- Email: [email protected]
Probing the potential of the Shark Panel InDel multiplex v2.0 on the forensic identification of batoid elasmobranchs
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/fsigss
Probing the potential of the Shark Panel InDel multiplex v2.0 on the forensic identification of batoid elasmobranchs ⁎
C.R.L. Amarala, , A. Bitencourta, C. Teixeiraa, F. Pereirab, D.A. Silvaa, A. Amorimc, E.F. Carvalhoa a
Laboratório de Diagnósticos por DNA, Universidade do Estado do Rio de Janeiro, Brazil Interdisciplinary Centre of Marine and Environmental Research, Universidade do Porto, Porto, Portugal c Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP)/Instituto de Investigação e Inovação em Saúde, Universidade do Porto/Faculdade de Ciências, Universidade do Porto, Portugal b
A R T I C L E I N F O
A B S T R A C T
Keywords: Elasmobranchii Batoids 16SrRNA InDels
The Elasmobranchii comprises the diverse and important group of sharks and rays. The group includes some of the ocean’s largest predatory fishes, suffering unsustainable fishing activities and is commercially overexploited for their meat and fins. Overfishing has resulted in significant population declines and several species are now considered under high threat and facing extinction, with about 93% of its nominal species included in the IUCN Red List. Molecular data have provided important information about these species, allowing the management of natural stocks and preventing their decline. Population genetics and connectivity data knowledge are now available and play an important role on establishing conservation policies. However, despite the ecological, commercial and conservation importance, no molecular method is available to identify sharks and rays in a forensic context. Following the development of the Shark Panel v1.0 which includes uniquely sharks, now we tested the effectiveness of the v2.0 multiplex on batoid elasmobranchs. We carried out a systematic molecular analysis using 85 previously published mitochondrial 16S rRNA gene sequences obtained from the NCBI database and found that indels in the 16S rRNA gene can be used to distinguish several analyzed species, including some of the most threatened according to IUCN Red List. The regions selected in this study can be used for the construction of molecular identification assays.
1. Introduction Representing one of the most sucessful vertebrate lineages with an evolutionary history dating back to the Devonian-Silurian boundary [1], the Elasmobranchii comprise the diverse and important group of sharks and rays. The Elasmobranchii is the most diverse clade of large predatory fishes with about 1200 species spread all over the world’s seas [2], including ocean- and freshwater-dwelling fishes, such as sharks, skates and rays, which play an important ecological role in marine ecosystems [3,4]. As a consequence of unsustainable fishing activities, a resultant population decline related to several elasmobranch species can be foreseen. Fished for their meat and fins, several species are considered under high threat and are now facing extinction [5], with several nominal species included on the IUCN Red List. Recent molecular analyses provided important information about several elasmobranch species, allowing the management of natural stocks and the establishing of conservation policies all around the world. However, despite the ecological, commercial and conservation importance, no robust
⁎
molecular method is available to identify batoid elasmobranchs. Following the development of the Shark Panel v1.0 [6], which includes uniquely sharks, now we tested the effectiveness of the v2.0 multiplex on batoid elasmobranchs. As for the first version of the multiplex, aiming the construction of a reliable method for batoid elasmobranchs identification, we carried out a molecular systematic analysis using previously published mitochondrial 16S rRNA gene sequences obtained from the NCBI database. 2. Methodology The database was constructed with 40 elasmobranch sequences of the 16S rRNA retrieved from the NCBI database. All sequences were aligned using the default parameters of KALIGN [7] and MUSCLE [8] running on XSEDE (Extreme Science and Engineering Discovery Environment) at CIPRES Science Gateway (http://www.phylo.org). The alignment was analyzed with the software Geneious v.4.8.2 and InDel regions were determined with the software SPInDel WorkBench [9].
Corresponding author. E-mail address: [email protected] (C.R.L. Amaral).
http://dx.doi.org/10.1016/j.fsigss.2017.09.106 Received 1 September 2017; Accepted 18 September 2017 1875-1768/ © 2017 Elsevier B.V. All rights reserved.
Please cite this article as: Amaral, C.R., Forensic Science International: Genetics Supplement Series (2017), http://dx.doi.org/10.1016/j.fsigss.2017.09.106
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
C.R.L. Amaral et al.
Fig. 1. 16S rRNA UPGMA tree of batoid elasmobranchs.
Fig. 2. 16S Maximum-Likelihood tree of batoid elasmobranchs.
3. Results and discussion
enforcement authorities still struggle when dealing with taxonomic uncertainties and fragmented/processed material. In this sense, trying to fill this gap and following the path for the Selachii species studied by [6], five hypervariable InDel regions were defined for the development of primers and now are being tested for several elasmobranch species including sharks and batoids. The 16S rRNA Maximum Likelihood (ML) and UPGMA trees that could be observed in Figs. 1 and 2clearly show that the mitochondrial 16S rRNA region is clearly able to identify all the analyzed batoid species and to reconstruct the overall phylogenetic history of the batoid clade.
As for several teleost fish species, batoids themselves represent a considerable fraction of the ornamental fish trade related with the elasmobranchs. Amazonian freshwater stingrays are one of the most traded and overexploited elasmobranchs, being freely commercialized in petshops and internet aquarium sites. Reliable species identification tools and systems, especially those for forensic purposes, are still unavailable for the vast majority of commercially important fish species, including batoid elasmobranchs. Mainly relying on DNA barcoding methods for molecular identification, both researchers and law
2
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
C.R.L. Amaral et al.
4. Conclusions
(NORTE-01-0145-FEDER-000031).
We found that InDels in the 16S rRNA gene can be used to distinguish all the analyzed batoid species. Five hypervariable InDel regions of the 16S rRNA gene were selected for flanking primers development and are now being tested for efficient elasmobranch species identification. As for the first version pof the multiplex, the selected regions can be used for the construction of molecular assays for batoid elasmobranchs identification in a forensic context with the use of the SPInDel methodology.
Conflict of interest None. References [1] S. Corrigan, L.B. Beheregaray, A recent shark radiation: molecular phylogeny, biogeography and speciation of wobbegong sharks (family: Orectolobidae), Mol. Phylogenet. Evol. 52 (2009) 205–216. [2] X. Vélez-Zuazo, I. Agnarsson, Shark tales: a molecular species-level phylogeny of sharks (Selachimorpha, Chondrichthyes), Mol. Phylogenet. Evol. 58 (2011) 207–217. [3] R.A. Myers, B. Worm, Rapid worldwide depletion of predatory fish communities, Nature 423 (2003) 280–283. [4] S. Libralato, V. Christensen, D. Pauly, A method for identifying keystone species in food web models, Ecol. Model. 195 (2005) 153–171. [5] W.D. Robbins, M. Hisano, S.R. Connolly, et al., Ongoing collapse of coral-reef shark populations, Curr. Biol. 16 (2006) 2314–2319. [6] F. Amaral, et al., The shark panel: an InDel multiplex for shark species identification, Forensic Sci. Int. Genet. Suppl. Ser. 5 (2015) e430–e432. [7] T. Lassmann, E.L.L. Sonnhammer, Kalign – an accurate and fast multiple sequence alignment algorithm, BMC Bioinform. 6 (298) (2005) 1–9. [8] R.C. Edgar, MUSCLE: multiple sequence alignment with high accuracy and high throughput, Nucleic Acids Res. 32 (5) (2004) 1792–1797. [9] J. Carneiro, F. Pereira, A. Amorim, SPInDel: a multifunctional workbench for species identification using insertion/deletion variants, Mol. Ecol. Resour. 6 (2012) 1190–1195, http://dx.doi.org/10.1111/1755-0998.12011.
Role of fundings This study was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro, FEDER-Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT-Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Inovação in the framework of the project “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274). FP was supported by Investigator FCT (IF/01356/2012) and MARINFO
3
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/fsigss
Probing the potential of the Shark Panel InDel multiplex v2.0 on the forensic identification of batoid elasmobranchs ⁎
C.R.L. Amarala, , A. Bitencourta, C. Teixeiraa, F. Pereirab, D.A. Silvaa, A. Amorimc, E.F. Carvalhoa a
Laboratório de Diagnósticos por DNA, Universidade do Estado do Rio de Janeiro, Brazil Interdisciplinary Centre of Marine and Environmental Research, Universidade do Porto, Porto, Portugal c Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP)/Instituto de Investigação e Inovação em Saúde, Universidade do Porto/Faculdade de Ciências, Universidade do Porto, Portugal b
A R T I C L E I N F O
A B S T R A C T
Keywords: Elasmobranchii Batoids 16SrRNA InDels
The Elasmobranchii comprises the diverse and important group of sharks and rays. The group includes some of the ocean’s largest predatory fishes, suffering unsustainable fishing activities and is commercially overexploited for their meat and fins. Overfishing has resulted in significant population declines and several species are now considered under high threat and facing extinction, with about 93% of its nominal species included in the IUCN Red List. Molecular data have provided important information about these species, allowing the management of natural stocks and preventing their decline. Population genetics and connectivity data knowledge are now available and play an important role on establishing conservation policies. However, despite the ecological, commercial and conservation importance, no molecular method is available to identify sharks and rays in a forensic context. Following the development of the Shark Panel v1.0 which includes uniquely sharks, now we tested the effectiveness of the v2.0 multiplex on batoid elasmobranchs. We carried out a systematic molecular analysis using 85 previously published mitochondrial 16S rRNA gene sequences obtained from the NCBI database and found that indels in the 16S rRNA gene can be used to distinguish several analyzed species, including some of the most threatened according to IUCN Red List. The regions selected in this study can be used for the construction of molecular identification assays.
1. Introduction Representing one of the most sucessful vertebrate lineages with an evolutionary history dating back to the Devonian-Silurian boundary [1], the Elasmobranchii comprise the diverse and important group of sharks and rays. The Elasmobranchii is the most diverse clade of large predatory fishes with about 1200 species spread all over the world’s seas [2], including ocean- and freshwater-dwelling fishes, such as sharks, skates and rays, which play an important ecological role in marine ecosystems [3,4]. As a consequence of unsustainable fishing activities, a resultant population decline related to several elasmobranch species can be foreseen. Fished for their meat and fins, several species are considered under high threat and are now facing extinction [5], with several nominal species included on the IUCN Red List. Recent molecular analyses provided important information about several elasmobranch species, allowing the management of natural stocks and the establishing of conservation policies all around the world. However, despite the ecological, commercial and conservation importance, no robust
⁎
molecular method is available to identify batoid elasmobranchs. Following the development of the Shark Panel v1.0 [6], which includes uniquely sharks, now we tested the effectiveness of the v2.0 multiplex on batoid elasmobranchs. As for the first version of the multiplex, aiming the construction of a reliable method for batoid elasmobranchs identification, we carried out a molecular systematic analysis using previously published mitochondrial 16S rRNA gene sequences obtained from the NCBI database. 2. Methodology The database was constructed with 40 elasmobranch sequences of the 16S rRNA retrieved from the NCBI database. All sequences were aligned using the default parameters of KALIGN [7] and MUSCLE [8] running on XSEDE (Extreme Science and Engineering Discovery Environment) at CIPRES Science Gateway (http://www.phylo.org). The alignment was analyzed with the software Geneious v.4.8.2 and InDel regions were determined with the software SPInDel WorkBench [9].
Corresponding author. E-mail address: [email protected] (C.R.L. Amaral).
http://dx.doi.org/10.1016/j.fsigss.2017.09.106 Received 1 September 2017; Accepted 18 September 2017 1875-1768/ © 2017 Elsevier B.V. All rights reserved.
Please cite this article as: Amaral, C.R., Forensic Science International: Genetics Supplement Series (2017), http://dx.doi.org/10.1016/j.fsigss.2017.09.106
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
C.R.L. Amaral et al.
Fig. 1. 16S rRNA UPGMA tree of batoid elasmobranchs.
Fig. 2. 16S Maximum-Likelihood tree of batoid elasmobranchs.
3. Results and discussion
enforcement authorities still struggle when dealing with taxonomic uncertainties and fragmented/processed material. In this sense, trying to fill this gap and following the path for the Selachii species studied by [6], five hypervariable InDel regions were defined for the development of primers and now are being tested for several elasmobranch species including sharks and batoids. The 16S rRNA Maximum Likelihood (ML) and UPGMA trees that could be observed in Figs. 1 and 2clearly show that the mitochondrial 16S rRNA region is clearly able to identify all the analyzed batoid species and to reconstruct the overall phylogenetic history of the batoid clade.
As for several teleost fish species, batoids themselves represent a considerable fraction of the ornamental fish trade related with the elasmobranchs. Amazonian freshwater stingrays are one of the most traded and overexploited elasmobranchs, being freely commercialized in petshops and internet aquarium sites. Reliable species identification tools and systems, especially those for forensic purposes, are still unavailable for the vast majority of commercially important fish species, including batoid elasmobranchs. Mainly relying on DNA barcoding methods for molecular identification, both researchers and law
2
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
C.R.L. Amaral et al.
4. Conclusions
(NORTE-01-0145-FEDER-000031).
We found that InDels in the 16S rRNA gene can be used to distinguish all the analyzed batoid species. Five hypervariable InDel regions of the 16S rRNA gene were selected for flanking primers development and are now being tested for efficient elasmobranch species identification. As for the first version pof the multiplex, the selected regions can be used for the construction of molecular assays for batoid elasmobranchs identification in a forensic context with the use of the SPInDel methodology.
Conflict of interest None. References [1] S. Corrigan, L.B. Beheregaray, A recent shark radiation: molecular phylogeny, biogeography and speciation of wobbegong sharks (family: Orectolobidae), Mol. Phylogenet. Evol. 52 (2009) 205–216. [2] X. Vélez-Zuazo, I. Agnarsson, Shark tales: a molecular species-level phylogeny of sharks (Selachimorpha, Chondrichthyes), Mol. Phylogenet. Evol. 58 (2011) 207–217. [3] R.A. Myers, B. Worm, Rapid worldwide depletion of predatory fish communities, Nature 423 (2003) 280–283. [4] S. Libralato, V. Christensen, D. Pauly, A method for identifying keystone species in food web models, Ecol. Model. 195 (2005) 153–171. [5] W.D. Robbins, M. Hisano, S.R. Connolly, et al., Ongoing collapse of coral-reef shark populations, Curr. Biol. 16 (2006) 2314–2319. [6] F. Amaral, et al., The shark panel: an InDel multiplex for shark species identification, Forensic Sci. Int. Genet. Suppl. Ser. 5 (2015) e430–e432. [7] T. Lassmann, E.L.L. Sonnhammer, Kalign – an accurate and fast multiple sequence alignment algorithm, BMC Bioinform. 6 (298) (2005) 1–9. [8] R.C. Edgar, MUSCLE: multiple sequence alignment with high accuracy and high throughput, Nucleic Acids Res. 32 (5) (2004) 1792–1797. [9] J. Carneiro, F. Pereira, A. Amorim, SPInDel: a multifunctional workbench for species identification using insertion/deletion variants, Mol. Ecol. Resour. 6 (2012) 1190–1195, http://dx.doi.org/10.1111/1755-0998.12011.
Role of fundings This study was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro, FEDER-Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT-Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Inovação in the framework of the project “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274). FP was supported by Investigator FCT (IF/01356/2012) and MARINFO
3