- Email: [email protected]
Nuclear DNA RFLP variation among Atlantic cod in south and south-east Icelandic waters
Fisheries Research 67 (2004) 227–233
Short communication
Nuclear DNA RFLP variation among Atlantic cod in south and south-east Icelandic waters A.K. Imsland a,∗,1 , Ó.D.B. Jónsdóttir b,1 , A.K. Dan´ıelsdóttir c a Akvaplan-niva, Iceland Office, Akralind 4, 201 Kópavogi, Iceland Department of Fisheries and Marine Science, University of Bergen, 5020 Bergen, Norway Marine Research Institute, Division of Population Genetics, Keldnaholti, 112 Reykjav´ık, Iceland b
c
Received 24 April 2003; received in revised form 12 September 2003; accepted 21 September 2003
Abstract The aim of this study was to describe the population structure of Atlantic cod, Gadus morhua, in Icelandic waters. A total of 404 cod was collected from five locations in Icelandic waters. The samples were analysed for allelic variation at five restriction fragment length polymorphism (RFLP) (GM862, GM860, GM738, GM777 and GM865) loci. Significant differences between sample sites were found at two loci (GM865 and GM777). The results are in line with other recent findings from this area indicating that the cod in south and south-east Icelandic waters do not belong to one panmictic population but may represent two genetically different reproductive stocks of cod. © 2003 Elsevier B.V. All rights reserved. Keywords: Atlantic cod; Gadus morhua; Nuclear RFLP; Population genetics; Iceland
1. Introduction From 38 years of tagging and recapture studies of cod in Iceland, Jónsson (1996) showed that the main migratory routes were related to spawning off the southwest coast. After spawning, the fish migrated northwards along the west coast to the rich feeding grounds off the northwest and north coast. Also prior to spawning, the fish migrated from the north along the west coast to the spawning area off the southwest coast, then continuing to the east along the south coast. Endemic populations were found in some of the fjords of the west and north coast. The lack of recaptured cod tagged at the northeast coast at the spawning grounds ∗ Corresponding author. Tel.: +354-562-5800; fax: +354-564-5801. E-mail address: [email protected] (A.K. Imsland). 1 Equal Authorship.
in the southwest might be due to a local spawning at the northeast coast. Studies of cod populations genetics at Iceland have lead to contradictory results. In a study of cod populations in Icelandic waters using transferrin and haemoglobin, Jamieson and Jónsson (1971) described spatial (both loci) and temporal (haemoglobin) variation. Hardy–Weinberg disequilibrium was found at some of the sampling sites, indicating that different units of cod are present in Icelandic waters. In contrast, Árnason et al. (1992), using restriction fragment analysis of mitochondrial DNA, found no evidence of population differentiation in Icelandic cod. Recently, the work of Jónsdóttir et al. (1999, 2001, 2002) indicated considerable population substructuring of cod in Icelandic waters. In the studies of Jónsdóttir et al. (1999, 2001, 2002), cod samples were analysed using the sequenced and analysed SypI locus presented by Fevolden and Pogson (1995,
0165-7836/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.fishres.2003.09.045
228
A.K. Imsland et al. / Fisheries Research 67 (2004) 227–233
1997), as well as the haemoglobin locus. Significant differences were detected at the SypI locus between two groups (Loftstaðahraun, Reykjanesgrunn and Eyrabakkabugur vs. Kantur and Austfjarðadjúp) in Icelandic waters. These results are consistent with tagging studies in Iceland (Jónsson, 1996). However, the discrepancy between the studies of Jamieson and Jónsson (1971), Jónsson (1996) and Jónsdóttir et al. (1999, 2001, 2002), and that of Árnason et al. (1992) shows that we still have limited knowledge of the structuring of Atlantic cod in Icelandic waters. The present paper is aimed at examining the genetic structure of Atlantic cod from south Icelandic waters using an analysis of five nuclear DNA RFLP loci. The study is a part of larger study where frequencies at the SypI and haemoglobin loci sampled at the same sampling sites have been described in details in earlier papers (Jónsdóttir et al., 1999, 2001, 2002). 2. Materials and methods 2.1. Sampling A total of 404 cod was collected from five locations in Icelandic waters (Fig. 1, Table 1). Two of
the samples were taken from known spawning sites (Loftstaðahraun [L], Kantur [K]) and three from feeding grounds (Reykjanesgrunn [R], Eyrabakkabugur [E], Austfjarðadjúp [A]). At Kantur samples taken ca. 13 nautical miles apart (called Kantur 1 and Kantur 2) showed no intra-area differences (Fisher’s exact test, P > 0.65), and so were pooled for inter-area comparisons (hereafter called Kantur). Further rationale of the sampling scheme is given in Jónsdóttir et al. (1999). 2.2. Genetic analysis The cod samples were examined using nuclear DNA RFLP analysis. The primary source of the nuclear DNA analysis was cod gill tissue (200 mg), collected from cod and preserved immediately in 96% ethanol. The DNA extraction was performed by phenol-extraction (Taggart et al., 1992). The five restriction fragment length polymorphism (RFLP) loci (GM842, GM860, GM738, GM777 and GM865) were analysed using cDNA probes and amplified by PCR conditions as described by Pogson et al. (1995). Each of the five RFLPs examined in the present study is characterised by a specific cDNA probe-restriction
Fig. 1. Collection sites of Atlantic cod in Icelandic waters. Abbreviations: L = Loftstaðahraun, E = Eyrabakkabugur, K = Kantur (Dyrh´olaey), R = Reykjanesgrunn, A = Austfjarðadj´up (see Table 1 for details).
A.K. Imsland et al. / Fisheries Research 67 (2004) 227–233
229
Table 1 Locations, number of specimens (n), location, date of collection, and sex-ratio of Atlantic cod in Icelandic waters used in this study Location Loftstaðahraun (L) Eyrabakkabugur (E) Reykjanesgrunn (R) Kantur 1 (Dyrh´olaey) (K 1) Kantur 2 (Dyrh´olaey) (K 2) Austfjarðadj´up (A)
n
Position
100 36 64 48 52 104
63◦ 47N, 63◦ 47N, 63◦ 47N, 63◦ 15N, 63◦ 19N, 64◦ 33N,
20◦ 51W 21◦ 40W 22◦ 50W 18◦ 54W 19◦ 24W 12◦ 20W
enzyme combination. Total DNA samples (7 g) from all specimens were digested with two restriction enzymes (DraI, TaqI). DraI digested samples were probed with cDNA clones GM842 and GM860 and TaqI digests were probed with clones GM738, GM777 and GM865. The cDNA probe-based loci were screened using non-radioactively labelled cDNA probes (Enhanced Chemiluminescence, ECL, Amersham, Buckinghamshire). The probes were labelled with the enzyme horseradish peroxidase according to instructions in the ECL protocol (Amersham). Probes were hybridized to total DNA samples (7 g) from each cod specimen that had been digested with either of the two restriction enzymes (DraI, TaqI) according to the manufacturer (Boehringer). The digested DNA was electrophoresed on 0.8% agarose gels in 1 × TBE buffer, pH 8.3, for 17–19 h and transferred to nylon membranes (Amersham, Hybond N+) in a Pharmacia VacuGene apparatus, after which it was fixed to the membranes by baking for 2 h at 80 ◦ C. Membranes were prehybridized for 2 h at 42 ◦ C in 50 ml hybridization buffer (50 ml Gold buffer, 5% Blocking reagent (both provided by Amersham), 0.5 M NaCl). The horseradish peroxidase labelled cDNA probes were added directly into the hybridization buffer. Hybridizations were carried out overnight at 42 ◦ C. Membranes were washed once in 80 ml of 5 × SSC at 42 ◦ C for 5 min, then twice in 0.125 ml/cm2 washing solution I (0.4 × SSC, 0.4% SDS) at 55 ◦ C for 10 min, and finally twice in 0.125 ml/cm2 washing solution II (2 × SSC) at room temperature for 5 min. Following washing, the membranes were soaked in equal volumes of detection reagents 1 and 2 (provided by Amersham) for 1 min. The membranes were then placed in a plastic envelope and exposed to X-ray films (Sterling, Diagnostic Imaging) overnight to
Date
Depth (m)
Sex (male:female)
3–4 April 1997 30 January 1998 31 January 1998 25–26 March 1997 25–26 March 1997 26–28 October 1997
82 54 110 253 262 355
67:33 11:25 43:21 28:20 28:24 43:61
detect band patterns. The membranes were stored in 2 × SSC between hybridizations. Restriction fragment sizes were estimated using DNAfrag 3.01 package (Schaffer and Sederoff, 1981) with reference to the HindIII ladder run alongside the samples in the electrophoresis gel. RFLP patterns were apportioned into three groups based on Pogson (1994). Each form of polymorphism was first assigned a letter and then broken down into allelic sizes. 2.3. Statistical methods Allele and genotype frequencies together with pairwise FST values (Reynolds et al., 1983), and exact test of population differentiation were calculated using the ARLEQUIN 2.0 computer package (Schneider et al., 1997). Genotypic disequilibrium between loci was tested with an extension of Fisher exact test on contingency tables (Schneider et al., 1997). Allele frequencies were bootstraped 1000 times and Nei’s (1972) genetic distances based on the allele frequencies were calculated using the SEQBOOT and GENDIST program in the PHYLIP package (Felsenstein, 1993) and a UPGMA dendrogram of the bootstraped Nei’s genetic distance matrix was constructed in the NEIGHBOR program in PHYLIP. A Bonferroni correction (Johnson and Field, 1993) of the significance level (α = 0.05) was applied when testing for significant differences in allele frequencies and for significant departures from Hardy–Weinberg expectations.
3. Results No differences between sexes were found in frequencies of any of the loci investigated (P > 0.25). Genotypic disequilibrium between loci was found
230
A.K. Imsland et al. / Fisheries Research 67 (2004) 227–233
Table 2 Allele frequencies at each locus for analysed cod samples from five locations in Icelandic watersa Locus/alleles (kb)
Loftstaðahraun (spawning area)
Eyrabakkabugur (feeding area)
Reykjanesgrunn (feeding area)
Kantur (spawning area)
Austfjarðadj´up (feeding area)
GM842 n 2.65 2.92 3.07 3.15 3.35 3.60 H
42 0.000 0.036 0.024 0.928 0.012 0.000 0.142 (0.112)
33 0.036 0.030 0.000 0.934 0.000 0.000 0.090 (0.112)
55 0.009 0.018 0.009 0.953 0.000 0.009 0.090 (0.085)
19 0.000 0.053 0.000 0.947 0.000 0.000 0.105 (0.100)
37 0.027 0.000 0.000 0.946 0.000 0.027 0.081 (0.051)
GM860 n 1.60 2.38 5.70 6.27 H
56 0.527 0.000 0.473 0.000 0.482 (0.498)
35 0.614 0.000 0.386 0.000 0.371 (0.474)
61 0.616 0.000 0.384 0.000 0.475 (0.473)
32 0.625 0.010 0.375 0.000 0.593 (0.468)
84 0.568 0.000 0.432 0.021 0.452 (0.490)
GM738 n 1.89 2.70 2.77 H
45 0.556 0.000 0.444 0.644 (0.493)
36 0.458 0.014 0.528 0.500 (0.484)
61 0.500 0.000 0.500 0.510 (0.500)
57 0.491 0.000 0.509 0.456 (0.500)
83 0.545 0.000 0.455 0.437 (0.495)
GM777 n 1.57 2.03 2.55 3.04 4.30 H
48 0.010 0.010 0.104 0.855 0.021 0.250 (0.189)
26 0.000 0.000 0.330 0.670 0.000 0.498 (0.442)
38 0.000 0.000 0.162 0.811 0.027 0.316 (0.262)
GM865 n 1.71 1.78 1.82 H
45 0.000 0.422 0.578 0.511 (0.487)
32 0.008 0.200 0.800 0.390 (0.32)
49 0.021 0.306 0.673 0.489 (0.465)
0b – – – – –
30 0.000 0.375 0.625 0.433 (0.468)
0b – – – – –
58 0.000 0.465 0.535 0.379 (0.497)
a n: number of specimens giving detectable bands at each site; allele sizes are given in kb; H: frequencies of heterozygotes, observed (expected). b Due to technical reasons no variation could be detected.
for all loci and sampling units (P < 0.01). Although observed frequencies of heterozygotes were in most cases higher than expected Hardy–Weinberg values (Table 2), all samples were in Hardy–Weinberg equilibrium (P > 0.05). The most common allele was the same at each of four loci (GM842, GM860, GM777 and GM865) for all sample units, with frequencies ranging from 0.53 to 0.95 (Table 2). Significant
differences in allelic frequencies were found at GM865 (K vs. E: FST = 0.1, P < 0.05; K vs. L: FST = 0.07, P < 0.05) and GM777 (K vs. L: FST = 0.09, P < 0.05). When tested separately each locus was found to be in Hardy–Weinberg equilibrium (P > 0.29). However, the total material (all samples compiled) was in Hardy–Weinberg disequilibrium (P < 0.05), suggesting that the samples were
A.K. Imsland et al. / Fisheries Research 67 (2004) 227–233
Fig. 2. A UPGMA dendrogram of the Nei’s (1972) genetic distance matrix among the cod populations in the present study. Values on the nodes represent the percentage of bootstrap samples (n = 1000).
drawn from more than one panmictic population. The highest level of differentiation was found between the L and the K samples (FST = 0.30, P < 0.001) and the L and the A samples (FST = 0.12, P < 0.001). The bootstrapped UPGMA dendrogram constructed using Nei’s (1972) genetic distances illustrated a clear differentiation between the sample sites analysed (Fig. 2). The largest genetic distance was found between Loftstaðahraun and Kantur (Fig. 2).
4. Discussion The present findings and the findings of Jónsdóttir et al. (1999, 2001, 2002), support the sub-structuring theory of cod in Icelandic waters. Significant differences were detected at the GM865 and GM777 (present study, see above) and the SypI (Jónsdóttir et al., 1999) loci between the sampling sites in Icelandic waters. Consistent with the findings of Fevolden and Pogson (1997) who studied cod in Norwegian waters, Jónsdóttir et al. (1999, 2001, 2002) detected significant differences at the SypI locus between two groups (Loftstaðahraun, Reykjanesgrunn and Eyrabakkabugur vs. Kantur and Austfjarðadjúp) of cod in Icelandic waters. The subdivision of cod
231
into the two groups supports earlier tagging studies of cod from the investigated areas. Cod spawning at Loftstaðahraun have been recaptured along the southwest coast (e.g. Eyrabakkabugur and Reykjanesgrunn) during the feeding period, but cod spawning at Kantur have been recaptured along the south-east and east coast (e.g. Austfjarðadjúp; Jónsson, 1996). When comparing the present findings with those reported for the same sampling locations applying different markers (Jónsdóttir et al., 1999) clear similarities are apparent. Both studies clearly differentiated the two spawning areas (Loftstaðahraun and Kantur) thus suggesting the occurrence of two reproductive stocks. However, the status of the feeding areas seems to differ between the studies. In the present study, Loftstaðahraun did not group with any of the feeding areas, whereas in the first study it grouped with Reykjanesgrunn and Eyrabakkabugur. However, based on FST and exact population tests both studies give similar results. The feeding ground samples (Reykjanesgrunn, Eyrabakkabugur and Austfjarðadjúp) are not different, but differ from both spawning grounds samples (Table 4 in Jónsdóttir et al., 1999) or Loftstaðahraun (present study). Why the Kantur sample does not differ from three feeding ground samples can at this stage only be speculated and might well be the result of rather limited data material in the present study. This is especially true for the GM777 locus as it was, due to technical reasons, not detected in Eyrabakkabugur and Reykjanesgrunn (see Table 2). Overall the material was found to be in Hardy– Weinberg disequilibrium indicating that the samples are drawn from more than one population (Wahlund effect), and that the deviation is due to an unusual, but random, combination of excesses or deficiencies of heterozygotes in different population units. Alternatively, deviation of the pooled data could be due to the presence of rare and private alleles or the cumulative effect of selection at individual loci. However, with the exception of GM842 and GM777 in Loftstaðahraun and GM842 in Austfjarðadjúp, the rare and private alleles represented only 1% or less of the total occurrence at each locus (Table 2). Also, earlier investigations have not indicated any selective effect on those loci presented here (Pogson and Fevolden, 1998). The present study indicates subdivision of Atlantic cod in Icelandic waters. This is in sharp contrast to studies based on mitochondrial DNA
232
A.K. Imsland et al. / Fisheries Research 67 (2004) 227–233
variation as these have shown limited or no differentiation of populations (e.g. Árnason et al., 1992, 2000). The question must arise why the RFLP analyses of mitochondrial, on one hand, and DNA on the other, give such contrasting results? Firstly, all samples in Árnason et al. (1992) study were taken from coastal areas making it impossible to test whether an off-coastal population might exist off Iceland. Further, the vast majority of samples were sampled from depths shallower than 90 m thus ignoring the fact that cod off Iceland is predominantly caught at depths exceeding 90 m. It is possible that this sampling procedure which is biased towards shallow depths and coastal areas might not be adequate if the aim is to describe the population structure of cod off Iceland. Secondly, the contrast between mitochondrial DNA and nuclear DNA analysis in resolving population structure may relate to different potential for detecting variation (Carvalho and Hauser, 1994; Ward and Grewe, 1994; Ruzzante et al., 1996). This is supported by the fact that in many cases mtDNA analysis has not lead to enhanced resolution of stock issues compared with other molecular methods (Ward and Grewe, 1994 and references therein). It has, thus, been suggested that analysis of frequencies distributions demands larger sample sizes than traditionally has been employed in many mtDNA studies, and that the use of too few individuals may contribute to the mtDNA homogeneity commonly observed (e.g. Árnason et al., 1992). The discrepancy between the studies of Jamieson and Jónsson (1971) and Jónsdóttir et al. (1999, 2001, 2002), and those of Árnason et al. (1992), shows that knowledge of the structuring of Atlantic cod in Icelandic waters is limited and emphasis the need for further research on this topic. In Norway the subdivision of Atlantic cod into Arcto-Norwegian (AN) cod and Norwegian coastal (NC) cod has been supported and discussed in many studies (Dahle, 1991; Fyhn et al., 1994; Fevolden and Pogson, 1995, 1997). The results of Fevolden and Pogson (1995, 1997) confirmed the existence of highly significant differences between AN and NC cod. Further, they found the SypI locus to suggest that genetic heterogeneity might exist among resident populations of cod in different fjords. Recent studies in the Northwest Atlantic have also revealed genetic subdivision of cod at localized scales. The studies Pogson et al. (1995) using nuclear DNA RFLPs,
and Galvin et al. (1995) using the Mmer-AMP2 minisatellite locus revealed clear genetic subdivision of cod off Newfoundland and on the Scotian Shelf. The results from microsatellite studies of Bentzen et al. (1996) and Ruzzante et al. (1996) supported the findings of three genetically distinct populations of cod (Flemish Cap, Scotian Shelf, Northern cod), and further division of Northern cod into North and South components. The indications of sub-structuring of cod in Icelandic waters (Jónsdóttir et al., 1999, 2001, 2002, present study) are consistent with earlier genetic studies of cod in Norwegian waters and northwest Atlantic (see references above). This may indicate that cod in the North Atlantic Ocean is sub-structured at small geographic scale. In conclusion, the present study indicates population differentiation between cod populations off Iceland and that there are (at least) two genetically different reproductive stocks of cod in Icelandic waters (i.e. Kantur and Loftstaðahraun). However, only few analyses have been performed on cod in Icelandic waters, and only few genetic markers have yet been used in these population genetic studies. In order to gain more information on the genetic structure of cod in Icelandic waters, the application of alternative genetic markers is necessary (e.g. microsatellites). Also, more samples, especially from the fishing grounds off northwest Iceland, need to be analysed to confirm our findings.
Acknowledgements The authors thank G. Pogson, S.E. Fevolden, P. Galvin, J. Coughlan and J. Solvang for help and advice during the analytical work. Financial support was given by the EU FAIR program (FAIR CT95-0282).
References Árnason, E., Pálsson, S., Arason, A., 1992. Gene flow and lack of population differentiation in Atlantic cod, Gadus morhua L., from Iceland, and comparison of cod from Norway and Newfoundland. J. Fish Biol. 40, 751–770. Árnason, E., Petersen, P.H., Kristinsson, K., Sigurg´ıslason, H., Pálsson, S., 2000. Mitochondrial cytochrome b DNA sequence variation of Atlantic cod from Iceland and Greenland. J. Fish Biol. 56, 409–430.
A.K. Imsland et al. / Fisheries Research 67 (2004) 227–233 Bentzen, P., Taggart, C.T., Ruzzante, E., Cook, D., 1996. Microsatellite polymorphism and population structure of Atlantic cod (Gadus morhua) in the northwest Atlantic. Can. J. Fish. Aquat. Sci. 53, 2706–2721. Carvalho, G.R., Hauser, L., 1994. Molecular genetics and the stock concept in fisheries. In: Carvalho, G.R., Pitcher, T.J. (Eds.), Molecular Genetics in Fisheries. Chapman & Hall, London, pp. 55–79. Dahle, G., 1991. Cod Gadus morhua L., population identified by mitochondrial DNA. J. Fish Biol. 38, 295–303. Felsenstein, J., 1993. PHYLIP (Phylogeny Inference Package), Version 3.5c. University of Washington, Seattle. Fevolden, S.E., Pogson, G.H., 1995. Differences in nuclear DNA RFLPs between the Norwegian coastal and the North-east Arctic population of Atlantic cod. In: Skjoldal, H.R., Hopkins, C., Erikstad, K.E., Leinaas, H.P. (Eds.), Ecology of Fjords and Coastal Waters. Elsevier, Amsterdam, pp. 403–415. Fevolden, S.E., Pogson, G.H., 1997. Genetic divergence at the synaptophysin (SypI) locus among Norwegian coastal and north-east Arctic populations of Atlantic cod. J. Fish Biol. 51, 895–908. Fyhn, U.E., Brix, O., Nævdal, G., Johansen, T., 1994. New variants of the haemoglobin of Atlantic cod: a tool to discriminate between coastal and Arctic cod populations. ICES Mar. Sci. Symp. 198, 666–670. Galvin, P., Sadusky, T., McGregor, D., Cross, T., 1995. Population genetics of Atlantic cod using amplified single locus minisatellite VNTR analysis. J. Fish Biol. 47 (Suppl. A), 200– 208. Jamieson, A., Jónsson, J., 1971. The Greenland component of spawning cod at Iceland. Conseil International pour l’exploration de la Mer. Rapports et Procès Verbaux 161, 65–72. Johnson, C.R., Field, C.R., 1993. Using fixed-effects model multivariate analysis of variance in marine biology and ecology. Mar. Biol. Ann. Rev. 31, 177–221. Jónsdóttir, Ó.D.B., Imsland, A.K., Dan´ıelsdóttir, A.K., Thorsteinsson, V., Nævdal, G., 1999. Genetic differentiation among Atlantic cod in south and south-east Icelandic waters: synaptophysin (SypI) and haemoglobin (HbI) variation. J. Fish Biol. 54, 1259–1274. Jónsdóttir, Ó.D.B., Dan´ıelsdóttir, A.K., Nævdal, G., 2001. Genetic differentiation among Atlantic cod (Gadus morhua L.) in Icelandic waters: temporal stability. ICES J. Mar. Sci. 58, 114– 122.
233
Jónsdóttir, Ó.D.B., Imsland, A.K., Marteinsdóttir, G., Dan´ıelsdóttir, A.K., Nævdal, G., 2002. Genetic heterogeneity and growth properties of different genotypes of Atlantic cod (Gadus morhua L.) at two spawning sites off south Iceland. Fish. Res. 55, 37– 47. Jónsson, J., 1996. Tagging of cod (Gadus morhua) in Icelandic waters 1948–1986. Rit Fiskideildar XIV, 7–82. Nei, M., 1972. Genetic distance between populations. Am. Nat. 106, 283–292. Pogson, G.H., 1994. Contrasting patterns of DNA polymorphism in the deep-sea scallop (Plagopecten magellanicus) and the Atlantic cod (Gadus morhua) revealed by random cDNA probes. In: Beaumont, A.R. (Ed.), Genetics and Evolution in Aquatic Organisms. Chapman & Hall, London, pp. 207– 217. Pogson, G.H., Fevolden, S.E., 1998. DNA heterozygosity and growth rate in the Atlantic cod Gadus morhua L. Evolution 52, 915–920. Pogson, G.H., Mesa, K.A., Boutilier, R.G., 1995. Genetics population structure and gene flow in the Atlantic cod Gadus morhua: a comparison of allozyme and nuclear RFLP loci. Genetics 139, 375–385. Reynolds, J., Weir, B.S., Cockerham, C.C., 1983. Estimation for the coancestry coefficient: basis for a short-term genetic distance. Genetics 105, 769–779. Ruzzante, D.E., Taggart, C.T., Cook, D., Goddard, S., 1996. Genetic differentiation between inshore and offshore Atlantic cod (Gadus morhua) off Newfoundland: microsatellite DNA variation and antifreeze level. Can. J. Fish. Aquat. Sci. 53, 634–645. Schaffer, H.E., Sederoff, R.R., 1981. Least squares fit of DNA fragment length to gel mobility. Anal. Biochem. 115, 113– 122. Schneider, S., Kueffer, J.M., Roessli, D., Excoffier, L., 1997. Arlequin ver. 1.1: A Software for Population Genetic Data Analysis. Genetics and Biometry Laboratory, University of Geneva, Switzerland. Taggart, J.B., Hynes, R.A., Prodöhl, P.A., Ferguson, A., 1992. A simplified protocol for routine total DNA isolation from salmonid fishes. J. Fish Biol. 40, 963–965. Ward, R.D., Grewe, P.M., 1994. Appraisal of molecular genetic techniques in fisheries. In: Carvalho, G.R., Pitcher, T.J. (Eds.), Molecular Genetics in Fisheries. Chapman & Hall, London, pp. 29–54.
Short communication
Nuclear DNA RFLP variation among Atlantic cod in south and south-east Icelandic waters A.K. Imsland a,∗,1 , Ó.D.B. Jónsdóttir b,1 , A.K. Dan´ıelsdóttir c a Akvaplan-niva, Iceland Office, Akralind 4, 201 Kópavogi, Iceland Department of Fisheries and Marine Science, University of Bergen, 5020 Bergen, Norway Marine Research Institute, Division of Population Genetics, Keldnaholti, 112 Reykjav´ık, Iceland b
c
Received 24 April 2003; received in revised form 12 September 2003; accepted 21 September 2003
Abstract The aim of this study was to describe the population structure of Atlantic cod, Gadus morhua, in Icelandic waters. A total of 404 cod was collected from five locations in Icelandic waters. The samples were analysed for allelic variation at five restriction fragment length polymorphism (RFLP) (GM862, GM860, GM738, GM777 and GM865) loci. Significant differences between sample sites were found at two loci (GM865 and GM777). The results are in line with other recent findings from this area indicating that the cod in south and south-east Icelandic waters do not belong to one panmictic population but may represent two genetically different reproductive stocks of cod. © 2003 Elsevier B.V. All rights reserved. Keywords: Atlantic cod; Gadus morhua; Nuclear RFLP; Population genetics; Iceland
1. Introduction From 38 years of tagging and recapture studies of cod in Iceland, Jónsson (1996) showed that the main migratory routes were related to spawning off the southwest coast. After spawning, the fish migrated northwards along the west coast to the rich feeding grounds off the northwest and north coast. Also prior to spawning, the fish migrated from the north along the west coast to the spawning area off the southwest coast, then continuing to the east along the south coast. Endemic populations were found in some of the fjords of the west and north coast. The lack of recaptured cod tagged at the northeast coast at the spawning grounds ∗ Corresponding author. Tel.: +354-562-5800; fax: +354-564-5801. E-mail address: [email protected] (A.K. Imsland). 1 Equal Authorship.
in the southwest might be due to a local spawning at the northeast coast. Studies of cod populations genetics at Iceland have lead to contradictory results. In a study of cod populations in Icelandic waters using transferrin and haemoglobin, Jamieson and Jónsson (1971) described spatial (both loci) and temporal (haemoglobin) variation. Hardy–Weinberg disequilibrium was found at some of the sampling sites, indicating that different units of cod are present in Icelandic waters. In contrast, Árnason et al. (1992), using restriction fragment analysis of mitochondrial DNA, found no evidence of population differentiation in Icelandic cod. Recently, the work of Jónsdóttir et al. (1999, 2001, 2002) indicated considerable population substructuring of cod in Icelandic waters. In the studies of Jónsdóttir et al. (1999, 2001, 2002), cod samples were analysed using the sequenced and analysed SypI locus presented by Fevolden and Pogson (1995,
0165-7836/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.fishres.2003.09.045
228
A.K. Imsland et al. / Fisheries Research 67 (2004) 227–233
1997), as well as the haemoglobin locus. Significant differences were detected at the SypI locus between two groups (Loftstaðahraun, Reykjanesgrunn and Eyrabakkabugur vs. Kantur and Austfjarðadjúp) in Icelandic waters. These results are consistent with tagging studies in Iceland (Jónsson, 1996). However, the discrepancy between the studies of Jamieson and Jónsson (1971), Jónsson (1996) and Jónsdóttir et al. (1999, 2001, 2002), and that of Árnason et al. (1992) shows that we still have limited knowledge of the structuring of Atlantic cod in Icelandic waters. The present paper is aimed at examining the genetic structure of Atlantic cod from south Icelandic waters using an analysis of five nuclear DNA RFLP loci. The study is a part of larger study where frequencies at the SypI and haemoglobin loci sampled at the same sampling sites have been described in details in earlier papers (Jónsdóttir et al., 1999, 2001, 2002). 2. Materials and methods 2.1. Sampling A total of 404 cod was collected from five locations in Icelandic waters (Fig. 1, Table 1). Two of
the samples were taken from known spawning sites (Loftstaðahraun [L], Kantur [K]) and three from feeding grounds (Reykjanesgrunn [R], Eyrabakkabugur [E], Austfjarðadjúp [A]). At Kantur samples taken ca. 13 nautical miles apart (called Kantur 1 and Kantur 2) showed no intra-area differences (Fisher’s exact test, P > 0.65), and so were pooled for inter-area comparisons (hereafter called Kantur). Further rationale of the sampling scheme is given in Jónsdóttir et al. (1999). 2.2. Genetic analysis The cod samples were examined using nuclear DNA RFLP analysis. The primary source of the nuclear DNA analysis was cod gill tissue (200 mg), collected from cod and preserved immediately in 96% ethanol. The DNA extraction was performed by phenol-extraction (Taggart et al., 1992). The five restriction fragment length polymorphism (RFLP) loci (GM842, GM860, GM738, GM777 and GM865) were analysed using cDNA probes and amplified by PCR conditions as described by Pogson et al. (1995). Each of the five RFLPs examined in the present study is characterised by a specific cDNA probe-restriction
Fig. 1. Collection sites of Atlantic cod in Icelandic waters. Abbreviations: L = Loftstaðahraun, E = Eyrabakkabugur, K = Kantur (Dyrh´olaey), R = Reykjanesgrunn, A = Austfjarðadj´up (see Table 1 for details).
A.K. Imsland et al. / Fisheries Research 67 (2004) 227–233
229
Table 1 Locations, number of specimens (n), location, date of collection, and sex-ratio of Atlantic cod in Icelandic waters used in this study Location Loftstaðahraun (L) Eyrabakkabugur (E) Reykjanesgrunn (R) Kantur 1 (Dyrh´olaey) (K 1) Kantur 2 (Dyrh´olaey) (K 2) Austfjarðadj´up (A)
n
Position
100 36 64 48 52 104
63◦ 47N, 63◦ 47N, 63◦ 47N, 63◦ 15N, 63◦ 19N, 64◦ 33N,
20◦ 51W 21◦ 40W 22◦ 50W 18◦ 54W 19◦ 24W 12◦ 20W
enzyme combination. Total DNA samples (7 g) from all specimens were digested with two restriction enzymes (DraI, TaqI). DraI digested samples were probed with cDNA clones GM842 and GM860 and TaqI digests were probed with clones GM738, GM777 and GM865. The cDNA probe-based loci were screened using non-radioactively labelled cDNA probes (Enhanced Chemiluminescence, ECL, Amersham, Buckinghamshire). The probes were labelled with the enzyme horseradish peroxidase according to instructions in the ECL protocol (Amersham). Probes were hybridized to total DNA samples (7 g) from each cod specimen that had been digested with either of the two restriction enzymes (DraI, TaqI) according to the manufacturer (Boehringer). The digested DNA was electrophoresed on 0.8% agarose gels in 1 × TBE buffer, pH 8.3, for 17–19 h and transferred to nylon membranes (Amersham, Hybond N+) in a Pharmacia VacuGene apparatus, after which it was fixed to the membranes by baking for 2 h at 80 ◦ C. Membranes were prehybridized for 2 h at 42 ◦ C in 50 ml hybridization buffer (50 ml Gold buffer, 5% Blocking reagent (both provided by Amersham), 0.5 M NaCl). The horseradish peroxidase labelled cDNA probes were added directly into the hybridization buffer. Hybridizations were carried out overnight at 42 ◦ C. Membranes were washed once in 80 ml of 5 × SSC at 42 ◦ C for 5 min, then twice in 0.125 ml/cm2 washing solution I (0.4 × SSC, 0.4% SDS) at 55 ◦ C for 10 min, and finally twice in 0.125 ml/cm2 washing solution II (2 × SSC) at room temperature for 5 min. Following washing, the membranes were soaked in equal volumes of detection reagents 1 and 2 (provided by Amersham) for 1 min. The membranes were then placed in a plastic envelope and exposed to X-ray films (Sterling, Diagnostic Imaging) overnight to
Date
Depth (m)
Sex (male:female)
3–4 April 1997 30 January 1998 31 January 1998 25–26 March 1997 25–26 March 1997 26–28 October 1997
82 54 110 253 262 355
67:33 11:25 43:21 28:20 28:24 43:61
detect band patterns. The membranes were stored in 2 × SSC between hybridizations. Restriction fragment sizes were estimated using DNAfrag 3.01 package (Schaffer and Sederoff, 1981) with reference to the HindIII ladder run alongside the samples in the electrophoresis gel. RFLP patterns were apportioned into three groups based on Pogson (1994). Each form of polymorphism was first assigned a letter and then broken down into allelic sizes. 2.3. Statistical methods Allele and genotype frequencies together with pairwise FST values (Reynolds et al., 1983), and exact test of population differentiation were calculated using the ARLEQUIN 2.0 computer package (Schneider et al., 1997). Genotypic disequilibrium between loci was tested with an extension of Fisher exact test on contingency tables (Schneider et al., 1997). Allele frequencies were bootstraped 1000 times and Nei’s (1972) genetic distances based on the allele frequencies were calculated using the SEQBOOT and GENDIST program in the PHYLIP package (Felsenstein, 1993) and a UPGMA dendrogram of the bootstraped Nei’s genetic distance matrix was constructed in the NEIGHBOR program in PHYLIP. A Bonferroni correction (Johnson and Field, 1993) of the significance level (α = 0.05) was applied when testing for significant differences in allele frequencies and for significant departures from Hardy–Weinberg expectations.
3. Results No differences between sexes were found in frequencies of any of the loci investigated (P > 0.25). Genotypic disequilibrium between loci was found
230
A.K. Imsland et al. / Fisheries Research 67 (2004) 227–233
Table 2 Allele frequencies at each locus for analysed cod samples from five locations in Icelandic watersa Locus/alleles (kb)
Loftstaðahraun (spawning area)
Eyrabakkabugur (feeding area)
Reykjanesgrunn (feeding area)
Kantur (spawning area)
Austfjarðadj´up (feeding area)
GM842 n 2.65 2.92 3.07 3.15 3.35 3.60 H
42 0.000 0.036 0.024 0.928 0.012 0.000 0.142 (0.112)
33 0.036 0.030 0.000 0.934 0.000 0.000 0.090 (0.112)
55 0.009 0.018 0.009 0.953 0.000 0.009 0.090 (0.085)
19 0.000 0.053 0.000 0.947 0.000 0.000 0.105 (0.100)
37 0.027 0.000 0.000 0.946 0.000 0.027 0.081 (0.051)
GM860 n 1.60 2.38 5.70 6.27 H
56 0.527 0.000 0.473 0.000 0.482 (0.498)
35 0.614 0.000 0.386 0.000 0.371 (0.474)
61 0.616 0.000 0.384 0.000 0.475 (0.473)
32 0.625 0.010 0.375 0.000 0.593 (0.468)
84 0.568 0.000 0.432 0.021 0.452 (0.490)
GM738 n 1.89 2.70 2.77 H
45 0.556 0.000 0.444 0.644 (0.493)
36 0.458 0.014 0.528 0.500 (0.484)
61 0.500 0.000 0.500 0.510 (0.500)
57 0.491 0.000 0.509 0.456 (0.500)
83 0.545 0.000 0.455 0.437 (0.495)
GM777 n 1.57 2.03 2.55 3.04 4.30 H
48 0.010 0.010 0.104 0.855 0.021 0.250 (0.189)
26 0.000 0.000 0.330 0.670 0.000 0.498 (0.442)
38 0.000 0.000 0.162 0.811 0.027 0.316 (0.262)
GM865 n 1.71 1.78 1.82 H
45 0.000 0.422 0.578 0.511 (0.487)
32 0.008 0.200 0.800 0.390 (0.32)
49 0.021 0.306 0.673 0.489 (0.465)
0b – – – – –
30 0.000 0.375 0.625 0.433 (0.468)
0b – – – – –
58 0.000 0.465 0.535 0.379 (0.497)
a n: number of specimens giving detectable bands at each site; allele sizes are given in kb; H: frequencies of heterozygotes, observed (expected). b Due to technical reasons no variation could be detected.
for all loci and sampling units (P < 0.01). Although observed frequencies of heterozygotes were in most cases higher than expected Hardy–Weinberg values (Table 2), all samples were in Hardy–Weinberg equilibrium (P > 0.05). The most common allele was the same at each of four loci (GM842, GM860, GM777 and GM865) for all sample units, with frequencies ranging from 0.53 to 0.95 (Table 2). Significant
differences in allelic frequencies were found at GM865 (K vs. E: FST = 0.1, P < 0.05; K vs. L: FST = 0.07, P < 0.05) and GM777 (K vs. L: FST = 0.09, P < 0.05). When tested separately each locus was found to be in Hardy–Weinberg equilibrium (P > 0.29). However, the total material (all samples compiled) was in Hardy–Weinberg disequilibrium (P < 0.05), suggesting that the samples were
A.K. Imsland et al. / Fisheries Research 67 (2004) 227–233
Fig. 2. A UPGMA dendrogram of the Nei’s (1972) genetic distance matrix among the cod populations in the present study. Values on the nodes represent the percentage of bootstrap samples (n = 1000).
drawn from more than one panmictic population. The highest level of differentiation was found between the L and the K samples (FST = 0.30, P < 0.001) and the L and the A samples (FST = 0.12, P < 0.001). The bootstrapped UPGMA dendrogram constructed using Nei’s (1972) genetic distances illustrated a clear differentiation between the sample sites analysed (Fig. 2). The largest genetic distance was found between Loftstaðahraun and Kantur (Fig. 2).
4. Discussion The present findings and the findings of Jónsdóttir et al. (1999, 2001, 2002), support the sub-structuring theory of cod in Icelandic waters. Significant differences were detected at the GM865 and GM777 (present study, see above) and the SypI (Jónsdóttir et al., 1999) loci between the sampling sites in Icelandic waters. Consistent with the findings of Fevolden and Pogson (1997) who studied cod in Norwegian waters, Jónsdóttir et al. (1999, 2001, 2002) detected significant differences at the SypI locus between two groups (Loftstaðahraun, Reykjanesgrunn and Eyrabakkabugur vs. Kantur and Austfjarðadjúp) of cod in Icelandic waters. The subdivision of cod
231
into the two groups supports earlier tagging studies of cod from the investigated areas. Cod spawning at Loftstaðahraun have been recaptured along the southwest coast (e.g. Eyrabakkabugur and Reykjanesgrunn) during the feeding period, but cod spawning at Kantur have been recaptured along the south-east and east coast (e.g. Austfjarðadjúp; Jónsson, 1996). When comparing the present findings with those reported for the same sampling locations applying different markers (Jónsdóttir et al., 1999) clear similarities are apparent. Both studies clearly differentiated the two spawning areas (Loftstaðahraun and Kantur) thus suggesting the occurrence of two reproductive stocks. However, the status of the feeding areas seems to differ between the studies. In the present study, Loftstaðahraun did not group with any of the feeding areas, whereas in the first study it grouped with Reykjanesgrunn and Eyrabakkabugur. However, based on FST and exact population tests both studies give similar results. The feeding ground samples (Reykjanesgrunn, Eyrabakkabugur and Austfjarðadjúp) are not different, but differ from both spawning grounds samples (Table 4 in Jónsdóttir et al., 1999) or Loftstaðahraun (present study). Why the Kantur sample does not differ from three feeding ground samples can at this stage only be speculated and might well be the result of rather limited data material in the present study. This is especially true for the GM777 locus as it was, due to technical reasons, not detected in Eyrabakkabugur and Reykjanesgrunn (see Table 2). Overall the material was found to be in Hardy– Weinberg disequilibrium indicating that the samples are drawn from more than one population (Wahlund effect), and that the deviation is due to an unusual, but random, combination of excesses or deficiencies of heterozygotes in different population units. Alternatively, deviation of the pooled data could be due to the presence of rare and private alleles or the cumulative effect of selection at individual loci. However, with the exception of GM842 and GM777 in Loftstaðahraun and GM842 in Austfjarðadjúp, the rare and private alleles represented only 1% or less of the total occurrence at each locus (Table 2). Also, earlier investigations have not indicated any selective effect on those loci presented here (Pogson and Fevolden, 1998). The present study indicates subdivision of Atlantic cod in Icelandic waters. This is in sharp contrast to studies based on mitochondrial DNA
232
A.K. Imsland et al. / Fisheries Research 67 (2004) 227–233
variation as these have shown limited or no differentiation of populations (e.g. Árnason et al., 1992, 2000). The question must arise why the RFLP analyses of mitochondrial, on one hand, and DNA on the other, give such contrasting results? Firstly, all samples in Árnason et al. (1992) study were taken from coastal areas making it impossible to test whether an off-coastal population might exist off Iceland. Further, the vast majority of samples were sampled from depths shallower than 90 m thus ignoring the fact that cod off Iceland is predominantly caught at depths exceeding 90 m. It is possible that this sampling procedure which is biased towards shallow depths and coastal areas might not be adequate if the aim is to describe the population structure of cod off Iceland. Secondly, the contrast between mitochondrial DNA and nuclear DNA analysis in resolving population structure may relate to different potential for detecting variation (Carvalho and Hauser, 1994; Ward and Grewe, 1994; Ruzzante et al., 1996). This is supported by the fact that in many cases mtDNA analysis has not lead to enhanced resolution of stock issues compared with other molecular methods (Ward and Grewe, 1994 and references therein). It has, thus, been suggested that analysis of frequencies distributions demands larger sample sizes than traditionally has been employed in many mtDNA studies, and that the use of too few individuals may contribute to the mtDNA homogeneity commonly observed (e.g. Árnason et al., 1992). The discrepancy between the studies of Jamieson and Jónsson (1971) and Jónsdóttir et al. (1999, 2001, 2002), and those of Árnason et al. (1992), shows that knowledge of the structuring of Atlantic cod in Icelandic waters is limited and emphasis the need for further research on this topic. In Norway the subdivision of Atlantic cod into Arcto-Norwegian (AN) cod and Norwegian coastal (NC) cod has been supported and discussed in many studies (Dahle, 1991; Fyhn et al., 1994; Fevolden and Pogson, 1995, 1997). The results of Fevolden and Pogson (1995, 1997) confirmed the existence of highly significant differences between AN and NC cod. Further, they found the SypI locus to suggest that genetic heterogeneity might exist among resident populations of cod in different fjords. Recent studies in the Northwest Atlantic have also revealed genetic subdivision of cod at localized scales. The studies Pogson et al. (1995) using nuclear DNA RFLPs,
and Galvin et al. (1995) using the Mmer-AMP2 minisatellite locus revealed clear genetic subdivision of cod off Newfoundland and on the Scotian Shelf. The results from microsatellite studies of Bentzen et al. (1996) and Ruzzante et al. (1996) supported the findings of three genetically distinct populations of cod (Flemish Cap, Scotian Shelf, Northern cod), and further division of Northern cod into North and South components. The indications of sub-structuring of cod in Icelandic waters (Jónsdóttir et al., 1999, 2001, 2002, present study) are consistent with earlier genetic studies of cod in Norwegian waters and northwest Atlantic (see references above). This may indicate that cod in the North Atlantic Ocean is sub-structured at small geographic scale. In conclusion, the present study indicates population differentiation between cod populations off Iceland and that there are (at least) two genetically different reproductive stocks of cod in Icelandic waters (i.e. Kantur and Loftstaðahraun). However, only few analyses have been performed on cod in Icelandic waters, and only few genetic markers have yet been used in these population genetic studies. In order to gain more information on the genetic structure of cod in Icelandic waters, the application of alternative genetic markers is necessary (e.g. microsatellites). Also, more samples, especially from the fishing grounds off northwest Iceland, need to be analysed to confirm our findings.
Acknowledgements The authors thank G. Pogson, S.E. Fevolden, P. Galvin, J. Coughlan and J. Solvang for help and advice during the analytical work. Financial support was given by the EU FAIR program (FAIR CT95-0282).
References Árnason, E., Pálsson, S., Arason, A., 1992. Gene flow and lack of population differentiation in Atlantic cod, Gadus morhua L., from Iceland, and comparison of cod from Norway and Newfoundland. J. Fish Biol. 40, 751–770. Árnason, E., Petersen, P.H., Kristinsson, K., Sigurg´ıslason, H., Pálsson, S., 2000. Mitochondrial cytochrome b DNA sequence variation of Atlantic cod from Iceland and Greenland. J. Fish Biol. 56, 409–430.
A.K. Imsland et al. / Fisheries Research 67 (2004) 227–233 Bentzen, P., Taggart, C.T., Ruzzante, E., Cook, D., 1996. Microsatellite polymorphism and population structure of Atlantic cod (Gadus morhua) in the northwest Atlantic. Can. J. Fish. Aquat. Sci. 53, 2706–2721. Carvalho, G.R., Hauser, L., 1994. Molecular genetics and the stock concept in fisheries. In: Carvalho, G.R., Pitcher, T.J. (Eds.), Molecular Genetics in Fisheries. Chapman & Hall, London, pp. 55–79. Dahle, G., 1991. Cod Gadus morhua L., population identified by mitochondrial DNA. J. Fish Biol. 38, 295–303. Felsenstein, J., 1993. PHYLIP (Phylogeny Inference Package), Version 3.5c. University of Washington, Seattle. Fevolden, S.E., Pogson, G.H., 1995. Differences in nuclear DNA RFLPs between the Norwegian coastal and the North-east Arctic population of Atlantic cod. In: Skjoldal, H.R., Hopkins, C., Erikstad, K.E., Leinaas, H.P. (Eds.), Ecology of Fjords and Coastal Waters. Elsevier, Amsterdam, pp. 403–415. Fevolden, S.E., Pogson, G.H., 1997. Genetic divergence at the synaptophysin (SypI) locus among Norwegian coastal and north-east Arctic populations of Atlantic cod. J. Fish Biol. 51, 895–908. Fyhn, U.E., Brix, O., Nævdal, G., Johansen, T., 1994. New variants of the haemoglobin of Atlantic cod: a tool to discriminate between coastal and Arctic cod populations. ICES Mar. Sci. Symp. 198, 666–670. Galvin, P., Sadusky, T., McGregor, D., Cross, T., 1995. Population genetics of Atlantic cod using amplified single locus minisatellite VNTR analysis. J. Fish Biol. 47 (Suppl. A), 200– 208. Jamieson, A., Jónsson, J., 1971. The Greenland component of spawning cod at Iceland. Conseil International pour l’exploration de la Mer. Rapports et Procès Verbaux 161, 65–72. Johnson, C.R., Field, C.R., 1993. Using fixed-effects model multivariate analysis of variance in marine biology and ecology. Mar. Biol. Ann. Rev. 31, 177–221. Jónsdóttir, Ó.D.B., Imsland, A.K., Dan´ıelsdóttir, A.K., Thorsteinsson, V., Nævdal, G., 1999. Genetic differentiation among Atlantic cod in south and south-east Icelandic waters: synaptophysin (SypI) and haemoglobin (HbI) variation. J. Fish Biol. 54, 1259–1274. Jónsdóttir, Ó.D.B., Dan´ıelsdóttir, A.K., Nævdal, G., 2001. Genetic differentiation among Atlantic cod (Gadus morhua L.) in Icelandic waters: temporal stability. ICES J. Mar. Sci. 58, 114– 122.
233
Jónsdóttir, Ó.D.B., Imsland, A.K., Marteinsdóttir, G., Dan´ıelsdóttir, A.K., Nævdal, G., 2002. Genetic heterogeneity and growth properties of different genotypes of Atlantic cod (Gadus morhua L.) at two spawning sites off south Iceland. Fish. Res. 55, 37– 47. Jónsson, J., 1996. Tagging of cod (Gadus morhua) in Icelandic waters 1948–1986. Rit Fiskideildar XIV, 7–82. Nei, M., 1972. Genetic distance between populations. Am. Nat. 106, 283–292. Pogson, G.H., 1994. Contrasting patterns of DNA polymorphism in the deep-sea scallop (Plagopecten magellanicus) and the Atlantic cod (Gadus morhua) revealed by random cDNA probes. In: Beaumont, A.R. (Ed.), Genetics and Evolution in Aquatic Organisms. Chapman & Hall, London, pp. 207– 217. Pogson, G.H., Fevolden, S.E., 1998. DNA heterozygosity and growth rate in the Atlantic cod Gadus morhua L. Evolution 52, 915–920. Pogson, G.H., Mesa, K.A., Boutilier, R.G., 1995. Genetics population structure and gene flow in the Atlantic cod Gadus morhua: a comparison of allozyme and nuclear RFLP loci. Genetics 139, 375–385. Reynolds, J., Weir, B.S., Cockerham, C.C., 1983. Estimation for the coancestry coefficient: basis for a short-term genetic distance. Genetics 105, 769–779. Ruzzante, D.E., Taggart, C.T., Cook, D., Goddard, S., 1996. Genetic differentiation between inshore and offshore Atlantic cod (Gadus morhua) off Newfoundland: microsatellite DNA variation and antifreeze level. Can. J. Fish. Aquat. Sci. 53, 634–645. Schaffer, H.E., Sederoff, R.R., 1981. Least squares fit of DNA fragment length to gel mobility. Anal. Biochem. 115, 113– 122. Schneider, S., Kueffer, J.M., Roessli, D., Excoffier, L., 1997. Arlequin ver. 1.1: A Software for Population Genetic Data Analysis. Genetics and Biometry Laboratory, University of Geneva, Switzerland. Taggart, J.B., Hynes, R.A., Prodöhl, P.A., Ferguson, A., 1992. A simplified protocol for routine total DNA isolation from salmonid fishes. J. Fish Biol. 40, 963–965. Ward, R.D., Grewe, P.M., 1994. Appraisal of molecular genetic techniques in fisheries. In: Carvalho, G.R., Pitcher, T.J. (Eds.), Molecular Genetics in Fisheries. Chapman & Hall, London, pp. 29–54.