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Revista de biología marina y oceanografía

versión On-line ISSN 0718-1957

Rev. biol. mar. oceanogr. vol.49 no.2 Valparaíso ago. 2014

http://dx.doi.org/10.4067/S0718-19572014000200015 

 

NOTA CIENTÍFICA

New contribution to the systematic status of various Mediterranean scorpionfish, as inferred from a mitochondrial DNA sequence

Nueva contribución al estado sistemático de diferentes peces escorpión del Mar Mediterráneo, inferida a partir de secuencias de ADN mitocondrial

 

Marco Arculeo1 and Sabrina Lo Brutto1

1Dipartimento STEBICEF, Università di Palermo, Via Archirafi 18, 90123 Palermo, Italia. marco.arculeo@unipa.it


ABSTRACT

This study investigated the molecular phylogeny of 6 Mediterranean species of scorpionfish, belonging to the Scorpaenidae and Sebastidae family. Neighbor-Joining and Maximum Parsimony phylogenetic analyse, based on 424 base pairs of partial mitochondrial DNA sequences of the 12S-rRNA gene, revealed 2 main clades. One clade is represented by the Scorpaena genera (with the species S. notata, S. porcus, and S. scrofa) and another clade consists of the genera Helicolenus, Pterois, and Scorpaenodes. The molecular phylogeny showed that the Scorpaenodes genus (sub-family Scorpaeninae) is found within the clade of the species belonging to the other two sub-families (Pteroninae and Sebastinae). This pattern is in contrast with current classification and it, therefore, poses a number of problems if using only morphological characters when classifying these families.

Key words: Scorpionfish, mtDNA, 12S-rRNA, Mediterranean Sea


INTRODUCTION

The Scorpaeniformes order is represented in the Mediterranean Sea by two families: Scorpaenidae and Sebastidae, and both families include two sub-families Scorpaeninae and Pteroninae, and Sebastinae and Sebastolobinae, respectively. The Scorpeninae sub-family includes 3 genera (Pontinus, Scorpaenodes and Scorpaena) and 9 species, while the Pteroninae sub-family comprises a single genus and one species (Pterois miles). The Sebastinae and Sebastolobinae sub-families are both represented by one genus and one single species Helicolenus dactylopterus and Trachyscopia cristulata echinata, respectively (Froese & Pauly 2013, WoRMS1).

Some of these species have recently entered into the Mediterranean Sea through the Suez Channel or the Strait of Gibraltar. This is the case with Pterois miles, a lessepsian migrant, which has recently colonised the southeast of the Mediterranean Sea, and Trachyscopia cristulata echinata and Scorpaena stephanica, the latter two which have migrated from the Atlantic Ocean (Ciesm 2002). Many species of the Scorpaenidae and Sebastidae families are often very difficult to identify morphologically because the characters used in their identification are not easy to use. For this reason, there are occasionally many problems with correctly identifying the species, especially regarding those belonging to the Scorpaena genera.

Until now, many of the studies regarding the systematics and phylogeny of this order have been conducted on species outside the Mediterranean, thus highlighting the complexity of clarifying the position of the high taxonominal categories, i.e., family and genera (Rocha-Olivares et al. 1999a, b; Kai et al. 2003, Kochzius et al. 2003, Shinohara et al. 2007). Only a few studies have been conducted on the Scorpaeniformes species in the Mediterranean Sea and these were addressed using cytogenetics (Caputo et al. 1998), meristic characters and a genetic analysis of the mitochondrial 16S rDNA gene (Turan et al. 2009). These studies have shown a discrepancy between morphological and genetic-cytogenetic results and highlighted the need for a taxonomical re-evaluation of the analysed species.

Hence, a sound knowledge of the systematics of Scorpaeniformes is important not only for taxonomic and evolutionary purposes but also regarding species delimitation in stock assessment studies. Furthermore, it is considered suitable to use molecular markers as a tool with which to discriminate each species and to understand the phylogenetic relationships within the Scorpaenidae family.

The aim of this paper was to use mitochondrial DNA, and especially the 12S-rRNA portion, in order to genetically characterise the species analyzed in this paper and to study their phylogenetic relationships. 12S-rDNA is recognised as an important marker for improving species delimitation and for resolving taxonomic relationships in many groups of vertebrate and invertebrate families (Maggio et al. 2005, Sirna Terranova et al. 2007).

 

MATERIALS AND METHODS

A total of 30 specimens (5 individuals per species) were collected and analysed. A small piece of caudal fin from each specimen was preserved in ethanol (90%) and the total genomic DNA was extracted using a DNeasy Tissue Kit (Qiagen). The extracted DNA was suspended in distilled water and stored at -20°C until required. Amplification of the mitochondrial encoded 12S-rDNA gene was obtained using universal primers (Kocker et al. 1989). PCR was carried out in a Perkin Elmer Cetus Thermal cycler in a 100-µl solution containing 1 ng genomic DNA, 0.2 µM each dNTPs, 0.1 µM of each primer, 10 mM Tris_HCl (pH 9), 50 µM KCl, 1.5 mM MgCl2 and 2.5 U of Perkin Elmer Taq polymerase. The thermal cycling profile for the 12S-rDNA portion began at 94°C for 2 min as a hot start, followed by 5 cycles of 94°C (60 s), 48°C (45 s), and 72°C (60 s), 35 subsequent cycles of 94°C (120 s), 60°C (60 s), and 72°C (60 s), with a final step of 10 min at 72°C for the termination of PCR. PCR products were separated on 2% agarose gel, which had been stained with ethidium bromide. Subsequently, the bands were purified using a Qiaquick PCR purification Kit (Qiagen) and the PCR product sequenced in two directions in an ABI Prism 310 automated sequencer (Applied Biosystem).

Nucleotide sequences were aligned by the ClustalW Multiple Sequence Alignment program (Thompson et al. 1994) with default settings. In order to facilitate sequence comparison, we used homologous sequences of Dicentrarchus labrax (Perciformes, Moronidae) from GenBank (Accession Number X81566) as an outgroup in the analysis. The sequence analyses were performed using a 4.1 DNAsp version (Rozas & Rozas 1999) and MEGA version 5 (Tamura et al. 2011). Phylogenetic analysis was performed with the MEGA and PAUP computer programs (Swofford 2003), using Neighbor-Joining and Maximum Parsimony methods respectively.

 

RESULTS AND DISCUSSION

A total of 424 base pairs of 12S-rDNA were aligned, 307 were conserved and 112 were variable, of which 93 were parsimony informative (Table 1). The average nucleotide distance (Kimura 2 parameters) varied between a maximum value of 0.219 (Scorpaena porcus vs. Scorpaenodes arenai) and a minimum value of 0.088 (Scorpaenodes arenai vs. Pterois miles) (Table 2). As expected, most of the observed nucleotide variation was due to transitions.

 

Table 1. Variable sites of the 12S rDNA sequences of the 6 species of Scorpaeniformes analysed

Tabla 1. Sitios variables de las secuencias de 12S rADN de las 6 especies de Scorpaeniformes analizadas

 

Table 2. Nucleotide distance among the 6 species analysed according to Kimura-2 parameters

Tabla 2. Distancia nucleotídica entre las 6 especies analizadas de acuerdo a los parámetros de Kimura-2

 

 

No difference was observed among individuals of the same analysed species. When comparing species, a greater number of transitions were observed between Scorpaena porcus and Scorpaenodes arenai (n. 49); this number was greater than that for transversions (n. 28). The average value of the transition/transversion ratio was 1.90, with a maximum value of 3.25 and a minimum value of 1.48. To estimate the phylogenetic relationships among species, phylogenetic trees were constructed, which were based on the methods of Neighbor-Joining and Maximum Parsimony. Both methods produced phylogenetic trees with the same topology and, for this reason we decided to show only the tree constructed by the Maximum Parsimony method (Fig. 1). The values allocated to the nodes were those calculated on 1,000 bootstrap replicates. All the analysed species were separated by significant bifurcations in which the values were always higher than 50%. The tree revealed two main clades, one represented by the Scorpaena genera (with the S. notata, S. porcus and S. scrofa species), and another consisting of the Helicolenus, Pterois, and Scorpaenodes genera.

 

Figure 1. Maximum Parsimony tree based on 12S-rDNA nucleotide sequences of the 6 species of Scorpaeniformes analyzed. Numbers at nodes are bootstrap values based on 1000 Replicates. Scales bar represents an interval of Tamura-Nei genetic distance

Figura 1. Árbol de Parsimonia Máxima basado en las secuencias de nucleótidos del 12S-rADN de las 6 especies de Scorpaeniformes analizadas. Los números de los nodos son los valores de los bootstrap basados en 1000 réplicas. La barra de escala representa un intervalo de la distancia genética de Tamura-Nei

 

The data reported here place the Scorpaenodes genera of the Scorpaeninae subfamily in the same clade as the species belonging to the other 2 subfamilies (Pteroninae and Sebastinae). This conclusion contrasts with the current classification (Froese & Pauly 20142) and reveals various problems with using only morphological characters. According to Turan et al. (2009), the use of only morphological characters to identify various species of Mediterranean Scorpaenidae is not sufficient. Indeed, morphology produces different results if compared with the sequences obtained from mitochondrial 16S-rDNA. Moreover, the data reported by Caputo et al. (1998) underline that differences in morphology in the Scorpaena species are limited, whereas the karyotype reorganisation is considerable. The analysis of our data with those reported in the literature revealed a weakness regarding the exclusive use of morphological characters or colour in some genera of Mediterranean scorpionfish. This has also been shown in other commercially important fish species in the Mediterranean, like groupers, in which the exclusive use of morphological traits is not satisfactory for identifying the species and establishing their phylogenetic relationships (Maggio et al. 2009). The use of the mitochondrial gene 12S-rRNA proved to be a valuable tool in analysing phylogenetic relationships among the examined species. Moreover, the use of mitochondrial gene 12S-rRNA highlighted that it should be used to analyse all the species belonging to different genera in order to obtain more satisfactory phylogenetic relationships within this family in the Mediterranean Sea. The marker used should be an important tool for the precise identification of the species and it should also be used for tracing individuals, thereby satisfying the provisions of the laws already in force in the European Union. These require (since 2011) that any fish available on the market is labelled with the species and region of origin. Finally, and considering the economical importance of scorpionfish, we would like to suggest that the 12S-rRNA mitochondrial marker should also be used as a tool for formulating management programmer for stock assessment.

 

ACKNOWLEDGMENTS

This study was supported by Fondi di Ateneo (ex 60%)

 

NOTES

1WoRMS Editorial Board. 2014. World Register of Marine Species, VLIZ. <http://www.marinespecies.org/index.php>
2Froese R & D Pauly. 2014. FishBase.<www.fishbase.org>

 

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Received 28 October 2013 and accepted 5 May 2014
Editor: Claudia Bustos