Int. J. Aquat. Biol. (2015) 3(5): 314-322 E-ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2015 Iranian Society of Ichthyology Original Article Combined mitochondrial DNA analysis of the Mesopotamian spiny eel, Mastacembelus mastacembelus (Banks & Solander 1794), and its phylogenetic position Esen Tutar*1 Department of Bioengineering and Sciences, Graduate School of Natural and Applied Sciences, Kahramanmaraş Sütçü İmam University, Kahramanmaraş, Turkey. Article history: Received 7 April 2015 Accepted 4 September 2015 Available online 2 5 October 2015 Keywords: Mastacembelus 16S rRNA 12S rRNA tRNAPhe tRNAVal Phylogenetic relationships Abstract: Nucleotide sequences of the 12S rRNA, 16S rRNA, tRNAPhe and tRNAVal genes of mtDNA of Mesopotamian spiny eel, M. mastacembelus, was determined for the first time. The comparison of the three populations of Mesopotamian spiny eel from Turkish part of the Tigris basin based on the combined mitochondrial DNA was performed. Based on the results, no differences were determined and the identity found to be 100% among three populations. Furthermore, the obtained results from molecular methods were compared with morphological findings to validate the position of the studied populations of M. mastacembelus. In addition, the phylogenetic position of the Mesopotamian spiny eel was examined among the Mastacembelidae and Synbranchioformes based on 12S rRNA and 16S rRNA. The constructed phylogenetic relationship between M. mastacembelus and some other members of Synbranchioformes order supported their taxonomic hierarchy. Introduction The order Synbranchioformes includes 120 species in three families, including Chaudhuridae (10 species), Synbranchidae (23 species) and Mastacembelidae (87 species) (Froose and Pauly, 2014). The members of the family Mastacembelidae, known as spiny eels, are found in freshwaters and distributed in tropical and subtropical Africa, the Middle East, South-East Asia and North of China (Coad, 2015). This family consists of three genera, including Mastacembelus (64 species), Macrognathus (22 species) and Sinobdella (1 species) (Vreven, 2005a; Froose and Pauly, 2014). Nine species of the genus Mastacembelus inhabit Asian inland waters, whereas 52 species occur in African inland waters and all members of the genus Macrognathus were recognized in Asian inland waters (Froose and Pauly, 2014). Mastacembelids can attain a maximum length of about 1 m. They are eel-like fishes having a long series of well-separated dorsal spines and a short * Corresponding author: Esen Tutar E-mail address: esentutar@gmail.com series of anal spines. They have no pelvic girdle and fins (Vreven, 2005b). More than 70 species of spiny eels are consumed as food fishes (Britz, 2007). Mastacembelus mastacembelus occurs in the river basins of the Tigris and Euphrates in the Middle East; Turkey, Syria, Iraq and Iran (Coad, 1996; Froose and Pauly, 2014) and is known as Mesopotamian spiny eel referring to its inhabiting area. This taxon is a typical species of the Mastacembelidae and contains all the characteristics of the family (Coad, 2015). The phylogenetic structure of the family Mastacembelidae is under debate and its classification has mainly been based on meristic and morphometric characters (Travers, 1984; Kottelat, 1991; Johnson and Patterson, 1993; Britz, 1996; Vreven and Teugles, 1996; Vreven, 2004; Vreven, 2005a; Vreven, 2005b; Britz, 2007; Çakmak and Alp, 2010; Plamoottil and Abraham, 2013). Although, the majority of the mastacembelids were morphologically described, a few species such as 315 Tutar/ Combined mitochondrial DNA of the Mesopotamian spiny eel Mastacembelus aculeatus, M. erythrotenia, M. armatus, Macrognathus aculeatus and M. pancalus were described based on molecular data (Miya et al., 2001; Chen et al., 2003; Smith and Wheeler, 2006). However, there is no molecular information for M. mastacembelus and many mastacembelid species. The mitochondrial DNA (mtDNA) is a circular and small molecule, self-replicating and usually about 15-18 kb in length. Mitochondrial genome contains two ribosomal RNA genes, which play primary role in protein synthesis (12S rRNA and 16S rRNA), 13 protein-coding genes (ATPase 6, ATPase 8, COI–III, Cytb, ND1-6 and 4L), 22 transfer RNA genes and a non-coding control region (D-loop) in charge of its replication and transcription factor as other vertebrates (Ishıguro et al., 2001; Kartavtsev et al., 2007). The gene content and organization of complete vertebrate mtDNA are quite conserved (Boore, 1999). The mitochondrial DNAs have been widely used as a marker for identification of species and phylogenetic researches, since a lot of characteristics are attributed to the maternal inheritance, high copy numbers in each cell, lack of recombination and high evolution rate (Kartavtsev et al., 2007; Cui et al., 2009; Cawthorn et al., 2012). In addition, the complete mtDNA has been widely used in the phylogenetic researches, partial gene fragments such as Cytb, 12S rRNA, 16S rRNA and the control region has become also very useful molecular tools for mitochondrial analysis (Cruz- Agüero et al., 2012). Therefore, the mitochondrial DNA has been considered a popular marker in many areas including fisheries biology, management and aquaculture, especially for population and evolutionary studies (Avise, 1994; Okumuş and Çiftci, 2003; Galtier et al., 2009; Lin et al., 2014). The aim of this study is to determine nucleotide sequences of the 12S rRNA, 16S rRNA, tRNAPhe and tRNAVal genes of the spiny eel, M. mastacembelus, and to determine its phylogenetic position among the mastacembelids and the members of Synbranchioformes. Furthermore, it is aimed to validate the obtained results from molecular methods with morphological findings for identification of this species. To my best knowledge, there is no report on molecular taxonomy of M. mastacembelus. This is the first report on molecular identification of this species. These findings can contribute to understanding of the evolution and phylogenetic characterization of the M. mastacembelus based on mtDNA. Materials and Methods Total DNA Extraction: A total of 57 individuals (36 of Karakaya Reservoir, 7 of Tohma Stream and 14 of Tigris River) of M. mastacembelus from three different locations at Tigris and Euphrates Rivers were sampled. Genomic DNA samples were obtained from ethanol preserved caudal fin tissues. Caudal fins of 20-30 mg were minced and 600 µL TEN (100 mM Tris, 10 mM EDTA and 250 mM NaCl), 40 µL 20% SDS (Sodium Dodecyl Sulphate) and 10 µL Proteinase K (10 mg/l) were added on the samples. They were incubated at 55°C for 24 hours. After the incubation, 10 µL RNase (5 mg/ml) was added and second incubation was applied at 55°C for 24 hours. The total DNA was purified by standard phenol:chloroform extraction and ethanol precipitation (Sambrook et al., 1989). Isolated DNA was inspected under UV light after 1% agarose gel electrophoresis. PCR and Sequencing: PCR amplifications were performed in 50 µL tubes containing 5 µl 10X reaction buffer (50 mM KCl, 10 mM Tris-HCl, 0.1% Triton-X1-100), 0.5 µL 1mM dNTP (250 μm from each of nucleotides), 1 µl each of 20 pmol forward and reverse primers, 1 U Taq DNA polymerase, 1 µl DNA and 42 µl ddH2O. Reaction mixtures were subjected to the following cycling protocol: Initial denaturation (94°C: 4 min), 30 cycles (94°C: 1 min; Primer Denaturation Annealing Extension E1-E8 1 min in 94oC 30s in 59oC 2 min in 72oC E2 1 min in 94oC 30s in 55oC 2 min in 72oC E3-E6 1 min in 94oC 30s in 58oC 2 min in 72oC E4 1 min in 94oC 30s in 64oC 2 min in 72oC E5-E7 1 min in 94oC 30s in 62oC 2 min in 72oC Table 1. Denaturation, annealing and extension temperature and times in PCR. 316 Int. J. Aquat. Biol. (2015) 3(5): 314-322 55°C: 30 s; 72°C: 2 min) and final extension (72°C: 5 min) (Table 1). In order to amplify mitochondrial DNA with standard PCR techniques, the new primers were designed because mtDNA of M. mastacembelus has not been determined so far. In order to design PCR and sequencing primers for mtDNA genes, sequence for each gene were retrieved from the mitochondrial genome data of M. favus (Accession No. NC_003193). Sequence length of 12S rRNA gene of M. favus was 947 bp and 16S rRNA gene was 1671 bp (http://www.ncbi.nlm.gov). The target DNA fragment had 3036 bp and contains the region of D- loop (last 100 bp), tRNAPhe, 12S rRNA, tRNAVal, 16S rRNA, tRNALeu, and ND1 (initial 100 bp) (Fig. 1). The primers were designed on the alignments of these sequences. DNA fragment was divided into 8 sections because 400-600 bp were desired to sequencing. Forward and reverse primers were designed for each section. The length and temperature of these primers were given in Table 2. For sequence analyses, three samples from each population were used for sequence of mitochondrial 16S rRNA, 12S rRNA and tRNAs genes, which sequenced in Iontek (http://www.iontek.com.tr). Data analysis and phylogenetic relationships: The 16S rRNA, 12S rRNA, tRNAPhe and tRNAVal sequences of nine samples of M. mastacembelus Figure 1. A diagram showing arrangement and position of all amplifying primers. NC 003193 Location Primer Sequence ( 5′ → 3′ ) Length Start End Tm (oC) E1-F CCGGAAACAGGAAAACCTCT 20 24 43 64.10 E1-R TAGCTTTCGTGGGGTCAGAA 20 565 582 64.56 E2-F CTACGGCGTAAAGAGTGGTT 20 453 472 60.71 E2-R CTTTAGAACCGGTTTCAGCA 20 976 995 61.72 E3-F CAAACGTCAGGTCGAGGTGTA 21 843 863 64.97 E3-R ATCATGATGCAAAAGGTACGAG 22 1396 1417 62.69 E4-F TGCAAGTCGGATCACCCTGA 20 1172 1191 69.20 E4-R CGCTTTCTATTGTGGTGGCTGC 22 1757 1778 69.61 E5-F ATAGCTGGTTGCCCGAGAACTG 20 1570 1591 68.31 E5-R GGTAAACAGGCGAGGCTTATAAGG 20 2087 2110 66.31 E6-F GCCAACCTCTCTCCAAACAC 20 1894 1918 63.62 E6-R GTGTCTAAAGCTCCACAGGG 20 2369 2388 61.27 E7-F CCCCAAGGAAAGGCTGAAAG 21 2042 2061 66.76 E7-R CTTGAAGGGGATTGCGCTG 22 2565 2584 67.93 E8-F CGGGGATAACTCCATAAGAC 20 2310 2329 64.20 E8-R GGATTTGAACCTCTGTGGTAAAGG 22 2903 2926 65.43 Table 2. Specific primers used for PCR amplification of target genes of M. mastacembelus. 317 Tutar/ Combined mitochondrial DNA of the Mesopotamian spiny eel from three different locations were analyzed to determinate nucleotide composition by MEGA 5.2 software (Tamura et al., 2011). A blast search was performed on NCBI to compare the sequences of M. mastacembelus populations from Karakaya Reservoir, Tohma Stream and Tigris River, and its phylogenetic tree were constructed based on maximum likelihood model using MEGA 5.2 software (Tamura et al., 2011). The 16S rRNA and 12S rRNA nucleotide sequences of seven species, including M. mastacembelus (in this study), M. armatus, M. erythrotaenia, M. favus, Monopterus albus (Synbranchidae) and Synbranchus marmoratus (Synbranchidae) from the order Synbranchioformes registered to GenBank as in- group and Acipenser stellatus as out-group was used to study the phylogenetic relationships of M. mastacembelus among the members of Synbranchioformes (Table 3). The 16S rRNA and 12S rRNA of these species were translated in different formats aligned using Clustal X (Thompson et al., 1997). The genes of tRNAphe and tRNAval were excluded from this step since there is no sequence knowledge found in public databases such as NCBI and EMBL. Phylogenetic trees were constructed using Maximum Likelihood (ML) (Felsenstein, 1981) and Neighbor Joining (NJ) (Saitou and Nei, 1987) methods using MEGA 5.2 software (Tamura et al., 2011). The robustness of the internal branches of trees was assessed by bootstrapping with 1000 replicates. Phylogenetic trees including nucleotide sequences of M. mastacembelus individuals and M. favus were similarly constructed using ML and NJ methods. Results In the present study, I provided complete sequences of the mitochondrial 16S rRNA, 12S rRNA and tRNA genes of M. mastacembelus. The total length of the 12S rRNA, 16S rRNA, tRNAphe and tRNAval genes of M. mastacembelus were found to be 947 bp, 1667 bp, 69 and 73 bp, respectively. The 12S rRNA, 16S rRNA, tRNAPhe and tRNAVal gene sequences were deposited in GenBank (with accession no GU174757, GU174759, KM211690 and GU174758, respectively). The nucleotide composition of Order Family Species Common names* Distribution* 12S rRNA gene 16S rRNA gene GenBank accession no. GenBank accession no. Synbranchiformes Mastacembelidae Mastacembelus mastacembelus Mesopotami an spiny eel Asia: Tigris and Euphrates basin GU174757 GU174759 Mastacembelidae Mastacembelus armatus Zig zag eel Asia: Pakistan to Viet Nam and Indonesia AF508066 DQ532904 Mastacembelidae Mastacembelus erythrotaenia Fire eel Asia:Thailand and Cambodia to Indonesia AY141349 AY141419 Mastacembelidae Mastacembelus favus Tire track eel Asia:Thailand to the Malay peninsula NC_003193 NC_003193 Synbranchidae Monopterus albus Swamp eel Asia:India to China, Japan, Malaysia and Indonesia NC_003192 NC_003192 Synbranchidae Synbranchus marmoratus Marbled Swamp eel Central and South America: Mexica to northern Argentine AP004439 AP004439 Acipenseriformes Acipenseridae Acipenser stellatus Starry sturgeon Eurasia: Caspain, Balck, Azov and Aegean Seas NC_005795 NC_005795 *Common names and distribution were taken from www.fishbase.org. Table 3. GenBank accession numbers and location of examined species for phylogenetic relationships 318 Int. J. Aquat. Biol. (2015) 3(5): 314-322 12S rRNA is A: 32.4%, C: 26.7%, G: 19.6% and T: 21.2%. The content of A+T (53.6%) is higher than that of C+G (46.3%). The nucleotide composition of 16S rRNA is A: 34.2%, C: 26.2%, G: 19.3% and T: 20.3%. The content of A+T (54.6%) is higher than that of C+G (45.5%). The base compositions of the 12S rRNA, 16S rRNA and tRNAs nucleotide sequences are given in Table 4. The phylogenetic relationships of the studied M. mastacembelus populations i.e. the Karakaya Reservoir, Tohma Stream and Tigris River populations were investigated using combined mitochondrial DNA sequence. For this purpose, their combined identified sequences were compared using NCBI blast software and based on the results no differences were determined between populations and the identity found to be 100% among all three populations. Furthermore, based on the combined target sequences i.e. 12S rRNA, 16S rRNA and tRNA genes of these populations and M. favus, the ML tree was constructed. In this tree, the members of three studied population were clustered together with M. favus in another branch (Fig. 2). The phylogenetic position of M. mastacembelus among the mastacembelids and members of Synbranchioformes that their 16S rRNA and 12S rRNA nucleotide sequences were available in GenBank was constructed (Figs. 3 and 4). Both ML and NJ phylogenetic trees showed two main branches viz. the members of Synbranchiformes as in-group and A. stellatus as out-group showing monophyly of the Synbranchiformes with the families Mastacembelidae and Synbranchidae in discrete clade as sister groups. Mastacembelus favus, M. erythrotaenia and M. mastacembelus formed a monophyletic group with high bootstrap value. Mastacembelus mastacembelus diverged with high bootstrap value from M. erythrotaenia in trees based on 12 rRNA (Fig. 3) whereas, phylogenetic trees of 16S rRNA showed that M. mastacembelus and M. armatus are the closest (Fig. 4). Discussion Identification of fish species is traditionally based on morphological methods i.e. morphometric, meristic and anatomical features. However, there are major Fragment A (%) C (%) G (%) T (%) Total (bp) tRNAPhe 39.1 23.2 20.3 17.4 69 12S rRNA 32.4 26.7 19.6 21.2 947 tRNAVal 30.1 31.5 21.9 16.4 73 16S rRNA 34.3 26.2 19.3 20.3 1667 Average 33.7 26.4 19.5 20.4 565.2 Table 4. Base compositions (% of total number) of target genes of M. mastacembelus. Figure 2. Phylogenetic trees constructed from combined target nucleotide sequences (12S rRNA, 16SrRNA and tRNAs genes) of different studied populations of M. mastacembelus and M. favus based on ML methods with bootstrap support values for each branch. Figure 3. Phylogenetic trees based on ML and NJ methods of 12S rRNA genes with bootstrap support values for each branch. Acipenser stellatus was used as out-group. 319 Tutar/ Combined mitochondrial DNA of the Mesopotamian spiny eel problems to identify the fish species solely based on the morphological characters due to different ecological conditions, which are lead to morphological variations (Lakra et al., 2009; Teletchea, 2009; Chen et al., 2012). Therefore, molecular methods especially based on mtDNA are used as alternative for their identifications. These methods are highly specific, sensitive and simple compared with morphological methods (Comesana et al., 2003). Based on the results, the sequences of 12S rRNA, 16S rRNA, tRNAPhe and tRNAVal genes in M. mastacembelus from different studied locations showed no differences. However, in a previous study significant differences among these populations were observed in terms of morphometric characters (Çakmak and Alp, 2010). Based on Çakmak and Alp (2010), the Karakaya Reservoir population was morphologically different than two other populations. Such condition was reported in sea- bass, Dicentrarchus labrax by Turan and Erguven (2005). They noted that molecular techniques have great potential to support the detected phenotypic differentiation. Furthermore, 12S rRNA and 16S rRNA genes are highly conserved in of animal kingdom (Cawthorn et al., 2012). These genes have been proven to be the powerful phylogenetic tools (Cruz-Agüero et al., 2012). The 12S rRNA has been used to higher categorical levels such as in phyla and 16S rRNA often used for studies at middle categorical levels such as families or genera (Arif and Khan, 2009). Therefore, the results also showed that this genes are not proper to study the genetic population of the genus Mastacembelus as well. The 12S rRNA and 16S rRNA genes were respectively bordered by the tRNAPhe and tRNAVal genes and by the tRNAVal and tRNALeu genes as in other vertebrates (Nagase et al., 2005). The location of these genes are conserved in vertebrates (Chang et al., 1994; Cui et al., 2009). The results of this study were supported by location of these genes. The 12S rRNA and 16S rRNA genes of M. mastacembelus exhibit A+T rich-content like as other bony fishes (Chang et al., 1994). Phylogenetic trees based on ML and NJ method using 12S rRNA and 16S rRNA gene sequences showed that members of the same genus have been clustered together confirming the current taxonomic classifications of the studied fish species (Vreven, 2005a; Froose and Pauly, 2014). Although little nucleotide sequences of the members of Synbranchioformes were available in GenBank. This study is the first attempt to identify phylogenetic position of Mesopotamian spiny eel, M. mastacembelus based on mitochondrial sequences. 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(2015) 3(5): 314-322 E-ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2015 Iranian Society of Ichthyology چکیده فارسی Mastacembelus mastacembelusالنهرین،میتوکندیای مارماهی خاردار بین DNAرکیب ت و جایگاه تبارزایی آن *اسن توتار ترکیه.، کاهرامان ماراش، امام سوتچو کاهرامان ماراشدانشگاه مدرسه علوم طبیعی و کاربردی، ، مهندسی زیستی و علومگروه چکیده: ،M. mastacembelusالنهرین، مینوکندریایی مارماهی خاردار بین valtRNA و rRNA12S ،16S rRNA ،phetRNAهای تیدی ژنوتوالی نوکلئ میتوکندریایی DNAحوضه تیگریس ترکیه براساس ترکیب بخش النهرینمارماهی خاردار بینمقایسه سه جمعیت برای اولین بار تعیین گردید. درصد یکسان 011طور ها بهیافت نشد و توالی آن M. mastacembelusهای مورد مطالعه حاصل انجام شد. براساس نتایج، تفاوتی بین جمعیت انجام M. mastacembelusهای مورد مطالعه جمعیت شناختیآرایه جایگاهاعتبارسنجی نتایج مولکولی و ریختی به منظور همچنین مقایسه. بود براساس Synbranchioformesو راسته Mastacembelidaeدر بین اعضای خانواده النهرینمارماهی خاردار بینبه عالوه جایگاه تبارزایی شد. 12S Rrna 16 وS rRNA مورد بررسی قرار گرفت. روابط تبارزایی حاصل بینM. mastacembelus راسته و برخی دیگر اعضای Synbranchioformes شناختی آنها را مورد تایید قرار داد.جایگاه آرایه .، روابط تبارزاییMastacembelus، S rRNA16، S rRNA12، hePtRNA ،alVtRNA کلمات کلیدی: