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Cytochrome oxidase subunit I (COI) gene - A method for species identification of the blackmouth catshark (Galeus melastomus)

Conference Paper · December 2018

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CYTOCHROMECYTOCHROME OXIDASEOXIDASE SUBUNITSUBUNIT II (COI)(COI) GENEGENE -- AA METHODMETHOD FORFOR SPECIESSPECIES IDENTIFICATION OF THE BLACKMOUTH CATSHARK (Galeus melastomus)

Karachaliou E.1, Hatzopoulos. P.1, Exadactylos A.3, Gkafas. G.A.3, Megalofonou P.2*

1 Department of Biotechnology, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece 2 Department of Biology, National and Kapodistrian University of Athens, Panepistimiopolis, Ilissia, 15784 Athens Greece 3 Department of Ichthyology and Aquatic Environment, School of Agricultural Sciences, University of Thessaly, Fytokou Str., 38446, Volos, Greece

Abstract Molecular techniques are a powerful method for fisheries management and conservation, particularly regarding Elasmobranch species which are vulnerable to anthropogenic pressures. They can be utilized to answer questions regarding population dynamics and life traits, information crucial to sustainable population management. The blackmouth catshark Galeus melastomus is an example of a case in which taxonomic confusion can be solved by molecular methods. The mitochondrial cytochrome oxidase subunit I (COI) gene, a preferred molecular marker for species identification, was used to compare the sequences of specimens morphologically identified as G. melastomus to sequences from the GenBank database by BLAST. All specimens were verified as G. melastomus, thus proving the usefulness of confirming morphological observations with molecular data. Keywords: Elasmobranchii, cytochrome oxidase, mtDNA, species identification, Galeus melastomus *Corresponding author: Megalofonou Persefoni ([email protected])

1. Introduction Elasmobranchii (sharks and batoids) are a highly diverse subclass of Chondrichthyes and, as apex predators, their welfare is considered highly important for a balanced marine ecosystem. Due to low fecundity, long period of maturation and gestation, they are considered vulnerable to anthropogenic influences such as habitat degradation, pollution and the pressure of increased fishing. Understanding more about their population dynamics and life traits is crucial, considering one quarter of cartilaginous fishes are listed as threatened under the IUCN Red List (2014, www.iucn.org). The utilization of molecular techniques for species identification in Elasmobranchii can be applied to many fields of fisheries management and conservation, whether it is distinguishing morphologically similar species, or the identification of cryptic species, hybrids, immature specimens, body parts and fins (Dudgeon et. al, 2012). Rapidly advancing technologies have lowered the cost of traditional methods and provided new techniques with fewer limitations. Mitochondrial genes are commonly used, owing to their high copy number, greater yield of mtDNA compared with nuclear DNA, and in most cases lack of recombination that promotes loss or fixation of mtDNA haplotypes, allowing species identification due to reduced within-species diversity (Dawnay et. al, 2007). The mitochondrial located cytochrome oxidase subunit I (COI) gene is widely used for species identification and is considered by many a universal 'barcode' for assignment to species level of most animal taxa, therefore it is useful when attempting to match specimen to reference sequences through DNA sequence similarity search tools due to the abundance of reference data. The blackmouth catshark, Galeus melastomus (Rafinesque, 1810) is a small, bottom-dwelling scyliorhinid shark widely distributed on the continental shelves and upper slopes of the North-eastern Atlantic Ocean and the Mediterranean basin (Compagno, 1984). Sympatric occurrence along parts of its geographical range and morphological resemblance to the closely related Atlantic sawtail shark Galeus atlanticus have prompted an ongoing debate about whether they should be considered separate species or re-described as one. Moreover, the morphological similarity and geographic overlap between the two species make species identifications difficult, distort the by catch fishery statistics and lead to uncertainty regarding population management (Erzini et al., 2002; Coelho et al., 2005, Castilho et al., 2007). It is cases like this where the use of molecular techniques can be used to support morphological data,‐ or to provide answers when morphological features cannot be discerned. The aim of this study is to develop a species identification oriented COI gene sequencing method for the high interest Galeus melastomus and to compare sequencing results with morphological data, as part of a larger COI species identification in Elasmobranchii project.

2. Material and Methods The blackmouth catsharks used in this study were by-catch specimens caught by a bottom trawler in the Gulf of Corinth during December of 2017. All specimens were preserved in ice until their transfer to the facilities of the Department of Biology in Athens where they were frozen at –200C. The identification of the specimens as Galeus melastomus was confirmed by specific diagnostic morphological features and measurements (Compagno, 1984) within three months from their collection and storage. Tissue samples consisted of pectoral fins clips were preserved in 95% Ethanol at -200C. Genomic DNA was extracted using a phenol-chloroform based protocol (Sambrook et al., 1989) with minor modifications. Amplification of PCR fragments was conducted via the use of two universal primer pairs that target the cytochrome oxidase subunit I (COI) gene (L5950, H7196) located in the mtDNA (Normark et al., 1991). The following

ORALS | 2nd Field - Fisheries 235 HydromediT 2018 | Book of Proceedings 3rd International Congress on 8-11 November 2018 Applied Ichthyology Volos, Greece & Aquatic Environment www.hydromedit.gr

primer sequences were used for PCR amplification and sequencing: L5950 (5'-ACAATCACAAAGA(CT)AT(CT)GG-3') forward, H7196 (5'-AGAAAATGTTG(AT)GGGAA(AG)AA-3') reverse. PCR reactions were conducted in 50 μl reaction mixtures containing 3 μl DNA, 5 μl of 10X PCR buffer (TaKaRa), 5 μl MgCl2 (TaKaRa), 5 μl of 2 mM deoxyribonucleotide triphosphate (dNTPs) (Jena Bioscience), 5 μl of each 3 mM primer (Eurofins Genomics) and 0.5 μl LA Taq DNA Polymerase (TaKaRa). PCR amplification was performed using a PTC-200 thermal cycler (MJ Research) under the following cycle conditions: initial denaturation at 950 C for 10 min followed by 40 cycles. Each cycle comprised the following: denaturation at 95 C for 45 sec, annealing at 50 C for 45 sec, and extension at 72 C for 2 min. A final extension at 72 C was applied for 10 min. The PCR amplification products were separated in 1.5% agarose gels and photographed on a UV transilluminator. PCR amplification products were purified using the NucleoSpin Gel and PCR Clean-up (Macherey-Nagel) and Sanger sequencing was performed by Eurofins Genomics using the same primers (L5950, H7196). Sequences were viewed using Clustal X v.2.1 and similarities between specimen sequences and sequences from the GenBank database were discerned by BLAST.

3. Results and Discussion COI gene sequencing using forward primer L5950 resulted in a high quality (clipped) sequence size of 908 bp for sample A1, 975 bp for sample A2 and 982 bp for sample A3. Reverse primer H7196 sequences were of low quality and could not be used, possibly due to low specificity of degenerate primers. A sequence length of around 900 bp is considered an adequate length for species identification, and BLAST search results assigned all 3 specimens to the species Galeus melastomus (Highest Identity Score: A1 98%, A2 99%, A3 99%), thus confirming morphological data. As G. melastomus is a taxonomically obscure species with difficulties concerning its conservation and management, next steps could include extensive research on population structure, cladistic analysis and defining the evolutionary relationships between G. melastomus and its sister taxa.

Figure 1. Cytochrome oxidase subunit I (COI) gene sequence – Specimen A1

Figure 2. Cytochrome oxidase subunit I (COI) gene sequence – Specimen A2

ORALS | 2 nd Field - Fisheries 236 HydromediT 2018 | Book of Proceedings 3rd International Congress on 8-11 November 2018 Applied Ichthyology Volos, Greece & Aquatic Environment www.hydromedit.gr

Figure 3. Cytochrome oxidase subunit I (COI) gene sequence – Specimen A3

References Castilho, R., Freitas, M., Silva, G., Fernadez-Carvalho, J., Coelho, R. (2007) - Morphological and mitochondrial DNA divergence validates blackmouth catshark, Galeus maelastomus, and Atlantic sawtail catsharks, Galeus atlanticus, as a separate species. Journal of Fish Biology (2007) 70 (Supplement C), 346-358. Coelho, R., Erzini, K., Bentes, L., Correia, C., Lino, P. G., Monteiro, P., Ribeiro, J. & Gonçalves, J. M. S. (2005). Semi pelagic longline and trammel net elasmobranch catches in Southern Portugal: catch composition, catch rates and discards. Journal of Northwest Atlantic Fishery Science 35, 531–537. ‐ Compagno, L.J.V. (1984) FAO species catalogue (Vol.4). Sharks of the world: An annotated and illustrated catalogue of shark species known to date. Part 2: Carchariniformes. FAO Fisheries Synopsis, Rome, no. 125, pp. 655. Dudgeon, C.L., Blower, D.C., Broderick, D., Giles, J.L., Holmes, B.J., Kashiwagi, T., Krück, N.C., Morgan, J.A.T., Tillett, B.J., Ovenden, J.R. (2012) – A review of the application of molecular genetics for fisheries management and conservation of sharks and rays. Journal of Fish Biology (2012) 80, 1789-1843. Erzini, K., Costa, M. E., Bentes, L. & Borges, T. C. (2002). A comparative study of the species composition of discards from five fisheries from the Algarve (southern Portugal). Fisheries Management and Ecology 9, 31–40. Normark, B.B., McCune, A.R., Harrison, R.G., 1991. Phylogenetic relationships of neopterygian fishes, inferred from mitochondrial DNA sequences. Mol. Biol. Evol. 8, 819e834. Sambrook, J., Fritsch E.F., Maniatis, T., 1989. Molecular Cloning: a Laboratory Manual, second ed., vol. I. Cold Spring Harbor Laboratory Press.

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