Eco. Env. & Cons. 25 (July Suppl. Issue) : 2019; pp.

(S194-S198)
Copyright@ EM International
ISSN 0971-765X

New haplotypes of black-bearded tomb bat

(Taphozous melanopogon) from Puncakwangi Cave
(East Java, Indonesia)
Muhammad Hilman Fu’adil Amin*, Nabilatun Nisa’ and Bambang Irawan

Department of Biology, Faculty of Science and Technology, Universitas Airlangga, Surabaya,

Indonesia

(Received 29 March, 2019; Accepted 10 April, 2019)

ABSTRACT
DNA barcoding is a new method for identifying species from any biological species using standardized
target gen and/or intergenic target. However, identification success is only as good as the underlying
database. In this study, cytochrome c oxidase I (COI) sequences of 6 individuals were generated and analyzed
from Taphozous melanopogon. Genetic distance, Neighbor-Joining (NJ) tree, and haplotype network analyses
were performed to identify new haplotype. COI sequence analysis from the present study, combined with
downloaded sequences from NCBI GenBank, revealed two new COI haplotypes from East Java, Indonesia.
Population of T. melanopogon in East Java have unique haplotypes which are discrete from the haplotypes
from Genbank (Myanmar/Vietnam) with pairwise distances 0.27-0.28 and number of segregating sites
among samples are 19 nucleotides and number of parsimony-informative sites are 17 nucleotides,
demonstrating that population in this study are highly isolated. The existence of isolated haplotypes
highlights the significance of considering genetic diversity in developing sustainable management and
conservation plans for biodiversity.

Key words : Chiroptera, COI, DNA Barcoding, New haplotype, Taphozous melanopogon

Introduction largest order in mammals, with more than 1300 spe-

Southeast Asia, including Sundaland, Wallacea,
Philippines, and Indo-Burma, has been recognized
as one of the world’s biodiversity hotspots based on
animal diversity. Sundaland, after tropical Andes,
also has been recognized as second leading hotspots
in terms of endemics, which contains endemic ver-
tebrates amounting to 2,6% of total species world-
wide (Myers et al., 2000).
In mainland Southeast Asia, almost 500 species of
mammals are identified at present time (Francis,
2017). If Indonesia archipelagos and Philippines are
involved, the numbers are nearly twofold (Corbet
and Hill, 1992). Bats (Chiroptera) are the second
*Corresponding author email: m-hilman-f-a@fst.unair.ac.id
cies, and the number of species still grow up (Wang
and Cowled, 2015).
In other hand, much of our biodiversity is now
jeopardized and in need of serious conservation ac-
tion. One-quarter of global mammalian species di-
versity is alarmed with extinction and half of the
species have deteriorating populations (Schipper et
al., 2008).
Starting of sixth era of mass extinction was
claimed by some researchers, new approach should
be developed to accelerate describing biological di-
versity. It becomes evident that describing
biodiversity with traditional approaches happens at
a much slower rate than the rate of species loss
MUHAMMAD HILMAN FU’ADIL AMIN ET AL
(Blaxter, 2003, Ceballos et al., 2015).
DNA barcoding is a new approach proposed to
present fast, precise, and automatable species iden-
tifications. This method use short DNA sequences
and standardized gene regions as genetic species
labels (Hebert and Gregory, 2005). Initially, it has
been suggested to overcome the limitation of tax-
onomists and available of instruments for taxa iden-
tification by upgrading our ability to specifically
document biodiversity and as such. At this time,
DNA barcoding was expected as a solution to accel-
erate the velocity of species discovery and unlocked
novel perspectives in conservation (Hubert and
Hanner, 2015).
DNA barcode standard for members of the
eumetazoan uses cytochrome c oxidase subunit I
(cox1 or COI) gene region (Hebert and Gregory,
2005). This gene profiles usually set recently exam-
ined specimen to the proper taxa levels. Species-
rank identification can be acquired by generating
extensive COI profiles. COI assignment system will
produce a valid, price-effective and available solu-
tion to the recent challenge of species identification.
The COI database could provide the basis for a glo-
bal system of DNA based bioidentification for ani-
mal (Hebert et al., 2003).
As identification tool, DNA barcoding was deter-
mined on the presence, completeness, and accuracy
of DNA sequences included in the reference library.
When DNA sequences for specimen are absent in
the database, the specimen potentially be
misrecognized (Kress et al., 2015; Kvist, 2013).
Misidentified also potentially occur when lacking a
database of geographical context for specimens
(Bergsten et al., 2012).
In this study, we examine COI sequence of T.
melanopogon specimens from East Java, Indonesia
and compared with COI database from Genbank,
which have been sampled from Vietnam/Myanmar
to increase COI database for this species (Francis et
al., 2010; Arai et al., 2019).
Materials and Methods
Sample Collection and Genomic DNA Extraction
Six specimens of T. melanopogon from Puncakwangi
cave, Lamongan, East Java were obtained in this
study. All samples were preserved in 95% ethanol
for further analysis.
Total genomic DNA was extracted from muscle
S195
tissue using Blood-Animal-Plant DNA Preparation
Kit (Jena Bioscience, Germany) according to the
manufacturer’s instructions. Total DNA was evalu-
ated for DNA quantity at 260 nm absorbance and
DNA purity was calculated by ratio 260 and 280 nm
absorbance (Barbas et al., 2007).
PCR Amplification
Folmer region of COI gene was targeted as DNA
barcoding sequence. This region was amplified us-
ing LCO HCO primer (Folmer, 1994). 25 µL total
volume of PCR reaction contained 12.5 µL MyTaq
Red Mix (Bioline, Singapore), 1 µL of each primer
(10 pmol), 2 µL DNA-template, and 8.5 µL deion-
ized water (Himedia, India).
The PCR was conducted in thermal cycler
(Eppendorf personal, USA), with the following pro-
file: pre-denaturation at 95 0C for 2 minutes, 35
cycles of denaturation at 95 0C for 45s, annealing at
54 0C for 1 min and extension at 72 0C for 1 min, then
followed by final extension at 72 0C for 10 min. 2%
agarose electrophoresis system used to evaluate all
of PCR products, and stained by peqGREEN DNA/
RNA dye (VWR International Ltd, UK).
DNA Sequencing and Data Analysis
The PCR products were sequenced in 1st Base DNA
Sequencing Service, Singapore. Multiple alignments
of 6 specimen sequences were performed with
Geneious v.9.1.8 (Kearse et al., 2012) to ensure that
all sequences of each marker gene provide a ho-
mologous fragment. DNA sequences from speci-
mens and 9 sequences downloaded from Genbank
(HM541969-HM541974, JF444115, MK410407-
MK410408) were aligned and perform Neighbor-
Joining tree for preliminary analysis and pairwise
Kimura 2-parameter (K2P) haplotypes sequence di-
vergence comparison (Kimura, 1980) using MEGA
v7.0 (Kumar et al., 2016). All DNA sequences also
analyzed by PopART (Population Analysis with
Reticulate Trees) to provides a comprehensive
implementation of haplotype network (Leigh and
Bryant, 2015).
Results and Discussion
The 676-682 bp of cytochrome c oxidase subunit I
gene sequences were obtained from 6 bat specimens
and submitted to Genbank with accession
MK728945- MK728950.
S196
The COI sequences contained 1 variable position
(A/G) at 320 th base in consensus sequence. T.
melanopogon MK728946 and MK728947 have identi-
cal sequences with guanine base at 320th base, and T.
melanopogon MK728945, MK728948, MK728949,
MK728950 have identical sequences with adenine
base at 320th base.
There were no changes in amino acid sequences
because of this mutation (glutamic acid to glutamic
acid). The average nucleotide base composition was
23.2% (158 bases) A, 22.7% (155 bases) C, 18.8% (128
bases) G, 35.2% (240 bases) T, and 0.1% (1 base) R
(A/G) with total GC content 41.6%. The aligned 15
sequences, including 9 sequences from Genbank,
resulted 654 bp COI sequence and revealed that
specimen from Puncakwangi have 97,25-97,63%
similarities with COI sequence database from
Genbank. Two major clusters from 15 sequences of
T. melanopogon with high bootstrap value was ob-
served by NJ tree (Figure 1).
Fig. 1. Neighbor-Joining (NJ) tree from sequences from
this study and originated COI sequences from
NCBI (*) showed the divergence from NCBI data-
base and demonstrate the distinct haplotypes for
present study. Kimura 2-parameter method was
applied to calculate evolutionary distances Num-
bers near nodes indicate NJ bootstrap percentages
(1000 bootstrap replicates).
Table 1. Pairwise Kimura 2-parameter (K2P) sequence divergence comparison between the new haplotypes and 4
haplotypes obtained from Genbank
(1)
(1) Genbank Haplotype 1
(2) Genbank Haplotype 2 0.002
(3) Genbank Haplotype 3 0.002
(4) Genbank Haplotype 4 0.002
(5) New Haplotype 1 0.025
(6) New Haplotype 2 0.027
Eco. Env. & Cons. 25 (July Suppl. Issue) : 2019
The first cluster contained all NCBI database, sec-
ond cluster contained specimens of this study.
Analysis of sequences generated in present study
and downloaded sequences from NCBI indicated
the presence of a new haplotype for T. melanopogon
species.
Haplotype network among 15 sequences showed
in Figure 2. Out of 15 sequences, there are 6
haplotypes were identified, 4 haplotypes from
Genbank and 2 new haplotypes from this study.
Genbank haplotype 1 consist of HM541969-
HM541974, Genbank haplotype 2 consist of
JF444115, Genbank haplotype 3 consist of
MK410407, Genbank haplotype 4 consist of
MK410408. New haplotype 1 generated from this
study consist of MK728946 and MK728947, and new
Fig. 2. Haplotype network among 15 sequences (9 from
Genbank Database and 6 from this study). Num-
ber in brackets indicates number site mutations
between haplotype.
(2) (3) (4) (5)
0.003
0.003 0.003
0.027 0.023 0.027
0.028 0.025 0.028 0.002
MUHAMMAD HILMAN FU’ADIL AMIN ET AL S197

C
C
C
C
T
T
haplotype 2 consist of MK728945, MK728948,

6
3
0
MK728949, and MK728950. Number of segregating

C
C
C
C
sites among samples are 19 nucleotides and number

T
T
5
6
7
of parsimony-informative sites are 17 nucleotides.
Consistent with sequence divergence comparison,

C
C
C
C
T
T
5
3
5
East Java population in this study clearly separated
with Genbank (Myanmar/Vietnam) population

A
A
G
G
G
G
5
1
0
was obtained from Genbank database. There are 16
site mutation which discrete two populations.
C
C
C
C
T
T
4
8
6
Table 2. The haplotype diversity and pure diagnostic characters at selected nucleotide positions of COI fragments in 6 haplotypes

Pairwise Kimura 2-parameter (K2P) sequence

divergence comparison between the 15 sequences
C
C
T
T
T
T
4
2
0

were 0.002 to 0.028 (Table 1). Character-based DNA

barcodes (COI) were distinguished using unique
A
A
A
A
G
G
4
0
5

combinations of character at 20 nucleotide positions

(Table 2). There are 634 bases conserved and 20
C
C
C
C
T
T
3
8
7

bases variable, including 16-17 bases variation from

our sequences compared to Genbank database.
C
C

C
C
C
Y
3
5
5
Nucleotide position

Haplotype analysis and discovery useful for

many aspects, e.g. reveals existence of cryptogenic
C

C
C
T
T

T
3
3
0

colonial ascidian Botryllus schlosseri in Northwest

Atlantic and hypothesized the invasion status and
G
G
G
G
A
A
3
1
8

source regions (Yund et al., 2015) and highly iso-

lated populations of Hediste diversicolor was prob-
A
A
A
A

A
G
3
0
6

ably created by the complete separation of the gulf

and periodic hypoxic conditions (Vasileiadou et al.,
C
C
T
T
T
T
2
6
2

2016), develops DNA barcode library to assess in-

traspecific variation in Coleoptera (Hendrich et al.,
A
A
G
G
G
G
2
2
5

2015), provides a measure of subtle biological re-

sponse to environmental changes in the Antarctic
A
A
G
G
G
G
1
9
8

terrestrial ecosystem (Collins and Hogg, 2016), and

identifies sympatrically and allopatrically distribu-
C
C
C
C
T
T
1
7
7

tion of Indo-Pacific Acropora (Robert et al., 2018).

Collecting new specimens and COI database
C
C
C
C
T
T
1
7
4

from different geographical range also important to

enhance precision for species identification. The
C
C
C

C
C
T
8
1

boundaries of the accuracy with which specimens

can be recognized will diverge from estimated in
C
C
T
T
T
T
2
1

different local or regional, because of missed appli-

cations a geographical context for specimens. The
A

A
A
A
A
G
3

intraspecific variation rises with the geographical

scale of sampling was expected as a result of isola-
tion by distance and phylogeographic structure
New haplotype 1 (this study)
New haplotype 2 (this study)

(Bergsten et al., 2012).

Genbank Haplotype 1
Genbank Haplotype 2
Genbank Haplotype 3
Genbank Haplotype 4

Conclusion

We identified two new haplotypes of T. melanopogon

from East Java, Indonesia. They have minimum 16
Haplotype

sites mutation compared from Genbank database.

S198
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