http://informahealthcare.com/mdn ISSN: 1940-1736 (print), 1940-1744 (electronic) 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
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Genetic Fingerprinting Laboratory, National Fisheries Research and Development Institute, Quezon City, Philippines
Abstract
Keywords
Illegal trade has greatly affected marine fish stocks, decreasing fish populations worldwide. Despite having a number of aquatic species being regulated, illegal trade still persists through 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 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 characters in juveniles and processed products. Here, live juvenile eels (elvers) and dried products of sharks and rays confiscated for illegal trade were identified. Twenty out of 23 (87%) randomly selected ‘‘elvers’’ were identified as Anguilla bicolor pacifica and 3 (13%) samples as 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. 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.
CO1, illegal trade, juvenile eel, Manta ray, processed products
Introduction Illegal trade of marine fish stocks has been a major challenge to marine biodiversity conservation since the continuous and heavy exploitation of this resource leads to the declines in marine populations or even near-collapses of it (Mullon et al., 2005). Even with established regulation efforts, reports on illegal, unreported and unregulated fisheries (IUU) and illegal trade are still existent (Maes & Volckaert, 2007). At present, illegal substitution and trade of certain fish species has been a rising global concern (Rasmussen & Morrissey, 2008). Southeast Asia in particular has been recognized as a ‘‘wildlife trade hotspot’’ because of its unsustainable and ill-regulated wildlife trade (Nijman, 2010). This has been a rising concern since the epicenter of marine biodiversity that urgently needs improvement of conservation efforts is found in this part of the world, the Coral Triangle, encompassing to a large extent Indonesia, Malaysia, Philippines, Papua New Guinea, Solomon Islands, Timor L’Este, and Brunei, with the Philippines and eastern Indonesia having the highest concentration of species richness within the Coral Triangle (Sanciangco et al., 2013).
Correspondence: Mudjekeewis D. Santos, National Fisheries Research and Development Institute, Room 601 Corporate 101 Building, Mo. Ignacia Avenue, Quezon City 1103, Philippines. Tel/Fax: +63 23725063. E-mail: [email protected]
History Received 20 January 2014 Revised 3 April 2014 Accepted 5 April 2014 Published online 19 May 2014
In the Philippines, a number of aquatic species such as eels of various life stages and sharks and rays are being utilized for various purposes resulting into the decline in their population (Crook, 2010; SEAFDEC, 2012). The Philippine eel culture industry started in 1972 when profitable quantities of juvenile eels called ‘‘elvers’’ were discovered in the Cagayan river system (Gutierrez, 1976). The Anguilla were seen as commercially important and treated as fish species with aquaculture potential (Briones et al., 2007). However, a significant decline in population resulted to a prohibition of ‘‘elvers’’ exportation in the country during the 1970s (Gutierrez, 1976). This ban has been reinstated today under the Philippines’ Fisheries Administrative Order (FAO) 242, which upholds FAO 107 and 107-1 series of 1986 that banned ‘‘elvers’’ exportation and revoked FAO 159 series of 1986, which allowed the exportation of the commodity. The reinstatement of the ban was recommended by the Philippine Bureau of Fisheries and Aquatic Resources (BFAR) Regional Office II in Cagayan after observing the excessive and non-stop exploitation of ‘‘elvers’’ due to its sharp price increase. 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 catch. The commercial exploitation of shark and ray species began in 1960s and landings of these aquatic species has then declined (Barut & Zartiga, 2002; Bonfil, 2002). Manta ray fishing has also expanded and is now overexploited in by-catch and
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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 been contributing to the decline of manta ray populations in the area. Perhaps the best indication of the declining population 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 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 morphologies (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, DNAbased 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, conservation, 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 randomly chosen by the Bureau of Customs and sent to the NFRDIGenetic 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 (blast.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.
A. marmorata GBJQ665824 A. marmorata GBGC1717-06 A. marmorata JQ431414.1 A. australi saustralis GBGCl A. bicolor bicolor G BGC171 A. bicolorpacifica GBGC171 A. bicolorpacifica GBAP007 A. celebesensis GBGC1714-06 A. japonica GBGC1517-06 A. japonica HQ339972.1 A. malgumora GBGC1713-06 A. luzonensis GBGC6906-09 N. forsteri GBGC0188-06 A. bengalensis bengalensis EL4A01 EL4A12 EL4A17 EL4A02 EL4A08 EL4A10 EL4A13 EL4A14 EL4A15 EL4A16 EL4A18 EL4A19 EL4A20 EL4A21 EL4A22 EL4A23 EL4A24 EL4A25 EL4A26 EL4A27 EL4A28 EL4A29 EL4A30
0.004 0.004 0.004 0.069 0.063 0.063 0.066 0.066 0.063 0.066 0.069 0.063 0.063 0.060 0.066 0.063 0.063 0.063 0.066 0.069 0.063 0.063 0.066
0.000 0.000 0.000 0.074 0.069 0.069 0.071 0.071 0.069 0.071 0.074 0.069 0.069 0.066 0.071 0.069 0.069 0.069 0.071 0.074 0.069 0.068 0.071
0.009 0.009 0.009 0.074 0.069 0.069 0.071 0.071 0.069 0.071 0.074 0.069 0.069 0.066 0.071 0.069 0.069 0.069 0.071 0.074 0.069 0.068 0.071
0.090 0.090 0.090 0.136 0.129 0.129 0.132 0.132 0.129 0.132 0.129 0.129 0.129 0.126 0.128 0.129 0.129 0.129 0.129 0.136 0.129 0.125 0.126
0.066 0.066 0.066 0.027 0.023 0.023 0.020 0.025 0.023 0.025 0.027 0.023 0.023 0.025 0.025 0.023 0.023 0.023 0.025 0.027 0.023 0.027 0.025
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 0.000 0.004 0.002
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 0.000 0.004 0.002
0.080 0.080 0.080 0.110 0.110 0.110 0.113 0.112 0.110 0.113 0.103 0.110 0.110 0.107 0.109 0.110 0.110 0.110 0.107 0.110 0.110 0.106 0.113
0.071 0.071 0.071 0.125 0.119 0.119 0.122 0.122 0.119 0.122 0.112 0.119 0.119 0.116 0.118 0.119 0.119 0.119 0.116 0.125 0.119 0.115 0.122
0.069 0.069 0.069 0.119 0.113 0.113 0.116 0.116 0.113 0.116 0.106 0.113 0.113 0.110 0.112 0.113 0.113 0.113 0.110 0.119 0.113 0.109 0.116
0.084 0.084 0.084 0.120 0.114 0.114 0.117 0.117 0.114 0.117 0.114 0.114 0.114 0.111 0.113 0.114 0.114 0.114 0.117 0.120 0.114 0.110 0.117
0.042 0.042 0.042 0.080 0.074 0.074 0.077 0.077 0.074 0.071 0.074 0.074 0.074 0.071 0.077 0.074 0.074 0.074 0.077 0.080 0.074 0.074 0.077
0.329 0.329 0.329 0.341 0.341 0.341 0.341 0.345 0.341 0.336 0.331 0.341 0.341 0.346 0.340 0.341 0.341 0.341 0.341 0.341 0.341 0.340 0.336
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 0.054 0.054 0.054 0.096 0.090 0.090 0.093 0.093 0.090 0.093 0.090 0.090 0.090 0.087 0.093 0.090 0.090 0.090 0.093 0.096 0.090 0.090 0.093 0.000 0.000 0.074 0.069 0.069 0.071 0.071 0.069 0.071 0.074 0.069 0.069 0.066 0.071 0.069 0.069 0.069 0.071 0.074 0.069 0.068 0.071
0.000 0.000 0.000 0.000 0.074 0.074 0.069 0.069 0.069 0.069 0.071 0.071 0.071 0.071 0.069 0.069 0.071 0.071 0.074 0.074 0.069 0.069 0.069 0.069 0.066 0.066 0.071 0.071 0.069 0.069 0.069 0.069 0.069 0.069 0.071 0.071 0.074 0.074 0.069 0.069 0.068 0.068 0.071 0.071
0.014 0.013 0.013 0.013 0.014 0.013 0.013 0.013 0.014 0.013 0.013 0.013 0.003 0.003 0.004 0.004 0.000 0.002 0.004 0.000 0.002 0.007 0.002 0.002 0.004 0.002 0.002 0.004 0.004 0.000 0.000 0.002 0.007 0.002 0.002 0.004 0.009 0.004 0.004 0.007 0.004 0.000 0.000 0.002 0.004 0.000 0.000 0.002 0.007 0.002 0.002 0.004 0.007 0.002 0.002 0.004 0.004 0.000 0.000 0.002 0.004 0.000 0.000 0.002 0.004 0.000 0.000 0.002 0.007 0.002 0.002 0.004 0.000 0.004 0.004 0.007 0.004 0.000 0.000 0.002 0.009 0.004 0.004 0.007 0.007 0.002 0.002 0.004
0.002 0.004 0.007 0.002 0.002 0.004 0.004 0.002 0.002 0.002 0.004 0.004 0.002 0.007 0.004
0.013 0.013 0.013 0.003 0.002 0.002 0.003
0.002 0.004 0.000 0.000 0.002 0.002 0.000 0.000 0.000 0.002 0.004 0.000 0.004 0.002
0.013 0.013 0.013 0.003 0.000 0.000 0.002 0.002
0.007 0.002 0.002 0.004 0.004 0.002 0.002 0.002 0.004 0.007 0.002 0.007 0.004
0.014 0.014 0.014 0.004 0.002 0.002 0.003 0.003 0.002
0.004 0.004 0.007 0.007 0.004 0.004 0.004 0.007 0.009 0.004 0.009 0.007
0.014 0.014 0.014 0.004 0.003 0.003 0.004 0.004 0.003 0.004
0.000 0.002 0.002 0.000 0.000 0.000 0.002 0.004 0.000 0.004 0.002
0.013 0.013 0.013 0.003 0.000 0.000 0.002 0.002 0.000 0.002 0.003
0.002 0.002 0.000 0.000 0.000 0.002 0.004 0.000 0.004 0.002
0.013 0.013 0.013 0.003 0.000 0.000 0.002 0.002 0.000 0.002 0.003 0.000
0.004 0.002 0.002 0.002 0.004 0.007 0.002 0.007 0.004
0.013 0.013 0.013 0.004 0.002 0.002 0.003 0.003 0.002 0.003 0.004 0.002 0.002
0.002 0.002 0.002 0.004 0.007 0.002 0.002 0.004
0.013 0.013 0.013 0.004 0.002 0.002 0.003 0.003 0.002 0.003 0.004 0.002 0.002 0.003
0.000 0.000 0.002 0.004 0.000 0.004 0.002
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 0.002 0.004 0.000 0.004 0.002
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
0.002 0.004 0.000 0.004 0.002
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
0.014 0.014 0.014 0.000 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
0.013 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
0.013 0.013 0.013 0.004 0.003 0.003 0.004 0.004 0.003 0.004 0.004 0.003 0.003 0.003 0.002 0.003 0.003 0.003 0.003 0.004 0.003 0.007 0.002 0.004 0.007 0.009 0.004 0.004 0.007 0.002 0.007
0.013 0.013 0.013 0.004 0.002 0.002 0.003 0.003 0.002 0.003 0.004 0.002 0.002 0.003 0.003 0.002 0.002 0.002
0.013 0.013 0.013 0.004 0.002 0.002 0.003 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
0.012 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
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
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
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
0.324 0.329 0.315 0.310 0.337 0.341 0.341 0.350 0.320 0.319 0.325 0.327
0.047 0.042 0.052 0.099 0.079 0.074 0.074 0.090 0.087 0.081 0.084
0.090 0.084 0.096 0.090 0.113 0.114 0.114 0.108 0.104 0.101
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
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
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
0.074 0.069 0.080 0.098 0.117 0.113 0.113 0.091 0.004
0.077 0.071 0.083 0.101 0.119 0.119 0.119 0.091
0.086 0.080 0.092 0.115 0.109 0.110 0.110
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
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
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
0.063 0.069 0.069 0.129 0.023 0.000
0.063 0.069 0.069 0.129 0.023
0.060 0.066 0.066 0.118
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
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
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
0.096 0.090 0.102
0.004 0.009
0.004
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
Table 1. K2P Pairwise Genetic Distances of individual juvenile eel samples with reference sequences from BOLD and GenBank.
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Table 2. K2P Pairwise Genetic Distances of individual elamobranch samples with reference sequences from BOLD and GenBank. BOLD_FOAD629-05_ Manta_birostris Sample_B Sample_K Sample_L Sample_Q BOLD_GBGC0188-06_ Neoceratodus_forsteri Sample_C Sample_J Sample_M Sample_O Sample_P Sample_R Sample_S BOLD_ESHKB025-07_ Alopias_pelagicus BOLD_ESHKC116-07_ Carcharhinus_falciformis BOLD_ANGBF2038-12_ Mobula_japanica BOLD_FOA216-04_ Himantura_fai GENBANK_JQ_765555_ Taeniura_meyeni
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 0.000 0.004 0.002 0.000 0.269
0.003 0.002 0.000 0.004 0.004 0.003 0.002 0.006 0.002 0.000 0.004 0.002 0.269 0.275 0.272 0.269
0.028 0.029 0.029 0.028
0.217 0.239 0.228 0.239 0.239 0.209 0.101 0.232
0.217 0.239 0.228 0.239 0.239 0.209 0.101 0.232
0.298 0.317 0.281 0.317 0.317 0.232 0.276 0.278
0.219 0.242 0.228 0.239 0.242 0.211 0.101 0.234
0.219 0.242 0.231 0.242 0.242 0.212 0.103 0.235
0.217 0.239 0.228 0.239 0.239 0.209 0.101 0.232
0.025 0.025 0.025 0.025 0.031
0.025 0.026 0.026 0.025 0.032
0.024 0.025 0.025 0.024 0.027
0.253 0.224 0.256 0.253 0.236 0.223 0.224
0.027 0.023 0.021 0.187 0.002 0.184 0.000 0.187 0.265 0.283 0.221 0.207 0.187 0.004
0.025 0.025 0.025 0.025 0.032
0.025 0.026 0.026 0.025 0.032
0.025 0.025 0.025 0.025 0.026
0.016 0.016 0.016 0.016 0.029
0.025 0.025 0.025 0.025 0.027
0.025 0.026 0.026 0.025 0.032
0.016 0.016 0.016 0.016 0.028
0.025 0.025 0.025 0.025 0.026
0.025 0.025 0.025 0.025 0.030
0.027 0.027 0.026 0.025 0.002 0.000 0.029 0.025 0.021 0.021 0.029 0.023 0.002 0.029 0.025 0.002 0.029 0.025 0.268 0.265 0.024 0.218 0.221 0.200 0.190 0.187 0.280 0.212
0.023 0.022 0.003 0.022 0.022 0.029 0.024
0.027 0.000 0.021 0.002 0.000 0.029 0.025 0.022
0.025 0.025 0.024 0.025 0.025 0.024 0.002 0.024
0.026 0.029 0.029 0.029 0.029 0.002 0.024 0.029
0.002 0.027 0.023 0.027 0.027 0.026 0.024 0.023
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
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
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
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
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 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.
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 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,
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DOI: 10.3109/19401736.2014.913138
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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 continuously 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.
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P
Sample_P
70
J
BOLD_ESHKC116-07_Carcharhinus_falciformis
Silky Shark
100 Sample_J
95
O
Sample_O
Sample_M
64 100
BOLD_ESHKB025-07_Alopias_pelagicus
Pelagic Thresher Shark
M
Sample_C
100 GENBANK_JQ_765555_Taeniura_meyeni
91
Spinetail Mobula
100 Sample_S
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Blotched Fantail Ray
BOLD_ANGBF2038-12_Mobula_japanica
C
S K
Sample_K
100
B
BOLD_FOAD629-05_Manta_birostris
100
67
Sample_B
Manta Ray
Sample_L
L Q
Sample_Q
R
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
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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 internationally (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 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 enforcement 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 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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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
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Genetic Fingerprinting Laboratory, National Fisheries Research and Development Institute, Quezon City, Philippines
Abstract
Keywords
Illegal trade has greatly affected marine fish stocks, decreasing fish populations worldwide. Despite having a number of aquatic species being regulated, illegal trade still persists through 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 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 characters in juveniles and processed products. Here, live juvenile eels (elvers) and dried products of sharks and rays confiscated for illegal trade were identified. Twenty out of 23 (87%) randomly selected ‘‘elvers’’ were identified as Anguilla bicolor pacifica and 3 (13%) samples as 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. 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.
CO1, illegal trade, juvenile eel, Manta ray, processed products
Introduction Illegal trade of marine fish stocks has been a major challenge to marine biodiversity conservation since the continuous and heavy exploitation of this resource leads to the declines in marine populations or even near-collapses of it (Mullon et al., 2005). Even with established regulation efforts, reports on illegal, unreported and unregulated fisheries (IUU) and illegal trade are still existent (Maes & Volckaert, 2007). At present, illegal substitution and trade of certain fish species has been a rising global concern (Rasmussen & Morrissey, 2008). Southeast Asia in particular has been recognized as a ‘‘wildlife trade hotspot’’ because of its unsustainable and ill-regulated wildlife trade (Nijman, 2010). This has been a rising concern since the epicenter of marine biodiversity that urgently needs improvement of conservation efforts is found in this part of the world, the Coral Triangle, encompassing to a large extent Indonesia, Malaysia, Philippines, Papua New Guinea, Solomon Islands, Timor L’Este, and Brunei, with the Philippines and eastern Indonesia having the highest concentration of species richness within the Coral Triangle (Sanciangco et al., 2013).
Correspondence: Mudjekeewis D. Santos, National Fisheries Research and Development Institute, Room 601 Corporate 101 Building, Mo. Ignacia Avenue, Quezon City 1103, Philippines. Tel/Fax: +63 23725063. E-mail: [email protected]
History Received 20 January 2014 Revised 3 April 2014 Accepted 5 April 2014 Published online 19 May 2014
In the Philippines, a number of aquatic species such as eels of various life stages and sharks and rays are being utilized for various purposes resulting into the decline in their population (Crook, 2010; SEAFDEC, 2012). The Philippine eel culture industry started in 1972 when profitable quantities of juvenile eels called ‘‘elvers’’ were discovered in the Cagayan river system (Gutierrez, 1976). The Anguilla were seen as commercially important and treated as fish species with aquaculture potential (Briones et al., 2007). However, a significant decline in population resulted to a prohibition of ‘‘elvers’’ exportation in the country during the 1970s (Gutierrez, 1976). This ban has been reinstated today under the Philippines’ Fisheries Administrative Order (FAO) 242, which upholds FAO 107 and 107-1 series of 1986 that banned ‘‘elvers’’ exportation and revoked FAO 159 series of 1986, which allowed the exportation of the commodity. The reinstatement of the ban was recommended by the Philippine Bureau of Fisheries and Aquatic Resources (BFAR) Regional Office II in Cagayan after observing the excessive and non-stop exploitation of ‘‘elvers’’ due to its sharp price increase. 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 catch. The commercial exploitation of shark and ray species began in 1960s and landings of these aquatic species has then declined (Barut & Zartiga, 2002; Bonfil, 2002). Manta ray fishing has also expanded and is now overexploited in by-catch and
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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 been contributing to the decline of manta ray populations in the area. Perhaps the best indication of the declining population 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 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 morphologies (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, DNAbased 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, conservation, 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
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(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 randomly chosen by the Bureau of Customs and sent to the NFRDIGenetic 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 (blast.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.
A. marmorata GBJQ665824 A. marmorata GBGC1717-06 A. marmorata JQ431414.1 A. australi saustralis GBGCl A. bicolor bicolor G BGC171 A. bicolorpacifica GBGC171 A. bicolorpacifica GBAP007 A. celebesensis GBGC1714-06 A. japonica GBGC1517-06 A. japonica HQ339972.1 A. malgumora GBGC1713-06 A. luzonensis GBGC6906-09 N. forsteri GBGC0188-06 A. bengalensis bengalensis EL4A01 EL4A12 EL4A17 EL4A02 EL4A08 EL4A10 EL4A13 EL4A14 EL4A15 EL4A16 EL4A18 EL4A19 EL4A20 EL4A21 EL4A22 EL4A23 EL4A24 EL4A25 EL4A26 EL4A27 EL4A28 EL4A29 EL4A30
0.004 0.004 0.004 0.069 0.063 0.063 0.066 0.066 0.063 0.066 0.069 0.063 0.063 0.060 0.066 0.063 0.063 0.063 0.066 0.069 0.063 0.063 0.066
0.000 0.000 0.000 0.074 0.069 0.069 0.071 0.071 0.069 0.071 0.074 0.069 0.069 0.066 0.071 0.069 0.069 0.069 0.071 0.074 0.069 0.068 0.071
0.009 0.009 0.009 0.074 0.069 0.069 0.071 0.071 0.069 0.071 0.074 0.069 0.069 0.066 0.071 0.069 0.069 0.069 0.071 0.074 0.069 0.068 0.071
0.090 0.090 0.090 0.136 0.129 0.129 0.132 0.132 0.129 0.132 0.129 0.129 0.129 0.126 0.128 0.129 0.129 0.129 0.129 0.136 0.129 0.125 0.126
0.066 0.066 0.066 0.027 0.023 0.023 0.020 0.025 0.023 0.025 0.027 0.023 0.023 0.025 0.025 0.023 0.023 0.023 0.025 0.027 0.023 0.027 0.025
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 0.000 0.004 0.002
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 0.000 0.004 0.002
0.080 0.080 0.080 0.110 0.110 0.110 0.113 0.112 0.110 0.113 0.103 0.110 0.110 0.107 0.109 0.110 0.110 0.110 0.107 0.110 0.110 0.106 0.113
0.071 0.071 0.071 0.125 0.119 0.119 0.122 0.122 0.119 0.122 0.112 0.119 0.119 0.116 0.118 0.119 0.119 0.119 0.116 0.125 0.119 0.115 0.122
0.069 0.069 0.069 0.119 0.113 0.113 0.116 0.116 0.113 0.116 0.106 0.113 0.113 0.110 0.112 0.113 0.113 0.113 0.110 0.119 0.113 0.109 0.116
0.084 0.084 0.084 0.120 0.114 0.114 0.117 0.117 0.114 0.117 0.114 0.114 0.114 0.111 0.113 0.114 0.114 0.114 0.117 0.120 0.114 0.110 0.117
0.042 0.042 0.042 0.080 0.074 0.074 0.077 0.077 0.074 0.071 0.074 0.074 0.074 0.071 0.077 0.074 0.074 0.074 0.077 0.080 0.074 0.074 0.077
0.329 0.329 0.329 0.341 0.341 0.341 0.341 0.345 0.341 0.336 0.331 0.341 0.341 0.346 0.340 0.341 0.341 0.341 0.341 0.341 0.341 0.340 0.336
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 0.054 0.054 0.054 0.096 0.090 0.090 0.093 0.093 0.090 0.093 0.090 0.090 0.090 0.087 0.093 0.090 0.090 0.090 0.093 0.096 0.090 0.090 0.093 0.000 0.000 0.074 0.069 0.069 0.071 0.071 0.069 0.071 0.074 0.069 0.069 0.066 0.071 0.069 0.069 0.069 0.071 0.074 0.069 0.068 0.071
0.000 0.000 0.000 0.000 0.074 0.074 0.069 0.069 0.069 0.069 0.071 0.071 0.071 0.071 0.069 0.069 0.071 0.071 0.074 0.074 0.069 0.069 0.069 0.069 0.066 0.066 0.071 0.071 0.069 0.069 0.069 0.069 0.069 0.069 0.071 0.071 0.074 0.074 0.069 0.069 0.068 0.068 0.071 0.071
0.014 0.013 0.013 0.013 0.014 0.013 0.013 0.013 0.014 0.013 0.013 0.013 0.003 0.003 0.004 0.004 0.000 0.002 0.004 0.000 0.002 0.007 0.002 0.002 0.004 0.002 0.002 0.004 0.004 0.000 0.000 0.002 0.007 0.002 0.002 0.004 0.009 0.004 0.004 0.007 0.004 0.000 0.000 0.002 0.004 0.000 0.000 0.002 0.007 0.002 0.002 0.004 0.007 0.002 0.002 0.004 0.004 0.000 0.000 0.002 0.004 0.000 0.000 0.002 0.004 0.000 0.000 0.002 0.007 0.002 0.002 0.004 0.000 0.004 0.004 0.007 0.004 0.000 0.000 0.002 0.009 0.004 0.004 0.007 0.007 0.002 0.002 0.004
0.002 0.004 0.007 0.002 0.002 0.004 0.004 0.002 0.002 0.002 0.004 0.004 0.002 0.007 0.004
0.013 0.013 0.013 0.003 0.002 0.002 0.003
0.002 0.004 0.000 0.000 0.002 0.002 0.000 0.000 0.000 0.002 0.004 0.000 0.004 0.002
0.013 0.013 0.013 0.003 0.000 0.000 0.002 0.002
0.007 0.002 0.002 0.004 0.004 0.002 0.002 0.002 0.004 0.007 0.002 0.007 0.004
0.014 0.014 0.014 0.004 0.002 0.002 0.003 0.003 0.002
0.004 0.004 0.007 0.007 0.004 0.004 0.004 0.007 0.009 0.004 0.009 0.007
0.014 0.014 0.014 0.004 0.003 0.003 0.004 0.004 0.003 0.004
0.000 0.002 0.002 0.000 0.000 0.000 0.002 0.004 0.000 0.004 0.002
0.013 0.013 0.013 0.003 0.000 0.000 0.002 0.002 0.000 0.002 0.003
0.002 0.002 0.000 0.000 0.000 0.002 0.004 0.000 0.004 0.002
0.013 0.013 0.013 0.003 0.000 0.000 0.002 0.002 0.000 0.002 0.003 0.000
0.004 0.002 0.002 0.002 0.004 0.007 0.002 0.007 0.004
0.013 0.013 0.013 0.004 0.002 0.002 0.003 0.003 0.002 0.003 0.004 0.002 0.002
0.002 0.002 0.002 0.004 0.007 0.002 0.002 0.004
0.013 0.013 0.013 0.004 0.002 0.002 0.003 0.003 0.002 0.003 0.004 0.002 0.002 0.003
0.000 0.000 0.002 0.004 0.000 0.004 0.002
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 0.002 0.004 0.000 0.004 0.002
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
0.002 0.004 0.000 0.004 0.002
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
0.014 0.014 0.014 0.000 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
0.013 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
0.013 0.013 0.013 0.004 0.003 0.003 0.004 0.004 0.003 0.004 0.004 0.003 0.003 0.003 0.002 0.003 0.003 0.003 0.003 0.004 0.003 0.007 0.002 0.004 0.007 0.009 0.004 0.004 0.007 0.002 0.007
0.013 0.013 0.013 0.004 0.002 0.002 0.003 0.003 0.002 0.003 0.004 0.002 0.002 0.003 0.003 0.002 0.002 0.002
0.013 0.013 0.013 0.004 0.002 0.002 0.003 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
0.012 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
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
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
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
0.324 0.329 0.315 0.310 0.337 0.341 0.341 0.350 0.320 0.319 0.325 0.327
0.047 0.042 0.052 0.099 0.079 0.074 0.074 0.090 0.087 0.081 0.084
0.090 0.084 0.096 0.090 0.113 0.114 0.114 0.108 0.104 0.101
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
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
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
0.074 0.069 0.080 0.098 0.117 0.113 0.113 0.091 0.004
0.077 0.071 0.083 0.101 0.119 0.119 0.119 0.091
0.086 0.080 0.092 0.115 0.109 0.110 0.110
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
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
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
0.063 0.069 0.069 0.129 0.023 0.000
0.063 0.069 0.069 0.129 0.023
0.060 0.066 0.066 0.118
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
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
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
0.096 0.090 0.102
0.004 0.009
0.004
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
Table 1. K2P Pairwise Genetic Distances of individual juvenile eel samples with reference sequences from BOLD and GenBank.
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Table 2. K2P Pairwise Genetic Distances of individual elamobranch samples with reference sequences from BOLD and GenBank. BOLD_FOAD629-05_ Manta_birostris Sample_B Sample_K Sample_L Sample_Q BOLD_GBGC0188-06_ Neoceratodus_forsteri Sample_C Sample_J Sample_M Sample_O Sample_P Sample_R Sample_S BOLD_ESHKB025-07_ Alopias_pelagicus BOLD_ESHKC116-07_ Carcharhinus_falciformis BOLD_ANGBF2038-12_ Mobula_japanica BOLD_FOA216-04_ Himantura_fai GENBANK_JQ_765555_ Taeniura_meyeni
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 0.000 0.004 0.002 0.000 0.269
0.003 0.002 0.000 0.004 0.004 0.003 0.002 0.006 0.002 0.000 0.004 0.002 0.269 0.275 0.272 0.269
0.028 0.029 0.029 0.028
0.217 0.239 0.228 0.239 0.239 0.209 0.101 0.232
0.217 0.239 0.228 0.239 0.239 0.209 0.101 0.232
0.298 0.317 0.281 0.317 0.317 0.232 0.276 0.278
0.219 0.242 0.228 0.239 0.242 0.211 0.101 0.234
0.219 0.242 0.231 0.242 0.242 0.212 0.103 0.235
0.217 0.239 0.228 0.239 0.239 0.209 0.101 0.232
0.025 0.025 0.025 0.025 0.031
0.025 0.026 0.026 0.025 0.032
0.024 0.025 0.025 0.024 0.027
0.253 0.224 0.256 0.253 0.236 0.223 0.224
0.027 0.023 0.021 0.187 0.002 0.184 0.000 0.187 0.265 0.283 0.221 0.207 0.187 0.004
0.025 0.025 0.025 0.025 0.032
0.025 0.026 0.026 0.025 0.032
0.025 0.025 0.025 0.025 0.026
0.016 0.016 0.016 0.016 0.029
0.025 0.025 0.025 0.025 0.027
0.025 0.026 0.026 0.025 0.032
0.016 0.016 0.016 0.016 0.028
0.025 0.025 0.025 0.025 0.026
0.025 0.025 0.025 0.025 0.030
0.027 0.027 0.026 0.025 0.002 0.000 0.029 0.025 0.021 0.021 0.029 0.023 0.002 0.029 0.025 0.002 0.029 0.025 0.268 0.265 0.024 0.218 0.221 0.200 0.190 0.187 0.280 0.212
0.023 0.022 0.003 0.022 0.022 0.029 0.024
0.027 0.000 0.021 0.002 0.000 0.029 0.025 0.022
0.025 0.025 0.024 0.025 0.025 0.024 0.002 0.024
0.026 0.029 0.029 0.029 0.029 0.002 0.024 0.029
0.002 0.027 0.023 0.027 0.027 0.026 0.024 0.023
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
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
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
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
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 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.
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 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,
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DOI: 10.3109/19401736.2014.913138
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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 continuously 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.
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P
Sample_P
70
J
BOLD_ESHKC116-07_Carcharhinus_falciformis
Silky Shark
100 Sample_J
95
O
Sample_O
Sample_M
64 100
BOLD_ESHKB025-07_Alopias_pelagicus
Pelagic Thresher Shark
M
Sample_C
100 GENBANK_JQ_765555_Taeniura_meyeni
91
Spinetail Mobula
100 Sample_S
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Blotched Fantail Ray
BOLD_ANGBF2038-12_Mobula_japanica
C
S K
Sample_K
100
B
BOLD_FOAD629-05_Manta_birostris
100
67
Sample_B
Manta Ray
Sample_L
L Q
Sample_Q
R
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
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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 internationally (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 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 enforcement 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 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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