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Mitochondrial DNA, Early Online: 1–8

! 2014 Informa UK Ltd. DOI: 10.3109/19401736.2014.913138

FULL LENGTH RESEARCH PAPER

Illegal trade of regulated and protected aquatic species in the

Philippines detected by DNA barcoding
Angelli Marie Jacynth M. Asis, Joanne Krisha M. Lacsamana, and Mudjekeewis D. Santos

Genetic Fingerprinting Laboratory, National Fisheries Research and Development Institute, Quezon City, Philippines

Abstract Keywords
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Illegal trade has greatly affected marine fish stocks, decreasing fish populations worldwide. CO1, illegal trade, juvenile eel, Manta ray,
Despite having a number of aquatic species being regulated, illegal trade still persists through processed products
the transport of dried or processed products and juvenile species trafficking. In this regard,
accurate species identification of illegally traded marine fish stocks by DNA barcoding is History
deemed to be a more efficient method in regulating and monitoring trade than by
morphological means which is very difficult due to the absence of key morphological Received 20 January 2014
characters in juveniles and processed products. Here, live juvenile eels (elvers) and dried Revised 3 April 2014
products of sharks and rays confiscated for illegal trade were identified. Twenty out of 23 (87%) Accepted 5 April 2014
randomly selected ‘‘elvers’’ were identified as Anguilla bicolor pacifica and 3 (13%) samples as Published online 19 May 2014
Anguilla marmorata. On the other hand, 4 out of 11 (36%) of the randomly selected dried
samples of sharks and rays were Manta birostris. The rest of the samples were identified as
Alopias pelagicus, Taeniura meyeni, Carcharhinus falciformis, Himantura fai and Mobula japonica.
For personal use only.

These results confirm that wild juvenile eels and species of manta rays are still being caught in
the country regardless of its protected status under Philippine and international laws. It is
evident that the illegal trade of protected aquatic species is happening in the guise of dried or
processed products thus the need to put emphasis on strengthening conservation measures.
This study aims to underscore the importance of accurate species identification in such cases of
illegal trade and the effectivity of DNA barcoding as a tool to do this.

Introduction In the Philippines, a number of aquatic species such as eels of

various life stages and sharks and rays are being utilized for
Illegal trade of marine fish stocks has been a major challenge to
various purposes resulting into the decline in their population
marine biodiversity conservation since the continuous and heavy
(Crook, 2010; SEAFDEC, 2012).
exploitation of this resource leads to the declines in marine
The Philippine eel culture industry started in 1972 when
populations or even near-collapses of it (Mullon et al., 2005).
profitable quantities of juvenile eels called ‘‘elvers’’ were
Even with established regulation efforts, reports on illegal,
discovered in the Cagayan river system (Gutierrez, 1976). The
unreported and unregulated fisheries (IUU) and illegal trade are
Anguilla were seen as commercially important and treated as fish
still existent (Maes & Volckaert, 2007). At present, illegal
species with aquaculture potential (Briones et al., 2007).
substitution and trade of certain fish species has been a rising
However, a significant decline in population resulted to a
global concern (Rasmussen & Morrissey, 2008). Southeast Asia
prohibition of ‘‘elvers’’ exportation in the country during the
in particular has been recognized as a ‘‘wildlife trade hotspot’’
1970s (Gutierrez, 1976). This ban has been reinstated today under
because of its unsustainable and ill-regulated wildlife trade
the Philippines’ Fisheries Administrative Order (FAO) 242, which
(Nijman, 2010). This has been a rising concern since the epicenter
upholds FAO 107 and 107-1 series of 1986 that banned ‘‘elvers’’
of marine biodiversity that urgently needs improvement of
exportation and revoked FAO 159 series of 1986, which allowed
conservation efforts is found in this part of the world, the Coral
the exportation of the commodity. The reinstatement of the ban
Triangle, encompassing to a large extent Indonesia, Malaysia,
was recommended by the Philippine Bureau of Fisheries and
Philippines, Papua New Guinea, Solomon Islands, Timor L’Este,
Aquatic Resources (BFAR) Regional Office II in Cagayan after
and Brunei, with the Philippines and eastern Indonesia having the
observing the excessive and non-stop exploitation of ‘‘elvers’’ due
highest concentration of species richness within the Coral
to its sharp price increase.
Triangle (Sanciangco et al., 2013).
Shark and ray fisheries in the Philippines have also been
expanding with an average annual production of 5882 t for the
past 20 years (Barut & Zartiga, 2002). According to the FAO
fisheries Department (2012), the average annual reported shark
catch for 2000–2010 was 5277 t or 0.65% of the global reported
Correspondence: Mudjekeewis D. Santos, National Fisheries Research
catch. The commercial exploitation of shark and ray species
and Development Institute, Room 601 Corporate 101 Building, Mo. began in 1960s and landings of these aquatic species has then
Ignacia Avenue, Quezon City 1103, Philippines. Tel/Fax: +63 23725063. declined (Barut & Zartiga, 2002; Bonfil, 2002). Manta ray fishing
E-mail: mudjiesantos@yahoo.com has also expanded and is now overexploited in by-catch and
 2 A. M. J. M. Asis et al.
targeted fisheries (Heinrichs et al., 2011; Homma et al., 1999;
Marshall et al., 2009). As such, manta rays are considered
globally Vulnerable as listed under the IUCN Red List of
Threatened Species, due to their very low reproductive capacity.
In the Philippines, manta rays are protected by virtue of the
country’s Fisheries Administrative Order (FAO) 193 series of
1998 banning the catching, selling, purchasing and exporting of
manta rays in the country. A report made by Alava & Dolumbalo
(2002) states that the trade of manta rays in Bohol Sea has
been operational for several generations and has already
threatened manta ray populations in the region. However due to
the difficulty of differentiating manta and mobula rays, a rapid
resource assessment in 2010 considered these species as one
and reported that the status of populations of rays in Bohol Sea
is unchanged compared to 2002 (Rayos et al., 2012) thus
presenting a misperception of the status of Manta rays in
Bohol sea. Manta rays and mobulid species have often been
mistaken as one due to similarities in its morphology hence
the continuing Mobula fisheries in the Bohol Sea has also
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been contributing to the decline of manta ray populations in
the area. Perhaps the best indication of the declining popula-
tion status of manta rays is its very recent addition to
the Appendix II of the Convention on the International Trade
in Endangered Species of Wild Flora and Fauna (CITES)
(CITES, 2013)
A fundamental requirement in effective implementation of
laws regulating and/or protecting aquatic species such as the eels
and manta rays is to know the accurate identity of the species
concerned. Unfortunately this is hindered by the difficulty in
species identification especially through the use of morphological
For personal use only.
characters (Bhattacharjee et al., 2012; Fowler, 2002; Lago et al.,
2012). Anguilla spp., for instance, are difficult to identify since
different species of freshwater eels have almost similar morphol-
ogies (Teng et al., 2009). This is the same with manta rays and
sharks, since they are difficult to identify morphologically even by
a shark and ray specialist (Fowler, 2002; Rayos et al., 2012;
SEAFDEC, 2012). Moreover, since processed IUU products are
all similar in appearance and taste, identification based on
morphological characters is impossible (Lago et al., 2012). Due to
these difficulties in morphology-based identifications, DNA-
based methods such as DNA barcoding are being utilized
worldwide as a more reliable means of confirming identities
(Hebert et al., 2003; Ko et al., 2013; Rasmussen & Morrissey,
2008; Smith et al., 2008).
DNA barcoding has been widely used in monitoring, conser-
vation, and management of fish species (Bhattacharjee et al.,
2012). DNA barcodes are particularly useful in taxonomy because
intraspecific phenotypic variation often overlaps that of sister taxa
in nature, which can lead to incorrect identifications if based on
phenotype only (Pfenninger et al., 2006). Also cryptic variation
and prominent levels of undetected taxonomic diversity are
detected by DNA barcodes (Hebert et al., 2003). In the
Philippines, DNA barcoding has been applied in identifying
juvenile tunas (Pedrosa-Gerasmio et al., 2012), in resolving
identities of sardine species (Willette & Santos, 2012; Willette
et al., 2011), and in detecting mislabelled and fish and fish
by-products (Maralit et al., 2013).
In this study, confiscated juvenile eels and dried by-products of
sharks and rays were identified using DNA barcoding. The export
items were suspected IUU fisheries products and were thus
confiscated by the Bureau of Fisheries and Aquatic Resources
(BFAR) and the Bureau of Customs. The confiscated products
consisted of 13 boxes of live juvenile eels with an estimated
worth of P75,000 (1690 USD) bound for Taiwan which was
intercepted at the NAIA MIASCOR Warehouse by BFAR; and
2300 kg of dried shark and rays approximately worth P10,000,000
Mitochondrial DNA, Early Online: 1–8
(222,000 USD) that came from Cebu City, intercepted by the
Bureau of Customs in Manila North Harbor. Detection of these
cases of illegal trade underscores the importance of species
identification in monitoring and conservation of illegally traded
aquatic species and the effectivity of DNA barcoding as a tool to
do this.
Materials and methods
Sample collection
Twenty three random samples were collected from one plastic bag
of the 13 confiscated boxes of live eel fry. On the other hand, the
dried shark and ray samples used in the analysis were ran-
domly chosen by the Bureau of Customs and sent to the NFRDI-
Genetic Fingerprinting Laboratory for genetic identification.
Approximately 150 mg of the muscle tissue was preserved in a
tube with 95% ethanol and stored at 20  C.
DNA extraction using CTAB
DNA was extracted using Cetyl Trimethyl Ammonium Bromide
(CTAB) Extraction Buffer following the methodology of Doyle &
Doyle (1987) with modifications (Santos et al., 2010). Dried
samples were subjected to rehydration for at least 1 hour prior to
extraction. Resulting stock DNA extracts were stored in cryovials
at 20  C.
COI amplification
A 10 ml reaction mixture was prepared containing water, 1x PCR
Buffer, 10 mM dNTP’s, 0.8 mM each of Forward primer LCO1490
and Reverse primer HCO2198 (Folmer et al., 1994), 10 mM
MgCl, 1 ml BSA, 0.2 unit Taq polymerase and 1 ml of DNA
template. The PCR profile for the reaction was: 94  C for 10 min,
followed by 35 cycles of 1 min at 94  C, 1 min at 48  C and
1.5 min at 72  C, and a final extension of 10 min at 72  C. COI
amplicons were electrophoresed through a 1% agarose gel stained
with GelRedÔ and submerged in 1 TAE buffer. Standard
sequencing and DNA purification was done and outsourced to
Macrogen, Inc., Korea.
Genetic analysis
Voucher CO1 sequences were obtained using BLAST (blas-
t.ncbi.nlm.nih.gov) and BOLD (www.boldsystems.org). DNA
sequences were edited and aligned using alignment explorer
packaged in MEGA 5.0 (Tamura et al., 2007). Corresponding
DNA sequence accession numbers of the samples analyzed were
reflected in Tables 1 and 2. Species classification was inferred
using the Neighbor-Joining method (Saitou & Nei, 1987). The
percentage of replicate trees in which the associated taxa clustered
together in the bootstrap test (1000 replicates) was shown next to
the branches (Felsenstein, 1985). The tree is drawn to scale, with
branch lengths in the same units as those of the evolutionary
distances used to infer the phylogenetic tree. The evolutionary
distances were computed using the Kimura 2-parameter method
(Kimura, 1980) and are in the units of the number of base
substitutions per site.
Results
Identification of juvenile fish species as well as processed fish
products through morphology-based methods has been proven to
be extremely difficult even for trained taxonomists since the key
characters for identification to the species level is lacking. Hence,
DNA based methods, particularly DNA barcoding, have been
employed in this study in order to identify the following illegally
traded fish species.
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Table 1. K2P Pairwise Genetic Distances of individual juvenile eel samples with reference sequences from BOLD and GenBank.

A. marmorata 0.003 0.003 0.017 0.012 0.013 0.013 0.017 0.015 0.015 0.016 0.010 0.041 0.013 0.003 0.003 0.003 0.013 0.013 0.013 0.013 0.013 0.013 0.013 0.014 0.013 0.013 0.012 0.013 0.013 0.013 0.013 0.013 0.013 0.013 0.013 0.013
GBJQ665824
A. marmorata 0.004 0.004 0.016 0.013 0.013 0.013 0.016 0.015 0.014 0.015 0.009 0.041 0.012 0.000 0.000 0.000 0.014 0.013 0.013 0.013 0.013 0.013 0.014 0.014 0.013 0.013 0.013 0.013 0.013 0.013 0.013 0.013 0.014 0.013 0.013 0.013
GBGC1717-06
A. marmorata 0.004 0.009 0.017 0.013 0.013 0.013 0.018 0.016 0.016 0.017 0.011 0.040 0.013 0.004 0.004 0.004 0.014 0.013 0.013 0.014 0.014 0.013 0.014 0.014 0.013 0.013 0.013 0.014 0.013 0.013 0.013 0.014 0.014 0.013 0.014 0.014
JQ431414.1
A. australi saustralis 0.096 0.090 0.102 0.019 0.020 0.020 0.019 0.017 0.017 0.017 0.017 0.039 0.019 0.016 0.016 0.016 0.021 0.020 0.020 0.021 0.021 0.020 0.021 0.020 0.020 0.020 0.020 0.020 0.020 0.020 0.020 0.020 0.021 0.020 0.020 0.020
GBGCl
A. bicolor bicolor 0.060 0.066 0.066 0.118 0.007 0.007 0.019 0.021 0.020 0.019 0.014 0.044 0.017 0.013 0.013 0.013 0.008 0.007 0.007 0.007 0.007 0.007 0.007 0.008 0.007 0.007 0.007 0.007 0.007 0.007 0.007 0.007 0.008 0.007 0.008 0.007
G BGC171
A. bicolorpacifica 0.063 0.069 0.069 0.129 0.023 0.000 0.018 0.020 0.019 0.019 0.014 0.044 0.016 0.013 0.013 0.013 0.003 0.000 0.000 0.002 0.002 0.000 0.002 0.003 0.000 0.000 0.002 0.002 0.000 0.000 0.000 0.002 0.003 0.000 0.003 0.002
GBGC171
A. bicolorpacifica 0.063 0.069 0.069 0.129 0.023 0.000 0.018 0.020 0.019 0.019 0.014 0.044 0.016 0.013 0.013 0.013 0.003 0.000 0.000 0.002 0.002 0.000 0.002 0.003 0.000 0.000 0.002 0.002 0.000 0.000 0.000 0.002 0.003 0.000 0.003 0.002
GBAP007
A. celebesensis 0.086 0.080 0.092 0.115 0.109 0.110 0.110 0.017 0.017 0.019 0.016 0.041 0.018 0.016 0.016 0.016 0.018 0.018 0.018 0.019 0.019 0.018 0.019 0.018 0.018 0.018 0.018 0.018 0.018 0.018 0.018 0.018 0.018 0.018 0.018 0.019
GBGC1714-06
A. japonica 0.077 0.071 0.083 0.101 0.119 0.119 0.119 0.091 0.003 0.019 0.016 0.040 0.018 0.015 0.015 0.015 0.021 0.020 0.020 0.020 0.020 0.020 0.021 0.019 0.020 0.020 0.020 0.020 0.020 0.020 0.020 0.020 0.021 0.020 0.020 0.021
GBGC1517-06
A. japonica 0.074 0.069 0.080 0.098 0.117 0.113 0.113 0.091 0.004 0.019 0.016 0.040 0.017 0.014 0.014 0.014 0.020 0.019 0.019 0.019 0.019 0.019 0.020 0.018 0.019 0.019 0.019 0.019 0.019 0.019 0.019 0.019 0.020 0.019 0.019 0.019
HQ339972.1
A. malgumora 0.090 0.084 0.096 0.090 0.113 0.114 0.114 0.108 0.104 0.101 0.015 0.040 0.017 0.015 0.015 0.015 0.020 0.019 0.019 0.019 0.019 0.019 0.019 0.019 0.019 0.019 0.018 0.019 0.019 0.019 0.019 0.019 0.020 0.019 0.019 0.019
GBGC1713-06
A. luzonensis 0.047 0.042 0.052 0.099 0.079 0.074 0.074 0.090 0.087 0.081 0.084 0.040 0.013 0.009 0.009 0.009 0.014 0.014 0.014 0.014 0.014 0.014 0.013 0.014 0.014 0.014 0.013 0.014 0.014 0.014 0.014 0.014 0.014 0.014 0.014 0.014
GBGC6906-09
N. forsteri 0.324 0.329 0.315 0.310 0.337 0.341 0.341 0.350 0.320 0.319 0.325 0.327 0.045 0.041 0.041 0.041 0.043 0.044 0.044 0.044 0.044 0.044 0.043 0.042 0.044 0.044 0.044 0.043 0.044 0.044 0.044 0.044 0.043 0.044 0.043 0.043
GBGC0188-06
A. bengalensis 0.060 0.054 0.065 0.123 0.097 0.090 0.090 0.096 0.102 0.096 0.112 0.064 0.359 0.012 0.012 0.012 0.017 0.016 0.016 0.016 0.016 0.016 0.017 0.016 0.016 0.016 0.016 0.017 0.016 0.016 0.016 0.016 0.017 0.016 0.016 0.016
bengalensis
EL4A01 0.004 0.000 0.009 0.090 0.066 0.069 0.069 0.080 0.071 0.069 0.084 0.042 0.329 0.054 0.000 0.000 0.014 0.013 0.013 0.013 0.013 0.013 0.014 0.014 0.013 0.013 0.013 0.013 0.013 0.013 0.013 0.013 0.014 0.013 0.013 0.013
EL4A12 0.004 0.000 0.009 0.090 0.066 0.069 0.069 0.080 0.071 0.069 0.084 0.042 0.329 0.054 0.000 0.000 0.014 0.013 0.013 0.013 0.013 0.013 0.014 0.014 0.013 0.013 0.013 0.013 0.013 0.013 0.013 0.013 0.014 0.013 0.013 0.013
EL4A17 0.004 0.000 0.009 0.090 0.066 0.069 0.069 0.080 0.071 0.069 0.084 0.042 0.329 0.054 0.000 0.000 0.014 0.013 0.013 0.013 0.013 0.013 0.014 0.014 0.013 0.013 0.013 0.013 0.013 0.013 0.013 0.013 0.014 0.013 0.013 0.013
EL4A02 0.069 0.074 0.074 0.136 0.027 0.004 0.004 0.110 0.125 0.119 0.120 0.080 0.341 0.096 0.074 0.074 0.074 0.003 0.003 0.004 0.003 0.003 0.004 0.004 0.003 0.003 0.004 0.004 0.003 0.003 0.003 0.004 0.000 0.003 0.004 0.004
EL4A08 0.063 0.069 0.069 0.129 0.023 0.000 0.000 0.110 0.119 0.113 0.114 0.074 0.341 0.090 0.069 0.069 0.069 0.004 0.000 0.002 0.002 0.000 0.002 0.003 0.000 0.000 0.002 0.002 0.000 0.000 0.000 0.002 0.003 0.000 0.003 0.002
EL4A10 0.063 0.069 0.069 0.129 0.023 0.000 0.000 0.110 0.119 0.113 0.114 0.074 0.341 0.090 0.069 0.069 0.069 0.004 0.000 0.002 0.002 0.000 0.002 0.003 0.000 0.000 0.002 0.002 0.000 0.000 0.000 0.002 0.003 0.000 0.003 0.002
EL4A13 0.066 0.071 0.071 0.132 0.020 0.002 0.002 0.113 0.122 0.116 0.117 0.077 0.341 0.093 0.071 0.071 0.071 0.007 0.002 0.002 0.003 0.002 0.003 0.004 0.002 0.002 0.003 0.003 0.002 0.002 0.002 0.003 0.004 0.002 0.004 0.003
EL4A14 0.066 0.071 0.071 0.132 0.025 0.002 0.002 0.112 0.122 0.116 0.117 0.077 0.345 0.093 0.071 0.071 0.071 0.004 0.002 0.002 0.004 0.002 0.003 0.004 0.002 0.002 0.003 0.003 0.002 0.002 0.002 0.003 0.003 0.002 0.004 0.003
EL4A15 0.063 0.069 0.069 0.129 0.023 0.000 0.000 0.110 0.119 0.113 0.114 0.074 0.341 0.090 0.069 0.069 0.069 0.004 0.000 0.000 0.002 0.002 0.002 0.003 0.000 0.000 0.002 0.002 0.000 0.000 0.000 0.002 0.003 0.000 0.003 0.002
EL4A16 0.066 0.071 0.071 0.132 0.025 0.002 0.002 0.113 0.122 0.116 0.117 0.071 0.336 0.093 0.071 0.071 0.071 0.007 0.002 0.002 0.004 0.004 0.002 0.004 0.002 0.002 0.003 0.003 0.002 0.002 0.002 0.003 0.004 0.002 0.004 0.003
EL4A18 0.069 0.074 0.074 0.129 0.027 0.004 0.004 0.103 0.112 0.106 0.114 0.074 0.331 0.090 0.074 0.074 0.074 0.009 0.004 0.004 0.007 0.007 0.004 0.007 0.003 0.003 0.004 0.004 0.003 0.003 0.003 0.004 0.004 0.003 0.004 0.004
EL4A19 0.063 0.069 0.069 0.129 0.023 0.000 0.000 0.110 0.119 0.113 0.114 0.074 0.341 0.090 0.069 0.069 0.069 0.004 0.000 0.000 0.002 0.002 0.000 0.002 0.004 0.000 0.002 0.002 0.000 0.000 0.000 0.002 0.003 0.000 0.003 0.002
EL4A20 0.063 0.069 0.069 0.129 0.023 0.000 0.000 0.110 0.119 0.113 0.114 0.074 0.341 0.090 0.069 0.069 0.069 0.004 0.000 0.000 0.002 0.002 0.000 0.002 0.004 0.000 0.002 0.002 0.000 0.000 0.000 0.002 0.003 0.000 0.003 0.002
EL4A21 0.060 0.066 0.066 0.126 0.025 0.002 0.002 0.107 0.116 0.110 0.111 0.071 0.346 0.087 0.066 0.066 0.066 0.007 0.002 0.002 0.004 0.004 0.002 0.004 0.007 0.002 0.002 0.003 0.002 0.002 0.002 0.003 0.004 0.002 0.003 0.003
EL4A22 0.066 0.071 0.071 0.128 0.025 0.002 0.002 0.109 0.118 0.112 0.113 0.077 0.340 0.093 0.071 0.071 0.071 0.007 0.002 0.002 0.004 0.004 0.002 0.004 0.007 0.002 0.002 0.004 0.002 0.002 0.002 0.003 0.004 0.002 0.002 0.003
EL4A23 0.063 0.069 0.069 0.129 0.023 0.000 0.000 0.110 0.119 0.113 0.114 0.074 0.341 0.090 0.069 0.069 0.069 0.004 0.000 0.000 0.002 0.002 0.000 0.002 0.004 0.000 0.000 0.002 0.002 0.000 0.000 0.002 0.003 0.000 0.003 0.002
EL4A24 0.063 0.069 0.069 0.129 0.023 0.000 0.000 0.110 0.119 0.113 0.114 0.074 0.341 0.090 0.069 0.069 0.069 0.004 0.000 0.000 0.002 0.002 0.000 0.002 0.004 0.000 0.000 0.002 0.002 0.000 0.000 0.002 0.003 0.000 0.003 0.002
EL4A25 0.063 0.069 0.069 0.129 0.023 0.000 0.000 0.110 0.119 0.113 0.114 0.074 0.341 0.090 0.069 0.069 0.069 0.004 0.000 0.000 0.002 0.002 0.000 0.002 0.004 0.000 0.000 0.002 0.002 0.000 0.000 0.002 0.003 0.000 0.003 0.002
EL4A26 0.066 0.071 0.071 0.129 0.025 0.002 0.002 0.107 0.116 0.110 0.117 0.077 0.341 0.093 0.071 0.071 0.071 0.007 0.002 0.002 0.004 0.004 0.002 0.004 0.007 0.002 0.002 0.004 0.004 0.002 0.002 0.002 0.004 0.002 0.003 0.003
EL4A27 0.069 0.074 0.074 0.136 0.027 0.004 0.004 0.110 0.125 0.119 0.120 0.080 0.341 0.096 0.074 0.074 0.074 0.000 0.004 0.004 0.007 0.004 0.004 0.007 0.009 0.004 0.004 0.007 0.007 0.004 0.004 0.004 0.007 0.003 0.004 0.004
EL4A28 0.063 0.069 0.069 0.129 0.023 0.000 0.000 0.110 0.119 0.113 0.114 0.074 0.341 0.090 0.069 0.069 0.069 0.004 0.000 0.000 0.002 0.002 0.000 0.002 0.004 0.000 0.000 0.002 0.002 0.000 0.000 0.000 0.002 0.004 0.003 0.002
EL4A29 0.063 0.068 0.068 0.125 0.027 0.004 0.004 0.106 0.115 0.109 0.110 0.074 0.340 0.090 0.068 0.068 0.068 0.009 0.004 0.004 0.007 0.007 0.004 0.007 0.009 0.004 0.004 0.007 0.002 0.004 0.004 0.004 0.007 0.009 0.004 0.004
EL4A30 0.066 0.071 0.071 0.126 0.025 0.002 0.002 0.113 0.122 0.116 0.117 0.077 0.336 0.093 0.071 0.071 0.071 0.007 0.002 0.002 0.004 0.004 0.002 0.004 0.007 0.002 0.002 0.004 0.004 0.002 0.002 0.002 0.004 0.007 0.002 0.007
 4 A. M. J. M. Asis et al. Mitochondrial DNA, Early Online: 1–8

Table 2. K2P Pairwise Genetic Distances of individual elamobranch samples with reference sequences from BOLD and GenBank.

BOLD_FOAD629-05_ 0.000 0.003 0.002 0.000 0.028 0.025 0.025 0.024 0.025 0.025 0.025 0.016 0.025 0.025 0.016 0.025 0.025
Manta_birostris
Sample_B 0.000 0.003 0.002 0.000 0.028 0.025 0.025 0.024 0.025 0.025 0.025 0.016 0.025 0.025 0.016 0.025 0.025
Sample_K 0.004 0.004 0.004 0.003 0.029 0.025 0.026 0.025 0.025 0.026 0.025 0.016 0.025 0.026 0.016 0.025 0.025
Sample_L 0.002 0.002 0.006 0.002 0.029 0.025 0.026 0.025 0.025 0.026 0.025 0.016 0.025 0.026 0.016 0.025 0.025
Sample_Q 0.000 0.000 0.004 0.002 0.028 0.025 0.025 0.024 0.025 0.025 0.025 0.016 0.025 0.025 0.016 0.025 0.025
BOLD_GBGC0188-06_ 0.269 0.269 0.275 0.272 0.269 0.031 0.032 0.027 0.032 0.032 0.026 0.029 0.027 0.032 0.028 0.026 0.030
Neoceratodus_forsteri
Sample_C 0.217 0.217 0.219 0.219 0.217 0.298 0.027 0.023 0.027 0.027 0.026 0.025 0.023 0.027 0.025 0.026 0.002
Sample_J 0.239 0.239 0.242 0.242 0.239 0.317 0.253 0.021 0.002 0.000 0.029 0.025 0.022 0.000 0.025 0.029 0.027
Sample_M 0.228 0.228 0.228 0.231 0.228 0.281 0.224 0.187 0.021 0.021 0.029 0.023 0.003 0.021 0.024 0.029 0.023
Sample_O 0.239 0.239 0.239 0.242 0.239 0.317 0.256 0.002 0.184 0.002 0.029 0.025 0.022 0.002 0.025 0.029 0.027
Sample_P 0.239 0.239 0.242 0.242 0.239 0.317 0.253 0.000 0.187 0.002 0.029 0.025 0.022 0.000 0.025 0.029 0.027
Sample_R 0.209 0.209 0.211 0.212 0.209 0.232 0.236 0.265 0.283 0.268 0.265 0.024 0.029 0.029 0.024 0.002 0.026
Sample_S 0.101 0.101 0.101 0.103 0.101 0.276 0.223 0.221 0.207 0.218 0.221 0.200 0.024 0.025 0.002 0.024 0.024
BOLD_ESHKB025-07_ 0.232 0.232 0.234 0.235 0.232 0.278 0.224 0.187 0.004 0.190 0.187 0.280 0.212 0.022 0.024 0.029 0.023
Alopias_pelagicus
BOLD_ESHKC116-07_ 0.239 0.239 0.242 0.242 0.239 0.317 0.253 0.000 0.187 0.002 0.000 0.265 0.221 0.187 0.025 0.029 0.027
Carcharhinus_falciformis
BOLD_ANGBF2038-12_ 0.098 0.098 0.103 0.101 0.098 0.273 0.223 0.221 0.209 0.221 0.221 0.200 0.002 0.213 0.221 0.024 0.024
Mobula_japanica
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BOLD_FOA216-04_ 0.212 0.212 0.214 0.215 0.212 0.235 0.232 0.262 0.287 0.265 0.262 0.002 0.203 0.284 0.262 0.203 0.025
Himantura_fai
GENBANK_JQ_765555_ 0.220 0.220 0.222 0.223 0.220 0.295 0.002 0.256 0.221 0.259 0.256 0.232 0.219 0.221 0.256 0.220 0.229
Taeniura_meyeni

Juvenile eels (elvers) Dried shark and ray products

Mitochondrial DNA CO1 sequences of 23 confiscated juvenile eel
samples were obtained and analyzed. The analysis, which
included reference sequences of all the eels that could be found
here in the Philippines from BOLD and GenBank and the sample
For personal use only.
All of the dried sample sequences were subjected to identification
engines such as BLAST and BOLD and resulted to 98–100%
scores. Mitochondrial DNA CO1 sequences of 11 samples of
confiscated dried sharks and rays were obtained and analyzed

CO1 sequences, involved 37 nucleotide sequences. There were a
total of 461 positions in the final dataset. The result of genetic
analysis using Neighbor Joining method and Kimura 2-parameter
model is shown in Figure 1. The phylogenetic tree inferred from
the CO1 sequences of the unidentified elvers sample confirms that
3 specimens (EL4A01, EL4A12, and EL4A17) belonged to the
same branch as A. marmorata and 20 specimens (EL4A02,
EL4A08, EL4A10, EL4A13, EL4A14, EL4A15, EL4A16,
EL4A18, EL4A19, EL4A20, EL4A21, EL4A22, EL4A23,
EL4A24, EL4A25, EL4A26, EL4A27, EL4A28, EL4A29,
EL4A30) belonged to the same branch as A. bicolor pacifica
indicating that these elvers are from the same lineage as that of
A. marmorata and A. bicolor pacifica respectively. The reliability
of the inferred tree topology was also tested by determining the
bootstrap values wherein values above 70% were considered
reliable. Results obtained show that the samples have above 83%
bootstrap values thus it can be inferred that these elvers are the
same species as the ones mentioned above.
To further support the inferred species identities of the elvers
samples, the genetic distances were also computed and are shown
in Table 1. Results of the evaluation of the genetic distances
strongly confirms the inferred identities since the computed mean
genetic distance between EL4A01, EL4A12, and EL4A17 with
3 reference sequences of A. marmorata (0.004, 0.000 &
0.009) was very low as compared to the other eel species
included in the analysis which has genetic distances that
ranged from 0.042–0.090. As for the elvers samples EL4A02,
EL4A08, EL4A10, EL4A13, EL4A14, EL4A15, EL4A16,
EL4A18, EL4A19, EL4A20, EL4A21, EL4A22, EL4A23,
EL4A24, EL4A25, EL4A26, EL4A27, EL4A28, EL4A29,
EL4A30, their computed mean genetic distances with 2 reference
sequences of A. bicolor pacifica (0.002 & 0.002) were also
very low as compared to another closely related species,
A. bicolor bicolor which is 0.024. The low values of nucleotide
differences therefore validate the inferred identities of elvers
samples.
using Neighbor Joining method and the Kimura 2-parameter
model (Figure 2). The analysis involved 18 nucleotide sequences
and a total of 588 positions in the final dataset. Results showed
that there are four samples (Q, B, K and L) which were identified
to be manta ray with genetic distances occurring from 0.00 to
0.004 (Manta birostris) (Table 2).
The rest of the samples were identified as elasmobranch
species. Sample P, J and O were identified as Silky Shark
(Carcharhinus falciformis) with a 100% bootstrap and genetic
distances occurring from 0.00 to 0.002. Pelagic thresher shark
(Alopias pelagicus) has also been identified to be present in the
dried samples. Sample M shows a 100% similarity and 0.004
genetic variations with the obtained reference sequence (BOLD
ESHKB025-07). On the other hand, Sample C showed a 100%
similarity and 0.002 genetic distance with Blotched Fantail Ray
(Taeniura meyeni) obtained reference sequence (GENBANK JQ
765555). Sample S appears to be another species of ray which is a
spinetail mobula (Mobula japanica) resulting from a 100%
bootstrap and 0.002 genetic distance to obtained reference
sequence (BOLD ANGBF2038-12). Lastly, Sample R showed a
100% similarity and 0.002 genetic distances with the obtained
Pink Whip Ray (Himantura fai) reference sequence (BOLD
FOA216-04). Voucher sequence of Neoceratodus forsteri was
used as outgroup. (Table 2 shows the pairwise distances.)
Discussion
Various aquatic organisms are being exploited by IUU fisheries
and illegally traded through shipment of unidentifiable juveniles
or processed and dried products. In this study illegal shipment of
juvenile Anguilla species and dried shark and ray products which
are protected under Philippine and international laws were
identified through the use of DNA barcoding. The species
which were identified in the confiscated shipments include the
eels Anguilla marmorata, and Anguilla bicolor pacifica; and
the sharks and rays Alopias pelagicus, Taeniura meyeni,
 DOI: 10.3109/19401736.2014.913138 Barcoding illegally traded aquatic species in Philippines 5
Mitochondrial DNA Downloaded from informahealthcare.com by 110.54.128.45 on 05/19/14
For personal use only.

Figure 1. Neighbor-Joining Tree of juvenile eels CO1 sequences using Kimura 2-parameter model. Voucher sequences of A. bicolor pacifica
(GBGC1712-06, GBAP007237), A. bicolor bicolor (GBGC1711-06), A. luzonensis (GBGC6906-09), A. bengalensis bengalensis (JX260629.1),
A. marmorata (GBJQ665824, GBGC1717-06), A. celebensis (GBGC1714-06), A. japonica (GBGC1517-06, HQ339972.1), A. malgumora
(GBGC1713-06), A. australis australis (GBGC1709-06) and the outgroup Neoceratodus forsteri (GBGC0188-06) from BOLD and GenBank were
included in the analysis.

Carcharhinus falciformis, Himantura fai, Manta birostris and
Mobula japonica.
The exploitation of juvenile eels of the Anguilla spp. due to
illegal trade has been evident worldwide because of the continu-
ously increasing demand for this commodity (Crook, 2010). The
species of Anguilla that have been identified in this study,
A. marmorata and A. bicolor pacifica, are both found in
abundance in Philippine waters (Han et al., 2012; Jamandre
et al., 2007; Sugeha & Suharti, 2009) and, apparently, are illegally
exported even with the existing bans in the country. The exact
location where the confiscated juvenile Anguilla species were
caught is not known. However, Anguilla spp. is known to thrive in
the Northern Philippines where the endemic A. luzonensis can
also be found. The exploitation of Anguilla species in this life
stage calls for a great concern in conservation since not only does
it contribute to growth overfishing of Anguilla species but it also
poses a great threat to endemic populations of A. luzonensis.
Primarily, knowing the correct identification of these illegally
traded Anguilla spp., as what was done here, will enable its
effective protection and monitoring for its conservation.
Shark and rays are also products of IUU fisheries in the
Philippines. However there is a poor understanding of the
country’s shark fisheries due to inadequate catch data and species
identification in catch and trade (Lack & Sant, 2012). The seas of
Cebu City, where the products have been intercepted, are known
for its rich marine life where sightings of different species of
sharks and rays often occur. Therefore there is a high probability
that the confiscated products came from the surrounding waters of
Cebu or its neighboring seas, such as the Bohol Sea that is also
well known for its Manta and Mobula rays.
 6 A. M. J. M. Asis et al. Mitochondrial DNA, Early Online: 1–8

P
Sample_P

70
BOLD_ESHKC116-07_Carcharhinus_falciformis J
100 Silky Shark
Sample_J

O
95 Sample_O

Sample_M
Pelagic M
64
100 BOLD_ESHKB025-07_Alopias_pelagicus
Thresher Shark

Sample_C
Blotched
Fantail Ray C
91 100 GENBANK_JQ_765555_Taeniura_meyeni

100 Sample_S Spinetail

Mobula S
BOLD_ANGBF2038-12_Mobula_japanica
Mitochondrial DNA Downloaded from informahealthcare.com by 110.54.128.45 on 05/19/14

Sample_K
K
100
BOLD_FOAD629-05_Manta_birostris B
100 Manta Ray
Sample_B
L
67 Sample_L
Q
Sample_Q

R
For personal use only.

Sample_R
Pink Whip Ray
100 BOLD_FOA216-04_Himantura_fai

BOLD_GBGC0188-06_Neoceratodus_forsteri

0.02

Figure 2. Neighbor-Joining Tree of dried sharks and rays (Sample B to Sample S) CO1 sequences using Kimura 2-parameter model. Voucher
sequences of Manta birostris (FOAD629-05), Mobula japanica (ANGBF2038-12), Alopias pelagicus (ESHKB025), Carcharhinus falciformis
(ESHKC116-07), Himantura fai (FOA216-04), Neoceratodus forsteri (GBGC0188-06) from BOLD, and Taeniura meyeni (JQ765555) from
GENBANK were included in the analysis.

Manta birostris or manta rays are considered protected species
under Philippine laws (Fisheries Administrative Order No. 193)
and international laws (Appendix II of CITES). However, due to
the increasing the demand of Manta ray in the black market
(Alava & Dolumbalo, 2002), manta rays are still being exported
even with existing bans, as what was shown in this study, through
shipment of its slaughtered and dried products. The identification
of manta rays in the illegal shipment using DNA barcodes verifies
the illegal export trade of the said species in the country.
Other species identified which are not protected under
Philippine laws but are considered as vulnerable species under
IUCN include the thresher shark (A. pelagicus) and blotched
fantail ray (T. meyeni) (Kyne & White, 2006; Reardon et al.,
2009). Meanwhile those identified that are considered near
threatened species by the IUCN include silky shark
(C. falciformis) and spinetail Mobula (M. japanica) (Bonfil
et al., 2009; White et al., 2006).
The utility of DNA barcoding in accurately identifying
otherwise unidentifiable species by morphological means, as
what was shown here, has also been presented in various studies
such as that of de Franco et al. (2012) and Holmes et al. (2009).
Also, aside from confirming the identities of shark and rays being
exploited for illegal trade, the use of DNA barcoding in this study
also confirms the occurrence of thresher sharks (A. pelagicus),
silky sharks (C. falciformis) and blotched fantail rays (T. meyeni)
in the Cebu seas. These species have been recorded in the
Philippines however A. pelagicus has only been recently recorded
in 2005. According to Compagno et al. (2005) the first records of
thresher shark, silky shark and blotched fantail ray were also from
Cebu City which may imply the abundance of these species in that
area. Still, significant data with regards to these species’ status and
distribution in the country is lacking. On the other hand, the
spinetail Mobula (M. japonica) which was also identified in the
illegal shipment was not previously recorded in the country
(Carpenter & Niem, 1999; Compagno et al., 2005; Herre, 1953)
hence the result of this study may also be suggestive of the
occurrence of M. japonica in Cebu or its neighboring seas.
However, further investigation is warranted to confirm the first
record of this species in the Philippines since only one sample has
been identified in this study.
The pink whip ray (Himantura fai) was also identified as
one of the confiscated products. This species is considered as
 DOI: 10.3109/19401736.2014.913138
‘‘Least Concern’’ globally but is considered as a Vulnerable
species in Southeast Asia by the IUCN due to the high levels of
exploitation in the region (Manjaji et al., 2009). According to
Compagno et al. (2005), the first record of H. fai in the
Philippines came from Negros Occidental and that this species,
although common in the Indo-Pacific region, was not commonly
found in regional fish markets in 1998. Yet results of this study
show that this species is now targeted in IUU fisheries and may be
a valuable export commodity.
Trade in aquatic species not only contributes to national
economy and income generation but also plays a significant role
in advancing food security and ensuring that the supply of
products meets the nutritional requirements for food fish inter-
nationally (SEAFDEC, 2012). Overfishing, IUU fisheries, and
illegal trade of these species however have become a threat to
species survival. Despite conservation and monitoring efforts
made to regulate trade and guarantee sustainability, such as listing
in the IUCN and CITES, and implementation of fishing ban, the
trade continues to expand and populations to decline. Since the
Mitochondrial DNA Downloaded from informahealthcare.com by 110.54.128.45 on 05/19/14
illegal trade of protected species is happening in the country
through the guise of unrecognizable products, DNA barcoding
proves to be an extremely reliable method for accurate species
identification of these IUU products thus aiding in the enforce-
ment of domestic bans by providing significant data on the
species composition of the catch and trade of IUU fisheries’
exploited organisms. The alarming decline in the populations of
the species mentioned calls for an effective conservation and
monitoring of trade and this study shows that DNA barcoding can
contribute to such efforts. The utility of DNA barcoding for
conservation have been well established, being an efficient way in
For personal use only.
identifying priority species for conservation (Krishnamurthy &
Francis, 2012) as well as identifying legal or illegal fish catch
such as in shark fisheries (Ward et al., 2008). However its use is
limited on relatively-well studied taxa (Krishnamurthy & Francis,
2012; Taylor & Harris, 2012).
Acknowledgements
Authors would also like to thank the Bureau of Customs (BOC) and the
Bureau of Fisheries and Aquatic Resources (BFAR) IV-A for entrusting
the confiscated samples to NFRDI through its Genetic Fingerprinting
Laboratory. Lastly, we extend our sincerest thanks to Ms. Lilibeth Abina
and Ms. Aron Alcantara for their utmost support and assistance especially
with regards to administrative matters.
Declaration of interest
The authors would like to acknowledge the National Fisheries Research
and Development Institute (NFRDI) and the Department of Agriculture –
Biotechnology Program (DA-Biotech Program) for giving the necessary
funding for the study. The authors report no conflicts of interest. The
authors alone are responsible for the content and writing of this article.
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