International Journal of Philippine Science and Technology ( formerly Philippine Science Letters )

 




Posted on in the International Journal of Philippine Science and Technology 2016 Volume 9 Issue Number 1 Page 2329

*Corresponding Author
Email Address: mudjiesantos@gmail.com
Submitted: September 4, 2015
Revised: June 22, 2016
Accepted: June 20, 2016
Published: September 23, 2016



SHORT COMMUNICATION

Identification of prohibited red piranha, Pygocentrus nattereri (Kner 1858) from confiscated juvenile fish in Manila, Philippines

by , , and *

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

Abstract—Trade has significantly influenced marine and freshwater biodiversity because of overexploitation, habitat degradation, and invasive species introduction. Despite the developed international control measures, cases of illegal trade were proven to be rampant by reported cases of wildlife trafficking. In this regard, species authentication in traded fishery products through DNA barcoding is considered to be a more efficient technique in the confirmation of species identity since morphological characterization is difficult in juvenile species. In this study, confiscated live fish fingerlings, declared as “pacu”, were brought to the Genetic Fingerprinting Laboratory (GFL) by the Manila Police District - District Special Operations Unit (MPD-DSOU) for species identification. Three random samples were subjected to DNA barcoding by the amplification of cytochrome oxidase I (COI) gene and alignment of generated sequence with reference sequences for Pygocentrus nattereri and Piaractus brachypomus in the GenBank database. The Kimura 2-parameter (K2P) distance model was used to compute genetic distances and to generate Neighbour-Joining (NJ) tree. The results confirm that the three samples are P. nattereri as shown by a monophyletic cluster of sample sequences with reference sequences for P. nattereri. The mean genetic distance between the samples and P. nattereri was 1.1%. This report shows that the trade of prohibited red piranha is still happening in spite of the ban. This study aims to emphasize the significance of species authentication in strengthening law implementation and the capability of DNA barcoding to support it.

Keywords—Trade, Juvenile, Red piranha, DNA barcoding, Cytochrome oxidase I



INTRODUCTION

Trading of various aquatic species serves as a major threat in biodiversity conservation due to endangering of wildlife species, threatening of wildlife stocks, and introduction of invasive species (Ferrier 2009). Trading was proven to be extensive through the reported cases of wildlife trafficking, disregarding the control measures generated for it (Lavorgna 2014). Illegal trade is fueled by the high demand of wildlife products used for various purposes such as food, commercial products, traditional Chinese medicine, or as personal pets (Hakken 2011). This high demand is directed at Southeast Asia, which is one of the key suppliers of the international market in wildlife trade, making it one of the world’s “wildlife trade hotspots” (Felbab-Brown 2013).

The Philippines prohibits the importation or possession of any live piranha under FAO No. 126 series of 1979. In the mentioned order, piranha is defined as: “fishes with lacerating teeth and strongly set on well- developed mandibles with which to take big bites out of the flesh of its victims, usually found in northern South America. They are strictly, freshwater species, sturdy and could adapt easily to new environment even in confinement under aquarium conditions”. This ban is supported by FAO No. 221 series of 2003 which is subjected to regulate the importation of live fish and fishery/aquatic products by the evaluation of its risk factors and potential occurrence of unfavorable consequences wherein piranhas are placed under prohibited species, defined as exotic species with known adverse effect on local fauna, human health and the environment.

There have been numerous reports on trading and selling of piranha in the Philippines signifying that importation of piranha is still functional despite the prohibition of the Philippine law. In fact, piranha was recently seen in an aquarium of an establishment in Pasig City (Guerrero 2014). Also, trading through the means of internet selling sites appears to be active up to date since there are advertisement posts marketing red piranhas (KP Sarmiento, personal observation).

Pygocentrus nattereri (Kner 1858), also known as “red piranha”, is one of the most common piranhas utilized in aquarium trade (Fuller et al. 1999). Red piranhas are high valued pets amounting to P600 (14USD) per piece of 1 inch-size piranha (Idolph 2009). It is considered as potentially invasive species with the score of 19, giving it a medium risk to become an invasive species according to the Fish Invasiveness Scoring Kit (FISK) (Almeida et al. 2013). Red piranhas have been observed to breed on flooded pasture with a depth of ~35 cm and water temperature of 27-28°C, in South-western Brazil near the Rio Vermelho stream (Uetanabaro et al. 1993). This has been supported by more recent studies suggesting that the reproductive season of red piranhas occur when there is high water level and increased flooding rates on grounds with good vegetation (Queiroz et al. 2010; Vicentin et al. 2013). Establishment of piranhas in its non-native grounds may create a great impact in the existing natural fauna because it may act as a voracious piscivore that may outcompete the other fishes in the area (Winemiller 1990).

Red piranha is often interchanged with Piaractus brachypomus (Cuvier 1818), also known as “pirapitinga or red-bellied pacu”, when morphologically identified due to their similar appearance (Nico et al. 2015). Dentition serves as a primary eminent characteristic in distinguishing the two, red piranha is notable by having a single-row of serrated incisor-like teeth, while the red-bellied pacu has two rows of molariform teeth (Nico et al. 2015). However, at earlier life stages of the two species, dentition characteristics are not very distinct. Moreover, red-bellied pacu, at juvenile stage, often mimics red piranha by displaying dark-grey to black spots in its body, a distinguishing characteristic of piranhas (Hintz 2012) hence making differentiation more difficult.

Accurate identification of species serves as an important tool for the establishment of marine protected areas, resource conservation, compensation of fishery, prevention of illegal trade, and ecological monitoring (Asis et al. 2014; Valdez-Moreno et al. 2010; Moura et al. 2008). Identification based on morphological characteristics becomes problematic when applied during early life stages of fishes, particularly in larval stage and juvenile stage (Graves et al. 1989). In fact, traditional morphological characterization was reported to result to different identification of fish larvae among different taxonomists and various laboratories (Ko et al. 2013). Because of this, DNA-based techniques, including DNA barcoding, are widely applied for more reliable species identification (Shen et al. 2013; Cawthorn et al. 2011).

The use of DNA barcoding was proven to be an effective tool in the assessment of marine biodiversity, conservation efforts, and fisheries management (Trivedi et al. 2015). Predominantly, DNA barcodes are applied in species authentication to prevent mislabeling of fishery products in various countries including the Philippines, South Africa, North America, and United States (Maralit et al. 2013; Cawthorn et al. 2011; Wong et al. 2011, Wong and Hanner 2008). In addition, DNA barcodes are utilized in the determination of specific predator-prey relationships (Jo et al. 2014).

In this study, confiscated live fish fingerlings, declared as “pacu”, were identified through the use of DNA barcoding. Four separate plastic bags, each containing twenty-five pieces of suspected piranha fingerlings, were confiscated by the Manila Police District - District Special Operations Unit (MPD-DSOU) while being sold along the sidewalk of Tayuman Street near the corner of Rizal Avenue at about 7:45 p.m. on January 08, 2015. The items were said to cost approximately P13,000 (285USD). Proper identification of aquatic species is highlighted as an important tool as a preventive measure to monitor and manage banned species, and this is achieved through the application of DNA barcoding.


MATERIALS AND METHODS
Sample collection

Three random samples (Figure 1) were forwarded to the Genetic Fingerprinting Laboratory from the Vertebrate Section of NFRDI. The samples were obtained from one plastic bag of twenty-five live fish fingerlings submitted by the MPD-DSOU, from United Nations Avenue, Ermita, Manila. The samples were submitted last January 09, 2015 for genetic identification. Approximately 150 mg of muscle tissue, obtained from the ventral side near the posterior end in the body of the fish, was preserved in 95% ethanol and stored in -20ºC.

Figure 1. Juvenile finfish samples submitted by MPD-DSOU last January 09, 2015. P1, P2 and P3 had weight of 1.79, 1.59 g and 1.51 g, respectively, and length of 4.5, 4.4, and 4.2 cm, respectively. Morphological measurements were provided by Ms. Eunice Bognot and Mr. Francisco Torres Jr. of the Vertebrate Section at NFRDI.

DNA extraction and COI amplification

DNA was extracted using 10% Chelex based on the protocol of Walsh et al. (1991). DNA extractions were carried out for each sample. A region of cytochrome oxidase I (~600 bp) was amplified using the primers as follows: VF2_t1 (5’TGTAAAACGACGGCCAGTCAACCAACCACAAAGACATTGGCAC3’), FishF2_t1 (5’TGTAA AACGACGGCCAGTCGACTAATCATAAAGATATCGGCAC3’), FishR2_t1 (5’CAGGAAACAG CTATGACACTTCAGGGTGACCGAAGAATCAGAA3’), and Fr1d_t1 (5’CAGGAAACAGCTA TGACACCTCAGGGTGTCCGAARAAYCARAA3’) (Ward et al. 2005; Ivanova et al. 2007). The 25 µl PCR reactions consisted of water, 0.5x PCR buffer, 0.08 mM dNTP’s, 1.4 mM MgCl2, 0.6 µM of each primers, 10 µl of 5x BSA, 0.1 unit Taq polymerase and 2 µl of template. The PCR cocktails were subjected to the following conditions: 94°C for 2 min, 38 cycles of 94°C for 30 sec, 52°C for 40 sec, 72°C for 60 sec, and a final extension of 72°C for 10 min. Product amplicons were electrophoresed in 1% agarose gel stained with ethidium bromide and submerged in 1x TAE buffer. Standard sequencing and DNA purification were outsourced to Macrogen Inc., Korea.

Genetic Analysis

BLAST analysis was carried out to identify sequences which are similar to the generated sample sequences (Altschul et al. 1990). Similar sequences were downloaded from GenBank together with their accession numbers (http://www.ncbi.nlm.nih.gov/genbank/) and were used as reference sequences for the generation of phylogenetic tree. MEGA 6.0 (Tamura et al. 2013) was used for aligning DNA sequences together with the reference sequences. Species identification was inferred using Neighbor-Joining (NJ) tree based on the Kimura 2-parameter (K2P) model with 500 bootstrap replications (Tamura et al. 2013). Genetic distance was computed between the sample and reference sequences using K2P model (Kimura 1980).


RESULTS AND DISCUSSION

Mitochondrial DNA CO1 sequences of the three juvenile fish samples were sequenced and analyzed. BLAST analysis revealed that the sample sequences (GenBank: KX396057, KX396058, KX396059) have >99% similarity with Pygocentrus nattereri (GenBank: AP012000). Other sequences which have 85-97% similarity with the sample sequences were detected from Pygocentrus piraya (Cuvier 1819), Serrasalmus marginatus (Valenciennes 1837), Piaractus mesopotamicus (Holmberg 1887), Leporinus friderici (Bloch 1794), and Trichomycterus giganteus (Lima and Costa 2004). CO1 sequences from the mentioned species and from the believed species "Red pacu" or Piaractus brachypomus (Cuvier 1818) was used for phylogenetic analysis. CO1 sequence from Apareiodon affinis (Steindachner 1879) was used as an outgroup because it belongs to the same order (Characiformes) as P. nattereri. Figure 2 shows the result of genetic analysis using Neighbour Joining (NJ) method and Kimura 2-parameter model. The phylogenetic tree suggested that the CO1 sequences of the samples belonged to the same monophyletic clade with P. nattereri, supported by a bootstrap value of 100%, confirming that the samples are from the same lineage as that of P. nattereri. The resulting tree also confirms that the fish are not P. brachypomus or "pacu", as declared. Complete multiple alignments of the sample sequences with reference sequences is shown in Figure 3.

Figure 2. Neighbour-Joining Tree of juvenile fish CO1 sequences using Kimura 2-parameter model. Bootstrap values from 500 bootstrap samples are indicated beside the nodes. The scale bar represents 2% genetic distance and GenBank accession numbers are shown next to species names.

Pygocentrus nattereri AP012000        1 : A - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - G T T G G A A C C G C C C T C A G C C T C T T A A T T C G A G C A G A G C T C A G T C A A : 45
Pygocentrus nattereri PH01 KX396057   1 : . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - G T T . G A A C C G . . C T T . A G C C T T C T . A . T . C G A G C A G A G C T . . . . . . . . : 48
Pygocentrus nattereri PH02 KX396058   1 : C - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - :
Pygocentrus nattereri PH03 KX396059   1 : . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - T A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 47
Piaractus brachypomus KP723348        1 : . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - . . . . . . . . . . . . . . . . G . . . . . A . . C . . . : 29
Pygocentrus piraya HM405211           1 : . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 45
Serrasalmus marginatus JN989232       1 : . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 45
Serrasalmus marginatus JN989233       1 : . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 45
Pygocentrus piraya HQ600844           1 : . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 45
Serrasalmus marginatus GU701508       1 : . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 45
Serrasalmus marginatus GU701507       1 : . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 45
Pygocentrus piraya HQ600843           1 : . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 45
Pygocentrus piraya HQ600849           1 : . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 45
Piaractus mesopotamicus GU701417      1 : . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - . . . . . . . . G . . . . . T . . . . . . . . . . . . . . . . . G . . . . . A . . C . . . : 45
Piaractus mesopotamicus KM897294      1 : . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - . . . . . . . . G . . . . . T . . . . . . . . . . . . . . . . . G . . . . . A . . C . . . : 45
Piaractus mesopotamicus GU701416.1    1 : . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - . . . . . . . . G . . . . . T . . . . . . . . . . . . . . . . . G . . . . . A . . C . . . : 45
Piaractus mesopotamicus KM897305      1 : . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - . . . . . . . . G . . . . . T . . . . . . . . . . . . . . . . . G . . . . . A . . C . . . : 45
Piaractus mesopotamicus KM897414      1 : . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - . . . . . . . . G . . . . . T . . . . . . . . . . . . . . . . . G . . . . . A . . C . . . : 45
Trichomycterus giganteus GU702303     1 : . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - . . . . . C . . . . . . . . T . . . T . G . . . . . . . . . . . . . . A T . A . . C . . . : 45
Trichomycterus giganteus GU702165     1 : . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - . . . . . C . . . . . . . . T . . . T . G . . . . . . . . . . . . . . A T . A . . C . . . : 45
Trichomycterus giganteus GU702160     1 : . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - . . . . . C . . . . . . . . T . . . T . G . . . . . . . . . . . . . . A T . A . . C . . . : 45
Trichomycterus giganteus GU702140     1 : . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - . . . . . C . . . . . . . . T . . . T . G . . . . . . . . . . . . . . A T . A . . C . . . : 45
Leporinus friderici KM897272          1 : . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - . . . . . T . . A . . . . . . . . . . . . C . . . . C . . . . . . . . . . . T . . C . . . : 45
Leporinus friderici KM897166          1 : . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - . . . . . T . . A . . . . . . . . . . . . C . . . . C . . . . . . . . . . . T . . . . . . : 45
Leporinus friderici KM897149          1 : . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - . . . . . T . . A . . . . . . . . . . . . C . . . . C . . . . . . . . . . . T . . . . . . : 45
Leporinus friderici KM897144          1 : . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - . . . . . T . . A . . . . . . . . . . . . C . . . . C . . . . . . . . . . . T . . . . . . : 45
Apareiodon affinis JN988674           1 : C T T T A T T T A G T A T T T G G T G C C T G A G C C G G A A T A . . . . . . . . A . . . . . . . . T . . . C . . . . . . . . . . . . . . . . G . . C . . . : 78

Pygocentrus nattereri AP012000       46 : C C C G G A T C C C T A C T G G G C G A C G A T C A G A T C T A T A A T G T T A T C G T T A C T G C A C A C G C C T T C G T A A T A A T T T T C T T T A T A : 124
Pygocentrus nattereri PH01 KX396057  49 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 127
Pygocentrus nattereri PH02 KX396058   1 : - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . : 33
Pygocentrus nattereri PH03 KX396059  48 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 126
Piaractus brachypomus KP723348       30 : . . . . . . . . . . . C T . A . . T . . T . . C . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . : 108
Pygocentrus piraya HM405211          46 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 124
Serrasalmus marginatus JN989232      46 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 124
Serrasalmus marginatus JN989233      46 : . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 124
Pygocentrus piraya HQ600844          46 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 124
Serrasalmus marginatus GU701508      46 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 124
Serrasalmus marginatus GU701507      46 : . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 124
Pygocentrus piraya HQ600843          46 : . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 124
Pygocentrus piraya HQ600849          46 : . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 124
Piaractus mesopotamicus GU701417     46 : . . . . . . . . . . . C T . A . . T . . T . . C . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . : 124
Piaractus mesopotamicus KM897294     46 : . . . . . . . . . . . C T . A . . T . . T . . C . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . : 124
Piaractus mesopotamicus GU701416.1   46 : . . . . . . . . . . . C T . A . . T . . T . . C . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . : 124
Piaractus mesopotamicus KM897305     46 : . . . . . . . . . . . C T . A . . T . . T . . C . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . : 124
Piaractus mesopotamicus KM897414     46 : . . . . . . . . . . . C T . A . . T . . T . . C . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . : 124
Trichomycterus giganteus GU702303    46 : . . . . . . G . . T . G . . . . . . . . . . . C . . A . . T . . . . . . . . . . . T . . . . . C . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . : 124
Trichomycterus giganteus GU702165    46 : . . . . . . G . . T . G . . . . . . . . . . . C . . A . . T . . . . . . . . . . . T . . . . . C . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . : 124
Trichomycterus giganteus GU702160    46 : . . . . . . G . . T . G . . . . . . . . . . . C . . A . . T . . . . . . . . . . . T . . . . . C . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . : 124
Trichomycterus giganteus GU702140    46 : . . . . . . G . . T . G . . . . . . . . . . . C . . A . . T . . . . . . . . . . . T . . . . . C . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . : 124
Leporinus friderici KM897272         46 : . . . . . G . . . . . T . . A . . T . . . . . C . . A . . T . . . . . . . . . . . . . . . . . C . . . . . T . . T . . . . . . . . . . . C . . . . . C . . . : 124
Leporinus friderici KM897166         46 : . . . . . G . . . . . T . . A . . T . . . . . C . . A . . T . . . . . . . . . . . . . . . . . C . . . . . T . . T . . . . . . . . . . . C . . . . . C . . . : 124
Leporinus friderici KM897149         46 : . . . . . G . . . . . T . . A . . T . . . . . C . . A . . T . . . . . . . . . . . . . . . . . C . . . . . T . . T . . . . . . . . . . . C . . . . . C . . . : 124
Leporinus friderici KM897144         46 : . . . . . G . . . . . T . . A . . T . . . . . C . . A . . T . . . . . . . . . . . . . . . . . C . . . . . T . . T . . . . . . . . . . . C . . . . . C . . . : 124
Apareiodon affinis JN988674          79 : . . . . . G . . . . . G . . A . . . . . T . . C . . A . . T . . . . . . . . . . . . . . . . . . . . . . . T . . T . . T . . . . . G . . . . . . . . . . . . : 157
                                      1
Pygocentrus nattereri AP012000      125 : G T A A T A C C A A T T A T G A T T G G G G G C T T C G G A A A C T G A C T A G T T C C C C T A A T G A T T G G C G C G C C C G A C A T A G C A T T T C C C : 203
Pygocentrus nattereri PH01 KX396057 128 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 206
Pygocentrus nattereri PH02 KX396058  34 : . G . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 112
Pygocentrus nattereri PH03 KX396059 127 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 205
Piaractus brachypomus KP723348      109 : . . . . . . . . . . . . . . . . . . . . A . . . . . . . . G . . T . . . T . . . . C . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . C . . . : 187
Pygocentrus piraya HM405211         125 : . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . : 203
Serrasalmus marginatus JN989232     125 : . . . . . . . . . . . C . . . . . . . . . . . . . . T . . . . . . . . G . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 203
Serrasalmus marginatus JN989233     125 : . . . . . . . . . . . C . . . . . . . . . . . . . . T . . . . . . . . G . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 203
Pygocentrus piraya HQ600844         125 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . : 203
Serrasalmus marginatus GU701508     125 : . . . . . . . . . . . C . . . . . . . . . . . . . . T . . . . . . . . G . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 203
Serrasalmus marginatus GU701507     125 : . . . . . . . . . . . C . . . . . . . . . . . . . . T . . . . . . . . G . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 203
Pygocentrus piraya HQ600843         125 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . : 203
Pygocentrus piraya HQ600849         125 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . : 203
Piaractus mesopotamicus GU701417    125 : . . . . . . . . . . . . . . . . . . . . A . . . . . . . . G . . T . . . T . G . . . . . . . . . . . . . . . . . T . . A . . . . . . . . . . . . . . C . . A : 203
Piaractus mesopotamicus KM897294    125 : . . . . . . . . . . . . . . . . . . . . A . . . . . . . . G . . T . . . T . G . . . . . . . . . . . . . . . . . T . . A . . . . . . . . . . . . . . C . . A : 203
Piaractus mesopotamicus GU701416.1  125 : . . . . . . . . . . . . . . . . . . . . A . . . . . . . . G . . T . . . T . G . . . . . . . . . . . . . . . . . T . . A . . . . . . . . . . . . . . C . . A : 203
Piaractus mesopotamicus KM897305    125 : . . . . . . . . . . . . . . . . . . . . A . . . . . . . . G . . T . . . T . G . . . . . . . . . . . . . . . . . T . . A . . . . . . . . . . . . . . C . . A : 203
Piaractus mesopotamicus KM897414    125 : . . . . . . . . . . . . . . . . . . . . A . . . . . . . . G . . T . . . T . G . . . . . . . . . . . . . . . . . T . . A . . . . . . . . . . . . . . C . . A : 203
Trichomycterus giganteus GU702303   125 : . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . G . . . . . T . . T . . . . . . . . G . . . . . A . . . . . G . . C . . . . . . : 203
Trichomycterus giganteus GU702165   125 : . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . G . . . . . T . . T . . . . . . . . G . . A . . A . . . . . G . . C . . . . . . : 203
Trichomycterus giganteus GU702160   125 : . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . G . . . . . T . . T . . . . . . . . G . . . . . A . . . . . G . . C . . . . . . : 203
Trichomycterus giganteus GU702140   125 : . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . G . . . . . T . . T . . . . . . . . G . . . . . A . . . . . G . . C . . . . . . : 203
Leporinus friderici KM897272        125 : . . G . . G . . G . . C . . A . . . . . . . . . . . . . . . . . . . . . . . . . . C . . G . . . . . . . . . . . . . . A . . . . . T . . G . . . . . . . . A : 203
Leporinus friderici KM897166        125 : . . G . . G . . G . . C . . A . . . . . . . . . . . . . . . . . . . . . . . . T . C . . G . . . . . . . . . . . . . . A . . . . . T . . G . . . . . . . . A : 203
Leporinus friderici KM897149        125 : . . G . . G . . G . . C . . A . . . . . . . . . . . . . . . . . . . . . . . . . . C . . G . . . . . . . . . . . . . . A . . . . . T . . G . . . . . . . . A : 203
Leporinus friderici KM897144        125 : . . G . . G . . G . . C . . A . . . . . . . . . . . . . . . . . . . . . . . . . . C . . G . . . . . . . . . . . . . . A . . . . . T . . G . . . . . . . . A : 203
Apareiodon affinis JN988674         158 : . . T . . . . . . . . . . . A . . C . . A . . . . . T . . . . . T . . . . . G A . . . . . . . . . . . . . . . . G . . A . . . . . . . . G . . . . . C . . T : 236

Pygocentrus nattereri AP012000      204 : C G A A T G A A T A A C A T A A G T T T C T G A C T T C T T C C C C C A T C C T T T C T T C T C C T C C T A G C A T C T T C A G G G G T T G A G G C T G G A : 282
Pygocentrus nattereri PH01 KX396057 207 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 285
Pygocentrus nattereri PH02 KX396058 113 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 191
Pygocentrus nattereri PH03 KX396059 206 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 284
Piaractus brachypomus KP723348      188 : . . . . . A . . . . . T . . . . . C . . . . . . . . C . . A . . . . . G . . . . . C . . . . . . . . G . . . . . . . . C . . . . . A A . C . . A . . C . . . : 266
Pygocentrus piraya HM405211         204 : . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 282
Serrasalmus marginatus JN989232     204 : . . . . . A . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . G : 282
Serrasalmus marginatus JN989233     204 : . . . . . A . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . G : 282
Pygocentrus piraya HQ600844         204 : . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 282
Serrasalmus marginatus GU701508     204 : . . . . . A . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . G : 282
Serrasalmus marginatus GU701507     204 : . . . . . A . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . G : 282
Pygocentrus piraya HQ600843         204 : . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . : 282
Pygocentrus piraya HQ600849         204 : . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . : 282
Piaractus mesopotamicus GU701417    204 : . . . . . A . . . . . T . . . . . C . . . . . . . . C . . A . . . . . . . . . . . C . . . . . T . . G . . . . . . . . C . . . . . A A . C . . A . . C . . . : 282
Piaractus mesopotamicus KM897294    204 : . . . . . A . . . . . T . . . . . C . . . . . . . . C . . A . . . . . . . . . . . C . . . . . T . . G . . . . . . . . C . . . . . A A . C . . A . . C . . . : 282
Piaractus mesopotamicus GU701416.1  204 : . . . . . A . . . . . T . . . . . C . . . . . . . . C . . A . . . . . . . . . . . C . . . . . T . . G . . . . . . . . C . . . . . A A . C . . A . . C . . . : 282
Piaractus mesopotamicus KM897305    204 : . . . . . A . . . . . T . . . . . C . . . . . . . . C . . A . . . . . . . . . . . C . . . . . T . . G . . . . . . . . C . . . . . A A . C . . A . . C . . . : 282
Piaractus mesopotamicus KM897414    204 : . . . . . A . . . . . T . . . . . C . . . . . . . . C . . A . . . . . . . . . . . C . . . . . T . . G . . . . . . . . C . . . . . A A . C . . A . . C . . . : 282
Trichomycterus giganteus GU702303   204 : . . . . . . . . C . . . . . . . . C . . T . . . . . C . . . . . T . . T . . . . . C . . C . . . . . G . . . . . C . . . . . T . . . . . A . . A . . A . . G : 282
Trichomycterus giganteus GU702165   204 : . . . . . . . . C . . . . . . . . C . . T . . . . . C . . . . . . . . T . . . . . C . . C . . . . . G . . . . . C . . . . . T . . . . . A . . A . . A . . G : 282
Trichomycterus giganteus GU702160   204 : . . . . . . . . C . . . . . . . . C . . T . . . . . C . . . . . T . . T . . . . . C . . C . . . . . G . . . . . C . . . . . T . . . . . A . . A . . A . . G : 282
Trichomycterus giganteus GU702140   204 : . . . . . . . . C . . . . . . . . C . . T . . . . . C . . . . . T . . T . . . . . C . . C . . . . . G . . . . . C . . . . . T . . . . . A . . A . . A . . G : 282
Leporinus friderici KM897272        204 : . . . . . A . . . . . . . . . . . C . . T . . . . . A . . G . . . . . . . . T . . C . . C . . . . . . . . . . . . . . . . . C . . A . . A . . . . . C . . G : 282
Leporinus friderici KM897166        204 : . . . . . A . . . . . . . . . . . C . . . . . . . . A . . G . . . . . . . . T . . C . . C . . . . . T . . . . . . . . . . . C . . A . . A C . . . . C . . G : 282
Leporinus friderici KM897149        204 : . . . . . A . . . . . . . . . . . C . . . . . . . . A . . G . . . . . . . . T . . C . . C . . . . . T . . . . . . . . . . . C . . A . . A . . . . . C . . G : 282
Leporinus friderici KM897144        204 : . . . . . A . . . . . . . . . . . C . . . . . . . . A . . G . . . . . . . . T . . C . . C . . . . . T . . . . . . . . . . . C . . A . . A . . . . . C . . G : 282
Apareiodon affinis JN988674         237 : . . G . . A . . . . . T . . G . . C . . T . . . . . A . . . . . A . . C . . A . . C . . C . . . . . . . . . . . T . . C . . T . . . . . . . . A . . C . . . : 315

Pygocentrus nattereri AP012000      283 : G C C G G G A C A G G T T G A A C C G T C T A C C C C C C A C T T G C C G G A A A C C T T G C A C A C G C C G G A G C T T C T G T A G A C C T A A C T A T T : 361
Pygocentrus nattereri PH01 KX396057 286 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 364
Pygocentrus nattereri PH02 KX396058 192 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 270
Pygocentrus nattereri PH03 KX396059 285 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 363
Piaractus brachypomus KP723348      267 : . . A . . . . . . . . C . . . . . T . . A . . . . . . . . T . . . . . . . . T . . T . . C . . . . . . . . G . . C . . C . . . . . T . . . T . . . . . . . C : 345
Pygocentrus piraya HM405211         283 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 361
Serrasalmus marginatus JN989232     283 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 361
Serrasalmus marginatus JN989233     283 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 361
Pygocentrus piraya HQ600844         283 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 361
Serrasalmus marginatus GU701508     283 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 361
Serrasalmus marginatus GU701507     283 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 361
Pygocentrus piraya HQ600843         283 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 361
Pygocentrus piraya HQ600849         283 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 361
Piaractus mesopotamicus GU701417    283 : . . A . . . . . . . . G . . . . . T . . A . . T . . . . . T . . . . . . . . T . . . . . C . . . . . . . . G . . C . . C . . . . . T . . . . . . . . C . . C : 361
Piaractus mesopotamicus KM897294    283 : . . A . . . . . . . . C . . . . . T . . A . . T . . . . . T . . . . . . . . T . . . . . C . . . . . . . . G . . C . . C . . . . . T . . . . . . . . C . . C : 361
Piaractus mesopotamicus GU701416.1  283 : . . A . . . . . . . . C . . . . . T . . A . . T . . . . . T . . . . . . . . T . . . . . C . . . . . . . . G . . C . . C . . . . . T . . . . . . . . C . . C : 361
Piaractus mesopotamicus KM897305    283 : . . A . . . . . . . . C . . . . . T . . A . . T . . . . . T . . . . . . . . T . . . . . C . . . . . . . . G . . C . . C . . . . . T . . . . . . . . C . . C : 361
Piaractus mesopotamicus KM897414    283 : . . A . . . . . . . . G . . . . . T . . A . . T . . . . . T . . . . . . . . T . . . . . C . . . . . . . . G . . C . . C . . . . . T . . . . . . . . C . . C : 361
Trichomycterus giganteus GU702303   283 : . . A . . A . . . . . A . . . . . A . . A . . . . . . . . T . . C . . . . . C . . T . . . . . C . . . . . A . . G . . C . . . . . T . . T . . . . . . . . C : 361
Trichomycterus giganteus GU702165   283 : . . A . . A . . . . . A . . . . . A . . A . . . . . . . . T . . C . . . . . C . . T . . . . . C . . . . . A . . G . . C . . . . . T . . T . . . . . . . . C : 361
Trichomycterus giganteus GU702160   283 : . . A . . A . . . . . A . . . . . A . . A . . . . . . . . T . . C . . . . . C . . T . . . . . C . . . . . A . . G . . C . . . . . T . . T . . . . . . . . C : 361
Trichomycterus giganteus GU702140   283 : . . A . . A . . . . . A . . . . . A . . A . . . . . . . . T . . C . . . . . C . . T . . . . . C . . . . . A . . G . . C . . . . . T . . T . . . . . . . . C : 361
Leporinus friderici KM897272        283 : . . . . . A . . . . . . . . . . . T . . . . . . . . G . . . . . . . . T . . . . . . T . G . . C . . . . . A . . C . . . . . C . . C . . . . . T . . C . . C : 361
Leporinus friderici KM897166        283 : . . . . . A . . . . . . . . . . . T . . . . . . . . G . . . . . . . . T . . . . . . T . G . . C . . . . . A . . C . . . . . C . . C . . . . . T . . C . . C : 361
Leporinus friderici KM897149        283 : . . . . . A . . . . . . . . . . . T . . . . . . . . G . . . . . . . . T . . . . . . T . G . . C . . . . . A . . C . . . . . C . . C . . . . . T . . C . . C : 361
Leporinus friderici KM897144        283 : . . . . . A . . . . . . . . . . . T . . . . . . . . G . . . . . . . . T . . . . . . T . G . . C . . . . . A . . C . . . . . C . . C . . . . . T . . C . . C : 361
Apareiodon affinis JN988674         316 : . . . . . . . . G . . . . . G . . T . . A . . T . . . . . C . . . . . A . . . . . . . . A . . T . . . . . A . . T . . C . . A . . . . . T T . . . . . . . C : 394

Pygocentrus nattereri AP012000      362 : T T T T C C C T A C A C C T C G C C G G G G T C T C T T C C A T C C T T G G G G C C A T T A A C T T T A T C A C A A C C A T C A T C A A C A T A A A A C C C : 440
Pygocentrus nattereri PH01 KX396057 365 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 443
Pygocentrus nattereri PH02 KX396058 271 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 349
Pygocentrus nattereri PH03 KX396059 364 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 442
Piaractus brachypomus KP723348      346 : . . . . . A . . C . . T . . T . . T . . . . . T . . . . . . . . . . . . . . . . . T . . . . . . . . C . . T . . . . . T . . T . . T . . . . . G . . G . . T : 424
Pygocentrus piraya HM405211         362 : . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 440
Serrasalmus marginatus JN989232     362 : . . . . . . . . G . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 440
Serrasalmus marginatus JN989233     362 : . . . . . . . . G . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 440
Pygocentrus piraya HQ600844         362 : . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 440
Serrasalmus marginatus GU701508     362 : . . . . . . . . G . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 440
Serrasalmus marginatus GU701507     362 : . . . . . . . . G . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 440
Pygocentrus piraya HQ600843         362 : . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 440
Pygocentrus piraya HQ600849         362 : . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 440
Piaractus mesopotamicus GU701417    362 : . . . . . A . . T . . T . . T . . T . . . . . T . . C . . . . . . . . . . . . . . T . . . . . . . . C . . . . . . . . T . . T . . T . . . . . G . . G . . T : 440
Piaractus mesopotamicus KM897294    362 : . . . . . A . . T . . T . . T . . T . . . . . T . . C . . . . . . . . . . . . . . T . . . . . . . . C . . T . . . . . T . . T . . T . . . . . G . . G . . T : 440
Piaractus mesopotamicus GU701416.1  362 : . . . . . A . . T . . T . . T . . T . . . . . T . . C . . . . . . . . . . . . . . T . . . . . . . . C . . T . . . . . T . . T . . T . . . . . G . . G . . T : 440
Piaractus mesopotamicus KM897305    362 : . . . . . A . . T . . T . . T . . T . . . . . T . . C . . . . . G . . . . . . . . T . . . . . . . . C . . T . . . . . T . . T . . T . . . . . G . . G . . T : 440
Piaractus mesopotamicus KM897414    362 : . . . . . A . . T . . T . . T . G T . . . . . T . . C . . . . . . . . . . . . . . T . . . . . . . . C . . T . . . . . T . . T . . T . . . . . G . . G . . T : 440
Trichomycterus giganteus GU702303   362 : . . C . . . . . C . . . . . G . . . . . T . . . . . . . . A . . . . . . . . . . . . . . . . . T . . . . . T . . . . . A . . T . . T . . . . . G . . . . . A : 440
Trichomycterus giganteus GU702165   362 : . . C . . . . . C . . . . . G . . . . . T . . . . . . . . A . . . . . . . . . . . . . . . . . T . . . . . T . . . . . A . . T . . T . . . . . G . . . . . A : 440
Trichomycterus giganteus GU702160   362 : . . C . . . . . C . . . . . G . . . . . T . . . . . . . . A . . . . . . . . . . . . . . . . . T . . . . . T . . . . . A . . T . . T . . . . . G . . . . . A : 440
Trichomycterus giganteus GU702140   362 : . . C . . . . . C . . . . . G . . . . . T . . . . . . . . A . . . . . . . . . . . . . . . . . T . . . . . T . . . . . A . . T . . T . . . . . G . . . . . A : 440
Leporinus friderici KM897272        362 : . . C . . . . . G . . T . . . . . . . . T . . . . . C . . . . . T . . . . . A . . . . . . . . T . . . . . T . . . . . A . . T . . T . . . . . . . . . . . . : 440
Leporinus friderici KM897166        362 : . . C . . . . . G . . T . . . . . . . . T . . . . . C . . . . . T . . . . . A . . . . . . . . T . . . . . T . . . . . A . . T . . T . . . . . . . . . . . . : 440
Leporinus friderici KM897149        362 : . . C . . . . . G . . T . . . . . . . . T . . . . . C . . . . . T . . . . . A . . . . . . . . T . . . . . T . . . . . A . . T . . T . . . . . . . . . . . . : 440
Leporinus friderici KM897144        362 : . . C . . . . . G . . T . . . . . . . . T . . . . . C . . . . . T . . . . . A . . . . . . . . T . . . . . T . . . . . A . . T . . T . . . . . . . . . . . . : 440
Apareiodon affinis JN988674         395 : . . C . . . . . . . . T . . T . . . . . . . . T . . C . . T . . T . . . . . C . . . . . . . . T . . . . . T . . T . . . . . T . . T . . T . . . . . . . . . : 473

Pygocentrus nattereri AP012000      441 : C C A G C C A T C T C A C A A T A T C A A A C A C C C C T A T T C G T C T G A G C C G T C C T A A T C A C T G C T G T T C T C C T C C T C C T T T C C C T G : 519
Pygocentrus nattereri PH01 KX396057 444 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 522
Pygocentrus nattereri PH02 KX396058 350 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 428
Pygocentrus nattereri PH03 KX396059 443 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 521
Piaractus brachypomus KP723348      425 : . . . . . . . . T . . . . . G . . . . . . . . . . . . . . . . . . . . G . . . . . A . . . . . . . . . . . . . . C . . . . . T . . T . . T . . C . . G . . A : 503
Pygocentrus piraya HM405211         441 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . : 519
Serrasalmus marginatus JN989232     441 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . G . . . . . . : 519
Serrasalmus marginatus JN989233     441 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . G . . . . . . : 519
Pygocentrus piraya HQ600844         441 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . : 519
Serrasalmus marginatus GU701508     441 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . : 519
Serrasalmus marginatus GU701507     441 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . G . . . . . . : 519
Pygocentrus piraya HQ600843         441 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . : 519
Pygocentrus piraya HQ600849         441 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . : 519
Piaractus mesopotamicus GU701417    441 : . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . T . . A . . . . . A . . . . . . . . . . . . . . C . . . . . T . . T . . T . . C . . G . . A : 519
Piaractus mesopotamicus KM897294    441 : . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . T . . A . . . . . A . . . . . . . . . . . . . . C . . . . . T . . T . . T . . C . . G . . A : 519
Piaractus mesopotamicus GU701416.1  441 : . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . T . . A . . . . . A . . . . . . . . . . . . . . C . . . . . T . . T . . T . . C . . G . . A : 519
Piaractus mesopotamicus KM897305    441 : . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . T . . A . . . . . A . . . . . . . . . . . . . . C . . . . . T . . T . . T . . C . . G . . A : 519
Piaractus mesopotamicus KM897414    441 : . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . T . . A . . . . . A . . . . . . . . . . . . . . C . . . . . T . . T . . T . . C . . G . . A : 519
Trichomycterus giganteus GU702303   441 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . G . . A . . G T . . . . T . . A . . . . . C . . A . . A . . G T . G . . T . . C : 519
Trichomycterus giganteus GU702165   441 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . G . . A . . G T . . . . T . . A . . . . . C . . A . . A . . G T . G . . T . . C : 519
Trichomycterus giganteus GU702160   441 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . G . . A . . G T . . . . T . . A . . . . . C . . A . . A . . G T . G . . T . . C : 519
Trichomycterus giganteus GU702140   441 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . G . . A . . G T . . . . T . . A . . . . . C . . A . . A . . G T . G . . T . . C : 519
Leporinus friderici KM897272        441 : . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . T . . . . . G . . . . . . . . . . . T . . G . . A . . A . . T . . T . . . . . C . . A . . A : 519
Leporinus friderici KM897166        441 : . . . . . . . . . . . . . . . . . . . . . . . C . . T . . . . . T . . . . . G . . . . . . . . . . . T . . G . . A . . A . . T . . T . . . . . C . . A . . A : 519
Leporinus friderici KM897149        441 : . . . . . . . . . . . . . . . . . . . . . . . C . . T . . . . . T . . . . . G . . . . . . . . . . . T . . G . . A . . A . . T . . T . . . . . C . . A . . A : 519
Leporinus friderici KM897144        441 : . . . . . . . . . . . . . . . . . . . . . . . C . . T . . . . . T . . . . . G . . . . . . . . . . . T . . G . . A . . A . . T . . T . . . . . C . . A . . A : 519
Apareiodon affinis JN988674         474 : . . G . . . . . T . . G . . . . . C . . . . . C . . . T . . . . T . . A . . . . . . . . . . . . . . T . . A . . C . . C . . . . . A . . . . . C . . T T . A : 552

Pygocentrus nattereri AP012000      520 : C C A G T C C T G G C C G C C G G G A T T A C A A T A C T T C T T A C A G A T C G G A A C C T A A A C A C C A C A T T C T T T G A C C C C G C G G G A G G G : 598
Pygocentrus nattereri PH01 KX396057 523 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 601
Pygocentrus nattereri PH02 KX396058 429 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 507
Pygocentrus nattereri PH03 KX396059 522 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 600
Piaractus brachypomus KP723348      504 : . . . . . T . . . . . T . . T . . A . . . . . C . . . . . . . . G . . . . . . . . A . . . . . T . . . . . . . . . . . . . . . . . . . . . . . A . . G . A . : 582
Pygocentrus piraya HM405211         520 : . . . . . T . . A . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . : 598
Serrasalmus marginatus JN989232     520 : . . . . . T . . A . . . . . . . . A . . C . . . . . G . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . A . . . . . A : 598
Serrasalmus marginatus JN989233     520 : . . . . . T . . A . . . . . . . . A . . C . . . . . G . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . A . . . . . A : 598
Pygocentrus piraya HQ600844         520 : . . . . . T . . A . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . - - - : 598
Serrasalmus marginatus GU701508     520 : . . . . . T . . A . . . . . . . . A . . C . . . . . G . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . A . . . . . A : 598
Serrasalmus marginatus GU701507     520 : . . . . . T . . A . . . . . . . . A . . C . . . . . G . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . A . . . . . A : 598
Pygocentrus piraya HQ600843         520 : . . . . . T . . A . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . - - - : 598
Pygocentrus piraya HQ600849         520 : . . . . . T . . A . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - - - - - - - : 598
Piaractus mesopotamicus GU701417    520 : . . . . . T . . . . . T . . T . . A . . . . . T . . . . . . . . G . . . . . . . . A . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . G . . A : 598
Piaractus mesopotamicus KM897294    520 : . . . . . T . . . . . T . . T . . A . . . . . T . . . . . . . . G . . . . . . . . A . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . G . . A : 598
Piaractus mesopotamicus GU701416.1  520 : . . . . . T . . . . . T . . T . . A . . . . . T . . . . . . . . G . . . . . . . . A . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . G . . A : 598
Piaractus mesopotamicus KM897305    520 : . . . . . T . . . . . T . . T . . A . . . . . T . . . . . . . . G . . . . . . . . A . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . G . . A : 598
Piaractus mesopotamicus KM897414    520 : . . . . . T . . . . . T . . T . . A . . . . . T . . . . . . . . G . . . . . . . . A . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . G . . A : 598
Trichomycterus giganteus GU702303   520 : . . T . . . . . . . . T . . A . . . . . . . . . . . G . . . . . A . . . . . C . . C . . . . . G . . . . . A . . . . . . . . . . . . . . A . . T . . C . . A : 598
Trichomycterus giganteus GU702165   520 : . . T . . . . . . . . T . . A . . . . . . . . . . . G . . . . . A . . . . . C . . C . . . . . G . . . . . A . . . . . . . . . . . . . . A . . T . . C . . A : 598
Trichomycterus giganteus GU702160   520 : . . T . . . . . . . . T . . A . . . . . . . . . . . G . . . . . A . . . . . C . . C . . . . . G . . . . . A . . . . . . . . . . . . . . A . . T . . C . . A : 598
Trichomycterus giganteus GU702140   520 : . . T . . . . . . . . T . . A . . . . . . . . . . . G . . . . . A . . . . . C . . C . . . . . G . . . . . A . . . . . . . . . . . . . . A . . T . . C . . A : 598
Leporinus friderici KM897272        520 : . . C . . T . . A . . T . . . . . A . . . . . T . . . . . . . . A . . . . . C . . A . . T . . T . . T . . . . . . . . . . . . . . . . . G . . A . . . . . A : 598
Leporinus friderici KM897166        520 : . . C . . T . . A . . T . . . . . A . . . . . T . . . . . . . . A . . . . . C . . A . . T . . T . . T . . . . . . . . . . . . . . . . . G . . A . . . . . A : 598
Leporinus friderici KM897149        520 : . . C . . T . . A . . T . . . . . A . . . . . T . . . . . . . . A . . . . . C . . A . . T . . T . . T . . . . . . . . . . . . . . . . . G . . A . . . . . A : 598
Leporinus friderici KM897144        520 : . . C . . T . . A . . T . . . . . A . . . . . T . . . . . . . . A . . . . . C . . A . . T . . T . . T . . . . . . . . . . . . . . . . . G . . A . . . . . A : 598
Apareiodon affinis JN988674         553 : . . T . . G . . . . . T . . G . . A . . . . . C . . G . . . . . A . . . . . C . . A . . . . . C . . . . . . . . . . . T - - - - - - - - - - - - - - - - - - : 614

Pygocentrus nattereri AP012000      599 : G G A G A C C C A A T C C T T T - A C C A A C A C C T A T T C T G G T T C T T T G G T C A C C C - - : 646
Pygocentrus nattereri PH01 KX396057 602 : . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . A . . . . . C . . C A C . . . C T : 653
Pygocentrus nattereri PH02 KX396058 508 : . . . . . . . . . . . . . . . . - . . . . . . . - - - - - - - - - - - - - - - - - - - - - - - - - - : 531
Pygocentrus nattereri PH03 KX396059 601 : . . . . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . A . . - - - - - - - - - - - - - - : 636
Piaractus brachypomus KP723348      583 : . . . . . . . . . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - : 592
Pygocentrus piraya HM405211         599 : . . . . . . . . . . . . . . . . A C . A . C A C . T A - - - - - - - - - - - - - - - - - - - - - - - : 650
Serrasalmus marginatus JN989232     599 : . . G . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . A . . . . . C . . . . . . . . - - : 646
Serrasalmus marginatus JN989233     599 : . . G . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . A . . . . . C - - - - - - - - - - : 639
Pygocentrus piraya HQ600844               - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Serrasalmus marginatus GU701508     599 : . . G . . . . . . . . . . . . . - . . . . . . . . . . . - - - - - - - - - - - - - - - - - - - - - - : 626
Serrasalmus marginatus GU701507     599 : . . G . . . . . . . . . . . . . - . . . . . . . . . . . . . . . . A - - - - - - - - - - - - - - - - : 632
Pygocentrus piraya HQ600843               - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Pygocentrus piraya HQ600849               - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Piaractus mesopotamicus GU701417    599 : . . . . . . . . . . . T . . C . - . . . . . . . - - - - - - - - - - - - - - - - - - - - - - - - - - : 622
Piaractus mesopotamicus KM897294    599 : . . . . . . . . . . . T . . C . - . . . . . . . . - - - - - - - - - - - - - - - - - - - - - - - - - : 623
Piaractus mesopotamicus GU701416.1  599 : . . . . . . . . . . . T . . C . - . . . . . . . - - - - - - - - - - - - - - - - - - - - - - - - - - : 622
Piaractus mesopotamicus KM897305    599 : . . . . . . . . . . . T . . C . - . . . . . . . . - - - - - - - - - - - - - - - - - - - - - - - - - : 623
Piaractus mesopotamicus KM897414    599 : . . . . . . . . . . . T . . C . - . . . . . . . - - - - - - - - - - - - - - - - - - - - - - - - - - : 622
Trichomycterus giganteus GU702303   599 : . . . . . . . . . . . T . . A . - . . . . . . . . . . G . . . - - - - - - - - - - - - - - - - - - - : 629
Trichomycterus giganteus GU702165   599 : . . . . . . . . . . . T . . A . - . . . . . . . . . . - - - - - - - - - - - - - - - - - - - - - - - : 626
Trichomycterus giganteus GU702160   599 : . . . . . . . . . . . T . . A . - . . . . . . . . . . - - - - - - - - - - - - - - - - - - - - - - - : 626
Trichomycterus giganteus GU702140   599 : . . . . . . . . . . . T . . A . - . . . . . . . . . . - - - - - - - - - - - - - - - - - - - - - - - : 650
Leporinus friderici KM897272        599 : . . . . . . . . . . . T . . C . - . . . . . . . . - - - - - - - - - - - - - - - - - - - - - - - - - : 623
Leporinus friderici KM897166        599 : . . . . . . . . . . . T . . C . - . . . . . . . . . . . - - - - - - - - - - - - - - - - - - - - - - : 626
Leporinus friderici KM897149        599 : . . . . . . . . . . . T . . C . - . . . . . . . . - - - - - - - - - - - - - - - - - - - - - - - - - : 623
Leporinus friderici KM897144        599 : . . . . . . . . . . . T . . C . - . . . . . . . . - - - - - - - - - - - - - - - - - - - - - - - - - : 623
Apareiodon affinis JN988674               - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Figure 3. Complete multiple alignments of juvenile fish CO1 sequences together with reference sequences. GenBank accession numbers are shown next to species names. Similar nucleotide sites are illustrated by dots and gaps or missing data are denoted by dashes.

Further analysis was carried out by computing genetic distances of the samples with the reference species (Table 1). Threshold value was set a 3.0% - 3.5% for species delineation among the included sequences (Ward et al. 2009). Genetic distances within the three CO1 sequences from the samples were observed at 0.0% - 1.1% (Table 1). Similar genetic distance range was also observed between the sample sequences and CO1 sequence from P. nattereri. These distances fall below the threshold value and are very low as compared to the genetic distance computed for the other reference samples, suggesting that they are the same species. In addition, the distance between the samples and P. brachypomus, were observed at 19.0% - 20.7% (Table 1) which are above the threshold value of 3.5% suggesting that the samples and Red pacu are two distinct species. The result of genetic distance analysis strongly suggests that the samples are P. nattereri. There were 482 positions in the final dataset for both the K2P genetic distance computation and generation of NJ tree. All positions containing gaps and missing data were eliminated for the generation of NJ tree and K2P genetic distance computation.

Table 1. Computed K2P pairwise genetic distances of piranha samples with reference sequences. GenBank accession numbers are shown next to species names.
  1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
1Pygocentrus nattereri AP012000 0.0000.0050.0000.0270.0080.0100.0100.0080.0100.0100.0080.0080.0280.0280.0280.0280.0280.0250.0250.0250.0250.0290.0310.0300.0300.036
2Pygocentrus nattereri PH01 KX3960570.000 0.0050.0000.0270.0080.0100.0100.0080.0100.0100.0080.0080.0280.0280.0280.0280.0280.0250.0250.0250.0250.0290.0310.0300.0300.036
3Pygocentrus nattereri PH02 KX3960580.0110.011 0.0050.0270.0090.0120.0120.0090.0110.0120.0100.0100.0290.0290.0290.0290.0290.0260.0270.0260.0260.0300.0320.0310.0310.037
4Pygocentrus nattereri PH03 KX3960590.0000.0000.011 0.0270.0080.0100.0100.0080.0100.0100.0080.0080.0280.0280.0280.0280.0280.0250.0250.0250.0250.0290.0310.0300.0300.036
5Piaractus brachypomus KP7233480.1900.1900.2070.190 0.0270.0280.0280.0270.0290.0280.0260.0260.0100.0090.0090.0090.0100.0300.0300.0300.0300.0360.0360.0360.0360.036
6Pygocentrus piraya HM4052110.0290.0290.0400.0290.190 0.0100.0100.0020.0100.0100.0030.0030.0280.0290.0290.0290.0290.0290.0290.0290.0290.0290.0300.0290.0290.036
7Serrasalmus marginatus JN9892320.0450.0450.0570.0450.2050.045 0.0000.0100.0020.0000.0100.0100.0290.0300.0300.0300.0300.0270.0270.0270.0270.0290.0310.0300.0300.038
8Serrasalmus marginatus JN9892330.0450.0450.0570.0450.2050.0450.000 0.0100.0020.0000.0100.0100.0290.0300.0300.0300.0300.0270.0270.0270.0270.0290.0310.0300.0300.038
9Pygocentrus piraya HQ6008440.0260.0260.0370.0260.1870.0020.0430.043 0.0090.0100.0020.0020.0280.0280.0280.0280.0280.0290.0290.0290.0290.0280.0290.0290.0290.037
10Serrasalmus marginatus GU7015080.0430.0430.0540.0430.2090.0430.0020.0020.040 0.0020.0100.0100.0300.0300.0300.0300.0300.0270.0270.0270.0270.0290.0300.0300.0300.038
11Serrasalmus marginatus GU7015070.0450.0450.0570.0450.2050.0450.0000.0000.0430.002 0.0100.0100.0290.0300.0300.0300.0300.0270.0270.0270.0270.0290.0310.0300.0300.038
12Pygocentrus piraya HQ6008430.0290.0290.0400.0290.1830.0040.0450.0450.0020.0430.045 0.0000.0270.0270.0270.0270.0270.0290.0290.0290.0290.0280.0290.0280.0280.037
13Pygocentrus piraya HQ6008490.0290.0290.0400.0290.1830.0040.0450.0450.0020.0430.0450.000 0.0270.0270.0270.0270.0270.0290.0290.0290.0290.0280.0290.0280.0280.037
14Piaractus mesopotamicus GU7014170.1960.1960.2130.1960.0400.1960.2110.2110.1930.2150.2110.1890.189 0.0030.0030.0030.0030.0320.0320.0320.0320.0340.0350.0340.0340.032
15Piaractus mesopotamicus KM8972940.2010.2010.2180.2010.0350.2010.2160.2160.1970.2200.2160.1930.1930.004 0.0000.0020.0030.0320.0310.0320.0320.0340.0350.0340.0340.032
16Piaractus mesopotamicus GU701416.10.2010.2010.2180.2010.0350.2010.2160.2160.1970.2200.2160.1930.1930.0040.000 0.0020.0030.0320.0310.0320.0320.0340.0350.0340.0340.032
17Piaractus mesopotamicus KM8973050.2040.2040.2210.2040.0380.2040.2190.2190.2000.2230.2190.1960.1960.0060.0020.002 0.0030.0320.0310.0320.0320.0340.0350.0340.0340.032
18Piaractus mesopotamicus KM8974140.2030.2030.2200.2030.0400.2030.2180.2180.2000.2220.2180.1960.1960.0040.0040.0040.006 0.0320.0320.0320.0320.0340.0350.0340.0340.032
19Trichomycterus giganteus GU7023030.2050.2050.2220.2050.2410.2350.2170.2170.2310.2170.2170.2350.2350.2530.2490.2490.2520.253 0.0030.0000.0000.0290.0310.0300.0300.031
20Trichomycterus giganteus GU7021650.2050.2050.2220.2050.2330.2350.2170.2170.2310.2170.2170.2350.2350.2450.2400.2400.2440.2440.004 0.0030.0030.0290.0310.0300.0300.031
21Trichomycterus giganteus GU7021600.2050.2050.2220.2050.2410.2350.2170.2170.2310.2170.2170.2350.2350.2530.2490.2490.2520.2530.0000.004 0.0000.0290.0310.0300.0300.031
22Trichomycterus giganteus GU7021400.2050.2050.2220.2050.2410.2350.2170.2170.2310.2170.2170.2350.2350.2530.2490.2490.2520.2530.0000.0040.000 0.0290.0310.0300.0300.031
23Leporinus friderici KM8972720.2200.2200.2380.2200.2700.2120.2270.2270.2080.2230.2270.2050.2050.2570.2540.2540.2530.2560.2370.2300.2370.237 0.0050.0030.0030.033
24Leporinus friderici KM8971660.2310.2310.2490.2310.2780.2230.2380.2380.2190.2340.2380.2150.2150.2640.2610.2610.2600.2630.2520.2440.2520.2520.011 0.0030.0030.035
25Leporinus friderici KM8971490.2240.2240.2420.2240.2700.2160.2320.2320.2120.2270.2320.2080.2080.2570.2540.2540.2530.2560.2450.2370.2450.2450.0060.004 0.0000.034
26Leporinus friderici KM8971440.2240.2240.2420.2240.2700.2160.2320.2320.2120.2270.2320.2080.2080.2570.2540.2540.2530.2560.2450.2370.2450.2450.0060.0040.000 0.034
27Apareiodon affinis JN9886740.2800.2800.2920.2800.2660.2760.2930.2930.2800.2890.2930.2850.2850.2510.2480.2480.2470.2510.2580.2540.2580.2580.2540.2690.2670.267

Red piranha or P. nattereri is primarily utilized as an aquarium pet fish (Singh and Lakra 2011; Guerrero 2014). It is known to prey on smaller fish, aquatic insects, mollusks and crustaceans; hence, it is considered as opportunistic and omnivorous feeders. These characids usually thrive in moving freshwater, such as rivers and streams, in tropical areas (Saint-Paul et al. 2000). Breeding season of red piranhas was observed during the wet season, particularly, when the water level is high and when flooding occurs (Uetanabaro et al. 1993). It is considered as the most aggressive species of piranha because it tends to compete for food during foraging with other fish species in its area (Nico et al. 2015). Red piranhas are considered as potential invasive species receiving a medium risk score of 19 according to Fish Invasiveness Scoring Kit (FISK) (Almeida et al. 2013). In this regard, P. nattereri has been banned in various countries, including the Philippines.

Breeding of red piranhas in captivity was reported in 1994 at the Dallas Aquarium wherein red piranhas are placed in a 2,000- gallon (7,600 L) aquarium (Schleser 1997). The capability of piranhas to breed in a controlled environment suggests its possible adaptation in aquariums in the Philippines. In this regard, juvenile piranhas are more preferred to be imported because of the difficulty in immediate identification and often interchangeable with pacu.

The current case of the presence of red piranha in the country calls out the attention for strengthening measures of assuring species identification since the Philippines have a wide marine and freshwater area to protect. Particularly, there are numerous rivers and streams present within the country, therefore release of these piranhas in the Philippine waters may cause chaos in our ecological biodiversity.

Species transfer from one country to another is considered as a rising international concern not only due to the high probability of diseases introduction, but also its possible disruptive effects on aquatic communities (de Silva 1989). In line with this, policies are carried out to be able to control the entrance of possible invasive species that may harm the natural fauna in the Philippine environment. DNA barcoding allows rapid and more accurate method for species identification, thereby providing reliable data regarding species to monitor and hold. The confirmation of the identity of fish samples in this study supports the prevention measures regarding the introduction of exotic fishes in the Philippines. DNA barcoding serves to be an effective tool in monitoring entrance of alien species contributing to increased efforts in wildlife conservation.


ACKNOWLEDGEMENTS

Authors would like to thank the Manila Police District - District Special Operations Unit (MPD-DSOU) for entrusting the confiscated samples to NFRDI through its Genetic Fingerprinting Laboratory (GFL). Also, we extend our sincerest thanks to the members of the GFL for the utmost support.


CONFLICTS OF INTEREST

The authors would like to acknowledge the National Fisheries Research and Development Institute (NFRDI) 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.


CONTRIBUTIONS OF INDIVIDUAL AUTHORS

Sarmiento wrote the manuscript. Sarmiento and Ventolero conducted laboratory experiments, particularly DNA extraction, PCR amplification, and analysis of DNA sequences. Santos provided overall concept of the paper, supervised the experiments and edited the manuscript.


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STRIDE Grants The next call for Grants will commence on February 27, 2015. To know more about the Grants program of STRIDE, you may visit our website at stride.org.ph.

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