Genetic identification of the sharks Rhizoprionodon porosus and R. lalandii by PCR-RFLP and nucleotide sequence analyses of 5S rDNA

  • Danillo Pinhal
  • Otto B. F. Gadig
  • Cesar Martins
Technical Note

Abstract

A molecular approach based on nuclear 5S rDNA sequence variability was applied successfully to correctly identify samples from the two Rhizoprionodon species collected in the wild or sold in markets. The sequence of the non-transcribed spacer (NTS) of the 5S rDNA showed high interspecific variability and no intraspecific polymorphism, making it a useful marker for sharpnose shark identification. Polymorphisms in the NTS sequences of Rhizoprionodon sharks also created unique restriction patterns for each species after PCR-RFLP analysis. The 5S rDNA polymorphism represents a fast and non expensive tool to access species identification when rapid and unequivocal identification of shark products is needed, particularly for future management and other investigations.

Keywords

Genetic markers Non-transcribed spacer Trade monitoring Sharks Species identification 

Introduction

Rhizoprionodon is a genus of the family Carcharhinidae represented worldwide by seven species of small coastal sharks (Compagno 1984) that together form the basis of important commercial and recreational fisheries, as well as substantial artisanal fisheries (Motta et al. 2005). In Brazilian coastal waters, two species have been identified: Rhizoprionodon lalandii (Brazilian sharpnose shark) and Rhizoprionodon porosus (Caribbean sharpnose shark). Few biological studies are currently available for these species and they are decreasing in number through overfishing in Brazil over the last decade (Lessa et al. 2005).

Sharks are not usually sold as whole animals, but as carcasses or processed in the form of fillets, making it difficult to identify habitual commercialized species such as those of genus Rhizoprionodon. Although R. lalandii and R. porosus have well-defined species boundaries, their natural morphological similarity makes it difficult to distinguish between them, even when the fish are still intact. However, the common fishery practice of removing head, tail, and most fins from sharks while still at sea in order to reduce required storage space for the captured animals, also called finning, removes such major morphological identifying characteristics. This limits the precise identification of each species and, consequently, results in problems regarding their proper management (Shivji et al. 2002). In this way, we have investigated the genomic organization 5S rDNA in order to identify genetic variation patterns that could be used to distinguish the two sharpnose sharks R. lalandii and R. porosus.

Materials and methods

Tissue samples were collected from fresh, frozen or ethanol-preserved specimens caught by commercial fisheries and from frozen fillets of Rhizoprionodon sharks sold in markets from six distinct sampling sites along the Brazilian coast (Fig. 1). Frozen tissues were also obtained from two Galeocerdo cuvier and Alopias superciliosus individuals for comparative analysis.
Fig. 1

Map with the sample sites of the four shark species evaluated in this study. Numbers indicate sample sizes from each location

Genomic DNA was extracted from the fin clip, gills or muscles of fresh, frozen or ethanol-preserved tissues (Aljanabi and Martinez 1997) to assess the best tissue for DNA extraction. DNA was also isolated from frozen fillets to test the use of this sample kind. An elasmobranch specific set of primers Cart5S1F (5′-CAC GCC CGA TCC CGT CCG ATC-3′) and Cart5S1R (5′-CAG GCT AGT ATG GCC ATA GGC-3′), based on the nucleotide sequences of the 5S rRNA gene from the skate Taeniura lymma (AY278251) and the shark Scyliorhinus caniculus (M24954), recovered in GenBank, were designed and used to amplify the repeat units of 5S rDNA that include the 5S rRNA gene and the non-transcribed spacer (NTS) of all sharks samples (Pinhal et al. 2009). PCR-amplified products were cloned into the plasmid pGEM-T (Promega) and used to transform competent cells of E. coli, DH5α strain (Invitrogen). The positive clones were sequenced on an ABI Prism 3100 automatic DNA Sequencer (Applied Biosystems), following the manufacturer’s instructions.

Nucleic acid sequences were subjected to BLASTN (Altschul et al. 1990) searches at the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/blast), and the sequence alignment was performed using the computer program BioEdit (Hall 1999) and checked manually. Neighbor- Joining (NJ) and Maximum Parsimony (MP) phylogenetic analyses employing the Kimura-2-parameter genetic distance model (Kimura 1980) were conducted using the software MEGA 3.1 (Kumar et al. 2004). Nucleotide sequences of the NTSs from R. pororus and R. lalandii were analyzed using the software NEBcutter version 2.0 (Vincze et al. 2003) to recognize specific restriction sites for each species. Digestion of PCR products with selected endonucleases were performed in 20 μl volumes with 10 μl amplified DNA, 5 U of enzyme and 2 μl of the recommended 10× digestion buffer. Reactions were incubated for 4 h at 37°C.

Results

PCR products of 5S rDNA repeats were successfully obtained and generated agarose gel bands of ~463 bp for R. lalandii and R. porosus, ~500 bp for G. cuvier, and ~990 bp for A. superciliosus. Comparative analysis of 5S rDNA sequences acquired from the four shark species confirmed a highly conserved region (98% similarity) corresponding to the 5S rRNA gene (120 bp) and a variable NTS. Unexpectedly, the NTSs sequences of Rhizoprionodon species retain a very high conservation level (about 97% of nucleotide identity), indicating that R. lalandii and R. porosus have a very short evolutionary time of divergence. When the NTSs of Rhizoprionodon sharks were compared to G. cuvier, this identity decreased to about 71%, and it further decreased to 39% compared to A. superciliosus. Spacer sequences of G. cuvier were 28% similar to those of A. superciliosus. A very low level of divergence was found in the NTS of the two Rhizoprionodon species independent of sample origin and site collection. However, intraspecific sequence polymorphisms occurred in all populations assessed from both species. It was possible to detect 29 polymorphic sites for the 32 NTS sequences analyzed. Of all the polymorphic sites, those at positions 98, 211, 254, 337 and 338 were species-specific. These five polymorphic sites, representing a low but consistent interspecific variability in NTS sequences, were sufficient to separate all R. lalandii from R. porosus in 99% of the recovered NJ and MP trees (Fig. 2). Furthermore, intraspecific base divergences in NTSs could not be correlated with sample origin, since it was found that in some cases there was greater similarity between sequences of individuals from distinct localities than those belonging to the same locality. This result shows that polymorphisms detected in the NTS of sharpnose sharks are not biased by sample site and thus are an informative species diagnostic.
Fig. 2

Trees of maximum parsimony (a) and neighbor joining (b) from NTS sequences of R. lalandii (RL) and R. porosus (RP). Branch lengths are proportional to evolutionary distance (scale bar), and the numbers at each node indicate the percentage recovery (>60%) of the particular node (1,000 bootstrap replicates) in which the same internal branch was recovered

The restriction site assessment by PCR-RFLP allowed the characterization of enzymes that had cleavage in only one species. Hence, 30 enzymes were characterized with particular cleavage sites in the R. porosus NTS sequences (PleI, AciI, BfaI, EcoP15I, PspGI, BstNI, FaiI, MnlI, BssSI, StyD4I, BssKI, PspGI, BstNI, ScrFI, AflIII, Bsp1286I, BsiHKAI, HpyCH4IV, PmlI, BmgBI, BsaAI, TstI, MboII, BbsI, AlwNI, BsaHI, ZraI, Hpy99I, MnlI and HpyCH4V), while 8 enzymes presented only cleavage in R. lalandii (EcoRV, HpyAV, BsmFI, AvaII, PpuMI, HinP1I, HpyCH4III and HhaI). Several of these enzymes were checked and always produced fragments of different sizes between the species (Fig. 3), undoubtedly distinguishing the two sharpnose species.
Fig. 3

PCR-RFLP profiles of the 5S rDNA amplicons digested with EcoRV. Rhizoprionodon porosus samples: lanes 1, 2, 3, 4; and R. lalandii samples: 5, 6, 7, 8; M molecular weight marker in bp

Discussion

True identification of species is a prerequisite for forensic investigations regarding species management and conservation as well as in taxonomic/systematic studies (Heist and Gold 1999). Among the many nuclear and mitochondrial markers so far studied, the 5S rDNA is of special interest in species identification because of its inherent characteristics, which make it an ideal species-specific marker in higher eukaryotes. The fact that the organization of 5S rDNA presents rapid rates of base substitution, no intraspecific polymorphism, and high interspecific variability makes it a very good candidate for comparison of close related species, such as Rhizoprionodon sharks (Pinhal et al. 2009).

In the present study, we used DNA sequencing and PCR- restriction length fragment techniques to discriminate between congeners Rhizoprionodon species. Previous 5S rDNA data have reported that sequence polymorphisms in the ribosomal NTS were sufficient to distinguish subspecies of mice Mus musculus domesticus from M. m. musculus (Suzuki et al. 1994), suggesting that this region was evolving at an appropriate rate for distinguishing close related species. In the same way, the NTS region has been extensively used for fish species discrimination, either through a simple PCR amplification analysis (Cespedes et al. 1999; Pendás et al. 1995; Pinhal et al. 2008) or in combination with PCR-RFLP analysis (Aranishi et al. 2005; Carrera et al. 2000). NTSs seem to bear consistently low intraspecific variability and high interspecific variability independent of the vertebrate group analyzed. Our data show that the NTS spacers of sharks seem to evolve as quickly as those of mice and other fish, since their polymorphisms allowed differentiation between congeners, representing the first report of genetic data analysis of 5S rDNA applied to Caribbean and Brazilian sharpnose sharks identification. In addition, the PCR-RFLP provides a practical and rapid species-specific diagnostic for these species and enlarges the forensic potential of NTSs as molecular markers.

Techniques here applied, 5S rDNA sequencing and PCR-RFLP, permit the tracking of two economically important species: the Brazilian sharpnose shark and the Caribbean sharpnose shark, currently one of the most exploited and common traded species of sharks. In addition, the analysis of relationships between such close related species by means of a species-specific element may be useful to infer evolutionary relationships between other members of the elasmobranch group. Given the relatively ancient phylogenetic radiation of elasmobranch species, and the consequent high levels of interspecific differentiation, the use of 5S rDNA sequences as molecular markers, particularly the highly polymorphic NTS regions, can provide secure and easily reproducible data. Thus, both techniques represent an efficient way to differentiate the two closely related shark species and can be used to reinforce morphological identification, allowing effective traceability of Rhizoprionodon sharks in future management studies.

Notes

Acknowledgment

The authors are grateful to Fundação de Amparo à Pesquisa do Estado de São Paulo for financial support.

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Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Danillo Pinhal
    • 1
  • Otto B. F. Gadig
    • 2
  • Cesar Martins
    • 1
  1. 1.Departamento de Morfologia, Instituto de BiociênciasUNESP—Universidade Estadual PaulistaBotucatuBrazil
  2. 2.Campus Litoral Paulista, UNESP—Universidade Estadual PaulistaSão VicenteBrazil

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