Abstract

Dolphins and other cetacean are protected species whose capture is illegal in many countries. Despite the high mercury content accumulated by these animals, occasional episodes of illegal fishing occur to supply certain black markets with dolphin meat. When fishing authority inspections detect meat portions presumably of cetacean origin, it is particularly necessary to identify the species before taking the case to court. The mitochondrial cytochrome b gene has been used extensively to identify species. In forensic cases, due to the degradation of the samples, short amplicons are preferable. The universal cyt b L15601 and H15748 primers, previously tested in 63 species, have been used in this work on individuals of seven species of cetaceans: Balaenoptera physalus, Delphinus delphis, Globicephala melas, Phocoena phocoena, Stenella coeruleoalba, Tursiops truncatus and Ziphius cavirostris. These universal primers, with a short amplicon of 148 bp, have the advantage of identifying postmortem samples even when showing obvious signs of degradation. Our results showed that all tested species were correctly identified, except S. coeruleoalba and S. clymene. This is because S. clymene arose by hybrid speciation between S. coeruleoalba and S. longirostris. Therefore, their identification requires the analysis of both mitochondrial DNA and nuclear DNA. In summary, the identification of illegally obtained cetacean meat can be easily performed in forensic laboratories by analyzing a short fragment of the mitochondrial cyt b in all the studied species except in S. coeruleoalba and S. clymene and even when the remains were in an advanced stage of degradation.

Keywords1. Introduction

Cetacean species identification is highly useful in forensic, ecologic and food security cases. Several cetacean species and cetacean habitats have special protections to avoid uncontrolled fishing and conserve endangered species. Dolphin species have selling restrictions because of their high amount of mercury could be a health issue for human consumption. Illegal catches of cetaceans are usually discovered some time later, generally in pieces that lack the morphological characteristics necessary for the identification of the species. This typology of cases requires a genetic identification tool, capable to identify among cetaceans species in a fast, reliable and cost effective way.

The mitochondrial cytochrome b (cyt b) gene that has been widely used in forensic and taxonomic studies [[1]Hsieh H.M. Chiang H.L. Tsai L.C. et al.Cytochrome b gene for species identification of the conservation animals.]. This gene provides a highly accurate reconstruction, on the phylogeny of mammals, in the levels of super-order, order and family [[2]Tobe S.S. Kitchener A.C. Linacre A.M. Reconstructing mammalian phylogenies: a detailed comparison of the cytochrome B and cytochrome oxidase subunit I mitochondrial genes.]. The use of mini barcodes with sizes below 200 bp is recommended to perform successful amplifications [[3]Schneider P.M. Bender K. Mayr W.R. et al.STR analysis of artificially degraded DNA-results of a collaborative European exercise.]. Universal primers cyt b L15601 and H15748, with a short amplicon of 148 bp, have been probed to identify forensic samples, even when showing obvious signs of degradation.The objective of this study was based in developing a rapid, simple and cost-effective method able to identify genetically several cetacean species, even in forensic cases involving highly degraded samples. In order to achieve this goal, we will assay the identification of seven cetacean species using a single pair of universal primers previously probed in more than 60 species by Lopez-Oceja et al. 2016 [[4]Lopez-Oceja A. Gamarra D. Borragan S. et al.New cyt b gene universal primer set for forensic analysis.], amplifying and analysing by Sanger sequencing a highly conserved small region of 148 bp of the mitochondrial cyt b gene.2. Material and methods

Eleven individuals from a total of seven species belonging to four families of the order Artiodactyla were analyzed; Balaenoptera physalus (Balaenopteridae, n = 1), Phocoena phocoena (Phocoenidae, n = 2), Ziphius cavirostris (Ziphiidae, n = 1), Delphinus delphis (Delphinidae, n = 2), Globicephala melas (Delphinidae, n = 1), Stenella coeruleoalba (Delphinidae, n = 2) and Tursiops truncatus (Delphinidae, n = 2). Muscle tissues, with different degradation levels, were obtained from one fish market in Japan and strandings in beaches from Basque County, collected by AMBAR, Society for the study and conservation of marine fauna. For the extraction of muscle tissues, 5 mg were taken, and DNA was extracted by the salting out method, following the protocol of the Gentra Puregene Tissue Kit (Qiagen).

Cyt b gene L15601 and H15748 Universal primers were used. The primers amplificate highly preserved areas to maximize the amplification and have been previously probed by Lopez-Oceja et al. 2016 [[3]Schneider P.M. Bender K. Mayr W.R. et al.STR analysis of artificially degraded DNA-results of a collaborative European exercise.] in 63 species belonging to 38 Families from 14 Orders and 5 different Classes. Forward primer L15601: 5´-TACGCAATCCTACGATCAATTCC-3´ and reverse primer H15748: 5´-GGTTGTCCTCCAATTCATGTTAG-3´.

PCR amplification was optimized under the following conditions: 1.5 mL of dNTPs (2.5 mM), 1 mL of Mg Cl2 (50 mM), 2.5 mL of buffer (10), 0.6 mL of each primer at a 5 mM concentration, 1 mL of bovine serum albumin (BSA, 10 ng/mL), and 0.3 mL of Taq polymerase (5 U/mL); sterile water was used to bring it to a final volume of 20 mL, and 5 mL of DNA (1 ng/mL) were added in a Thermal Cycler C1000 (Bio-RadLab., Hercules, CA) under the following conditions: initial denaturalization for 11 min at 95 oC; 32 cycles of 30 s at 95 oC, 30 s at 50 oC, and 45 s at 72 oC; then a final extension for 5 min at 72 oC. Direct cycle sequencing was performed with the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and cyt b primers L15601 and H15748. The amplified DNA products were sequenced on an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems).

The sequences were edited with Chromas Pro v.1.34 software (Technelysium Pty Ltd.). Once edited, they were aligned using Clustal X v.2.0.11 [[5]Larkin M.A. Blackshields G. Brown N.P. et al.Clustal W and Clustal X version 2.0.]. The edited sequences were loaded in GenBank with accession numbers MN330613-MN330623. NJ phylogenetic trees were constructed using the FigTree v.1.4.2 software (tree.bio.ed.ac.uk/software/).3. Results and discussion

Samples from seven species of four cetacean families where analyzed. Cyt b gene universal primers L15601 and H15748 were capable to amplify and sequence all the analyzed species. The 148 bp amplicons permit to classify correctly each species in a cetacean phylogenetic NJ tree.

The samples 02CSO and 11CSO were classified as S. coeruleoalba, but shown high coincidence with some S. clymene sequences when the NCBI nucleotide BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was executed. In order to analyze these similarities more deeply, we downloaded every cyt b gene sequences of S. coeruleoalba and S. clymene hosted in GenBank (88 complete and partial sequences). The sequences were aligned, and a NJ phylogenetic tree was performed (Supp Fig. 1). The NJ phylogenetic tree presents tree haplogroups, one with only S. coeruleoalba, a second one only with S. Clymene, and a third one with both species, harboring samples 02CSO and 11CSO. This is because S. clymene arose by hybrid speciation between S. coeruleoalba and S. longirostris. This is a very rare mechanism of speciation that requires additional analysis of both mitochondrial DNA and nuclear DNA to obtain the complete identification of these species [[6]Amaral A.R. Lovewell G. Coelho M.M. et al.Hybrid speciation in a marine mammal: the clymene dolphin (Stenella clymene).].4. Conclusion

The universal primers cyt b L15601 and H15748 have demonstrated to be useful to identify cetacean species involved in forensic, ecologic and food security cases. Amplification of a 148 bp amplicon have been probed advantageous when amplifying degraded samples, and only presents limitations when identifying species arisen by hybrid speciation.

Declaration of Competing Interest

The authors report no conflicts of interest.

Acknowledgements

The authors are grateful to PhD Maite Alvarez for her technical and human support provided by the DNA Bank Service (SGIker) of the University of the Basque Country (UPV/EHU), Spain.

Funding

This study was funded by the Basque Government (Consolidated Group Grants IT-998-16 and IT-1271-19 ).

Appendix A. Supplementary dataThe following is Supplementary data to this article:ReferencesHsieh H.M. Chiang H.L. Tsai L.C. et al.

Cytochrome b gene for species identification of the conservation animals.

Forensic Sci. Int. 122: 7-18Tobe S.S. Kitchener A.C. Linacre A.M.

Reconstructing mammalian phylogenies: a detailed comparison of the cytochrome B and cytochrome oxidase subunit I mitochondrial genes.

PLoS One. 5e14156Schneider P.M. Bender K. Mayr W.R. et al.

STR analysis of artificially degraded DNA-results of a collaborative European exercise.

Forensic Sci. Int. 139: 123-134Lopez-Oceja A. Gamarra D. Borragan S. et al.

New cyt b gene universal primer set for forensic analysis.

Forensic Sci. Int. Genet. 23: 159-165Larkin M.A. Blackshields G. Brown N.P. et al.

Clustal W and Clustal X version 2.0.

Bioinformatics. 23: 2947-2948Amaral A.R. Lovewell G. Coelho M.M. et al.

Hybrid speciation in a marine mammal: the clymene dolphin (Stenella clymene).

PLoS One. 9e83645Article InfoPublication History

Published online: November 12, 2019

Accepted: October 16, 2019

Received in revised form: October 14, 2019

Received: September 15, 2019

Identification

DOI: https://doi.org/10.1016/j.fsigss.2019.10.171

Copyright

© 2019 Elsevier B.V. All rights reserved.

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