Complete mitochondrial DNA sequences of the frigate

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DNA Sequence, June 2008; 19(3): 159–166

FULL LENGTH RESEARCH PAPER

Complete mitochondrial DNA sequences of the frigate tuna Auxis thazard and the bullet tuna Auxis rochei GAETANO CATANESE†, CARLOS INFANTE, & MANUEL MANCHADO‡ IFAPA Centro El Torunõ, 11500 El Puerto de Santa Marıá, Ca´diz, Spain (Received 19 September 2006)

Abstract The complete mitochondrial DNA sequence of the frigate tuna Auxis thazard and two divergent mitotypes (Mitotype I and Mitotype II) of the bullet tuna Auxis rochei have been determined. The total length of the mitogenomes was 16,506, 16,501 and 16,503 bp, respectively. All mitogenomes had a gene content (13 protein-coding, 2 rRNAs and 22 tRNAs) and organization similar to those observed in most other vertebrates. The major non-coding region (control region) ranged between 843 and 847 bp in length, and showed the typical conserved blocks. Phylogenetic analyses revealed a monophyletic origin of Auxis with respect to other tuna fish. Molecular data here presented provide a useful tool for evolutionary as well as population genetic studies.

Keywords: Auxis thazard, Auxis rochei, mitogenome, mitotype, phylogeny

Introduction Mitochondrial DNA (mtDNA) is commonly used in population genetic surveys and molecular phylogenetic studies due to its high abundance in the cell, high mutation rate, and maternal inheritance (Curole and Kocher 1999). The gene content of vertebrate mtDNA is a nearly identical set of 13 proteins, 22 tRNAs and 2 rRNAs, and a large non-coding region (control region) known to contain replication and transcription regulatory elements (Boore 1999). A considerable progress in the sequencing of complete mtDNA genomes (mitogenomes) has been observed during the last years, becoming useful genetic tools in resolving persistent controversies over higher-level relationships of teleosts (Inoue et al. 2001a; Miya et al. 2001; Lavoue et al. 2005). Tunas of the genus Auxis are cosmopolitan species and the smallest members of the tribe Thunnini, the true tunas. It is currently recognized the existence

of two distinct species, the narrow-corseleted Auxis thazard (Lacepe`de 1800) and the wide-corseleted Auxis rochei (Risso 1810) (Collette and Aadland 1996; Collette et al. 2001). Morphologically, they are differentiated primarily by the width of the corselet under the origin of the second dorsal fin and by the anterior extent of the dorsal scaleless area above the pectoral fin. In A. thazard, the corselet has five or fewer scales under the second dorsal fin, and the dorsal scaleless area extends anterior to the tip of the pectoral fin. On the contrary, A. rochei has six or more scales and the dorsal scaleless area does not reach the tip of the pectoral fin (Collette and Aadland 1996). Both frigate and bullet tuna support very important commercial fisheries throughout the world. In fact, the total world catch of A. thazard and A. rochei in 1996 was higher than 172,000 ton (FAO Fishery Statistics). Such commercial interest is particularly high in Andalucıá (Southern Spain), with nearly 8000 ton off-loaded at Andalusian fishing ports from 1995

Correspondence: C. Infante, IFAPA Centro El Torunõ, Camino Tiro de Pichoń s/n. 11500 El Puerto de Santa Marıá, Ca´diz, Spain. Tel: 34 956011315. Fax: 34 956011324. E-mail: [email protected] † E-mail: [email protected] ‡ E-mail: [email protected] ISSN 1042-5179 print/ISSN 1029-2365 online q 2008 Informa UK Ltd. DOI: 10.1080/10425170701207117


160 G. Catanese et al. to 2002 (Fishery Statistics of Consejerıá de Agricultura y Pesca, Junta de Andalucıá, Spain). They have been largely exploited mainly as canned products due to the excellent properties of the meat, with its mild taste and low cholesterol content. In spite of the high commercial importance of these species, only partial mtDNA sequences are available. So, a fragment of cytochrome b(cytb) (Block et al. 1993) and a flanking region between ATPase6 and COIII (Chow et al. 2003) genes have been employed as diagnostic DNA markers in Auxis identification. Moreover, partial sequences of cytb and ATPase6 genes have been used for the authentication of A. thazard and A. rochei commercial canned products (Infante et al. 2004). However, complete nucleotide sequences of both A. thazard and A. rochei mitochondrial genomes remain to be described. Interestingly, the existence of two mitochondrial lineages in A. rochei (referred to as Mitotype I and Mitotype II) has been recently reported (Infante et al. 2004). Mitotype I (Mit I) was detected exclusively in individuals from the Mediterranean – Atlantic area, while Mitotype II (Mit II) was found in specimens from both the Pacific (Pac-Mit II) and Mediterranean –Atlantic (MA-Mit II) ocean basins. Taking into account these considerations, the aim of this work was to describe the complete mtDNA sequence of A. thazard as well as both A. rochei mitotypes and compare them with those of other teleosts. All the information reported here may facilitate further studies on population structuring, genetic diversity as well as Auxis identification and authentication in raw or processed products.

The four specimens analyzed in this survey (one individual of A. thazard, and one example of each of the A. rochei mitotypes: Mit I, MA-Mit II and Pac-Mit II) were provided by Conservas Ubago, S.L. (Spain). They were collected in the Mediterranean Sea, Eastern Atlantic and Eastern Pacific oceans. Taxonomic classification of A. thazard and A. rochei examples was carried out in basis to morphologic and meristic features as described by Collette and Aadland (1996).

et al. (2004). In order to perform long PCR, four primers were designed using Oligo v6.82 software (Medprobe). For this purpose, known teleost sequences for the 16S rRNA and tRNA-Leu genes were aligned using the program Megalign v5.05 (DNASTAR). Primers were located in conserved regions. Primers leuz1 and leuz2 were overlapping while the primers ARN16Sz1 and ARN16Sz2 formed an amplicon of about 550 bp. The mitochondrial genome was amplified by long PCR in two fragments of , 10 and , 7 kb, respectively. Reactions were carried out using Elongase Enzyme Mix (Invitrogen) according to supplier’s recommendations. One primer set consisted of ARN16Sz1 (50 -CCTCGCCTGTTTACCAAAAACATCGCCTC-30 ) and leuz2 (50 -GACCAATGGGTGAGCTGTTATCCTTTAGAAGC-30 ). The other primer set consisted of leuz1 (50 -GCTTCTAAAGGATAACAGCTCATCCATTGGTC-30 ) and ARN16Sz2 (50 -TAATAGCGGCTGCACCATTAGGATGTCCTG-30 ). PCR reactions were carried out in a 50 ml reaction volume containing 32 ml of sterile distilled water, 1 ml of dNTP mix 10 mM, 10 ml of 5 £ buffer B, 2 ml of each primer (10 mM), 2 ml of Elongase Enzyme Mix, and 1 ml of DNA template containing approximately 20 ng of DNA. The thermal cycle profile was: an initial denaturation step of 968C for 2 min was followed by 30 cycles of denaturation at 968C for 30 s, annealing at 648C for 30 s, and extension at 728C for 10 min. Long PCR products were electrophoresed on a 1% agarose gel and visualized via ultraviolet transillumination, and further purified with CONCERT Rapid PCR Purification System (Invitrogen) and then cloned using the TOPO XL PCR cloning kit (Invitrogen). Plasmids were purified with CONCERT Rapid Plasmid Purification System (Invitrogen) and both strands of template DNA were sequenced by primer walking. Sequencing reactions were carried out with BigDye Terminator v3.1 kit (Applied Biosystems) on an ABI PRISM 377 automatic sequencer. DNA sequences were analyzed using Sequencing Analysis v3.4.1 (Applied Biosystems) and Seqman v5.51 (DNASTAR) programs. From sequence analysis of cloned products, a new pair of primers was employed to PCR amplify and analyze the overlapping tRNA-Leu gene region.

DNA isolation, amplification and purification

Sequence and phylogenetic analyses

A portion of the musculature of each sample was excised, frozen in liquid nitrogen and kept at 2 808C. Total genomic DNA was isolated from 150 mg of tissue using FastDNA kit for 40 s and speed setting 5 in the FastPrep FG120 instrument (Bio101 Inc.). All DNA isolation procedures were performed following the manufacturer’s protocol. PCR reactions and mitogenomes sequencing strategies were carried out as described in Manchado

Sequences were aligned using Megalign v5.51 software (DNASTAR). DnaSP v4.10.3 (Rozas et al. 2003) was used to estimate the number of polymorphic sites among Auxis sequences. Tamura – Nei genetic distances (Tamura and Nei 1993) were calculated with PAUP 4.0b10 (Swofford 2000). The Modeltest Version 3.06 software (Posada and Crandall 1998) was employed as a guide to determine the best-fit maximum likelihood (ML) model. Additionally, ML,

Material and methods Fish sampling


Complete mitochondrial DNA sequences of the frigate tuna Auxis maximum parsimony (MP), and neighbor-joining (NJ) analyses were performed using PAUP 4.0b10 in order to build-up phylogenetic trees. The degree of confidence assigned to nodes in trees was determined by bootstrapping with 2000 replicates (Felsenstein 1985). ML analysis was performed using the fast stepwise-addition search with random addition sequence. For NJ, the ML distance settings were employed. The MP tree was found by using the treebisection-reconnection (TBR) branch-swapping algorithm, with randomized stepwise addition of taxa under the heuristic search method. The mitogenome sequence of Scomber scombrus was used as out-group to root trees (Accession No. AB120717). The entire nucleotide sequence of A. thazard, and the mitogenome sequences corresponding to A. rochei Mit I, Pac-Mit II and MA-Mit II have been deposited in the GenBank/EMBL/DDBJ under Accession numbers AB105447 (A. thazard), AB103467 (A. rochei Pac-Mit II), AB105165 (A. rochei Pac Mit II) and AB103468 (A. rochei MA-Mit II).

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between 47.9 and 48.2%, being the highest among the tuna species analyzed: Thunnus thynnus (46.6%), Thunnus alalunga (46.8%), Katsuwonus pelamis (47.8%) and Euthynnus alletteratus (47.1%). These values were also higher than in other teleosts, such as Engraulis japonicus (45.9%) (Inoue et al. 2001b), Salmo salar (45.2%) (Hurst et al. 1999) and Danio rerio (39.9%) (Broughton et al. 2001). It has been hypothesized that the high G þ C content is associated with higher temperatures in tropical waters (Dalgaard and Garrett 1993; Galtier and Lobry 1997; Wang and Hickey 2002). The cosmopolitan distribution of A. thazard and A. rochei in tropical and subtropical warm waters (Collette and Aadland 1996) agrees with such hypothesis. Base composition of Auxis mitogenomes was analyzed separately for protein-coding genes. A strong anti-G bias was observed in the third codon positions, ranging between 12.2 and 12.7% (not shown). Moreover, pyrimidines were over-represented in the second codon positions (near 64%), as has been reported for other vertebrate mitogenomes, owing to the hydrophobic character of the proteins (Naylor et al. 1996).

Results and discussion Genome organization, genetic code and base composition

Ribosomal RNA genes

Total length for A. rochei mitogenomes was determined to be 16,501 bp for Mit I, and 16,503 bp for both MA-Mit II and Pac-Mit II. A. thazard mitogenome was 16,506 bp in length. These values were similar to those determined for several other Scombridae fish (Manchado et al. 2004, 2005) as well as for other teleosts (Broughton et al. 2001; Inoue et al. 2001b; Miya et al. 2001). The organization and location of different features in the genomes fit to the common vertebrate mitogenome model (Miya et al. 2001) (Table I). All protein-coding genes in A. thazard and A. rochei mitotypes had a methionine (ATG) start codon except for COI, which started with GTG. A GTG codon has also been reported at the beginning of COI genes in other bony fishes (Broughton et al. 2001; Inoue et al. 2001b; Manchado et al. 2004, 2005). Open reading frames ended with TAA (ND1, ND2, COI, ATPase8, ATPase6, COIII, ND4L and ND5) and TAG (ND3 and ND6). The three remaining genes (COII, ND4 and cytb) had an incomplete stop codon, T. Entire stop codons are probably generated by polyadenylation of the corresponding mRNAs (Clayton 1984). Moreover, the pair of genes ATPase8–ATPase6, ATPase6 – COIII, ND4L – ND4 and ND5 –ND6 overlapped ten, one, seven and four nucleotides, respectively (Table I). The overall base composition values for the light strand (L-strand) of Auxis mitogenomes are shown in Table II. Similarly to other teleosts, two main features were found. First, the most represented base in all cases was C; secondly, a bias against G was observed. The G þ C content of Auxis mitogenomes ranged

The 16S rRNA gene was 1692 and 1693 nucleotides long in A. thazard and A. rochei, respectively. With regard to 12S rRNA gene, it was 946, 944 and 945 nucleotides long in A. thazard, A. rochei Mit I, and both A. rochei mitotypes II, respectively. As in other vertebrates, these rRNA genes were located between tRNA-Phe and tRNALeu(UUR), being separated by the tRNA-Val (Hurst et al. 1999; Manchado et al. 2004; Parma et al. 2004; Lin et al. 2006). A typical pattern of conserved segments interrupted by variable ones was found after alignment of ribosomal genes. Nucleotide identity values were always higher than 99%, indicating that rRNA sequences are highly conserved. Transfer RNA genes The 22 tRNA genes in Auxis mitogenomes ranged in size from 67 to 74 nucleotides. They were interspersed between rRNA and protein-coding genes. All tRNAs could be folded into the typical cloverleaf secondary structure as determined by tRNAscan-SE software (Lowe and Eddy 1997). The majority of these postulated secondary structures contained 7 bp in the amino acid stem, 5 bp in the TYC stem, 5 bp in the anticodon stem and 4 bp in the DHU stem. The most different was tRNA-Ser(AGY), with 6 bp in the TYC stem, 6 bp in the anticodon stem and 3 bp in the DHU stem. Single nucleotide variable positions were detected in the DHU arm of tRNA-Trp in A. rochei Mit I, and in the TYC arm of tRNA-Asn in both A. rochei mitotypes II. Nevertheless, these substitutions did not impose changes in the secondary structure.

162 G. Catanese et al.


Table I. Characteristics of A. rochei and A. thazard mitochondrial genomes. Position A. rochei Mit I

Gene/Element

Strand

Control region

–

1 –843

tRNA-Phe 12S rRNA

H H

844 –911 912 –1855

tRNA-Val 16S rRNA tRNA-Leu (UUR) NADH dehydrogenase subunit 1 (ND1) tRNA-Ile tRNA-Gln tRNA-Met NADH dehydrogenase subunit 2 (ND2) tRNA-Trp tRNA-Ala tRNA-Asn OL tRNA-Cys tRNA-Tyr Cytochrome c oxidase subunit 1 (COI) tRNA-Ser(UCN) tRNA-Asp Cytochrome c oxidase subunit 2 (COII) tRNA-Lys ATPase subunit 8 (ATPasa8) ATPase subunit 6 (ATPasa6) Cytochrome c oxidase subunit 3 (COIII) tRNA-Gly NADH dehydrogenase subunit 3 (ND3) tRNA-Arg NADH dehydrogenase subunit 4L (ND4L) NADH dehydrogenase subunit 4 (ND4) tRNA-His tRNA-Ser (AGY) tRNA-Leu(CUN) NADH dehydrogenase subunit 5 (ND5) NADH dehydrogenase subunit 6 (ND6) tRNA-Glu Cytochrome b (cytb) tRNA-Thr tRNA-Pro

H H H H H L H H H L L – L L H L H H H H H H H H H H H H H H H L L H H L

1856–1927 1928–3620 3621–3694 3695–4669 4674–4744 4744–4814 4814–4882 4883–5929 5930–6000 6002–6070 6073–6145 6142–6190 6181–6248 6249–6315 6317–7867 7868–7938 7942–8014 8023–8713 8714–8787 8789–8956 8947–9630 9630–10415 10,415–10,485 10,486–10,836 10,835–10,903 10,904–11,200 11,194–12,574 12,575–12,644 12,645–12,712 12,717–12,789 12,790–14,628 14,625–15,146 15,147–15,215 15,220–16,360 16,361–16,432 16,432–16,501

Size (bp)

Start

844 A. rochei Mit II 847 A. thazard 68 945 A. rochei Mit II 946 A. thazard 72 1692 A. thazard 74 975 71 71 69 1047 71 69 73 49 68 67 1551 71 73 691 74 168 684 786 71 351 69 297 1381 70 68 73 1839 522 69 1141 72 70

End

30 0 0 0

ATG

TAA

ATG

TAA

GTG

TAA

ATG

T...

ATG ATG ATG

TAA TAA TAA

ATG

TAG

ATG ATG

TAA T...

ATG ATG

TAA TAG

ATG

T...

0 0 0 þ4 21 21 0 0 þ1 þ2 24 210 0 þ1 0 þ3 þ8 0 þ1 210 21 21 0 22 0 27 0 0 þ4 0 24 0 þ4 0 21 0

Location of features in the mitogenome of A. rochei Mit I. Size differences of control region and rRNAs in A. thazard and both A. rochei mitotypes II are shown. L and H signify that the indicated gene is transcripted from L-strand or H-strand, respectively. The numbering of positions starsts with the 50 position of control region. Start and stop codons are indicated for protein-coding genes. The 30 column indicates the number of nucleotides downstream to the start of the next gene. The value is negative for genes with overlapping reading frames.

Table II. Overall base composition (%) for the L-strand of Auxis mitogenomes.

Auxis rochei Mit I Auxis rochei MA-Mit II Auxis rochei Pac-Mit II Auxis thazard Thunnus thynnus Thunnus alalunga Katsuwonus pelamis Euthynnus alletteratus Engraulis japonicus Salmo salar Danio rerio

A

C

G

T

26 26 26 25.8 26.2 26.6 26.8 26.5 24.8 28.5 32

32.1 32.1 32 32.2 30.5 31.4 32.6 31.5 27.8 29 23.8

15.8 15.9 15.9 16 16.1 15.4 15.2 15.6 18.2 16.2 16

26.1 26 26.1 26 27.2 26.6 25.4 26.4 29.2 26.3 28.2

Accession No. AB 103467 AB 103468 AB 105165 AB 105447 AB 097669 AB 101291 AB 101290 AB 099716 AB 040676 U 12143 AC 024175

Values for other bony fish mitogenomes are shown for comparison. GeneBank/EMBL/DDBJ accession numbers are indicated in each case.


Complete mitochondrial DNA sequences of the frigate tuna Auxis

Figure 1. Complete nucleotide sequences of mtDNA control region of Auxis mitogenomes. Termination associated sequences (TAS1, TAS2 and TAS3) as well as conserved sequence blocks (CSB-D, CSB-1, CSB-2 and CSB-3) are shaded in gray. Numbers above sites indicate arbitrary positions with respect to the first sequence (A. thazard) in the alignment. Dots indicate identity with reference sequence; dashes represent indels.

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164 G. Catanese et al.


Non-coding sequences L-strand replication origin (OL) was determined to be 49 bp in length in all Auxis mitogenomes, and it was located as in most vertebrates within a cluster of five tRNA genes (tryptophan, alanine, asparagine, OL, cysteine and tyrosine) known as WANCY region. This region was predicted to form a typical secondary structure with a stem of 13 paired nucleotides and a loop of 11 nucleotides rich in T. The conserved sequence motif 50 -GCCGG-30 was found at the base of the stem within tRNA-Cys. The major non-coding region in Auxis mitogenomes was located between tRNA-Pro and tRNA-Phe. Length was determined to be 847, 843 and 844 bp in A. thazard, A. rochei Mit I, and both A. rochei mitotypes II, respectively. In each case, this non-coding sequence appeared to correspond to the control region because it exhibited the typical tripartite structure with a central domain and two adjacent variable domains (ETAS and CSB) (Sbisa` et al. 1997). All blocks of conserved sequences, TAS (TAS1, TAS2 and TAS3), CSB-D, CSB-1, CSB-2 and CSB-3 could be identified. All these regulatory elements were highly conserved among Auxis mitogenomes, with only single substitutions (Figure 1). The identification of a central conserved section, variable left and right domains (Lee et al. 1995), and the high degree of sequence conservation of the putative TAS sequences (Doda et al. 1981), and conserved blocks CSB (Sbisa` 1997) indicate that the mode of DNA replication is also conserved in Auxis. Tandemly repeated sequences present in the control region of some teleosts (Lee et al. 1995) were not observed in Auxis mitogenomes. Minor but highly conserved non-coding sequences varying from one to eight nucleotides were also localized in Auxis mitogenomes between some coding regions (see Table I, positive values in the last column).

Sequence variation and phylogenetic analyses Comparisons between A. thazard and A. rochei mitogenomes resulted in 770, 770 and 767 polymorphic sites with respect to Mit I, MA-Mit II and Pac-Mit II, respectively. Additionally, 265 polymorphic positions between A. rochei Mit I and

MA-Mit II, and 259 between Mit I and Pac-Mit II were determined. Finally, only 94 sites revealed as polymorphic between both A. rochei mitotypes II. These polymorphisms were reflected in Tamura –Nei genetic distances among sequences. As it is shown in Table III, distances among A. rochei and A. thazard mitogenomes were lower than those obtained for other tuna fish (with the exception of T. thynnus/T. alalunga), revealing their closer relatedness. The nearest sequences were found to be A. rochei MA-Mit II and Pac-Mit II (0.005), suggesting minimum evolutionary divergence between both mitotypes. We investigated the phylogenetic position of Auxis species relative to four other tuna species (T. thynnus, T. alalunga, E. alletteratus and K. pelamis) using the concatenated sequence corresponding to tRNA, rRNA and protein-coding genes. Previously, no saturation of transitions/transversions was confirmed by plotting the number of substitutions against uncorrected genetic p-distance for each pairwise ingroup comparison (not shown). The Transversional model þ Invariable sites þ Gamma model (TVM þ I þ G) of DNA sequence evolution was the most appropriate as selected by Modeltest software. The parameters of the ML method were: base frequencies A ¼ 0.2668, C ¼ 0.3237, G ¼ 0.1536, T ¼ 0.2559; proportion of invariable sites was 0.5636, and gamma distribution (a) ¼ 0.8108. With these values a ML tree was built, and the topology was compared with trees obtained by MP and NJ. Largely congruent topological trees were generated using the three methods (Figure 2). A consistent clustering of Auxis sequences showing a monophyletic origin was evident, with a highly significant bootstrap support (100%) in all cases. Similarly, A. rochei sequences were grouped together as a distinct lineage with respect to A. thazard (100% of bootstrap replicates). In addition, both A. rochei mitotypes II were significantly clustered (100% of bootstrap support) in relation to A. rochei Mit I. The presence of two divergent sets of mtDNA lineages (or mitotypes) is not restricted to A. rochei. In fact, it has been also reported in other scombroid fishes, including T. thynnus (Chow and Kishino 1995; Alvarado Bremer et al. 1997), Thunnus obesus (Alvarado Bremer et al. 1998; Chow et al. 2000), T. alalunga (Vinãs et al. 2004a), Sarda sarda

Table III. Pairwise Tamura–Nei genetic distances among Auxis mitogenomes.

A. rochei MA-Mit II A. rochei Pac-Mit II A. thazard T. thynnus T. alalunga K. pelamis E. alletteratus

A. rochei Mit I

A. rochei MA Mit II

A. rochei Pac Mit II

A. thazard

T. thynnus

T. alalunga

K. pelamis

0.016 0.015 0.048 0.117 0.118 0.084 0.097

0.005 0.048 0.117 0.117 0.084 0.097

0.048 0.117 0.117 0.084 0.096

0.119 0.118 0.085 0.098

0.031 0.107 0.120

0.110 0.117

0.084

Other tuna mitogenomes have been included for comparison.


Complete mitochondrial DNA sequences of the frigate tuna Auxis

165

Figure 2. Phylogenetic relationships among Auxis sequences. Four different tuna mitogenomes have been also included in the analyses. The species Scomber scombrus was used as outgroup. ML/MP/NJ bootstrap values are indicated above nodes.

(Vinãs 2004b) and Xiphia gladius (Alvarado Bremer et al. 1996; Rosel and Block 1996). The presence of divergent sympatric mitotypes within species has been interpreted as evidence of vicariance followed by reinvasion in highly migratory species (Avise 2000). Given the migratory character of the bullet tuna this hypothesis cannot be ruled out, although further population genetic analysis will be necessary to clarify this issue. As conclusion, in this survey we have presented the sequences of A. thazard and A. rochei mitochondrial genomes. Two different mitotypes in A. rochei have been also characterized. All these sequences will be useful for further surveys of the evolution of the frigate and bullet tuna, as well as for population studies aimed to determine the degree of genetic structuring between them.

Acknowledgements We wish to thank Conservas Ubago, S.L. for kindly providing samples. We also thank Direccioń General de Pesca (Consejerıá de Agricultura y Pesca, Junta de Andalucıá), Empresa Pu´blica para el Desarrollo Agrario y Pesquero de Andalucıá (D.a.p.), and Jose´ Marıá Naranjo Ma´rquez for their continuous support to the laboratory. This work has been funded by IFOP (EU).

References Alvarado Bremer JR, Mejuto J, Greig TW, Ely B. 1996. Global population structure of the swordfish (Xiphias gladius L.) as revealed by analysis of the mitochondrial DNA control region. J Exp Mar Biol Ecol 197:295–310. Alvarado Bremer JR, Naseri I, Ely B. 1997. Orthodox and unorthodox phylogenetic relationships among tunas revealed by the nucleotide sequence analysis of the mitochondrial DNA control region. J Fish Biol 50:540–554.

Alvarado Bremer JR, Stequert B, Robertson NW, Ely B. 1998. Genetic evidence for inter-oceanic subdivision of bigeye tuna (Thunnus obesus) populations. Mar Biol 132:547–557. Avise JC. 2000. Phylogeography: The history and formation of species. Cambridge: Harvard University Press. Block BA, Finnerty JR, Stewart AF, Gidd J. 1993. Evolution of endothermy in fish: Mapping physiological traits on a molecular phylogeny. Science 260:210–214. Broughton RE, Milam JE, Roe BA. 2001. The complete sequence of the zebrafish (Danio rerio) mitochondrial genome and evolutionary patterns in vertebrate mitochondrial DNA. Genome Res 11: 1958–1967. Boore JL. 1999. Animal mitochondrial genomes. Nucleic Acids Res 27:1767–1780. Chow S, Kishino H. 1995. Phylogenetic relationships between tuna species of the genus Thunnus (Scombridae: Teleostei): Inconsistent implications from morphology, nuclear and mitochondrial genomes. J Mol Evol 41:741–748. Chow S, Nohara K, Tanabe T, Itoh T, Tsuji S, Nishikawa Y, Uyeyanagi S, Uchikawa K. 2003. Genetic and morphological identification of larval and small juvenile tunas (Pisces: Scombridae) caught by a mid-water trawl in the western Pacific. Bull Fish Res Agen 8:1–14. Chow S, Okamoto H, Miyabe N, Hiramatsu K, Barut N. 2000. Genetic divergence between Atlantic and Indo-Pacific stocks of bigeye tuna (Thunnus obesus) and admixture around South Africa. Mol Ecol 9:221–227. Clayton DA. 1984. Transcription of the mammalian mitochondrial genome. Annu Rev Biochem 53:573–594. Collette BB, Aadland CR. 1996. Revision of the frigate tunas (Scombridae, Auxis), with descriptions of two new subspecies from the eastern Pacific. Fish Bull 94:423–441. Collette BB, Reeb C, Block B. 2001. Systematics of the tunas and mackerels (Scombridae). Fish Physiol 19:1 –33. Curole JP, Kocher TD. 1999. Mitogenomics: Digging deeper with complete mitochondrial genomes. Tree 14:394–398. Dalgaard JZ, Garret TA. 1993. In: Kates M, Kushner DJ, Matheson AT, editors. Archaeal hyperthermophile genes. Amsterdam: Elsevier. p 535–563. Doda JN, Wright CT, Clayton DA. 1981. Elongation of displacement-loop strands in human and mouse mitochondrial DNA is arrested near specific template sequences. Proc Natl Acad Sci USA 78:6116 –6120. Felsenstein J. 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39:783– 791.


166 G. Catanese et al. Galtier N, Lobry JR. 1997. Relationships between genomic G þ C content, RNA secondary structures and optimal growth temperture in prokaryotes. J Mol Evol 44:632–663. Hurst CD, Barlett SE, Davidson WS, Bruce IJ. 1999. The complete mitochondrial DNA sequence of the Atlantic Salmon, Salmo salar. Gene 239:237–242. Infante C, Catanese G, Ponce M, Manchado M. 2004. Novel method for the authentication of frigate tunas (Auxis thazard and Auxis rochei) in commercial canned products. J Agric Food Chem 52:7435 –7443. Inoue JG, Miya M, Tsukamoto K, Nishida M. 2001a. A mitogenomic perspective on the basal teleostean phylogeny: Resolving higher-level relationships with longer DNA sequences. Mol Phylogenet Evol 20:275–285. Inoue JG, Miya M, Tsukamoto K, Nishida M. 2001b. Complete mitochondrial DNA sequence of the Japanese anchovy Engraulis japonicus. Fish Sci 67:828–835. Lavoue S, Miya M, Inoue JG, Saitoh K, Ishiguro NB, Nishida M. 2005. Molecular systematics of the gonorynchiform fishes (Teleostei) based on whole mitogenome sequences: Implications for higher-level relationships within the Otocephala. Mol Phylogenet Evol 37:165–177. Lee WJ, Conroy J, Howell WH, Kocher TD. 1995. Structure and evolution of teleost mitochondrial control region. J Mol Evol 41:54–66. Lin G, Lo LC, Zhu ZY, Feng F, Chou R, Yue GH. 2006. The complete mitochondrial genome sequence and characterization of single-nucleotide polymorphisms in the control region of the Asian seabass (Lates calcarifer). Mar Biotechnol 8:71–79. Lowe TM, Eddy SR. 1997. tRNAscan-SE: A program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 25:955–964. Manchado M, Catanese G, Infante C. 2004. Complete mitochondrial DNA sequence of the Atlantic bluefin tuna Thunnus thynnus. Fish Sci 70:68–73. Manchado M, Catanese G, Infante C. 2005. Estudio comparativo del ADN mitocondrial en miembros de la familia Scombridae Consejerıá de Agricultura y Pesca de la Junta de Andalucıá, editor. Sevilla: Libro de Actas IX Congreso Nacional de Acuicultura Ca´diz 2003. p 149–153.

Miya M, Kawaguchi A, Nishida M. 2001. Mitogenomic exploration of higher teleostean phylogenies: A case study for moderate-scale evolutionary genomics with 38 newly determined complete mitochondrial DNA sequences. Mol Biol Evol 18:1993–2009. Naylor GJ, Collins TM, Brown WM. 1996. Hydrophobicity and phylogeny. Nature 373:565–566. Parma P, Erra-Pujada M, Feligini M, Greppi G, Enne G. 2004. Water buffalo (Bubalus bubalis): Complete nucleotide mitochondrial genome sequence. DNA Seq 15:369–373. Posada D, Crandall KA. 1998. Modeltest: Testing the model of DNA substitution. Bioinformatics 14:817–818. Rosel PE, Block BA. 1996. Mitochondrial control region variability and global population structure in the swordfish, Xiphias gladius. Mar Biol 125:11–22. Rozas J, Sańchez-DelBarrio JC, Messeguer X, Rozas R. 2003. DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 19:2496–2497. Sbisa` E, Tanzariello F, Reyes A, Pesole G, Saccone C. 1997. Mammalian mitochondrial D-loop region structural analysis: Identification of new conserved sequences and their functional and evolutionary implications. Gene 205:125–140. Swofford DL. 2000. PAUP* phylogenetic analysis using parsimony (*and other methods) version 4.0b2. Sunderland, Mass. Sinauer. Tamura K, Nei M. 1993. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in human and chimpanzees. Mol Biol Evol 10:512–526. Vinãs J, Alvarado Bremer J, Pla C. 2004a. Inter-oceanic genetic differentiation among albacore (Thunnus alalunga) populations. Mar Biol 145:225–232. Vinãs J, Alvarado Bremer J, Pla C. 2004b. Phylogeography of the Atlantic bonito (Sarda sarda) in the northern Mediterranean: The combined effects of historical vicariance, population expansion, secondary invasion, and isolation by distance. Mol Phylogenet Evol 33:32–42. Wang HC, Hickey DA. 2002. Evidence for strong selective constraint acting on nucleotide composition of 16S ribosomal RNA genes. Nucleic Acids Res 30:2501 –2507.