesies kerapu dari wilayah perairan
Indonesia yang termasuk ke dalam 5 genera (Anyperodon, Cephalopholis,
Epinephelus, Plectropomus, dan Variola) berhasil diperoleh dan dipelajari
hubungan kekerabatannya dalam penelitian ini. Beberapa sekuen juga telah
didepositkan ke GenBank. Sebuah pohon filogeni yang dibangun menggunakan
metode Neighbor-Joining (NJ) (Kimura 2-parameter) berhasil diperoleh
berdasarkan runutan parsial gen CO1 dengan menggunakan Haemulon scuderii
sebagai outgroup. Kemunculan Anyperodon dalam kelompok Epinephelus
menyebabkan kelompok ini menjadi tidak monofiletik. Genus Cephalopholis
lebih primitif dibandingkan genus Epinephelus. Plectropomus dan Variola
menempati posisi basal dalam pohon filogeni, hal ini memungkinkan keduanya
merupakan kelompok yang yang primitif diantara kelompok subfamili
Epinephelinae yang lain. Hingga saat ini identifikasi fenotipik terhadap kelompok
ikan serranid dilakukan berdasarkan perbedaan pola warna dan beberapa karakter
morfologi, seperti jumlah jari-jari keras dan lunak, serta bentuk sirip ekor. Namun

identifikasi berdasarkan pola warna dan beberapa karakter morfologi tersebut
seringkali terkendala oleh adanya variasi intraspesifik serta perbedaan morfologi
yang sangat mencolok pada satu spesies yang sama antara individu yang masih
juvenil dengan individu yang telah dewasa sehingga menimbulkan kebingungan

dalam penentuan spesies kelompok ikan serranid. Berkaitan dengan hal tersebut,
salah satu contoh kasus yang diungkap dalam penelitian ini adalah penentuan
spesies dari individu yang masih juvenile pada genus Epinephelus (Epinephelus
erythtrurus). Analisis dilakukan dengan mengombinasikan identifikasi secara
morfologi dan secara molekuler (CO1).
Hasil penelitian ini diharapkan dapat berguna untuk dunia taksonomi dalam
memahami analisis hubungan filogenetik antara beberapa jenis kerapu Indo
Pasifik Barat di perairan Indonesia.
Kata kunci: CO1, Epinephelinae, Filogenetik, Indo Pasifik Barat, Kerapu

Copyright © 2015 Bogor Agricultural University
All rights reserved
It is prohibited to cite all or a part of this thesis without referring to and
mentioning the source. Citation only permitted for the purpose of education,
research, scientific paper, report, or critical writing only; and it does not defame
the name and honour of Bogor Agricultural University.
It is prohibited to publish or to reproduce all or a part of this thesis without the
written permission from Bogor Agricultural University.

PHYLOGENETIC ANALYSIS OF SOME INDO-WEST

PACIFIC GROUPER SUBFAMILY EPINEPHELINAE
(SERRANIDAE) FROM INDONESIAN WATERS

YANTI ARIYANTI

Thesis
As partial fulfilment of the requirements for a Master Degree in
Animal Biosciences Master Program of Graduate School of
Bogor Agricultural University

GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2015

Thesis Examiners: Dr Ir Mohammad Mukhlis Kamal, MSc

PREFACE
This thesis would not have been possible without the help and support of
many people. I would like to thank my committee supervisors: Dr Ir Achmad

Farajallah M.Si, my advisor, allowed me to pursue my research interests and
provided me with endless support and knowledge throughout my graduate thesis,
to Dr Irma Shita Arlyza for support and a chances how to learn become a real
researcher, and to my examiner Dr. Ir. Mohammad Mukhlis Kamal, M.Sc for a
great discussion. Thanks to many people who help me in collecting samples;
Irwan Hikmawan, Mihwan Sataral, Maulana Syafril Yusuf, Meutya Agustina, and
Vice Tantri. I am deeply indebted to Mrs. Maria Ulfah for all her help that has
been given. Also to my partner in laboratory for their kindness and sincerity; Mrs.
Tini, Mr. Adi Surahman, Sister Sianturi, Asri Febriana, Puji Utari Ardika, Novita
Anggraeni, and Esa Ayu Pratama. Thanks to all people in Zoo Corner and BSH
2013 have I regard as my second family. Lastly, I would like to thank all my
family and I am especially grateful for my parents and the loved ones, who have
always been my biggest fans and have encouraged me by all means to achieve my
success. Above all, I thank God Allah Subhanahu Wa Ta’ala for His almighty.

Bogor, October 2015
Yanti Ariyanti
G352130011

CONTENTS

LIST OF TABLES

xi

LIST OF FIGURES

xi

LIST OF FIGURES

vi

INTRODUCTION

1

Background
Research objective
MATERIALS AND METHOD

1
2
2

Study Site and Time

2

Sample Collection
Morphological identification

2
2

DNA Extraction and PCR Reaction

2

Visualization
Data Analysis

3
4

RESULT AND DISCUSSION
Result
Discussion

4
4
6

CONCLUSION

17

LIST OF REFERENCES

17

APPENDICES

20

AUTHOR’S BIOGRAPHY

36

LIST OF TABLES
1
2
3
4
5
6
7
8

Sampling location and GenBank Accession number of species
grouper in this study
Average nucleotide frequencies of CO1 sequences in present study
Maximum Likelihood Estimate of Substitution Matrix
Mean percentage group distance (Kimura 2-parameter)
Genetic distance of CO1 sequences in present study
Morphometric comparison of the E. erythrurus specimens in
the present study with those in the literature
Genetic distance of CO1 sequences from Indonesian E. erythrurus
and reference sequences from GenBank
DNA Sequences details of Epinephelus erythrurus in GenBank
file version

3
4
5
5
9
13
14
16

LIST OF FIGURES
1 Schematic structure of E. erythrurus (107 mm standard length)
6
2 Neighbour-joining tree based on the CO1 nucleotide sequences of the
grouper specimens analysed in the present work and of species from
GenBank.
7
3 Representative illustration congruence between tree topological and shape
types of generas
4 Neighbour-joining tree based on the CO1 nucleotide sequences
8
of the E. erythrurus specimens analysed in the present work and of
species from GenBank (without Thai E. erythrurus)
15
5 Neighbour-joining tree based on the CO1 nucleotide sequences of the
E. erythrurus specimens analysed in the present work and of species
from GenBank (with Thai E. erythrurus)
15

APPENDICES
Juveniles of E. erythrurus photograph at present study
20
Type shapes of 5 genera sub family Epinephelinae at present study based
on Heemstra and Randall 1993
21
3 List of GenBank Accession number for references sequences in the present
study
22
4 Alignment of partial CO1 gene sequences in the present study and
homologous sequences from GenBank
23

1
2

1

INTRODUCTION
Background
The Serranid subfamily Epinephelinae comprises about 159 species of marine
fishes in 15 genera, commonly known as groupers, rockcods, hinds, and seabasses
(Heemstra and Randall 1993). Various fishes of this family are known as Kerapu
or Belong in Bahasa (Burhanudin et al. 1980). Serranidae are demersal fish that
occupies coral reefs and rocky substrates. Most members of Serranidae inhabit sea
water, while others inhabit freshwater in tropical and temperate regions in around
the world (Craig and Hastings 2007).
Indonesia has a vast water areas with coral reefs, so that there are potentially
groupers (Syaifudin et al. 2007). According to Allen (2000), Indonesia has
become the leading country for endemism and also boast the highest overall
species diversity including subfamily Epinephelinae. The Indo-Pacific region has
the most diverse types of grouper than the other areas such as Western Atlantic,
Eastern Atlantic, and Eastern Pacific. Therefore, it is becoming interesting to
understanding genetic relationship of groupers in this region particularly in
Indonesian water.
Recently, the reef fishes of the family Serranidae especially subfamily
Epinephelinae still continues studied, however, the classification and their
evolutionary relationship is often constrained by the incredible number of species,
wide distribution, and lack of morphological characters that it’s used in
classification. Phenotypic identification of the grouper commonly based on color
pattern and some morphological characters. The colour pattern is usually
distinctive enough to identify large adult groupers at the species level, but intraspecific variations in colour pattern exist for each species (Heemstra and Randall
1993; Govindaraju and Jayasankar 2004). In many cases, fishes, and especially
their diverse developmental stages, are difficult to identify using morphological
characters (Teletchea 2009). Furthermore, difficulties in reconstructing the
evolutionary relationship among grouper species is due to their homogeneous
morphology (Smith 1971).
In this present work, we have used cytochrome oxidase c subunit 1 (CO1)
gene as a molekular marker in order to investigate the molecular relationship
among some West Indo-Pacific grouper. A partial sequence of the mitochondrial
cytochrome oxidase c subunit 1 (CO1) gene is commonly used as a barcode with a
size of about 650 bp, has been used in several animal taxa such as insects, birds,
and fish (Hebert et al. 2007). CO1 gene has been used in rapid analyses for
commercial purposes, especially for the confirmation of fish species (Ward et al.
2005; Barber and Boyce 2006; Wong and Hanner 2008; Sachithanandam et al.
2012).
Molecular techniques, such as DNA barcoding using CO1 genes are known to
complement morphological identification and reduce identification errors
(misidentification) on commercially important fish such as grouper in every stage
of the development (Chow et al. 1994; Hebert et al. 2003; Sachithanandam et al.
2011). In case, intraspesific variation and extremely different phenotypic traits
between juvenile and adult individual in same species still become serious
problem on species determining. So that, we sought to determine species on

2

juvenile individual of Epinephelus genus (Epinephelus erythrurus) by combining
morphological identification and molecular based analysis (CO1).
Research objective
This study aimed to analyse the molecular characteristics of grouper species
(subfamily Epinephelinae) collected from several major islands in Indonesia and
to understanding phylogenetic relationship analyses among some Indo WestPacific grouper in Indonesian waters by mean of phylogenetic tree construction
through the use of partial CO1 gene segment.

MATERIALS AND METHOD
Study Site and Time
The research was conducted in April 2014 - April 2015 in the Laboratory of
Molecular, Research Center for Oceanography, Indonesian Institute of Sciences
(LIPI-P2O) and the Laboratory of Animal Function and Behaviour, Departement of
Biology.
Sample Collection
Several collection samples of fishes is belonging to Dr. Achmad Farajallah
and Dr. Irma Shita Arlyza (LIPI, Oceanography). The specimens were collected
from 2013-2015 were obtained from various sources including fishing rod,
seafood market, and marine fishery station in several sampling point as follow
Aceh (Sumatera), Luwuk (Sulawesi), Kupang (East Nusa Tenggara), Pangandaran
(West Java), Raja Ampat (Papua), Sinjai, and Selayar Island (South Sulawesi)
(Table 1). All samples for molecular analysis were stored in 95% alcohol. Tissue
samples were used as the source of DNA is part of the dorsal muscle, gill and fins
tissue.
Morphological identification
Phenotypic characterization was conducted using the FAO species catalogue
of groupers of the world. The length and the morphometric parameters (body
shape, colour and the rays of the dorsal fins etc.) was measured and counted by
the calliper, lup, and counter.
DNA Extraction and PCR Reaction
Total DNA was extracted from ethanol preserved muscle using DNA
Extraction Kit for animal tissue (Qiagen and Geneaid) by following the
manufacturer's protocol. Approximately 648 bp were amplified from the 5′ region
of the COI gene using combinations of the fish-specific primers FishF1 (5’TCAACCAACCACAAAGACATTGGCAC-3’)
and
FishR1
(5’TAGACTTCTGGGTGGCCAAAGAATCA-3’) described in Ward et al. (2005)
and pair of AF282 (5’-TCTACCAACCACAAAGACATCGG-3’) and AF283
(5’TACTTCTGGGTGTCCRAAGAATCA-3’) with some modification from
Ivanova (2007, FishBol). The 25 μL PCR mixes included 18.75 μL of nuclease
free water, 2.25 μL of 10× PCR buffer, 1.25 μL of MgCl2, 0.25 μL of each primer
(0.1 mm), 0.125 μL of each dNTP (0.05 mm), 0.625 U of Taq polymerase, and
0.5–2.0 μL of DNA template. The thermal regime consisted of an initial step of 2
min at 95 °C followed by 35-40 cycles of 0.5 min at 94 °C, 0.5 min at 56 °C, and
1 min at 72 °C, followed in turn by 10 min at 72 °C and then held at 4.0 °C.

3

Table 1 Sampling location and GenBank Accession number of species grouper in this study
Species
Anyperodon
Anyperodon leucogrammicus
Epinephelus
E. coeruleopunctatus
E. coioides

E. epistictus
E. erythrurus

E. fasciatus
E. melanostigma
E. ongus
E. quoyanus
Cephalopholis
Cephalopholis miniata
Cephalopholis urodeta

Plectropomus
Plectropomus leopardus
Variola
Variola albimarginata

Variola louti

Code of
Speciment

Collection site

RJ3

Raja Ampat

RJ1
K1
K2
K3
K4
K5
K6
RJ4
KA2
K7
K8
K9
K10
RJ6
RJ7
RJ5
RJ9

Raja Ampat
Pangandaran
Pangandaran
Pangandaran
Pangandaran
Pangandaran
Pangandaran
Raja Ampat
Banda Aceh
Pangandaran
Pangandaran
Pangandaran
Pangandaran
Raja Ampat
Raja Ampat
Raja Ampat
Raja Ampat

RJ11
IA23
IA24
IA25

Raja Ampat
Sinjai
Sinjai
Selayar Island

KL3

Luwuk (Sulawesi)

KL1

Luwuk (Sulawesi)

KK2

Kupang (NTT)

KK1

Kupang (NTT)

GenBank Accession
number

KP998435
KP998436
KP998437
KP998438
KP998439
KP998440

KP998441
KP998442

KP998444

KP998443

Visualization
Separation of the products was performed using two methods. Amplicon was
performed using 1 % agarose gel that run at a voltage of 100 volts for 30 minutes.
Then proceed with Ethidium Bromide and visualized under Ultra Violet light.
While separation of the products using 6% polyacrilamide gel was run at a voltage
of 200 V for 40 min. Visualization was facilitated by silver staining (Byun et al.

4

2009). (Kimura 1980), including genetic distance calculations and neighbourjoining (NJ) analysis.
Data Analysis
All amplicons were sequenced commercially following the manufacturer’s
protocol. The DNA sequences were proofread, aligned and edited using MEGA6
(Tamura et al. 2013) and BioEdit (Hall 1999). A Kimura 2-parameter metric was
employed for sequence comparisons (Kimura 1980), including genetic distance
calculations and to generate neighbour-joining trees based on the CO1 region,
with node frequencies calculated based on 1000 bootstrap replicates.

RESULT AND DISCUSSION
Result
We obtained 5 genera and 14 species of some Indo West-Pacific grouper from
several places in Indonesia. Cytochrome oxidase subunit 1 (CO1) were partially
sequenced at least 1 specimens for each species. To form the analysis matrix, the
resulting data were combined with the 14 homologous sequences of the grouper
species downloaded from the GenBank. A total of 47 taxa were used for the
analysis of which 26 amplified and sequenced in this study, while the rest
(including outgroup) are reference sequence acquired from GenBank database.
Sequence characters
The partial CO1 sequences was 520 base pairs (bp) which translated to 173
amino acids with 200 variable sites, and 182 parsimony informative sites. The
frequencies of mean base composition of all codon was 31.4%, 27.2%, 25.1% and
16.3% for thymine, cytosine, arginine, and guanine, respectively. The content of
A+T (56.5%) was higher than that of C+G (43.5%). The three codon position
differed greatly in their base composition. The nucleotides frequencies of base
composition were almost similar for C, A, and G at the first codon positions,
while T was 18%. At the second position T was 43% and at third G was 6.9%. An
anti-guanine bias was observed for the second (G=12.7%) and third codon
(G=6.9%), it exihibited a strong bias anti-G (Table 2). The estimated
transition/tranversion bias (R) was 3.23 (Kimura 2-parameter), which showed that
transition was obviously more than tranversion (Table 3).
Table 2 Average nucleotide frequencies of CO1 sequences in present study
Base content of CO1 (%)
1

Average

st

2nd

3rd

T

C

A

G

T

C

A

G

T

C

A

G

18

24.1

28.6

29.4

43

29.5

14.5

12.7

33

28.1

32

6.9

5
Table 3 Maximum Likelihood Estimate of Substitution Matrix
A

T/U

C

G

-

2.96

2.96

19.09

T/U

2.96

-

19.09

2.96

C

2.96

19.09

-

2.96

G

19.09

2.96

2.96

-

Original
nucleotide
A

Substitution pattern and rates were estimated under the Kimura (1980) 2-parameter model.
Rates of different transitional substitutions are shown in bold and those of transversional
substitutions are shown in italics.

Phylogenetic relationship
Except the outgroup, the mean percentage divergence among those 5 genera
was 16.3%. The mean percentage group distance between Epinephelus and
Anyperodon was 13.5% as the lowest, while between Cephalopholis and
Plectropomus was 24% as the highest (Table 4). The minimum pairwise
nucleotide divergence value in CO1 among all taxa was 0.081 between E.
erythrurus and E. coeruleopunctatus, while the maximum pairwise nucleotide
divergence value was 0.262 between Epinephelus ongus and Variola louti (Table
5).
Table 4 Mean percentage group distance (Kimura 2-parameter)
Genera
1. Epinephelus

Mean percentage of group distance (%)
1
2
3
4
5
-

2. Anyperodon

13.5

3. Cephalopholis

18.5

19.1

4. Variola

21.9

22.1

21.7

5. Plectropomus

19.9

20.1

24.0

21.6

-

Based on the partial Cytochorome oxidase subunit 1 and using Haemulon
scuderii as outgroup, a molecular phylogenetic tree was constructed by NeighborJoining (NJ) method (Kimura 2-parameter). The values of bootsrap confidence
level of nodes were indicated above the branch. Fig. 2 shows that all GenBank
sequences and sequences of subfamily Epinephelinae acquired in this study. NJ
tree clearly exhibited 4 separate groups. The representative illustration congruence
between tree topological and shape types of generas are shown in Fig. 3.
Based on molecular data that the minimum pairwise nucleotide divergence
value in CO1 among all taxa was 0.08 between E. erythrurus and E.
coeruleopunctatus. According to Noitokr et al. (2013), the top ten homologous
analysis results (BLAST) showed that the sequences of E. erythrurus were highly
similar to those of E. coeruleopunctatus. The other distinctive genus which
comprises two very similar species V. albimarginata and V. louti has pairwise
nucleotide divergence ranged from 0.09-0.10. Whereas pairwise nucleotide
divergence between C. urodeta and C. miniata ranged is 0.09. Epinephelus cluster

6

become not monophyletic because appearance of the Anyperodon within the
group. Plectropomus and Variola stand on the other clade basal position and it is
seem like the primitive group among the subfamily Epinephelinae in this study.
Morphological and molecular based analysis (CO1) identification to confirm
juveniles of Epinephelus erythrurus
Diagnostic features.
Morphometric comparisons of E. erythrurus with the existing literature are
shown in Table 6, while the Fig. 1 shows schematic structure of E. erythrurus.
Specimen voucher K7_PND has a body depth 2.74 times in the standard length
(SL) and a head length of 2.37 in SL, with SL and total length of 107 mm and of
135 mm, respectively. Specimen voucher K8_PND has a body depth of 2.58 in SL
and a head of length 2.33 in SL, with SL and of 93 mm and 125 mm, respectively.
Both specimens have dorsal fins with XI spines and 16 rays, anal fins with III
spines and 8 rays, pectoral fins with 19 rays, rounded caudal fins and a dark gray
body.
Dorsal
fin
with
XI
spines;
insterspinous membranes of dorsal fin
not incised or moderately incised

Pelvic fins not reaching
anus

Caudal fin well
rounded

Anal fin with III spines
and 8 rays

Figure 1 Schematic structure of E. erythrurus (107 mm standard length)
CO1-based analysis.
The two E. erythrurus DNA sequences were submitted individually to
GenBank under accession numbers KP998441 for the K7_PND specimen voucher
and KP998442 for the K8_PND specimen voucher (Table 8). Of the 640–651-bp
basic taxonomic sequence length, we were able to obtain 548 bp.

Discussion
Based on sequence character analysis of the CO1 demonstrate that there is
transition and transversion (Table 2). An anti-guanine bias was observed for the
second (G=12.7%) and third codon (G=6.9%) position commonly observed in
fishes (Cantatore et al. 1994). As Magio et al. (2005) stated that the presence of
compositional bias in every case causes the transition/transversion ratios to vary
for different positions within the codons due to differences in selection pressure,
the third codon position is more likely to be silent than the first and second.
Epinephelinae sequence analysis showed values and characteristic which similar
to data reported on other fishes (Cantatore et al. 1994; Ward et al. 2005).

7

JN208608_E. erythrurus
JN208612_E. erythrurus
K10_E. erythrurus
E. erythrurus

K9_E. erythrurus
K8_E. erythrurus
K7_E. erythrurus
JQ349961_E. coeruleopunctatus
RJ1_E. coeruleopunctatus

E. coeruleopunctatus

KF929848_E. coeruleopunctatus
RJ5_E. ongus
E. ongus

DQ107858_E. ongus
RJ4_E. coioides
NC011111_E. coioides
DQ107890_E. coioides
K6_E. coioides
K5_E. coioides

E. coioides

K2_E. coioides
K1_E. coioides
K3_E. coioides
K4_E. coioides
NC012709_Anyperodon leucogrammicus
RJ3_Anyperodon leucogrammicus

A. leucogrammicus

KM077918_Anyperodon leucogrammicus
KA2_E. epistictus
NC021462_E. epistictus

E. epistictus

FJ237768_E. epistictus
RJ6_E. fasciatus
E. fasciatus

EU392207_E. fasciatus
RJ7_E. melanostigma
E. melanostigma

JQ349966_Epinephelus melanostigma
RJ9_E. quoyanus
E. quoyanus

NC021450_E. quoyanus
RJ11_Cephalopholis miniata
KM077909_Cephalopholis miniata

C. miniata

NC024100_C. miniata
IA23_C. urodeta
IA24_C. urodeta
C. urodeta

IA25_C. urodeta
FJ583013_C. urodeta
NC022139_Variola albimarginata
KK2_Variola albimarginata

V. albimarginata

KL1_Variola albimarginata
NC022138_Variola louti

V. louti

KK1_Variola louti
NC008449_Plectropomus leopardus
P. leopardus

KL3_Plectropomus leopardus
EU697542 Haemulon scudderi

(Photo credits: A. Farajallah, IS. Arlyza, M. Agustina, MS. Yusuf, Y. Ariyanti)

Figure 2 Neighbour-joining tree based on the CO1 nucleotide sequences of the grouper
specimens analysed in the present work and some sequences from GenBank. The
numbers at the nodes indicate bootstrap values for 1000 replicates.

8

Figure 3 Representative illustration congruence between tree topological and
shape types of generas
The phylogentic analysis showed the lowest mean group distance between
Epinephelus and Anyperodon (Table 4). It shows in the Fig. 2 that Anyperodon
leucogrammicus was grouped among Epinephelus genera, so that the cluster
become not monophyletic. This fact confirm the paraphyletic status of the
Epinephelus (Craig et al. 2001; Zhu et al. 2008; You et al. 2013). Currrent
classification, Anyperodon is distinctive monotypic genus is probably most closely
related to Epinephelus, with which it shares XI dorsal-fin spines and the absence
of trisegmental ptetrygiophores, but it differs from Epinephelus (and all other
groupers) in its lacking teeth on the palatines. Anyperodon is also unique among
groupers in its elongate groupers, but none of these are as compressed as
Anyperodon (Hemstra & Randall 1993).
The genera position between Epinephelus and Cephalopholis was also similar
with Craig et al (2001), using 16S gene, and then confirmed by Craig and Hasting
(2006) that support the valid genus of the Cephalopholis separate from
Epinephelus. Cephalopholis is more primitive than genus Epinephelus.
Cephalopholis is one of the largest genera (besides Mycteroperca and
Epinephelus) which has various species.

Table 5 Genetic distance of CO1 sequences in present study (below the diagonal), with standard error estimates (above the diagonal)
Sequence

RJ1

RJ1

RJ5

RJ6

RJ7

RJ9

KA2

RJ4

K1

K2

K3

K4

K5

K6

K7

K8

K9

K10

RJ3

RJ11

IA23

IA24

IA25

KK1

KK2

KL1

KL3

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.01

0.01

0.01

0.01

0.02

0.02

0.02

0.02

0.02

0.03

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.03

0.03

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.03

0.03

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.00

0.00

0.00

0.00

0.00

0.00

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.03

0.02

0.02

0.02

0.00

0.00

0.00

0.00

0.00

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.03

0.02

0.02

0.02

0.00

0.00

0.00

0.00

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.03

0.02

0.02

0.02

0.00

0.00

0.00

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.03

0.02

0.02

0.02

0.00

0.00

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.03

0.02

0.02

0.02

0.00

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.03

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.03

0.02

0.02

0.02

0.00

0.00

0.00

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.00

0.00

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.00

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.03

0.02

0.02

0.02

0.01

0.01

0.01

0.02

0.02

0.02

0.02

0.00

0.01

0.02

0.02

0.02

0.02

0.00

0.02

0.02

0.02

0.02

0.03

0.02

0.02

0.02

0.01

0.01

0.02

0.01

0.02

RJ5

0.10

RJ6

0.18

0.17

RJ7

0.15

0.13

0.14

RJ9

0.17

0.18

0.16

0.16

KA2

0.15

0.15

0.18

0.17

0.17

RJ4

0.13

0.12

0.15

0.15

0.17

0.14

K1

0.13

0.12

0.15

0.16

0.17

0.14

0.00

K2

0.13

0.12

0.15

0.16

0.17

0.14

0.00

0.00

K3

0.13

0.12

0.15

0.16

0.17

0.15

0.01

0.00

0.00

K4

0.13

0.12

0.15

0.16

0.17

0.15

0.01

0.00

0.00

0.00

K5

0.13

0.12

0.15

0.16

0.17

0.14

0.00

0.00

0.00

0.00

0.00

K6

0.13

0.12

0.15

0.16

0.17

0.14

0.00

0.00

0.00

0.00

0.00

0.00

K7

0.08

0.11

0.15

0.15

0.17

0.15

0.12

0.12

0.12

0.12

0.12

0.12

0.12

K8

0.08

0.11

0.15

0.15

0.17

0.15

0.12

0.12

0.12

0.12

0.12

0.12

0.12

0.00

K9

0.08

0.11

0.15

0.15

0.17

0.15

0.12

0.12

0.12

0.12

0.12

0.12

0.12

0.00

0.00

K10

0.08

0.11

0.15

0.15

0.17

0.15

0.12

0.12

0.12

0.12

0.12

0.12

0.12

0.00

0.00

0.00

RJ3

0.14

0.14

0.16

0.17

0.17

0.13

0.12

0.12

0.12

0.12

0.12

0.12

0.12

0.14

0.14

0.14

0.14

RJ11

0.17

0.20

0.21

0.21

0.18

0.18

0.22

0.22

0.22

0.22

0.22

0.22

0.22

0.17

0.17

0.17

0.17

0.21

IA23

0.15

0.20

0.20

0.19

0.19

0.18

0.18

0.18

0.18

0.18

0.18

0.18

0.18

0.17

0.17

0.17

0.17

0.18

0.09

IA24

0.15

0.20

0.20

0.19

0.19

0.19

0.18

0.19

0.19

0.19

0.19

0.19

0.19

0.17

0.17

0.17

0.17

0.19

0.09

0.00

IA25

0.15

0.20

0.20

0.19

0.19

0.18

0.18

0.19

0.19

0.19

0.19

0.19

0.19

0.17

0.17

0.17

0.17

0.19

0.09

0.01

0.01

KK1

0.24

0.26

0.24

0.23

0.24

0.20

0.24

0.25

0.25

0.25

0.25

0.25

0.25

0.22

0.22

0.22

0.22

0.24

0.21

0.23

0.22

0.23

KK2

0.22

0.25

0.24

0.22

0.23

0.20

0.22

0.22

0.22

0.22

0.22

0.22

0.22

0.20

0.20

0.20

0.20

0.22

0.22

0.22

0.22

0.22

0.10

KL1

0.21

0.23

0.21

0.21

0.21

0.19

0.21

0.21

0.21

0.21

0.21

0.21

0.21

0.19

0.19

0.19

0.19

0.21

0.21

0.21

0.21

0.20

0.09

0.02

KL3

0.20

0.21

0.19

0.18

0.22

0.20

0.20

0.20

0.20

0.20

0.20

0.20

0.20

0.20

0.20

0.20

0.20

0.20

0.24

0.24

0.24

0.24

0.23

0.22

0.02
0.20

9

10

Cephalopholis spp. has often been misidentified as Epinephelus spp., for
example, species of Chepalopholis have only IX dorsal-fin spines, and
Epinephelus acanthistius of the Eastern Pacific also has the same dorsal-fin
spines. According to Hemstra and Randall (1993), another useful generic
character separating both genera is the presence of 3 to 6 trisegmental
pterygiophores in the dorsal fin of Cephalopholis species.
In the separate clade, there was genus of Variola and Plectropomus that
occupied the basal position among all taxa. This molecular analysis concordant
with previous studies that said all genera with VIII-IX spines includes
Aetheloperca, Cephalopholis, Gracila, Paranthias, Plectropomus, Saloptia, and
Variola occupy basal positions in both the ML and MP analyses (Craig and
Hasting 2007; Zhu and Yue 2008). Position of Epinephelus is located at the top of
the phylogenetic tree indicating that is the most recently diverged species, which
is in concordant with the fact that it is also the most advanced genus in
Epinephelinae (Craig et al. 2001; Craig and Hasting 2006, Ding et al. 2006). E.
erythrurus and E. coeruleopunctatus has the lowest value in pairwise nucleotide
divergence (0.079) among all taxa. According to Noitokr et al. (2013), the top ten
homologous alignment results in GenBank showed that the sequences of E.
erythrurus were highly similar to those of E. coeruleopunctatus.
The E. erythrurus-E. coeruleopunctatus group has E. ongus as its sister taxa in
the CO1 tree. E. quoyanus is basal to the other species representing a sister taxa.
E. quoyanus is one of 9 shallow-water coral reef species that have a rounded
caudal fin and close-set dark brown spots with the pale interspaces forming a
network on the body. This reticulated groupers have been much confused in the
literature, and many museum specimen have been misidentified with the other
species such as E. bilobatus, E. faveatus, E. hexagonatus, E. macrospilos, E.
maculatus, E. melanostigma, E. merra, and E. spilotoceps (Hemstra and Randall
1993). The representative illustration congruence between tree topological and
shape types of generas are shown in Fig. 3. The shape of grouper illustrated in Fig.
3 which is shows 5 shape types representation to recognize each generas. There
are 5 symbols to illustrate the shape types of grouper:
= Epinephelus

= Anyperodon

= Cephalopholis

= Variola

= Plectropomus

In the current classification, E. coioides, E. epistictus, and E. fasciatus has a
dorsal fin with XI spines and finrays ranged from 14-17. E. coioides known as
orange-spotted grouper is one of the most importance fish species which has a
wide distribution in the world. This species is common and expensive fish in the
markets of the Persian Gulf, India, Singapore, Hongkong and Taiwan. Currently
in China, the orange-spotted grouper has become a major food in live fish markets
and is an important cultured fish for commercial sale in Guangdong, Hainan and

11

Fujian provinces of China (You et al. 2013). Whereas, E. epistictus (dotted
grouper) and E. fasciatus (black tip grouper) is known from continental localities
in the tropical Indo-West Pacific region. E. epistictus probably of some
commercial importance fish, while the E. fasciatus is abundance in shallow water.
Morphological and molecular based analysis (CO1) identification to confirm
juveniles of Epinephelus erythrurus
Based on the criteria shown in Table 6, both of our specimens diagnosed as
juvenile E. erythrurus. According to Carpenter and Niem (1999), that the female
species become mature at 15 cm of the SL. Also, the adult colour pattern of this
species is usually irregular pale spots and blotches that join randomly to form an
irregular dark reticulum of the background colour. Some specimens, especially
larger ones, are nearly uniform brown or have pale blotches on the body that are
only faintly visible (Randall and Heemstra 1993).
According to Noitokr et al. (2013), the top ten homologous analysis results
(BLAST) showed that the sequences of E. erythrurus were highly similar to those
of E. coeruleopunctatus. In the current classification an adult of E.
coeruleopunctatus has XI dorsal spines and 15 to 17 rays, the third or fourth spine
longest, its length contained 2.7 to 3.6 times in head length; anal fin with III
spines and 8 rays; pectoral fins large and fleshy, with 17-19 rays; caudal fin
rounded. The colour is brownish grey, the body covered with small pale spots
overlain with large pale blotches; oblique black saddle on rear half of peduncle; 4
to 5 indistinct black blotches at baseof dorsal fin, prominent black streak on
maxillary groove. While juveniles (less than 20 cm standard length) dark grey to
black, covered with prominent pupil-size white spots and smaller white dots
(Randall and Heemstra 1993).
BLAST searches using the two sequences indicated only nine sequences of E.
erythrurus from Thailand and Malaysia in GenBank with the query coverage was
91% and the maximum identity was 99–100% with existing E. erythrurus
sequences in GenBank. So that, we used those of nine sequences as the references
to construct phylogenetic tree and to understand genetic distances.
Based on the partial CO1 sequences, a molecular phylogenetic tree was
constructed using the neighbour-joining method (Kimura 2-parameter). The
bootstrap confidence values of the nodes are indicated above each branch.
Interestingly, Thai E. erythrurus had the smallest number of nucleotides (420 bp).
We constructed two phylogenetic trees, with and without the Thai E. erythrurus
sequences (Figures 4 and 5). Intra- and inter-specific genetic distances are shown
in Table 7.
Figure 4 show a phylogenetic tree without the Thai E. erythrurus sequence.
The two sequences in the present study were grouped with similar Malaysian E.
erythrurus sequences. Some E. coeruleopunctatus sequences formed a sister
group with E. erythrurus, while the other sequences formed a separate clade.
Furthermore, the intra-specific genetic distance of E. erythrurus ranged from 0.00
to 0.02 with the same species from other countries, while the inter-specific genetic
distance between E. erythrurus and E. coeruleopunctatus ranged from 0.01 to
0.07. However, the phylogenetic tree with the first barcode for Thai E. erythrurus
(JQ268576) (Figure 5) formed a species complex group with the distinction of

12

some E. coeruleopunctatus from the other separated clades of E. erythrurus and E.
coeruleopunctatus. It was suggested that E. erythrurus and E. coeruleopunctatus
are not monophyletic.
The genetic distance between pairs of Indonesian E. erythrurus and Thai E.
erythrurus ranged from 0.066 to 0.068 (Table 7), suggesting two main genotypes
of this species. Based on the two phylogenetic trees, both of our samples were
grouped with the Indonesian and Malaysian E. erythrurus that possessed a
genotype different from the Thai E. erythrurus.
E. erythrurus is a fish of minor commercial importance (Heemstra and
Randall 1993) that is often caught with other grouper species. The species is found
in Pakistan, India, Laccadive Island, Sri Lanka, the Gulf of Thailand, Indonesia,
Singapore, Borneo and the Malaysian Peninsula (Heemstra and Randall 1993;
Carpenter and Niem 1999; Allen et al. 2003). Based on the FishBase database, E.
erythrurus was recorded in Indonesia from Sulawesi to Java. In museums, we
identified RMNH 13525 (Java, Batavia), RMNH 13524 (Surabaya market), SU
61470 (Sangi Island), FMNH 22515-17 (Borneo, Kalimantan, Balikpapan
Harbour), AMS I.19355-039 (Sabah, Sandakan Island), USNM 183241 (North
Borneo) and FMNH 51717. In 2003, Allen and Adrim stated that the distribution
of the species in Indonesia stretched from Sulawesi to Sumatra, and specimens
were stored in the Western Australia Museum. In Thailand, these species was
recorded in a preliminary checklist of coral reef fishes of the Gulf of Thailand
(Satapoomin 2000). Hegde et al. (2013) reported that these species included in the
list of the new record along with their habitats from Goa, West coast of India.
Sluka (2013) also stated that E. erythrurus was recorded in the three locations
in nearshore rocky or corral habitats of western India, further that report about
these species can be used as an opportunity to remedy it is Data Deficient (DD)
status in IUCN Red List.
The samples in the present study originated from Bojongsalawe Beach
(7o43’8.31”S 108o30’11.59”E) in the Pangandaran district of West Java,
Indonesia. The shoreline of Bojongsalawe Beach directly faces the Indian Ocean.
Due to a lack of data, the species has not yet been assessed for the IUCN Red List
and also is not included in the Catalogue of Life. Another important result from
this research is that the barcode sequence for Indonesian E. erythrurus, which was
previously absent from GenBank, is presented here for the first time.
These studies may have not always permitted the reconstruction of
phylogenetic relationship among all the West Indo-Pacific grouper, however, this
result will be helpful in taxonomy and to understanding phylogenetic relationship
analyses among some West Indo-Pacific grouper in Indonesian waters.

Table 6 Morphometric comparison of the E. erythrurus specimens in the present study with those in the literature
Morphometric
characters

Body depth
Standard length
Total length
Head length
Dorsal fin
Dorsal rays
Anal spines
Anal rays
Caudal fin
Pectoral fins
Lateral scales series
Colour

Geographical
distribution

E. erythrurus
(KP998441
[K7_PND])
(Present study)
2.74 times in SL
107 mm
135 mm
2.37 times in SL
XI
16
III
8
Rounded
19
97
Dark gray

E. erythrurus
(KP998442
[K8_PND])
(Present study)
2.58 times in SL
93 mm
125 mm
2.33 times in SL
XI
16
III
8
Rounded
19
92
Dark gray

Bojongsalawe
beach Pangandaran
district,
Indonesia

Habitat

Depth

E. erythrurus
(Hemstra & Randall 1993)

E. erythrurus
(Carpenter & Niem
1998)

2.8-3.2 times in SL
110-280 mm
430 mm
2.4-2.7 times in SL
XI
15 or 17
III
8
Rounded
17 - 19
92 - 107
Olive to reddish brown, usually with
irregular pale spots and blotches that join
randomly to form an irregular dark
reticulum of the background colour. Some
specimens, especially the larger ones,
nearly uniform brown or with the pale
blotches on body only faintly visible
Pakistan, India, Laccadive Island, Sri
Pakistan, India,
Lanka, Gulf of Thailand, Indonesia,
Laccadive Is. Sri Lanka,
Singapore, and Borneo
Gulf of Thailand,
Indonesia, and Singapore

Harbours and estuaries with muddy or
silty-sand bottoms

In harbours and estuaries
with muddy or silty-sand
bottoms

E. erythrurus
(Allen et al. 2003)

To 430 mm

Dark gray with irregular pale
spots and randomly joined to
form maze-like pattern

Pakistan, Laccadive Is. off
India to Malaysian Peninsula
and W. Indonesia

Solitary, turbid harbours and
estuaries with mudy or siltysand bottoms
1-20

Abbreviation: SL,Standard Length; mm, milimetre; m, metre

13

JN208613

JN208612

JN208609

JN208611

JN208614

JN208608

JN208607

JN208610

JQ268576

JQ349961

JQ349962

JX674992

JX674993

JX674990

JX674991

KF929848

JF493438

JX093908

KP998441 E. erythrurus (Indonesia)

KP998442

KP998441

Accession number

0.00

0.00

0.00

0.01

0.01

0.01

0.00

0.00

0.00

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.00

0.00

0.01

0.01

0.01

0.00

0.00

0.00

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.00

0.01

0.01

0.01

0.00

0.00

0.00

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.00

0.00

0.00

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.00

0.00

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.00

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.00

0.00

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.00

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.00

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.01

0.00

0.01

0.00

0.00

0.00

0.01

0.01

0.01

0.00

0.00

0.01

0.01

0.01

0.00

0.01

0.01

0.01

0.01

0.01

0.01

0.00

0.01

KP998442 E. erythrurus (Indonesia)

0.00

JN208613 E. erythrurus (Malaysia)

0.00

0.01

JN208612 E. erythrurus (Malaysia)

0.00

0.00

0.00

JN208609 E. erythrurus (Malaysia)

0.01

0.01

0.01

0.01

JN208611 E. erythrurus (Malaysia)

0.01

0.01

0.02

0.01

0.00

JN208614 E. erythrurus (Malaysia)

0.01

0.01

0.02

0.01

0.00

0.00

JN208608 E. erythrurus (Malaysia)

0.00

0.00

0.01

0.00

0.01

0.01

0.01

JN208607 E. erythrurus (Malaysia)

0.00

0.01

0.00

0.00

0.01

0.02

0.02

0.01

JN208610 E. erythrurus (Malaysia)

0.00

0.01

0.00

0.00

0.01

0.02

0.02

0.01

0.00

JQ268576 E. erythrurus (Thailand)

0.07

0.07

0.07

0.07

0.07

0.07

0.07

0.07

0.07

0.07

JQ349961 E. coeruleopunctatus

0.07

0.07

0.08

0.07

0.07

0.07

0.07

0.07

0.08

0.08

0.02

JQ349962 E. coeruleopunctatus

0.06

0.06

0.07

0.06

0.06

0.06

0.06

0.06

0.07

0.07

0.01

0.01

JX674992 E. coeruleopunctatus

0.02

0.02

0.03

0.02

0.01

0.01

0.01

0.02

0.03

0.03

0.05

0.06

0.05

JX674993 E. coeruleopunctatus

0.02

0.02

0.03

0.02

0.01

0.01

0.01

0.02

0.03

0.03

0.05

0.06

0.05

0.00

JX674990 E. coeruleopunctatus

0.02

0.02

0.03

0.02

0.01

0.01

0.01

0.02

0.03

0.03

0.05

0.06

0.05

0.00

0.00

JX674991 E. coeruleopunctatus

0.02

0.02

0.03

0.02

0.01

0.01

0.01

0.02

0.03

0.03

0.05

0.06

0.05

0.00

0.00

0.00

KF929848 E. coeruleopunctatus

0.06

0.06

0.07

0.06

0.06

0.06

0.06

0.06

0.07

0.07

0.01

0.02

0.01

0.05

0.05

0.05

0.05

JF493438 E. coeruleopunctatus

0.06

0.06

0.06

0.06

0.06

0.06

0.06

0.06

0.06

0.06

0.01

0.01

0.00

0.05

0.05

0.05

0.05

0.01

JX093908 E. corallicola

0.06

0.06

0.06

0.06

0.06

0.06

0.06

0.06

0.06

0.06

0.06

0.06

0.05

0.06

0.06

0.06

0.06

0.06

0.01
0.05

14

Table 7 Genetic distance of CO1 sequences from Indonesian E. erythrurus and reference sequences from GenBank (below the diagonal),
with standard error estimates (above the diagonal)

Figure 4 Neighbour-joining tree based on the CO1 nucleotide sequences of
the E. erythrurus specimens analysed in the present work and of species from
GenBank (without Thai E. erythrurus). The numbers at the nodes indicate
bootstrap values for 1000 replicates.

Figure 5 Neighbour-joining tree based on the CO1 nucleotide sequences of
E. erythrurus specimens analysed in the present work and of species fr
GenBank (with Thai E. erythrurus). The numbers at the nodes indica
bootstrap values for 1000 replicates.

15

16

Table 8 DNA Sequences details of Epinephelus erythrurus in GenBank file version
Definition Locus
Accession Number
Submitted Date
Classification

K7_PND Epinephelus erythrurus CO1 gene CDS
KP998441
VRT 20-Mar-2015
Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;
Actinopterygii; Neopterygii; Teleostei; Euteleostei; Neoteleostei;
Acanthomorpha; Eupercaria; Perciformes; Serranoidei; Serranidae;
Epinephelinae; Epinephelini; Epinephelus

Alignment of Partial
aaccacaaagacatcggcaccctttatcttgtatttggtgcctgagccggtatagtaggaacggc
Sequences of the CO1 tctcagcctgcttattcgagccgagcttagccaaccaggggctctactaggtgacgatcagatct
Gene
ataatgtaattgttacagcacacgcttttgtaataatcttttttatagtaataccaatcatgattggtgg
ctttggaaactgactcatcccgctaataattggtgccccagatatagcattccctcgaataaataat
atgagcttctgacttctccccccatccttcttacttcttctcgcttcttctggagtagaagccggtgct
ggtactggctgaacggtctacccacccctagccggaaacctagcccatgcaggtgcatctgta
gacttaactatcttctcattacatttagcaggaatctcatcaattctaggtgcaatcaattttatcaca
actattattaatatgaaacccccagctatctcccaataccaaacacctttatttgtatgagcggtact
aattacagcagtgctcctgctcctctccctt