A mitogenomic approach to the taxonomy of pollocks: Theragra chalcogramma and T. finnmarchica represent one single species
© Ursvik et al; licensee BioMed Central Ltd. 2007
Received: 03 November 2006
Accepted: 07 June 2007
Published: 07 June 2007
The walleye pollock (Theragra chalcogramma) and Norwegian pollock (T. finnmarchica) are confined to the North Pacific and North Atlantic Oceans, respectively, and considered as distinct species within the family Gadidae. We have determined the complete mtDNA nucleotide sequence of two specimens of Norwegian pollock and compared the sequences to that of 10 specimens of walleye pollock representing stocks from the Sea of Japan and the Bering Sea, 2 specimens of Atlantic cod (Gadus morhua), and 2 specimens of haddock (Melanogrammus aeglefinus).
A total number of 204 variable positions were identified among the 12 pollock specimens, but no specific substitution pattern could be identified between the walleye and Norwegian pollocks. Phylogenetic analysis using 16.500 homologous mtDNA nucleotide positions clearly identify the Norwegian pollock within the walleye pollock species cluster. Furthermore, the Norwegian pollock sequences were most similar to mitochondrial genotypes present in walleye pollock specimens from the Sea of Japan, an observation supported both by neighbor-joining, maximum parsimony, and maximum likelihood analyses.
We infer that walleye pollock and Norwegian pollock represent one single species and that Norwegian pollock has been recently introduced from the Pacific to the Atlantic Oceans.
The walleye pollock (Theragra chalcogramma) is a commercially important codfish species confined to the North Pacific Ocean from the Sea of Japan to the Gulf of Alaska . The population structure of walleye pollock has been investigated by the use of various genetic markers including allozymes, microsatellites, and mitochondrial DNA sequences. The structuring is still unclear despite identification of distinct stocks in geographic regions including Sea of Japan, Sea of Okhotsk, Bering Sea, and Gulf of Alaska [2–5]. The Norwegian pollock (T. finnmarchica) is a very rare codfish species that was first discovered and described in 1932, and with a geographical distribution restricted to coastal regions of northern Norway for all the ca 50 specimens so far recorded . The Norwegian pollock closely resemble the walleye pollock, but previous comparative examinations of morphological features have concluded that the two pollocks represent distinct species [7, 8].
Mitogenomics is a high-resolution molecular genetic approach that includes the complete mitochondrial genome sequence (ca 16.500 bp) in the analyses. Mitogenomics, combined with molecular phylogenetics, has successfully resolved controversial issues of the origin and genetic variation of modern humans [9, 10], as well as phylogenetic relationships among closely related fish species [11, 12]. In the present study we have performed analysis that includes the complete mitochondrial genome sequences from multiple individuals of Norwegian pollock (T. finnmarchica), walleye pollock (T. chalcogramma), Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) in order to resolve the controversial relationship of the Theragra species.
Key features of Gadidae specimens and complete mtDNA sequences
mtDNA accession no.
Theragra finnmarchica (Norwegian pollock)
Tf 19; Norwegian coastal
Tf 21; Norwegian coastal
T. chalcogramma (Walleye pollock)
J1; Sea of Japan
J2; Sea of Japan
J3; Sea of Japan
J4; Sea of Japan
J5; Sea of Japan
B1; Bering Sea
B2; Bering Sea
B3; Bering Sea
B4; Bering Sea
B5; Bering Sea
Gadus morhua (Atlantic cod)
NC1; Norwegian coastal
Melanogrammus aeglefinus (Haddock)
NS; North Sea
No1; Norwegian coastal
Nucleotide substitutions and deletions were assessed by comparing the complete mtDNA sequence of the 12 pollock specimens. The total number of variable sites identified was 204, and include all protein coding and ribosomal RNA genes, the control region (D-loop), and 2 of the 22 transfer RNA genes (Fig 1B). Transition substitutions at third codon positions of protein coding genes were the most common changes, and nucleotide deletions were only observed at one site in the SSU rRNA gene as well as in the ETAS region of the control region. However, no specific substitution feature could be identified in any of the Norwegian pollock sequences in comparison to that of the walleye pollock sequences, including no unique sites that distinguished the two proposed pollock species. Yanagimoto et al.  identified a single nucleotide polymorphism in the ND4 gene as a diagnostic marker of pollocks from the Sea of Japan and the Bering Sea. Interestingly, both the Norwegian pollock specimens harbour the Sea of Japan-type of nucleotide at this position (C11578 in the TF 19 sequence, Fig 1B).
Summary of p-distances between and within species based on 16.500 nt positions
T. finnmarchica/T. chalcogramma
G. morhua/M. aeglefinus
Our finding that the Norwegian pollock appears recently derived from walleye pollock lineages within the Sea of Japan, is puzzling. One possibility is that a subpopulation of walleye pollock migrated from the North Pacific Ocean into the North Atlantic Ocean through the Bering Strait and Arctic Ocean, the only plausible connection between the Pacific and Atlantic Oceans in the Northern Hemisphere. If so, the Norwegian pollock would be expected to be more genetically similar to walleye pollock from the Bering Sea than that from the Sea of Japan due to geographical distances. However, this assumption is not supported in our analysis. Furthermore, walleye pollock has not been reported in the Arctic Ocean, between the Bering Strait and coast of Norway, despite intense fishery activities over years . Alternatively, walleye pollock could have been introduced (intentionally or unintentionally) by human to the northeast Atlantic from the Sea of Japan. In fact, the Soviet Russian authorities did some experiments around 1930 on transfer of marine species by railway from Vladivostok to Murmansk (see http://www.kartesh.com/texts/text.php?uid=46773), but currently there are no available documentations that include walleye pollock. Whatever the reason may be, the presence of a small population of pollock in the North Atlantic Ocean is a very interesting observation and should be included in ecosystem monitoring approaches  of the Arctic Ocean.
Based on the complete mitochondrial genome sequences we conclude that the walleye pollock (Theragra chalcogramma) and Norwegian pollock (T. finnmarchica) represent one single species, and that Norwegian pollock has been recently introduced from the Pacific to the Atlantic Oceans.
Fish samples and DNA extraction
Specimens of T. finnmarchica (Tf. 19 and Tf. 21) were collected at May 11, 2003 and April 29, 2003, respectively, in Norwegian coastal waters east of the North Cape. The G. morhua specimen was collected off Newfoundland, Canada (NF1), and the M. aeglefinus specimen (No1) was collected off the coast of northern Norway. Key-features of fish samples and mitochondrial DNA sequences used in this study are listed in Table 1. DNA was extracted from muscle tissue by using the mtDNA Extractor CT Kit from Wako. The method makes use of differential centrifugation steps to obtain a crude isolation of the mitochondria in the membrane fraction, lysis of the mitochondria, and subsequent precipitation of the supernatant with sodium iodide and isopropanol.
PCR amplification, cloning, and DNA sequencing
PCR and DNA sequencing primers
Sequence (5', 3')
GAT GGA CCC TAG AAA GTC C
AGA TGT GCC TGA TAC CTG CT
GAA AGC TTG ACT TAG TTA AG
TGA CTT CGG ATG CGT ATA AC
TAG AAC AGG CTC CTC TAG
CGC AAG GGA ACG CTG AAA
AGG TAC GAG TAG AAA ACT CTG
CTT ACC AGG CTG TCT TAT GC
GTC CGT TCC GAC TTA CAC
ATT ACA TAA GAC GAG AAG AC
GCC AGT ACT TGC ACT AAC TC
AGC CCA GAA ATA GTA CAG CT
TGG CAC TAG TGA TTT GAC AT
GTA CAC TTC TGG TTA CCA GA
AGC CAA GAT GTG CGA TTG AT
TAA GCC TTT ACT TTT ATC T
CTC TTA GTT AAC AGC TAA GC
AGG GTG CCA ATG TCT TTG TG
TCG AGC AGA GCT AAG TCA AC
ATG TAT AGG AGC TGT CTT TG
TCA ACT GCT ATT ACT TCC CG
ATC ACC CGT AAT AGA AGA GT
TGG AAG CAG GTG ACT CCC AA
ATG ACC TAG TGC ATG AGT TG
TTA CAG CTA ATC TTA CAG CA
TTC AAG GAC TGG AGT ACT AT
TGA CTT GCA AGG AGT ATT AG
GAC CAC ATG ATG ATT TAT TG
TAT AAA CCG CCA ACG TA
ATC TAA TGT CTT GGT TA
ACT TGG AGT TGC ACC AAG AG
TAT AGA GGC TGT AAC TTC TT
GTG AGT ACC TGT AGA TGA GT
CCA CCG TTG TTA TTC AAC T
AAT TAC GGT AGC TCC TCA GAA TGA TAT TTG TCC TCA
TAT TCT CCA TTC TAG TCC TT
CTC GAT TCT AGT CCT CAT GG
ACT GAG CTA CTA GGG CAG TTT C
GTT TAA TTT AGA ATT CTA GCT TTG G
GAA TAG CCA GGA AAC GTG TTA
AAC CGA GGA CTA GCT CCA CC
In general, computer analyses of DNA sequences were performed using software package programs from DNASTAR Inc. For the phylogenetic analyses, nucleotide sequences based on 16.500 positions covering the complete mitochondrial genomes except the highly variable ETAS, were used to make a multiple alignment as one single dataset using ClustalX version 1.81  and manual refinements. MEGA version 3.1  was used to estimate pairwise distances using the uncorrected p-distance model. Furthermore, MEGA was used to construct trees with the methods of neighbor joining (NJ) using different distance matrices, and maximum parsimony (MP) with heuristic searches using close-neighbor-interchang (CNI) level 3 and production of initial trees by of random addition of sequences (100 replicates). Maximum likelihood (ML) analyses, based on the sequence evolution model TMV+G+I selected by the program WinModeltest version 4b , were conducted with PAUP* (version 4.0 b10) . The reliability of tree branching points was assessed by bootstrapping (2000 replications).
We thank Marianne Nymark for technical assistance, and Dr. Sherrylynn Rowe, Bedford Institute of Oceanography, for providing the Newfoundland Atlantic cod sample. This work was supported by grants from the Norwegian Research Council, University of Tromsø, Bodø University College, and the MABIT (Marine Biotechnology in Northern Norway) program.
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