Anoa, dwarf buffalo from Sulawesi, Indonesia: Identification based on DNA barcode

Abstract

Priyono DS, Solihin DD, Farajallah A, Arini DID. 2018. Anoa, dwarf buffalo from Sulawesi, Indonesia: Identification based on DNA barcode. Biodiversitas 19: 1985-1992. Anoa is an endangered endemic species in Sulawesi. The controversial issue of anoa conservation until now is the taxonomic status of lowland and mountain anoa. This study aims to test the ability of DNA barcoding techniques to identify the taxonomy between mountain anoa and lowland anoa. A 681bp fragment of cytochrome oxidase subunit 1 (COI) gene was obtained and used to solve the molecular taxonomic problem and to resolve the phylogenetic relationships of the two types of anoa. Our results showed that the DNA barcode is useful in assigning the taxonomic position of anoa. In the phylogenetic tree, we found that the two types of anoa were in separate clades. We also found that based on the Kimura-2 parameter (K2P), the genetic distance between the two types of anoa showed higher values (3.4%) than the threshold of the separating species level. We, therefore, proposed that the binomial nomenclature for both types of lowland and mountain anoa are respectively Bubalus depressicornis and Bubalus quarlesi. We suggest that the use of DNA barcode techniques in anoa taxonomic studies and their implementation will be useful in conservation management

Full text

B I O D I V E R S IT A S
ISSN: 1412-033X
Volume 19, Number 6, November 2018 E-ISSN: 2085-4722
Pages: 1985-1992 DOI: 10.13057/biodiv/d190602
Anoa, dwarf buffalo from Sulawesi, Indonesia: Identification based on
DNA barcode
DWI SENDI PRIYONO1,♥, DEDY D. SOLIHIN1,♥♥, ACHMAD FARAJALLAH1, DIAH IRAWATI DWI ARINI2
1Department of Biology, Faculty of Mathematics and Natural Sciences, Institut Pertanian Bogor. Jl. Raya Dramaga, Bogor 16680, West Java, Indonesia.
Tel./fax.: +62-251-8622642, email: dwisendi92@yahoo.com, ♥♥dduryadi@yahoo.com
2Anoa Breeding Center of Manado's, Center for Environment and Forestry Research and Development. Jl. Raya Adipura, Manado 95119, North
Sulawesi, Indonesia
Manuscript received: 12 February 2018. Revision accepted: 4 October 2018.
Abstract. Priyono DS, Solihin DD, Farajallah A, Arini DID. 2018. Anoa, dwarf buffalo from Sulawesi, Indonesia: Identification based
on DNA barcode. Biodiversitas 19: 1985-1992. Anoa is an endangered endemic species in Sulawesi. The controversial issue of anoa
conservation until now is the taxonomic status of lowland and mountain anoa. This study aims to test the ability of DNA barcoding
techniques to identify the taxonomy between mountain anoa and lowland anoa. A 681bp fragment of cytochrome oxidase subunit 1
(COI) gene was obtained and used to solve the molecular taxonomic problem and to resolve the phylogenetic relationships of the two
types of anoa. Our results showed that the DNA barcode is useful in assigning the taxonomic position of anoa. In the phylogenetic tree,
we found that the two types of anoa were in separate clades. We also found that based on the Kimura-2 parameter (K2P), the genetic
distance between the two types of anoa showed higher values (3.4%) than the threshold of the separating species level. We, therefore,
proposed that the binomial nomenclature for both types of lowland and mountain anoa are respectively Bubalus depressicornis and
Bubalus quarlesi. We suggest that the use of DNA barcode techniques in anoa taxonomic studies and their implementation will be
useful in conservation management
Keywords: Anoa, COI, DNA barcoding, phylogenetic, molecular taxonomy
INTRODUCTION
Anoa is an endemic animal in Sulawesi Island. Anoa
has a unique characteristic as compared to other Bovidae
family members, in which anoa has a smaller size. The
population of anoa in the wild continues to decline (Broto
2015). The main causes of anoa population decrease are
due to poaching, conversions of forests into agricultural
land, industrial areas and human habitats (Burton et al.
2005). The IUCN Redlist has categorized anoa as an
endangered species since 1986, and The CITES included
anoa in Appendix I since 1975, meaning that anoa is
protected and cannot be traded. Various efforts in anoa
conservation have been taken including captive breeding
programs.
The success of species captive breeding program is not
only supported by the large population but also on the
information of the variation and genetic status of the
species. Information about the genetic status of species in
captivity provides records and guidelines in case of
transportation between different breeding populations (Xie
and Gipps 2012). If the genetic condition of the species is
ignored, extinction will occur, and the most significant loss
is the loss of germplasm of this endemic species in
Indonesia. In addition to genetic information, one of the
main problems in anoa conservation studies is the
taxonomic status of anoa (Burton et al. 2005).
Until now, the status of anoa taxonomy is still much
debated. Anoa is classified by habitat and some of its
morphological characters, such as lowland anoa and
mountain anoa. The varying number of chromosomes in
these two types of anoa (2N = 44, 45, 47, 48) shows
phylogenetic and taxonomic problems (Schreiber et al.
1993). At the subgenus level, the identification of the skull
(Groves 1969), the hemoglobin α amino acid sequence
analysis (Kakoi et al. 1994), and the identification of
cytochrome b (Tanaka et al. 1996) suggest that both types
of anoa are categorized under the anoa subgenus. However,
nuclear DNA analysis (cytochrome P450 aromatase partial
sequence and lactoferrin gene) shows that lowland anoa is
closer to the genus of Boselaphus, not Bubalus (Pitra et al.
1997). At the species level, the anoa taxonomic position
also encounters problems. A few researchers (Lydekker
1905; Dolan 1965) claimed that the anoa is divided into
two species, Bubalus depressicornis, and Bubalus quarlesi,
or as a species with three subspecies which is Bubalus d.
depressicornis, Bubalus d. quarlesi, and Bubalus d.
fergusoni. Schreiber et al. (1993) conducted allozyme
analysis on 25 anoas in European zoos. The allozyme
distance formed is between D=0.0206 to D=0.0505. The
range of allozyme distance is more likely to indicate a
separation of subspecies level (Nei 1987). Hartl et al.
(1988) previously also analyzed the greater distance of
allozyme (D=0.1389 to D=0.7621) to classify many species
in Bovini. Based on the taxonomic ambiguities that appear
at the subgenus, species and subspecies levels, further
research is needed to diagnose taxonomy of anoa using a
more robust marker, such as mitochondrial DNA markers
(Solihin 1994).
One of the most widely used mitochondrial DNA

B I O D I V E R S I T A S
19 (6): 1985-1992, November 2018
1986
markers in identifying taxonomic units is the cytochrome c
oxidase subunit I (COI) gene marker. The used of COI
gene as DNA barcoding marker has been declared as an
accurate tool in the identification and phylogenetic
inferences of a species (Hebert et al. 2003). Some recent
studies in Bovidae family member show that the use of
DNA barcode has also solved various problems in
determining a more appropriate taxonomic unit (Yang et al.
2013; Barmann et al. 2013).
Certainty in determining taxonomic units is necessary
to support the success of breeding in conservation.
Valuable data from genetic approaches such as genetic
information, evolution, and phylogenetics are required for
species monitoring and conservation management
(Schwartz et al. 2007). In such cases, conservation efforts
for 'endangered species' may be misdirected concerning the
goal of protecting biodiversity. This study aims to apply
DNA barcode techniques and to identify genetic
differences and taxonomic status of lowland anoa and
mountain anoa based on the mtDNA COI gene. These
results are used as a baseline for future planning and
management of anoa conservation management.
MATERIALS AND METHODS
Sample collection
A total of 10 samples of anoa was obtained, consisting
of 5 lowland anoa and five mountain anoa. Lowland anoa
were obtained from Palu (n=2), Gorontalo (n=1), Bolaang
Mongondow (n=1), and Toli-toli (n=1). Mountain anoa was
obtained from Sidenreng Rappang (n=2), Luwu (n=2) and
Seko (n=1) (Figure 1).
Figure 1. The location of origin of anoa used in this study: A. Ambo Sidenreng, B. Indo Sidenreng, C. Indo Luwu, D. Ambo Luwu, E.
Indo Seko, F. Denok, G. Manis, H. Rambo, I. Rita, J. Rocky
F
G
H
I
J
E
D
C
B
A

PRIYONO et al. – DNA barcode of anoa
1987
DNA isolation, PCR amplification, DNA sequencing
Total genomic DNA from blood was extracted by using
DNeasy Blood & Tissue Kit (Qiagen) following
manufacturer protocols with some modifications of the
original protocol to improve the yield and quality of the
extracted DNA. Modification of the protocol consisted of
adding proteinase-k to 40 µL and the length of the
incubation time was overnight after addition of ethanol.
The COI gene was amplified using the Polymerase chain
reaction (PCR) technique. PCR reaction was carried out
with a total mixture of 25 µL, with the composition of 1x
PCR buffer (Promega), GC 1x enhancer, 0.2 mM dNTP
(Qiagen), 10pg DNA template, 0.02 U/µL Taq polymerase
(BioLabs, England), 0.02 µM of each primer (COI
B_Depress (F)-5’GGCACCCTGTATTTGCTGTT3’, COI
B_Depress (R)-5’GCCGGAACATCATACTTCGT3’). The
amplification condition consisted an initial step for 5 min at
94°C followed by 35 cycles of 45 seconds at 94°C, 45
seconds at 53.4°C, and 6 min at 72°C, followed by a final
incubation at 72°C for 10 min. The 1.2% agarose gel was
used to visualize PCR amplicons. PCR products were then
sent for sequencing using an ABI3730 sequencing machine
provided by the First Base Laboratories (Singapore).
Data analysis
Alignment and sequence visualization was carried out
using ClustalW on MEGA ver. 6 (Tamura et al. 2013) and
manually checked in the BIOEDIT program ver 7.0.9 (Hall
1999). The final alignment consisting of 681 base pairs was
then verified into the Barcode of Life Data System (BoLD
System) (www.barcodinglife.org) to ensure the identity of
the samples and to test homology with the available
sequences in GenBank. Conspecific COI sequences of
lowland anoa species that have been obtained from
GenBank with the accession number of EF536351,
NC020615 (Hassanin et al. 2012) were also included in the
analysis and compared with the aligned sequences. The
sequence was derived from anoa that the origin of the anoa
was unknown, and the individual was housed at the
Ménagerie du Jardin des Plantes of the Museum national
d'Histoire Naturelle of Paris (Hassanin, pers. comm.). The
Kimura-2 Parameters method (K2P) was used to calculate
intra-and inter-species genetic distances (Kimura 1980). In
a case for overlooked species, we employed a sequence
divergence of 2,5 % as a screening threshold for mammals
as recommended by Tobe et al. (2010). Phylogenetic trees
were constructed with a combination of three models:
Maximum Likelihood in the Treefinder (Jobb et al. 2004)
with 1000 replications, Bayesian analysis using MrBayes
3.1.2 with Markov Chain Monte Carlo (MCMC) 10 million
(Huelsenbeck and Ronquist 2001) and 2500 burn-in, and
Neighbour Joining using Mega 6 (Tamura et al. 2013) with
1000 bootstrap replicates.
Currently, the anoa is in captivity of Anoa Breeding
Center (ABC), Center for Environment and Forestry
Research and Development (BP2LHK), Manado, North
Sulawesi, Indonesia, and Conservation Center of
Bontomarannu Education Park (BEP), Gowa, South
Sulawesi, Indonesia. As much as 5-10 mL of blood was
taken from the jugular vein from each specimen using the
EDTA vacutainer tube and then stored at-40ºC.
RESULTS AND DISCUSSION
BoLD identification
The 681 bp sequence was obtained and then analyzed
using the BoLD system to ensure the conformity of
samples with available databases (Table 3). The similarity
of the lowland anoa in this study with those of anoa in
BoLD system showed high similarity, ranged between
97.64-98.08%. Whereas for mountain anoa case, the
highest similarity was not with those of lowland anoa, but
with the Buffalo group (Bubalus bubalis) with the
similarity level ranging from 97.96-97.22% or divergence
ranging from 2.04-2.78%. The high similarity between the
mountain anoa and the buffalo (Bubalus bubalis and
Bubalus carabanesis) in BoLD system perhaps due to
misidentification occurred in the BOLD system as the
range of available base in genebank database (query cover)
did not reach the base length of anoa to be identified. The
previous case of BoLD misidentification has also been
described, especially in sequence coverage for
identification (Kwong et al. 2012). On the other hand, a
previous DNA barcode study, buffalo groups had huge
divergence that separate buffalo groups and buffalo group
classification is still a matter of debate (Cai et al. 2011,
Kochar et al. 2002). To clarify this case, it is important to
reconstruct a phylogenetic tree by including buffalo group.
Nucleotide variations
The concern when using mitochondrial DNA
amplification was the amplification of "COI-like
sequences" or nuclear pseudogenes of mitochondrial origin
(numt) (Buhay 2009). To confirm this sequence was utterly
originated from mitochondrial DNA COI gene, we
conducted several investigations. One of the numt
characters of numt is the presence of insertion and deletion
in DNA sequences (NUMTs, Bensasson et al. 2001).
However, the results of anoa COI gene alignment in this
study found no insertion-deletion (indel) event. Also, to
ensure in filtering numt, we carefully examined
heterozygosity of peak sequences (Buhay 2009), and the
obtained sequence did not contain any overlapping peaks.
So it could be ascertained that sequences used in the DNA
barcode technique in this study are COI genes in the
mitochondrial DNA.
Variations in base transitional mutations in COI genes
were occurring more than transversions. In all anoa
samples, the number of transition mutations sites was 33,
while transversion mutation sites were 14 or with ratio 2.3
(Table 2). The number of these transition mutations is also
found in mitochondrial DNA as reported Lakra et al.
(2009). The results of their research suggested that the ratio
of transition with transversion were 2.0 in the COI gene.
Although the phenomenon of transition bias was a few
little understood, it was suggested that there are two
contributing factors. First, spontaneous mutations rate
involving transitional mutations was much greater than
transversion mutations. Second, purification selection
affected the transition bias because transitional mutations
are more likely to be synonymous than transverse
mutations (Beckenbach et al. 1990). COI anoa gene

B I O D I V E R S I T A S
19 (6): 1985-1992, November 2018
1988
sequence showed less GC base composition than AT base
composition. GC base compositions range from 45.6-
45.8%. In many cases in mammals, GC base composition
was <50% (Martin 1995) and also found in other Bovidae
species (Hassanin et al. 2009). Each taxon had different GC
base composition but almost all show base composition
<50%, for example in other mammal groups: Bos taurus
33.1%, Homo sapiens 47.2%, Mus musculus 26.6%, (Perna
and Kocher 1995). The difference in GC base composition
probably related to metabolic physiology.
Closed examination on the aligned DNA sequences of
12 anoa samples (2 additional from GenBank) showed that
there are 47 variable sites and 634 conserved sites (Table
3). Of all the variable sites, there were unique sites that
found specifically for each type of anoa. For example,
lowland anoa has base A at position 60, while mountain
anoa only has base G. These unique sites are at the position
of 24, 30, 43, 60, and 384, with nucleotide T, A, G, A, and
G recorded for the lowland anoa, while G, C, T, G, and A
for the mountain anoa. These unique nucleotide sites can be
used as diagnostic characters for discriminating the
lowland and mountain anoa. Character-based identification
method (in this case, nucleotide DNA) was one of the
alternatives proposed in identification because they retain
lost information inherently in distance approach (Kelly et
al. 2007; Waugh et al. 2007). Such characters can be
regarded as a simple diagnostic nucleotide, sND). Sarkar et
al. (2002) used this term to describe the diagnostic
character that consists of several shared nucleotide sites.
ND, or character attribute (CA) in terms of Sarkar et al.
(2002), has been widely applied in molecular identification
studies in species (Wong et al. 2008; Wong et al. 2009;
Kelly et al. 2007; Rach et al. 2008). In this study, the
potential use of ND is for identification of anoa using COI
barcode sequence, especially in conservation forensics.
This identification method can also be a quick
identification alternative for both types of anoa, providing
the presence of ambiguity in the identification of distance-
based on BoLD.
Genetic distance
The genetic distance (Table 4) within and between
lowland and mountain anoa (including GenBank
sequences) was calculated based on the Kimura-2
parameter (K2P). The lowest genetic distance for all
specimens was 0.00, while the highest genetic distance was
0.039 (Table 4). Interspecific genetic ranges between the
two types of anoa ranged from the lowest (0.030) to the
highest (0.039) with an average of 3.4%. The average
interspecific genetic distance was lower than previous
DNA barcode study in the Bovidae group, i.e., 6.3% (Cai et
al. 2010). Cai et al. (2010) indicated that the high mean of
genetic distance in the study (6.3%) was due to the high
maximum genetic distance in the Bubalus bubalis group
(12.44%). The classification of buffalo that consists of 4
groups as a single species or not, is still being debated
(Kochar et al. 2002). The problem of buffalo classification
may because of ambiguity occurs when identifying
mountain anoa using the BoLD system.
Table 1. The top three BoLD identification result of anoa species
with respective percentage similarity
Name of
specimens
Putative
species
identification
Top three BOLD
identification result
Similarity
(%)
Rambo
Lowland anoa
B. depressicornis
98.08
B. bubalis
97.22
B. bubalis bubalis
97.17
Rita
Lowland anoa
B. depressicornis
98.08
B. bubalis
97.41
B. bubalis bubalis
97.02
Manis
Lowland anoa
B. depressicornis
98.08
B. bubalis
97.41
B. bubalis bubalis
97.02
Denok
Lowland anoa
B. depressicornis
97.64
B. bubalis
97.59
B. carabanesis
97.16
Rocky
Lowland anoa
B. depressicornis
98.82
B. bubalis
97.96
B. bubalis bubalis
97.77
Ambo Sidenreng
Mountain anoa
B. bubalis
97.78
B. carabanesis
97.4
B. depressicornis
96.76
Indo Seko
Mountain anoa
B. bubalis
97.22
B. carabanesis
96.93
B. depressicornis
96.61
Ambo Luwu
Mountain anoa
B. bubalis
97.78
B. carabanesis
97.4
B. depressicornis
97.05
Indo Luwu
Mountain anoa
B. bubalis
97.78
B. carabanesis
97.16
B. depressicornis
96.61
Indo Sidenreng
Mountain anoa
B. bubalis
97.96
B. carabanesis
97.4
B. depressicornis
97.05
Rambo
Lowland anoa
B. depressicornis
98.08
B. bubalis
97.22
B. bubalis bubalis
97.17
Rita
Lowland anoa
B. depressicornis
98.08
B. bubalis
97.41
B. bubalis bubalis
97.02
Manis
Lowland anoa
B. depressicornis
98.08
B. bubalis
97.41
B. bubalis bubalis
97.02
Denok
Lowland anoa
B. depressicornis
97.64
B. bubalis
97.59
B. carabanesis
97.16
Rocky
Lowland anoa
B. depressicornis
98.82
B. bubalis
97.96
B. bubalis bubalis
97.77
Ambo Sidenreng
Mountain anoa
B. bubalis
97.78
B. carabanesis
97.4
B. depressicornis
96.76
Indo Seko
Mountain anoa
B. bubalis
97.22
B. carabanesis
96.93
B. depressicornis
96.61
Ambo Luwu
Mountain anoa
B. bubalis
97.78
B. carabanesis
97.4
B. depressicornis
97.05
Indo Luwu
Mountain anoa
B. bubalis
97.78
B. carabanesis
97.16
B. depressicornis
96.61
Indo Sidenreng
Mountain anoa
B. bubalis
97.96
B. carabanesis
97.4
B. depressicornis
97.05

PRIYONO et al. – DNA barcode of anoa
1989
Table 2. Nucleotides variations, mutations types, and base composition of COI gene
Species
Conserved sites
Variation
Variable sites
si
sv
Base composition (%)
Pi
s
AT
GC
Gen COI
Bubalus depressicornis
655
18
8
26
19
7
54,2
45,8
Bubalus quarlessi
658
7
16
23
19
4
54,4
45,6
All specimen
634
36
11
47
33
14
54,4
45,6
Note: Pi: Parsimony-informative site, s: singleton site, si: transition pair, sv: transversi pair dan R: ratio of number of transitions to
number of transversions
Table 3. Variable sites in anoa based on COI gene. Bold indicates the unique nucleotide variations of each anoa type.
Specimen
Variable sites
1
1
1
1
1
2
2
2
2
2
3
3
3
4
4
4
4
4
6
6
8
9
1
2
2
4
5
0
0
1
4
9
0
2
5
2
3
4
6
7
0
3
1
0
4
0
4
7
9
4
7
B.depreesicornis_Rambo
T
T
G
A
C
G
A
G
G
A
C
A
G
T
G
G
C
G
A
C
T
T
B.depreesicornis_Rita
.
.
.
.
.
.
.
.
A
.
.
.
.
.
T
.
.
.
.
.
.
.
B.depreesicornis_Rocky
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
C
.
.
.
.
C
.
B.depreesicornis_Manis
.
.
.
.
.
T
.
.
.
.
.
.
.
.
.
.
.
A
.
.
C
.
B.depreesicornis_Denok
.
.
T
.
G
T
.
.
A
G
.
.
.
.
.
C
.
A
.
.
C
.
B.quarlessi_AmboSidenreng
G
G
T
C
G
.
.
T
A
G
A
G
A
.
.
C
T
A
G
.
C
.
B.depreesicornis_IndoSeko
G
G
T
C
G
.
.
T
A
G
A
G
A
.
.
C
T
A
G
.
C
.
B.depreesicornis_AmboLuwu
.
G
T
C
.
.
T
T
.
G
A
G
A
.
.
C
T
A
G
.
C
.
B.depreesicornis_IndoLuwu
.
G
T
C
G
.
T
T
A
G
G
G
A
.
.
C
T
A
G
.
C
.
B.depreesicornis_IndoSidenreng
.
G
C
C
.
.
T
T
.
G
A
G
.
C
.
C
.
.
.
T
C
C
Specimen
Variable sites
2
2
2
2
2
2
2
2
3
3
3
4
4
4
4
4
5
5
5
5
6
0
1
2
3
3
4
5
8
6
8
8
1
4
5
7
9
3
5
8
9
7
8
0
2
1
7
6
2
5
0
1
4
4
4
9
4
2
4
9
3
1
3
B.depreesicornis_Rambo
G
A
T
G
C
G
T
G
C
A
G
A
T
T
C
T
G
C
C
T
C
B.depreesicornis_Rita
C
.
.
.
.
.
.
.
.
G
.
.
.
.
.
.
.
.
.
.
.
B.depreesicornis_Rocky
C
.
C
A
.
.
.
A
.
G
.
.
.
.
T
C
.
.
T
C
.
B.depreesicornis_Manis
.
.
.
.
T
.
.
A
.
G
.
T
.
.
T
C
A
.
.
.
.
B.depreesicornis_Denok
.
.
.
.
T
.
.
A
.
G
.
T
.
.
T
C
A
.
.
.
.
B.quarlessi_AmboSidenreng
C
.
.
A
.
A
C
A
.
.
A
.
C
.
T
C
.
.
.
.
.
B.depreesicornis_IndoSeko
C
.
.
A
.
.
C
A
.
G
A
.
C
C
T
.
.
T
.
.
.
B.depreesicornis_AmboLuwu
C
.
.
A
.
A
C
A
.
.
A
.
C
.
T
C
.
.
.
.
.
B.depreesicornis_IndoLuwu
C
.
.
A
.
A
C
A
.
.
A
.
C
.
T
C
.
.
.
.
A
B.depreesicornis_IndoSidenreng
C
G
.
A
.
.
.
A
T
G
A
.
.
.
T
C
.
.
.
.
A
Table 4. Genetic distance in mountain and lowland anoa based on K2P method. Bold indicates the high genetic distances between two
type of anoa.
Specimen
1
2
3
4
5
6
7
8
9
10
11
12
1
EF536351_B.depress
2
NC020615_B.depres
0.000
3
B.depress_Rambo
0.019
0.019
4
B.depress _Rita
0.019
0.019
0.006
5
B.depress _Rocky
0.012
0.012
0.016
0.016
6
B.depress _Manis
0.019
0.019
0.015
0.018
0.016
7
B.depress _Denok
0.024
0.024
0.022
0.022
0.021
0.007
8
B.quarles_AmboSidenreng
0.033
0.033
0.038
0.038
0.033
0.038
0.030
9
B.quarles _IndoSeko
0.035
0.035
0.039
0.036
0.035
0.039
0.032
0.007
10
B.quarles _AmboLuwu
0.030
0.030
0.035
0.038
0.030
0.035
0.033
0.006
0.013
11
B.quarles _IndoLuwu
0.035
0.035
0.039
0.039
0.035
0.039
0.032
0.006
0.013
0.006
12
B.quarles _IndoSidenreng
0.030
0.030
0.035
0.035
0.027
0.035
0.035
0.028
0.029
0.022
0.026

B I O D I V E R S I T A S
19 (6): 1985-1992, November 2018
1990
Table 5. Intra-species and interspecies genetic distances in
mountain and lowland anoa based on the K2P method
Genetic distance
Bubalus
depressicornis
Bubalus
quarlessi
Gen COI
Minimum intraspecies distance
0.0
0.6
Maximum intraspecies distance
2.9
2.6
Average of intraspecies distance
1.6
1.6
Average of interspecies distance
3.4
Interesting result was found in genetic distance between
mountain and lowland anoa. The genetic distance between
these two species of anoa was 3.4% (Table 5). This genetic
distance is higher than 3% threshold for species-level
separation as mentioned by Hebert et al. (2003) in the study
of DNA barcodes, and also more than 2.5% threshold of
species-level separation for mammals according to Tobe et
al. (2010) for COI gene. Based on these higher distance, it
was suggested that both types of anoa have high genetic
differences and may have been even two different species.
Reconstruction of phylogenetic trees
A phylogenetic tree was reconstructed by including
domestic buffaloes to clarify the relationship between anoa
and domestic buffaloes regarding the ambiguity of BoLD
results, as well as Boselaphus tragocamelus. Previous
research by Pitra et al. (1997) suggested that anoa is
included within the Boselaphus group based on the nuclear
gene sequence. The phylogenetic reconstruction showed
that there are three main monophyletic clades (Figure 2).
Clade I consists of lowland anoa, Clade II consists of
mountain anoa, and Clade III consists of domestic buffalo.
Finally, results of this phylogenetic analysis have
successfully clarified that the mountain anoa was separated
from buffalo group and closer to the lowland anoa. In
addition, the anoa was included within the Bubalus group,
not within the Boselaphus group. These results support
previous studies on grouping anoa into Bubalus group
(Kakoi et al. 1994; Schreiber et al. 1995; Burton et al.
2005). The branch supports between groups of anoa had a
high percentage of posterior probability, bootstrap, and a
high ML (1/100/100), thus supports the separation and
polarity of two types anoa into different groups. These
results were similar to the results of genetic distance
analysis. A genetic richness that existed in anoa species had
previously been reported by Sugiri and Hidayat (1996) who
suggested that there were more than two species in anoa
based on karyotype of wild anoa from mountains of Central
Sulawesi. Three individuals from one region have a
chromosome number of 2n = 46, yet another anoa had 2n =
44. Recent research (Rozzi 2017) on fossil analysis
suggested there were three species of anoa, B. grovesi, B.
depressicornis and B. quarlesi.
Figure 2. Reconstruction of anoa phylogenetic tree with the addition of domestic buffaloes, and Boselaphus tragocamelus from
Genbank (node value from left-right: BA probability, NJ bootstrap, and ML percentage)

PRIYONO et al. – DNA barcode of anoa
1991
The endemicity of Anoa in Sulawesi island may be the
results of adaptive radiation process, a process by which
organisms diversify rapidly from ancestor species into
many new forms. This may be implied by the results of our
genetic study that discriminate anoa and the buffalo. Other
similar studies include an examination of seven Sulawesi
black ape morphotypes (Cynopithecus spp., Fooden 1969),
tarsiers (Tarsius spp., Merker et al. 2010), birds (White and
Bruce 1986), insects (Knight and Holloway 1990),
gastropods (Glaubrecht and Rintelen 2008), and frogs
(Setiadi et al. 2011). Such studies have provided examples
and cases that animals have undergone adaptive radiation
into various forms of species in Sulawesi.
There are several genetic information and molecular
taxonomy of anoa that have been identified in this study
that can become baseline information for further studies.
Conservation management of anoa can be carried out in
captive breeding with the aim is to increase quantity and
quality of breeding population/individual by correctly
grouping animal units according to their species and
genetic entity, as well as developing cryopreservation of
germplasm for its sustainability. A broader genetic study is
still needed to be carried out, especially by including more
samples and the use of the more advanced molecular
technique.
ACKNOWLEDGEMENTS
We acknowledged Ministry of Research, Technology
and Higher Education of the Republic of Indonesia for
providing research funding through Master Program of
Education Leading to Doctoral Degree for Excellent
Graduates (PMDSU) program, as well as to the staff and
researchers at Anoa Breeding Center, Manado, Indonesia
and Bontomarannu Education Park, Gowa, Indonesia.
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