Download Application of DNA barcoding for the control of the species from the

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Application of DNA barcoding for the control of the species from the Octopus genus.
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Francesco Debenedetti1, Alessandra Dalmasso1, Maria Teresa Bottero1, Maurizio Gilli3, Stefano Gili3,
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Valentina Tepedino2, Tiziana Civera1*
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(TO), Italy
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Eurofishmarket, Piazza Sergio Vieira De Mello 4, 40128 Bologna, Italy
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ASLTO1, Via della Consolata 10, 10122 Torino
Department of Veterinary Sciences, Università degli Studi di Torino, largo Braccini 2, 10095 Grugliasco
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Correspondence: * Tiziana Civera, Department of Veterinary Sciences, Università degli Studi di Torino,
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largo Braccini 2, 10095 Grugliasco (TO), Italy.
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Phone:+39. 011.67092
- Fax: +39.011.6709224
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Key words: DNA barcoding; Octopus spp.; commercial frauds
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Conflict of interests: the authors declare no potential conflict of interests.
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Abstract
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The DNA barcoding proposes the use of a particular sequence from a single genomic region as the base for
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an identifying system capable to determine all animal species. This methodology comprises the analysis of a
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655 base-pair region from the mithocondrial cytochrome C oxidase gene (COI). Its application in the species
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identification of fishery products has been very promising, however in the last years some doubts about its
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usage have emerged.
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In this work, we make use of the DNA barcoding for the identification of some of the octopus species with
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higher commercial interest (Octopus membranaceus, Octopus vulgaris, Octopus aegina, Octopus cyanea)
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focusing the attention on the reliability and completeness of the available information on the databases.
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The study looked over 51 individuals apparently belonging to the Octopus genus.
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For the identification of O.aegina, O.cyanea, O.vulgaris species no particular problems were found. On the
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other hand most of the samples of O.membranaceus, despite they clearly presented the morphological
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characteristics of the species, were not identified with the biomolecular analyses.
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Riassunto
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Il DNA barcoding propone l'utilizzo di una sequenza di una singola porzione genica come base di un sistema
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identificativo valido per l’identificazione di tutti gli animali. Questa tecnica prevede l’analisi di un
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frammento di 655 bp della subunità I della citocromo C ossidasi (COI). La sua applicazione nel campo
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dell’identificazione delle specie ittiche si è rilevata molto promettente fin dall'inizio, tuttavia sono emersi, nel
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corso degli anni, alcuni dubbi sul suo utilizzo.
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In questo lavoro abbiamo voluto impiegare il DNA barcoding per l'identificazione di alcune specie di polpi
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di maggior interesse commerciale (Octopus membranaceus, Octopus vulgaris, Octopus aegina, Octopus
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cyanea) focalizzando l’attenzione sull'attendibilità e la completezza delle banche dati
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Sono stati analizzati 51 soggetti di appartenenti al genere Octopus.
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Per le specie
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nell’identificazione. Viceversa la maggior parte dei campioni di O.membranaceus, nonostante presentasse
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inequivocabilmente le caratteristiche morfologiche della specie, non è stata identificata con le analisi
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biomolecolari.
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O.aegina , O.cyanea , O.vulgaris non sono state riscontrate particolari difficoltà
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Introduction
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The DNA barcoding puts forward the use of a sequence from a single genomic region (defined as
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“barcode”), as the base of a recognition system capable to identify all animal species (Hebert et al., 2003).
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This biomolecular methodology consists on the identification of the belonging species through the
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sequencing of a fragment of mitochondrial genes, its alignment and comparison with the information
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available on the databases.
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In particular, the barcoding approach comprises the analysis of a 655 bp region located at the 5’ end of the
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subunit I of the mithocondrial cytochrome C oxidase gene (COI). This gene codes for one part of the
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terminal enzyme of the mithocondrial respiratory chain. The COI gene has been chosen as a universal
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molecular target since it allows the design of universal, robust and functional primers for almost all the
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member of the animal Phyla (Folmer et al., 1994).
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The effectiveness of this fragment for the identification of species. has been demonstrated for several animal
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species, from vertebrates to invertebrates (Waugh, 2007; Ward and Holmes, 2007).
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Recently, this technique was recognized as the best method in Forensics for species’ identification, and was
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proposed by the United States Food and Drug Administration (USFDA) as the methodology for the
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authentication of commercial fish products (Dawnay et al., 2007).
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The US agency has also the intention of introduce the DNA barcoding data in the Regulatory Fish
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Encyclopedia (RFE) to support the investigations of mislabeling and the substitution of the fish species
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(Yancy et al., 2008). In May 2004, the Consortium for the Barcoding of Life (CBOL) was formed,
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comprising several international patterns. Since the beginning, its mission has been the exploration and the
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development of the potentiality of the DNA barcoding as a practical and research tool for the species’
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identification. The use of this methodology for the species identification of fishery products has been very
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promising from the outset, given the large number of species already identified. Actually it has been shown
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that 98% and 93% of the marine and fresh water species, respectively, can be differentiated using barcodes
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(Savolainen et al., 2005; Ward et al., 2009).
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This favorable outcome as well as the need of a complete and reliable instrument for identification of the
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species, have derived into the formation of the initiative Fish Barcode of Life (FISH-BOL)
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(www.fishbol.org).
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This campaign, launched in 2005, has as main objective the collection of DNA barcodes of all the fish in the
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world, equivalent to 31,000 species approximately. It endorses the FishBase (www.fishbase.org) as the
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taxonomic authority, and the BOL database (BOLD) as the working bioinformatic platform. FISH-BOL
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represents one of the most complete resources for the species identification of the fishery products (Ward et
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al., 2009).
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However, in the past years, some doubts regarding the use of DNA barcoding have emerged because of the
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difficulties to discriminate recently spread species, or species with a wide spatial differentiation; as well as
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the inability to differentiate new and hybrid species (Moritz and Cicero, 2004; Hickerson et al., 2006;
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Rubinoff et al., 2006).
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Furthermore, if on one hand this technique has been extensively evaluated for the identification of fish
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species, there are still few data supporting the applicability in the identification of the most common species
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of cephalopods and octopus.
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In this work we test the DNA barcoding for the identification of some octopus species of greatest
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commercial interest (Octopus membranaceus, Octopus vulgaris, Octopus aegina, Octopus cyanea) focusing
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the attention on the reliability and completeness of the available databases.
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Materials and Methods
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The sampling involved the collection of 51 specimens belonging to the genus Octopus: 1 O.dollfusi, 2
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O.aegina, 2 O.cyanea, 24 O.vulgaris and 22 O.membranaceus. All samples, collected in 2012 and 2013
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from different suppliers, were morphologically identified and kept at -20°C.
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The DNA extraction was done using the DNeasy Tissue and Blood Kit (Qiagen) according to the
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manufacturer’s instructions for animal tissue. The extracted DNA was quantified with NanoDrop 1000
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(Thermo Scientific).
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For all samples, segments of the COI genes were amplified following the protocol proposed by
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Folmer et al., (1994). Since there were no available reference sequences for O.membranaceus in FISH-BOL,
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a second sequencing protocol was introduced. It contemplated the usage of cytochrome b (cytb), a historical
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target used for the species identification (Espineira et al., 2010).
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Amplicons were visualized by electrophoresis on a 2% agarose gel and colored with Eurosafe Nucleic Acid
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Stain.
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The amplified products were purified with the enzyme ExoSap-IT (USB) and sequence with the BigDye
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terminator kit (Applied Biosystems) according to the manufacturer’s instructions. The extension products
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were purified using the DyeEx 2.0 spin kit (Qiagen), denatured in formamide and analyzed with the ABI
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Prism 310 Genetic Analyzer (Applied Biosystems).
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The obtained sequences were examined, corrected and analyzed with the MEGA5 software and subjected to
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identification with BOLD-IDS (Bold) for the COI gene, and to BLASTN (GenBank) for both genes.
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Finally phylogenetic relationships among the studied samples were investigated with differences method
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(Nei and Kumar, 2000). For the distance matrix, phylogenetic trees were constructed using the Neighbour-
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Joining method. The bootstrap method (500 replicates) was used to obtain the support of different groups
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included in the phylogenetic (Felsenstein, 1985).
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Results
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All samples originated an amplicon of 655 bp for the COI gene and of 651 bp for the cytb gene.
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The sequencing results are presented on Table 1.
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For the species O.aegina, O.cyanea, O.vulgaris and O.dollfusi there were no identification problems.
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Actually we were able to identify 27 samples, out of 29, with a similarity of 99-100%. Out of these, 22
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samples were in accordance with the labels of the products, while 5 didn’t correspond to what was declared
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in the product description.
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Finally, for 2 samples labeled as O.vulgaris it was not possible to obtain the biomolecular identification,
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since they presented a low identity (91%) with Amphioctopus rex and Amphioctopus marginatus, species not
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reported in the FAO catalogue.
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Further difficulties were encountered in the identification of samples labeled as O.membranaceus.
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Actually only one sample was identified from both databases in a non ambiguous way (100% similarity) as
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O.aegina, while for the other 21 samples it was not possible to obtain a certain identification. In particular,
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regarding the cytb, the similarity obtained values ranged from 93-94% with O.membranaceus, 93% with
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O.aegina and 90% with Cistopus taiwanicus (Fig.1).
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The results were even more complex for the COI gene because of the lack of reference sequences of
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O.membranaceus in both GenBank and BOLD databases (Fig.2).
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Discussion
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The expansion in food preservation, processing technologies and market liberalization has contributed
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significantly to the globalization of fish trade, both in terms of species and products.
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In a globalized market, the eagerness to offer leading products appreciated and recognized to derive
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maximum revenue, can introduce the habit of replacing valuable species with others very similar but
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economically or qualitatively inferior, incurring into commercial fraud (Jacquet and Pauly, 2008).
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The identification of species in fisherystock products, essential at the moment of the commercialization, has
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been based for a long time solely on morphological characteristics, and is still the official method for the
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taxonomic classification. However, in the last years this morphological classification has been supported by
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biomolecular laboratory techniques based on the sequencing of several mitochondrial genes (cytb, 16S
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rRNA, 12SrRNA, NADH).
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Particularly, the DNA barcoding, based on the sequencing of the COI gene, might be a reliable tool used in
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the daily practice for the control of commercial fraud.
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The ability of barcoding to provide unambiguous designation of species from a complete specimen or part of
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it, has important implications in several fields: in the retail (accidental or intentional replacement, consumer
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protection and the regulation of the commerce); in fisheries management (monitoring of fishing stocks,
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sustainable fishing); in the conservation of fish stock (identification of endangered species, protected,
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damaged or parts of them) (Costa and Carvalho, 2007).
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Given the limited applications of DNA barcoding in the field of cephalopods, the present work evaluated the
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possibility of using it to control the Octopus genus and the most commonly found species in the Italian fish
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markets.
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In fact octopus phylogeny has been subjected to confusion and controversy throughout the past years (Carlini
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et al., 2001).
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The Octopus genus is the largest within the family Octopidae, it comprises more than 200 species. Of these,
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90% were inserted just in the above-mentioned genus, for this reason it is called "catch-all" (Lü et al., 2013).
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Several taxonomic studies based on morphology, have recognized more complexity within the genus
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Octopus spp. ("Species group"). At the beginning there were identified 9 groups of species, many of which
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are under review. For example, the O.aegina complex was classified as Amphioctopus spp.. Currently many
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species, previously classified as Octopus spp., were included in the Amphioctopus genus (Huffard and
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Hochberg, 2005). Furthermore, the analysis of morphological parameters does not seem sufficient to
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describe a so complex variability, confirmed also by the data obtained in this work.
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If for the species O.aegina, O.vulgaris and O.cyanea there were no identification problems, the same cannot
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be said for O.membranaceus, important species of commercial value, fished in the Indo-Pacific area and
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widely spread in Italian fisheries.
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From the analysis of the COI gene, the majority of the O.membranaceus samples, despite they clearly
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presented the morphological characteristics of the species (a conspicuous, dark, ringed ocellus on web at
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base of arms II, enteroventral to the eyes) (FAO Fisheries synopsis), were not identified with molecular
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analyses. In particular regarding the gene COI, there were no reference sequences, while in the case of cytb
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the samples showed values of similarity with O.membranaceus, unexpectedly low (93-94%).
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From the alignments of the sequences of the O.membranaceus samples, both the COI and cytb presented a
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great variability not only with respect to the sequences deposited in GenBank, but also between the same
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samples. The only exception, represented by a group of homologous samples (identity of 99-100%) is
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justified by the fact that the 12 specimens were from the same batch (Fig. 1 and 2).
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Moreover this considerable variability was also found in O. vulgaris species: in fact from the alignment of
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the sequences of the COI gene a range of similarity between the 96% and 100% was observed. As example,
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in table 2, ten representative sequences of this gene were aligned, highliting the variable sites between them.
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There were found show 35 SNPs out of 655 bp, which demonstrate a higher variability.
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These results showed, on one hand that the extreme genotypic variability is not correlated with the
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phenotypic variability, and on the other hand are confirmed by the literature related to the Octopus genus.
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Actually the polyphyletic of the Octopus genus has been demonstrated by a number of molecular studies (Lü
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et al., 2013). The reconsideration of generic names and the major revision of these taxa have been proposed
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by some authors (Guzik et al., 2005;)
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Conclusion
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The DNA barcoding applied in the control of Octopus spp. highlights the need to enlarge the information in
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the database by increasing the number of species and the number of sequences available for each one. In
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addition, from this works emerges too the complexity in the interpretation of the data, pointing out the
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importance of the training and experience of the operator to avoid making mistakes.
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References
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Table 1: Results of sequencing identification
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Number of samples
Labeled
Species
Similarity (%)
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O.aegina
O.aegina
99
2
O.cyaneus
O.cyanea
99-100
1
O. dollfusi
O.aegina
100
4
O.vulgaris
O.cyanea
99-100
18
O.vulgaris
O.vulgaris
99-100
2
O.vulgaris
n.a.
n.a.
1
O.membranaceus
O.aegina
99-100
21
O.membranaceus
n.a.
n.a.
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
n.a.: identification not available
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Figure 1
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Phylogenetic tree showing the relationship among the studied samples carried from the alignment of the cytb
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gene
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The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test are
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shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the
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evolutionary distances used to infer the phylogenetic tree. The distances were computed using the number of
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differences method and are in the units of the number of base differences per sequence.
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Figure 2
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Phylogenetic tree showing the relationship among the studied samples carried fron the alignment of the COI
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gene.
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The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test are
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shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the
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evolutionary distances used to infer the phylogenetic tree. The distances were computed using the number of
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differences method and are in the units of the number of base differences per sequence.
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Table 2: Example of variable sites in COI gene from the alignment of ten O.vulgaris representative sequences.
SNPs position (referred to GenBank acc. n° HQ908427)
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Octopus vulgaris 1
Octopus vulgaris 2
Octopus vulgaris 3
Octopus vulgaris 4
Octopus vulgaris 5
Octopus vulgaris 6
Octopus vulgaris 7
Octopus vulgaris 8
Octopus vulgaris 9
Octopus vulgaris 10
1
0
7
T
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1
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4
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Dots indicate homolog nucleotides
2
1
2
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2
4
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C
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3
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5
3
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C
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5
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2
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5
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0
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6
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6
1
6
A
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6
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