© 2004 by ICES/CIEM International Council for the Exploration of the Sea/Conseil International pour l'Exploration de la Mer
A mitochondrial DNA PCR–RFLP marker for population studies of the black scabbardfish (Aphanopus carbo)
Instituto Nacional Investigação Agrária e das Pescas/Instituto de Investigação das Pescas e do Mar Av. de Brasilia, 1449-006 Lisboa, Portugal
*Correspondence to R. Quinta: tel: +351 21 3027025; fax: +351 21 3015948. e-mail: rquinta{at}ipimar.pt.
Black scabbardfish (Aphanopus carbo), a commercially valuable marine fish off Portugal and the Madeira Archipelago, was surveyed for mitochondrial DNA variation of part of the cytochrome b gene. In all, 51 fish from three Northeast Atlantic localities were examined using ten restriction enzymes. Overall nucleon diversity was 0.180. Genetic differentiation (
=0.25) was significant; the Madeira Archipelago sample was distinguishable from samples from the other two localities. The approach should be useful for a more extensive study of black scabbardfish populations.
Keywords: Aphanopus carbo, PCR–RFLP, stock identification
Received 12 January 2004; accepted 20 March 2004.
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Introduction |
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 Top Introduction MethodsResults and discussionReferences |
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Black scabbardfish (Aphanopus carbo Lowe, 1839) are marine fish found on the continental shelf and slope between 200 and 1600 m deep. They are widespread in the eastern Atlantic along the continental shelf and around several groups of islands and underwater rises (Parin, 1986; Sanches, 1991), are benthopelagic, and migrate to feed in midwater at night (Sanches, 1991). Juveniles are pelagic, but little is known about them because small fish are seldom captured (Martins et al., 1994; Morales-Nin and Sena-Carvalho, 1995; Haedrich, 1997; Vinnichenko, 2002).
The spawning areas of black scabbardfish in the eastern Atlantic are not well known, but several fish were in post-spawning condition between October and December off Madeira (Morales-Nin and Sena-Carvalho, 1995). According to Nakamura and Parin (1993), there is also a spawning season from November to April west of the British Isles, months also mentioned as the spawning season for the Azores Archipelago by Vinnichenko (2002).
Black scabbardfish are commercially important in Spain, the United Kingdom, Ireland, and especially Portugal, where total landings reached approximately 7000 t in 2001 (FAO, 2002). Most landings are made in Sesimbra (mainland Portugal), and Madeira, near the two main fishing areas for the species (Figueiredo et al., 1994). The species is an important part of the Madeiran diet, and is now the largest catch by the island's commercial fishing fleet, accounting for 55% of total landings.
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Methods |
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TopIntroductionMethodsResults and discussionReferences |
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Samples of Aphanopus carbo were collected from three localities (Figure 1) in the northeastern Atlantic Ocean: the Hatton Bank (59.25°N 15.21°W), Sesimbra (38.24°N 09.80°W), and the Madeira Archipelago (32.44°N 16.35°W). Samples were frozen at –20°C. Later, tissue was removed, placed into individual cryopreservation tubes, and again held at –20°C. The mitochondrial DNA was isolated from muscle using the Tetradecyl-Trimethyl-Ammonium bromide (CTAB) technique described by Milligan (1998), with some modification. About 25 mg of muscle tissue was removed from the dorsal white muscle of frozen specimens and dissolved in 300 µl of CTAB buffer (100 mM Tris–HCl, 20 mM EDTA, 2% Tetradecyl-Trimethyl-Ammonium bromide (CTAB), 0.2% mercaptoethanol), followed by 1 h of incubation with 25 µl of Proteinase K (20 mg ml–1) at 60°C. The DNA was purified using phenol–chloroform–isoamyl alcohol (25:24:1) and centrifuging at 13500 rpm for 10 min. The DNA was precipitated using ethanol, 25 µl of 3 M sodium acetate and guanidine chloride, and re-suspended in 100 µl TE.
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Part of the mtDNA region coding for cytochrome b was amplified using puReTaq Ready-To-Go PCR Beads (Amersham Biosciences), to which was added 25 ng of DNA, 10 pmol of primers L15162 [GenBank] and H15573 [GenBank] and water to a volume of 25 µl. Primer sequences (Taberlet et al., 1992) were:
- L15162, 5'-GCAAGCTTCTACCATGAGGACAAATATC-3'; H15573
[GenBank]
, 5'-AATAGGAAGTATCATTCGGGTTTGATG-3'.
The amplification protocol consisted of a denaturing step at 95°C for 3 min, followed by 35 cycles (93°C for 1 min, 55°C for 90 s, 72°C for 2 min), and a final extension step of 72°C for 10 min, after which the samples were allowed to cool. After amplification in a PCT-100TM thermal cycler (MJ Research, Inc.), the 411-bp fragment was sequenced in an ABI PRISM 310 automatic sequencer. The sequence was imported into the GCG-Wisconsin software package to obtain a list of enzymes that cut the fragment. Ten of these enzymes were chosen (AluI, BamHI, BsaI, BsaJI, Bsma, HaeIII, HinfI, HphI, MboII, RsaI). Then, 10 µl of DNA was digested with 5 U of endonuclease under conditions recommended by the manufacturer (New England Biolabs).
The restriction fragments were electrophoresced in acrylamide gels (GeneExcel gel 12.5/24 from Amersham Biosciences). The gels were run in a GenePhor apparatus (Amersham Biosciences), the running conditions being 200 V, 400 mA, 100 W for 2 h. The gel was stained with Silver Staining Colouration Kit (Amersham Biosciences). Analyses of mtDNA data were performed using the software TFPGA of Miller (1997).
A pairwise population comparison following the methodology of Raymond and Rousset (1995), using a Monte Carlo randomization procedure with 1000 dememorization steps, 100 batches, and 2000 permutations by batch, was carried out, and F-statistics were estimated using the approach of Weir and Cockerham (1984). Nucleon diversity was calculated with the formulations of Nei (1987).
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Results and discussion |
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TopIntroductionMethodsResults and discussionReferences |
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Of the ten restriction enzymes assayed, only two (BsaJI and MboII) showed multiple restriction patterns, two for each enzyme (Table 1). Haplotype A was composed of three bands of approximately 230, 130, and 51 bp, for BsaJI, and 281, 80, and 50 bp, for MboII. Haplotype B was composed of two bands of approximately 230 and 181 bp, for BsaJI, and 281 and 130 bp, for MboII.
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Most fish shared the most common restriction pattern (A) in each endonuclease digestion. All fish from the Hatton Bank and Sesimbra, and about 70% of fish from Madeira, showed this pattern. The remaining 30% of Madeira's sample showed pattern B, for both enzymes.
The nucleon diversity (h) of black scabbardfish, 0.180 (Table 1), is low compared with pelagic species such as jackass morwong, Nemadactylus macropterus (h=0.670, Grewe et al., 1994), bluefish, Pomatomus saltatrix (h=0.696, Graves et al., 1992), and yellowfin tuna, Thunnus albacares (h=0.724, Ward et al., 1994), but is consistent with other demersal species such as Atlantic cod, Gadus morhua (h=0.360, Carr and Marshall, 1991; h=0.100, Smith et al., 1989) and American plaice, Hippoglossoides platessoides (h=0.350, Stott et al., 1992). However, nucleon diversity is a crude measure of variability. It is based on haplotype frequencies alone, and as more restriction enzymes are employed in a study and additional variable cut sites detected, the number of haplotypes, and therefore the value of h, increases.
The test (Raymond and Rousset, 1995) for population differentiation gave a p-value of <0.001, indicating that the composite haplotypes were not distributed randomly with respect to locality. F-statistics (Weir and Cockerham, 1984) yielded a -value of 0.25. Following Hartl (1988), >0.05 means that the population is structured, so the value of 0.25 obtained for A. carbo indicates structuring into genetically differentiated subpopulations.
To identify the sample responsible for this great differentiation, a pairwise population comparison was performed. Clearly, the set of Madeiran samples was responsible for the heterogeneity, the two other samples not showing any differentiation (the two pairwise population comparisons with Madeira have a p-value of 0.015, and that without Madeira gave a p-value of 1.000).
Although there is no literature on population studies of black scabbardfish, ICES (2000) lists a single stock extending from the Faroe Islands to Madeira (though there is no evidence in the literature to support this statement). The present study suggests that the black scabbardfish population in the northeastern Atlantic is genetically structured. Based on the frequency of the restriction patterns, the samples can be divided into two groups, those from the eastern Atlantic (Sesimbra and the Hatton Bank) and the one from around the Madeira Archipelago.
Fish in spawning condition are captured from November to April, and fish captured off Scotland are generally considerably smaller than those caught off Sesimbra (ICES, 2000). In contrast, fish landed at Sesimbra are mainly immature, even the largest ones. Therefore, it would seem that only a few fish mature off Portugal, and that such females exhibit atresia (Bordalo-Machado et al., 2001). Some Sesimbra-caught fish are mature, but no juveniles or post-spawning fish are caught there.
One hypothesis is that the black scabbardfish feed in southern areas (off mainland Portugal), migrating north to spawn. Such a hypothesis is consistent with the results obtained here, namely no differences between the samples from the Hatton Bank and Sesimbra, suggesting a single population from Scotland to the coast of Portugal.
According to Morales-Nin and Sena-Carvalho (1995), the waters around the Madeira Archipelago constitute a spawning area for black scabbardfish from October to December, their evidence being the several mature and post-spawning fish caught then. The same authors caught several juveniles, but do not believe that such catches mean seasonality in the juvenile occurrence, because they are found in small numbers throughout the year. Those results are consistent with the present data, and suggest a different and possibly isolated population of black scabbardfish in Madeiran waters.
The technique used was simple, and these markers provide a powerful tool for further studies of the population structure of this species.
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Acknowledgements |
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We are grateful to the European Commission for funding this work within the BASBLACK–EU study project CT 97/0084, and to Bob Ward and an anonymous reviewer for helpful comments on the submitted manuscript.
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References |
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