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Nuclear DNA RFLP variation of Atlantic cod in the North Atlantic Ocean
Fisheries Research 63 (2003) 429–436
Short communication
Nuclear DNA RFLP variation of Atlantic cod in the North Atlantic Ocean Ó.D.B. Jónsdóttir a,1 , A.K. Imsland b,∗,1 , Ó.Ý. Atladóttir c , A.K. Dan´ıelsdóttir c a
c
Department of Fisheries and Marine Biology, University of Bergen, 5020 Bergen, Norway b Akvaplan-niva Iceland Office, Akralind 4, 210 Kópavogi, Iceland Marine Research Institute, Division of Population Genetics, Keldnaholti, 112 Reykjav´ık, Iceland
Received 13 August 2002; received in revised form 14 February 2003; accepted 27 February 2003
Abstract A total of 551 specimens of Atlantic cod was collected from six locations across the North Atlantic Ocean. The samples were analysed for allelic variation at six nuclear restriction fragment length polymorphism (RFLP) loci scored by five anonymous cDNA clones, and the sequenced and identified synaptophysin (Syp I). Significant differences in allelic variation were found between the sampling localities. The greatest degree of differentiation was detected between the Barents Sea and Scotian Shelf samples vs. the remaining sample units and a clear differentiation between west and east Atlantic samples was found. © 2003 Elsevier Science B.V. All rights reserved. Keywords: Atlantic cod; Gadus morhua; Genetic differentiation
1. Introduction Considerable effort has been made in the past to elucidate the possible subdivision of the Atlantic cod (Gadus morhua) (Cross and Payne, 1978; Carr and Marshall, 1991; Galvin et al., 1995; Pogson et al., 1995; Ruzzante et al., 1996). However, these studies have not led to a clear-cut conclusion about population structure of cod and some results are contradictory (e.g. Árnason et al., 1992; Pepin and Carr, 1993; Ruzzante et al., 1996; Jónsdóttir et al., 1999). Examination of temporal variability in gene frequencies is essential for the proper understanding of population genetic structure as significant temporal genetic differentiation might indicate the existence of stochastic ∗ Corresponding author. Tel.: +354-564-5800; fax: +354-564-5801. E-mail address: [email protected] (A.K. Imsland). 1 Equal Authorship.
effects due to small effective population size or subpopulations of stocks (Gold et al., 1993). Studies based on restriction fragment length polymorphism (RFLP) of nuclear DNA (Pogson et al., 1995; Fevolden and Pogson, 1997; Jónsdóttir et al., 1999, 2001) have displayed considerable population substructuring of cod both macro- and microgeographically. In the present study we have focused on the genetic structure of Atlantic cod collected from both sides of the North Atlantic Ocean, using nuclear DNA RFLP analysis.
2. Materials and methods 2.1. Sampling Between June 1994 and December 1997 cod samples from pre- and postspawning aggregations was collected from six locations in the North Atlantic (Fig. 1
0165-7836/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0165-7836(03)00098-5
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Fig. 1. Sampling locations of Atlantic cod in the North Atlantic Ocean.
and Table 1). A total of 551 specimens were gathered throughout the geographical range of the species, from the Scotian Shelf in the west to the North Sea, Celtic Sea and Norwegian waters (Trondheimsfjorden) in the east, as well as Icelandic waters (Loftstaðahraun), and the Barents Sea (Fig. 1). Between 93 and 100 samples were collected from each sample location (Scotian Shelf and Loftstaðahraun/Celtic Sea/North Sea, respectively), except in the Barents Sea where 62 samples were gathered (Table 1). The fish were collected using bottom trawls, at depths between 32 and 117 m (Loftstaðahraun and Scotian Shelf, respectively; Table 1). Sex of each specimen was recorded. Since the collection positions of the samples at Scotian Shelf, North Sea and Celtic Sea were separated by several nautical miles within each location, and over periods from approximately 3 to 4 weeks (North Sea and Scotian Shelf, respectively; Fig. 1 and Table 1), intra-area FST tests were performed. No differences were detected (Scotian Shelf and North Sea, P > 0.9, Celtic Sea, P > 0.13) thus samples from each location were pooled for inter-area comparisons. The results from the Syp I analysis of Loftstaðahraun sample has been examined in previous studies (Jónsdóttir et al., 1999, 2001).
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), which was collected from live or recently dead cod and was preserved immediately in 96% ethanol. The DNA extraction was performed by phenol-extraction (Taggart et al., 1992). The Syp I locus was analysed with a PCR-based method using the reaction conditions described by Jónsdóttir et al. (1999) modified by Fevolden and Pogson (1997). The five remaining loci 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 characterized by a specific cDNA proberestriction enzyme combination. Total DNA samples (7 g) from all specimens were digested with two restriction enzymes (DraI and TaqI). DraI digested samples (one restriction site) were probed with cDNA clones GM842 and GM860 and TaqI digests (one restriction site) were probed with clones GM738, GM777 and GM865. In accordance with Pogson et al. (1995), the name of each cDNA probe is synonymous with the name of the cDNA “locus” for the five clones
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Table 1 The collection location of Atlantic cod samples, number of specimens collected (N), collection position of the samples, date of collection, mean depth (m) at collection site, mean length (mm), mean mass (g) and mean age (years) of specimens and proportion of females (%) in the catch for each sample unit Location
N
Position of sample units
Date
Depth (m)
Scotian Shelf-Browns Bank
6 8 5 16 10
42◦ 76 N, 42◦ 61 N, 42◦ 45 N, 42◦ 52 N, 42◦ 62 N,
66◦ 16 W 66◦ 25 W 65◦ 36 W 64◦ 99 W 64◦ 77 W
7 July 1994 7 July 1994 12 July 1994 12 July 1994 12 July 1994
58 86 106 115 117
552 537 565 378 413
Scotian Shelf-Western Bank
5 6 15 16 6
43◦ 17 N, 43◦ 41 N, 43◦ 56 N, 43◦ 83 N, 43◦ 87 N,
61◦ 65 W 61◦ 35 W 60◦ 46 W 60◦ 25 W 60◦ 48 W
13 14 27 27 27
Loftstaahraun
16 26 22 18 6 12
63◦ 46 N, 63◦ 45 N, 63◦ 48 N, 63◦ 46 N, 63◦ 48 N, 63◦ 45 N,
20◦ 54 W 20◦ 49 W 20◦ 01 W 20◦ 58 W 20◦ 56 W 20◦ 49 W
3 4 4 4 4 4
Trondheimsfjorden
96
63◦ 51 N, 10◦ 44 E
62
74◦ 11 N,
34◦ 31 E
Celtic Sea
22 10 23 15 18 12
51◦ 42 N,
6◦ 28 W
North Sea
26 25 6 18 5 5 8 7
57◦ 39 N, 58◦ 28 N, 61◦ 02 N, 59◦ 12 N, 58◦ 21 N, 54◦ 53 N, 54◦ 56 N, 54◦ 54 N,
Barents Sea
a
51◦ 06 N, 51◦ 42 N, 51◦ 27 N, 51◦ 46 N, 51◦ 22 N,
Length (mm)
Mass (g)
Age (years)
Females (%)
1619 1479 1683 651 799
3.5 3.2 4.1 2.4 2.4
50 63 100 47 67
June June June June June
1994 1994 1994 1994 1994
88 75 77 46 35
494 377 367 357 385
1200 504 442 373 480
5.6 3.8 4.1 3.7 4.3
20 67 47 50 67
April April April April April April
1997 1997 1997 1997 1997 1997
47 34 58 57 42 32
1095 1067 1073 1069 1058 1095
17366 15953 16277 15682 16952 17669
8.3 8.3 8.5 8.6 7.5 7.9
19 23 46 39 50 33
17 April 1996
100
634
3118
6
77
13 August 1995
NAa
413
811
NA
48
8◦ 23 W 8◦ 01 W 7◦ 35 W 6◦ 42 W 5◦ 84 W
28 29 30 30 30 31
97 114 73 87 72 93
664 442 414 535 571 541
3588 1541 1208 2840 3223 3269
2.7 1.8 1.8 2.4 2.7 2.5
73 70 44 53 56 50
4◦ 40 E 3◦ 53 E 1◦ 12 W 3◦ 01 W 2◦ 34 W 1◦ 65 W 1◦ 77 W 1◦ 66 W
21 August 1997 22 August 1997 1 September 1997 2 September 1997 4 September 1997 5 September 1997 5 September 1997 5 September 1997
NA NA NA NA NA NA NA NA
391 467 602 351 318 436 445 370
779 1221 3036 600 380 1095 1123 634
1.7 2.2 2.8 1.3 1.2 2 2.6 1.7
40 52 50 65 80 80 50 71
March March March March March March
1997 1997 1997 1997 1997 1997
Not available.
used to score the observed polymorphism. 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 et al. (1995). Each form of polymorphism was first assigned a letter and then broken down into allelic sizes. The loci were screened using nonradioactively 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 and 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
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(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 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 a 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 detect band patterns. The membranes were stored in 2× SSC between hybridizations. 2.3. Statistical methods Allele frequencies for each locus, estimates for observed and expected heterozygosities, tests for heterozygote deficiency and calculations for allelic differentiation over all cod sample pairs for each loci were provided by the GENEPOP software (Raymond and Rousset, 1995). Population pairwise FST statistics were calculated for all sample units using the Arlequin, Version 1.1 computer package, and the significance was tested with permutation (1000 times, Schneider et al., 1997). Genotype frequencies were tested for conformity with expected Hardy–Weinberg proportions using GENEPOP, and separate tests were performed for each locus. 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). A UPGMA dendrogram of the bootstraped Nei’s genetic distance matrix was constructed in the NEIGHBOR program in PHYLIP. Bonferroni correction of the significance level (α = 0.05) were applied when testing for significant departures from Hardy–Weinberg expectations and for significance of FST values.
Mean age at the different sample locations was tested with one-way ANOVA and log-linear analysis (Zar, 1996) was used to test for sex- and age-specific frequencies of all loci in all sample locations.
3. Results All examined loci were polymorphic in all sample units, with the exception of Syp I at Celtic Sea (Table 2). The most common allele was the same for each of four loci (GM842, GM860, GM777 and GM865) for all sample units, with frequencies ranging from 0.53 to 0.93 (Table 2). For the remaining loci (GM738 and Syp I), the most common alleles differed between sample units (Table 2). For all sample units except the Barents Sea, the Syp IA allele was the most common one (frequencies from 0.81 to 1, Trondheimsfjorden and Celtic Sea, respectively, Table 2). No deviations from Hardy–Weinberg equilibrium were found within each sample unit (P > 0.34). A comparison of observed and expected heterozygosity and an exact probability test searching specifically for heterozygote deficiencies revealed heterozygote deficiencies only at the GM860 and GM738 loci in the Barents Sea and Trondheimsfjorden samples, respectively. Calculations for allelic differentiation over all cod sample pairs for each locus revealed significant differences at all loci for different pairs of sample units (χ2 -tests, P < 0.05). The most pronounced differences were found at the Syp I and the GM738 loci. The population pairwise FST test revealed highly significant differences (P < 0.001) between the Barents Sea sample vs. all sample units, and between the Scotian Shelf sample vs. all sample units except the Celtic Sea (Table 3). The bootstrapped UPGMA dendrogram constructed using Nei’s (1972) genetic distances (Fig. 2) illustrated a clear differentiation between the sample units analysed. The genetic distance between Barents Sea and the remaining sample units was several times greater than detected for any other sample pairs (Fig. 2). The mean age of the specimens in the different samples varied (One-way ANOVA, F7,381 = 19.2, P < 0.01) with the overall highest mean age found in the Loftstaðahraun and the Trondheimsfjorden samples (8.2 and 6 years, respectively). These two samples differed from the three other samples (age was not
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Table 2 Allele frequencies at each locus for analysed cod samples from six locations in the North Atlantic Ocean Locus/alleles (kb)
Trondheimsfjorden
Barents Sea
North Sea
Celtic Sea
Scotian Shelf
Loftstaahraun
58 0.069 0.026 0 0.905 0 0 0
15 0.067 0 0 0.900 0.033 0 0
31 0.032 0.016 0 0.823 0 0.032 0.097
42 0.036 0.024 0 0.928 0.012 0 0
GM842 na 2.65 2.92 3.07 3.15 3.35 3.60 4
26 0 0.019 0.019 0.924 0 0.038 0
GM860 n 1.60 2.08 2.38 2.67 5.70 6.27
49 0.561 0 0.010 0 0.429 0
29 0.638 0 0 0 0.362 0
59 0.551 0.008 0 0 0.441 0
24 0.583 0 0 0 0.396 0.021
75 0.680 0 0 0.007 0.313 0
56 0.527 0 0 0 0.473 0
GM738 n 1.89 2.70 2.77
48 0.479 0 0.521
27 0.537 0 0.463
54 0.556 0.009 0.435
58 0.474 0 0.526
69 0.116 0 0.884
45 0.556 0 0.444
GM777 n 1.57 2.03 2.55 3.04 4.30
11 0 0 0.227 0.773 0
0 – – – – –
14 0.036 0.036 0.178 0.750 0
10 0.050 0.100 0.250 0.600 0
GM865 n 1.71 1.78 1.82 7.18
63 0.008 0.294 0.698 0
6 0 0.417 0.583 0
19 0 0.210 0.790 0
62 0 0.218 0.782 0
96 0.813 0.187 293
50 0.020 0.980 112
96 0.917 0.083 300
Syp I n Syp IA Syp IB Total n a
0 – – – – – – –
84 1 0 253
0 – – – – –
48 0.010 0.010 0.104 0.855 0.021
51 0 0.196 0.794 0.010
45 0 0.422 0.578 0
86 0.948 0.052 312
97 0.876 0.124 333
Number of specimens giving detectable bands for each analysis.
recorded for Barents Sea) whereas no differences were found between the Celtic Sea (2.7), North Sea (2.9), and Scotian Shelf (3.6). Genotype proportions did not differ between sex or age groups for any locus and sample (contingency log-linear tests, Pearson χ2 > 4.1, P > 0.18).
4. Discussion The present study supports previous indications of genetic subdivision of Atlantic cod in the North Atlantic. In particular, these results support the studies showing genetic differentiation between eastern and
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Table 3 Population pairwise FST estimates among all sample units (permuting the individuals between the sampling sites tested the significance of the pairwise FST values)a
Barents Sea Celtic Sea Loftstaahraun North Sea Scotian Shelf Trondheimsfjorden
Barents Sea
Celtic Sea
Loftstaahraun
North Sea
Scotian Shelf
– 0.67311∗∗∗ 0.56711∗∗∗ 0.60539∗∗∗ 0.59959∗∗∗ 0.47570∗∗∗
– – −0.02088b −0.20421b 0.00031 0.01979
– – – −0.04334b 0.13091∗∗∗ −0.00227b
– – – – 0.10587∗∗∗ −0.08588b
– – – – – 0.07684∗∗∗
a
Thousand permutation were performed for all pair of sampling sites. F-statistic estimators in this model are random variables and as such may take either positive or negative values (Long, 1986; Excoffier et al., 1992). Such negative estimates should be interpreted as zero (Long, 1986) in the FST model there are no differences between those sampling pairs where negative FST values are found. ∗∗∗ P < 0.001 (Bonferroni correction for simultaneous tests). b
western Atlantic cod (Carr and Marshall, 1991; Galvin et al., 1995; Pogson et al., 1995) and between cod inhabiting the Barents Sea and other areas (Dahle, 1991; Bentzen et al., 1996). When comparing our results and those of Pogson et al. (1995) to previously published patterns of variation at allozyme loci obtained from cod collected from a range of similar localities (Mork et al., 1985), a considerable difference is detected in the magnitude of observed genetic differentiation, as the allele frequency variation at the nuclear RFLP loci
significantly exceeded that observed at the protein loci. This observation has at least two possible interpretations. First, allele scoring for the RFLP method can be seen as more uncertain compared to protein loci scoring. However, we applied a computerized sizing (Schaffer and Sederoff, 1981) combined with running a size ladder alongside the samples minimize the possibility of erroneous allele sizing. Secondly, the RFLP markers may be under selection. Fevolden and Pogson (1995) suggested that the polymorphism
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).
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revealed by one of the RFLP markers (GM798 cDNA probe), might represent promising marker to distinguish north-east Arctic cod (AC) from Norwegian coastal cod (NCC). Particularly the GM798C locus (synaptophysin, Syp I locus, Fevolden and Pogson, 1997) showed a considerable population substructuring of cod in the North Atlantic and was found to be a distinct genetic marker for distinguishing between AC and NCC (Fevolden and Pogson, 1995, 1997). Furthermore, Jónsdóttir et al. (1999, 2001) found an explicit difference between samples collected at two adjacent spawning areas (Loftstaðahraun and Kantur) off south Iceland, using the Syp I locus. These studies revealed a distinctive pattern of the locus, i.e. either high frequency of the Syp IA allele or high frequency of the Syp IB allele (NCC and Loftstaðahraun vs. AC and Kantur, respectively; Fevolden and Pogson, 1995, 1997; Jónsdóttir et al., 1999, 2001). When all sample pairs were compared for each locus in the present study, the striking features of the Syp I locus became apparent. With only three exceptions (Trondheimsfjorden vs. Loftstaðahraun, North Sea vs. Scotian Shelf and Loftstaðahraun) the sample units were significantly different from each other. Specifically, the Barents Sea sample differed from all other sample units. Such indications of genetic distinctiveness of Barents Sea fish populations have also been reported for other gadoid species, i.e. blue whiting Micromesistius poutassou (Risso) (Giæver and Stein, 1998), as well as for Atlantic halibut Hippoglossus hippoglossus (L.) (Foss et al., 1998), and long rough dab Hippoglossoides platessoides (Bloch) (Walsh, 1994). It should be noted that apart from the possible selection of the Syp I locus no indication of selection of the other RFLP markers have been found (Fevolden and Pogson, 1995). The sharp differences observed by Pogson et al. (1995) between populations calculated by the FST test were not seen in the present study as only the Barents Sea and the Scotian Shelf samples displayed significant genetic differences from other samples using the FST test. The lack of significant FST values in our study compared to the study of Pogson et al. (1995) might be explained by fewer nuclear RFLPs scored and less information gathered from each locus in our study. In conclusion the nuclear DNA RFLP analysis of Atlantic cod collected from six locations across the
435
North Atlantic revealed a genetic substructure of the species across its range. In particular our results indicate a clear differentiation between west and east Atlantic population units and between Barents Sea population and other population in the east Atlantic.
Acknowledgements We want to thank G. Pogson, P. Galvin, L. Bourke and J. Coughlan for contribution to the analytical work; C.T. Taggart, J.E. Eliassen, A. Aglen, J. Mork, and R. Millner for providing cod samples and biological information of cod from the west and the east Atlantic; S.L. Jónsdóttir for skilled technical assistant during the analytical work; G. Nævdal, T. Cross and S.E. Fevolden for valuable comments on the manuscript. Financial support was given by the EU FAIR programme (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. 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. Carr, S.M., Marshall, H.D., 1991. Detection of intraspecific DNA sequence variation in the mitochondrial cytochrome b gene of Atlantic cod (Gadus morhua) by the polymerase chain reaction. Can. J. Fish. Aquat. Sci. 48, 48–52. Cross, T.F., Payne, R.H., 1978. Geographic variation in Atlantic cod, Gadus morhua, off eastern North America: a biochemical systematic approach. Can. J. Fish. Aquat. Sci. 35, 117–123. Dahle, G., 1991. Cod, Gadus morhua L., population identified by mitochondrial DNA. J. Fish Biol. 38, 295–303. Excoffier, L., Smouse, P.E., Quattro, J.M., 1992. Analysis of molecular variance inferred form metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics 131, 479–491. 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 (Syp I) locus among Norwegian coastal and
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Short communication
Nuclear DNA RFLP variation of Atlantic cod in the North Atlantic Ocean Ó.D.B. Jónsdóttir a,1 , A.K. Imsland b,∗,1 , Ó.Ý. Atladóttir c , A.K. Dan´ıelsdóttir c a
c
Department of Fisheries and Marine Biology, University of Bergen, 5020 Bergen, Norway b Akvaplan-niva Iceland Office, Akralind 4, 210 Kópavogi, Iceland Marine Research Institute, Division of Population Genetics, Keldnaholti, 112 Reykjav´ık, Iceland
Received 13 August 2002; received in revised form 14 February 2003; accepted 27 February 2003
Abstract A total of 551 specimens of Atlantic cod was collected from six locations across the North Atlantic Ocean. The samples were analysed for allelic variation at six nuclear restriction fragment length polymorphism (RFLP) loci scored by five anonymous cDNA clones, and the sequenced and identified synaptophysin (Syp I). Significant differences in allelic variation were found between the sampling localities. The greatest degree of differentiation was detected between the Barents Sea and Scotian Shelf samples vs. the remaining sample units and a clear differentiation between west and east Atlantic samples was found. © 2003 Elsevier Science B.V. All rights reserved. Keywords: Atlantic cod; Gadus morhua; Genetic differentiation
1. Introduction Considerable effort has been made in the past to elucidate the possible subdivision of the Atlantic cod (Gadus morhua) (Cross and Payne, 1978; Carr and Marshall, 1991; Galvin et al., 1995; Pogson et al., 1995; Ruzzante et al., 1996). However, these studies have not led to a clear-cut conclusion about population structure of cod and some results are contradictory (e.g. Árnason et al., 1992; Pepin and Carr, 1993; Ruzzante et al., 1996; Jónsdóttir et al., 1999). Examination of temporal variability in gene frequencies is essential for the proper understanding of population genetic structure as significant temporal genetic differentiation might indicate the existence of stochastic ∗ Corresponding author. Tel.: +354-564-5800; fax: +354-564-5801. E-mail address: [email protected] (A.K. Imsland). 1 Equal Authorship.
effects due to small effective population size or subpopulations of stocks (Gold et al., 1993). Studies based on restriction fragment length polymorphism (RFLP) of nuclear DNA (Pogson et al., 1995; Fevolden and Pogson, 1997; Jónsdóttir et al., 1999, 2001) have displayed considerable population substructuring of cod both macro- and microgeographically. In the present study we have focused on the genetic structure of Atlantic cod collected from both sides of the North Atlantic Ocean, using nuclear DNA RFLP analysis.
2. Materials and methods 2.1. Sampling Between June 1994 and December 1997 cod samples from pre- and postspawning aggregations was collected from six locations in the North Atlantic (Fig. 1
0165-7836/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0165-7836(03)00098-5
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Fig. 1. Sampling locations of Atlantic cod in the North Atlantic Ocean.
and Table 1). A total of 551 specimens were gathered throughout the geographical range of the species, from the Scotian Shelf in the west to the North Sea, Celtic Sea and Norwegian waters (Trondheimsfjorden) in the east, as well as Icelandic waters (Loftstaðahraun), and the Barents Sea (Fig. 1). Between 93 and 100 samples were collected from each sample location (Scotian Shelf and Loftstaðahraun/Celtic Sea/North Sea, respectively), except in the Barents Sea where 62 samples were gathered (Table 1). The fish were collected using bottom trawls, at depths between 32 and 117 m (Loftstaðahraun and Scotian Shelf, respectively; Table 1). Sex of each specimen was recorded. Since the collection positions of the samples at Scotian Shelf, North Sea and Celtic Sea were separated by several nautical miles within each location, and over periods from approximately 3 to 4 weeks (North Sea and Scotian Shelf, respectively; Fig. 1 and Table 1), intra-area FST tests were performed. No differences were detected (Scotian Shelf and North Sea, P > 0.9, Celtic Sea, P > 0.13) thus samples from each location were pooled for inter-area comparisons. The results from the Syp I analysis of Loftstaðahraun sample has been examined in previous studies (Jónsdóttir et al., 1999, 2001).
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), which was collected from live or recently dead cod and was preserved immediately in 96% ethanol. The DNA extraction was performed by phenol-extraction (Taggart et al., 1992). The Syp I locus was analysed with a PCR-based method using the reaction conditions described by Jónsdóttir et al. (1999) modified by Fevolden and Pogson (1997). The five remaining loci 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 characterized by a specific cDNA proberestriction enzyme combination. Total DNA samples (7 g) from all specimens were digested with two restriction enzymes (DraI and TaqI). DraI digested samples (one restriction site) were probed with cDNA clones GM842 and GM860 and TaqI digests (one restriction site) were probed with clones GM738, GM777 and GM865. In accordance with Pogson et al. (1995), the name of each cDNA probe is synonymous with the name of the cDNA “locus” for the five clones
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Table 1 The collection location of Atlantic cod samples, number of specimens collected (N), collection position of the samples, date of collection, mean depth (m) at collection site, mean length (mm), mean mass (g) and mean age (years) of specimens and proportion of females (%) in the catch for each sample unit Location
N
Position of sample units
Date
Depth (m)
Scotian Shelf-Browns Bank
6 8 5 16 10
42◦ 76 N, 42◦ 61 N, 42◦ 45 N, 42◦ 52 N, 42◦ 62 N,
66◦ 16 W 66◦ 25 W 65◦ 36 W 64◦ 99 W 64◦ 77 W
7 July 1994 7 July 1994 12 July 1994 12 July 1994 12 July 1994
58 86 106 115 117
552 537 565 378 413
Scotian Shelf-Western Bank
5 6 15 16 6
43◦ 17 N, 43◦ 41 N, 43◦ 56 N, 43◦ 83 N, 43◦ 87 N,
61◦ 65 W 61◦ 35 W 60◦ 46 W 60◦ 25 W 60◦ 48 W
13 14 27 27 27
Loftstaahraun
16 26 22 18 6 12
63◦ 46 N, 63◦ 45 N, 63◦ 48 N, 63◦ 46 N, 63◦ 48 N, 63◦ 45 N,
20◦ 54 W 20◦ 49 W 20◦ 01 W 20◦ 58 W 20◦ 56 W 20◦ 49 W
3 4 4 4 4 4
Trondheimsfjorden
96
63◦ 51 N, 10◦ 44 E
62
74◦ 11 N,
34◦ 31 E
Celtic Sea
22 10 23 15 18 12
51◦ 42 N,
6◦ 28 W
North Sea
26 25 6 18 5 5 8 7
57◦ 39 N, 58◦ 28 N, 61◦ 02 N, 59◦ 12 N, 58◦ 21 N, 54◦ 53 N, 54◦ 56 N, 54◦ 54 N,
Barents Sea
a
51◦ 06 N, 51◦ 42 N, 51◦ 27 N, 51◦ 46 N, 51◦ 22 N,
Length (mm)
Mass (g)
Age (years)
Females (%)
1619 1479 1683 651 799
3.5 3.2 4.1 2.4 2.4
50 63 100 47 67
June June June June June
1994 1994 1994 1994 1994
88 75 77 46 35
494 377 367 357 385
1200 504 442 373 480
5.6 3.8 4.1 3.7 4.3
20 67 47 50 67
April April April April April April
1997 1997 1997 1997 1997 1997
47 34 58 57 42 32
1095 1067 1073 1069 1058 1095
17366 15953 16277 15682 16952 17669
8.3 8.3 8.5 8.6 7.5 7.9
19 23 46 39 50 33
17 April 1996
100
634
3118
6
77
13 August 1995
NAa
413
811
NA
48
8◦ 23 W 8◦ 01 W 7◦ 35 W 6◦ 42 W 5◦ 84 W
28 29 30 30 30 31
97 114 73 87 72 93
664 442 414 535 571 541
3588 1541 1208 2840 3223 3269
2.7 1.8 1.8 2.4 2.7 2.5
73 70 44 53 56 50
4◦ 40 E 3◦ 53 E 1◦ 12 W 3◦ 01 W 2◦ 34 W 1◦ 65 W 1◦ 77 W 1◦ 66 W
21 August 1997 22 August 1997 1 September 1997 2 September 1997 4 September 1997 5 September 1997 5 September 1997 5 September 1997
NA NA NA NA NA NA NA NA
391 467 602 351 318 436 445 370
779 1221 3036 600 380 1095 1123 634
1.7 2.2 2.8 1.3 1.2 2 2.6 1.7
40 52 50 65 80 80 50 71
March March March March March March
1997 1997 1997 1997 1997 1997
Not available.
used to score the observed polymorphism. 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 et al. (1995). Each form of polymorphism was first assigned a letter and then broken down into allelic sizes. The loci were screened using nonradioactively 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 and 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
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(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 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 a 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 detect band patterns. The membranes were stored in 2× SSC between hybridizations. 2.3. Statistical methods Allele frequencies for each locus, estimates for observed and expected heterozygosities, tests for heterozygote deficiency and calculations for allelic differentiation over all cod sample pairs for each loci were provided by the GENEPOP software (Raymond and Rousset, 1995). Population pairwise FST statistics were calculated for all sample units using the Arlequin, Version 1.1 computer package, and the significance was tested with permutation (1000 times, Schneider et al., 1997). Genotype frequencies were tested for conformity with expected Hardy–Weinberg proportions using GENEPOP, and separate tests were performed for each locus. 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). A UPGMA dendrogram of the bootstraped Nei’s genetic distance matrix was constructed in the NEIGHBOR program in PHYLIP. Bonferroni correction of the significance level (α = 0.05) were applied when testing for significant departures from Hardy–Weinberg expectations and for significance of FST values.
Mean age at the different sample locations was tested with one-way ANOVA and log-linear analysis (Zar, 1996) was used to test for sex- and age-specific frequencies of all loci in all sample locations.
3. Results All examined loci were polymorphic in all sample units, with the exception of Syp I at Celtic Sea (Table 2). The most common allele was the same for each of four loci (GM842, GM860, GM777 and GM865) for all sample units, with frequencies ranging from 0.53 to 0.93 (Table 2). For the remaining loci (GM738 and Syp I), the most common alleles differed between sample units (Table 2). For all sample units except the Barents Sea, the Syp IA allele was the most common one (frequencies from 0.81 to 1, Trondheimsfjorden and Celtic Sea, respectively, Table 2). No deviations from Hardy–Weinberg equilibrium were found within each sample unit (P > 0.34). A comparison of observed and expected heterozygosity and an exact probability test searching specifically for heterozygote deficiencies revealed heterozygote deficiencies only at the GM860 and GM738 loci in the Barents Sea and Trondheimsfjorden samples, respectively. Calculations for allelic differentiation over all cod sample pairs for each locus revealed significant differences at all loci for different pairs of sample units (χ2 -tests, P < 0.05). The most pronounced differences were found at the Syp I and the GM738 loci. The population pairwise FST test revealed highly significant differences (P < 0.001) between the Barents Sea sample vs. all sample units, and between the Scotian Shelf sample vs. all sample units except the Celtic Sea (Table 3). The bootstrapped UPGMA dendrogram constructed using Nei’s (1972) genetic distances (Fig. 2) illustrated a clear differentiation between the sample units analysed. The genetic distance between Barents Sea and the remaining sample units was several times greater than detected for any other sample pairs (Fig. 2). The mean age of the specimens in the different samples varied (One-way ANOVA, F7,381 = 19.2, P < 0.01) with the overall highest mean age found in the Loftstaðahraun and the Trondheimsfjorden samples (8.2 and 6 years, respectively). These two samples differed from the three other samples (age was not
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Table 2 Allele frequencies at each locus for analysed cod samples from six locations in the North Atlantic Ocean Locus/alleles (kb)
Trondheimsfjorden
Barents Sea
North Sea
Celtic Sea
Scotian Shelf
Loftstaahraun
58 0.069 0.026 0 0.905 0 0 0
15 0.067 0 0 0.900 0.033 0 0
31 0.032 0.016 0 0.823 0 0.032 0.097
42 0.036 0.024 0 0.928 0.012 0 0
GM842 na 2.65 2.92 3.07 3.15 3.35 3.60 4
26 0 0.019 0.019 0.924 0 0.038 0
GM860 n 1.60 2.08 2.38 2.67 5.70 6.27
49 0.561 0 0.010 0 0.429 0
29 0.638 0 0 0 0.362 0
59 0.551 0.008 0 0 0.441 0
24 0.583 0 0 0 0.396 0.021
75 0.680 0 0 0.007 0.313 0
56 0.527 0 0 0 0.473 0
GM738 n 1.89 2.70 2.77
48 0.479 0 0.521
27 0.537 0 0.463
54 0.556 0.009 0.435
58 0.474 0 0.526
69 0.116 0 0.884
45 0.556 0 0.444
GM777 n 1.57 2.03 2.55 3.04 4.30
11 0 0 0.227 0.773 0
0 – – – – –
14 0.036 0.036 0.178 0.750 0
10 0.050 0.100 0.250 0.600 0
GM865 n 1.71 1.78 1.82 7.18
63 0.008 0.294 0.698 0
6 0 0.417 0.583 0
19 0 0.210 0.790 0
62 0 0.218 0.782 0
96 0.813 0.187 293
50 0.020 0.980 112
96 0.917 0.083 300
Syp I n Syp IA Syp IB Total n a
0 – – – – – – –
84 1 0 253
0 – – – – –
48 0.010 0.010 0.104 0.855 0.021
51 0 0.196 0.794 0.010
45 0 0.422 0.578 0
86 0.948 0.052 312
97 0.876 0.124 333
Number of specimens giving detectable bands for each analysis.
recorded for Barents Sea) whereas no differences were found between the Celtic Sea (2.7), North Sea (2.9), and Scotian Shelf (3.6). Genotype proportions did not differ between sex or age groups for any locus and sample (contingency log-linear tests, Pearson χ2 > 4.1, P > 0.18).
4. Discussion The present study supports previous indications of genetic subdivision of Atlantic cod in the North Atlantic. In particular, these results support the studies showing genetic differentiation between eastern and
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Table 3 Population pairwise FST estimates among all sample units (permuting the individuals between the sampling sites tested the significance of the pairwise FST values)a
Barents Sea Celtic Sea Loftstaahraun North Sea Scotian Shelf Trondheimsfjorden
Barents Sea
Celtic Sea
Loftstaahraun
North Sea
Scotian Shelf
– 0.67311∗∗∗ 0.56711∗∗∗ 0.60539∗∗∗ 0.59959∗∗∗ 0.47570∗∗∗
– – −0.02088b −0.20421b 0.00031 0.01979
– – – −0.04334b 0.13091∗∗∗ −0.00227b
– – – – 0.10587∗∗∗ −0.08588b
– – – – – 0.07684∗∗∗
a
Thousand permutation were performed for all pair of sampling sites. F-statistic estimators in this model are random variables and as such may take either positive or negative values (Long, 1986; Excoffier et al., 1992). Such negative estimates should be interpreted as zero (Long, 1986) in the FST model there are no differences between those sampling pairs where negative FST values are found. ∗∗∗ P < 0.001 (Bonferroni correction for simultaneous tests). b
western Atlantic cod (Carr and Marshall, 1991; Galvin et al., 1995; Pogson et al., 1995) and between cod inhabiting the Barents Sea and other areas (Dahle, 1991; Bentzen et al., 1996). When comparing our results and those of Pogson et al. (1995) to previously published patterns of variation at allozyme loci obtained from cod collected from a range of similar localities (Mork et al., 1985), a considerable difference is detected in the magnitude of observed genetic differentiation, as the allele frequency variation at the nuclear RFLP loci
significantly exceeded that observed at the protein loci. This observation has at least two possible interpretations. First, allele scoring for the RFLP method can be seen as more uncertain compared to protein loci scoring. However, we applied a computerized sizing (Schaffer and Sederoff, 1981) combined with running a size ladder alongside the samples minimize the possibility of erroneous allele sizing. Secondly, the RFLP markers may be under selection. Fevolden and Pogson (1995) suggested that the polymorphism
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).
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revealed by one of the RFLP markers (GM798 cDNA probe), might represent promising marker to distinguish north-east Arctic cod (AC) from Norwegian coastal cod (NCC). Particularly the GM798C locus (synaptophysin, Syp I locus, Fevolden and Pogson, 1997) showed a considerable population substructuring of cod in the North Atlantic and was found to be a distinct genetic marker for distinguishing between AC and NCC (Fevolden and Pogson, 1995, 1997). Furthermore, Jónsdóttir et al. (1999, 2001) found an explicit difference between samples collected at two adjacent spawning areas (Loftstaðahraun and Kantur) off south Iceland, using the Syp I locus. These studies revealed a distinctive pattern of the locus, i.e. either high frequency of the Syp IA allele or high frequency of the Syp IB allele (NCC and Loftstaðahraun vs. AC and Kantur, respectively; Fevolden and Pogson, 1995, 1997; Jónsdóttir et al., 1999, 2001). When all sample pairs were compared for each locus in the present study, the striking features of the Syp I locus became apparent. With only three exceptions (Trondheimsfjorden vs. Loftstaðahraun, North Sea vs. Scotian Shelf and Loftstaðahraun) the sample units were significantly different from each other. Specifically, the Barents Sea sample differed from all other sample units. Such indications of genetic distinctiveness of Barents Sea fish populations have also been reported for other gadoid species, i.e. blue whiting Micromesistius poutassou (Risso) (Giæver and Stein, 1998), as well as for Atlantic halibut Hippoglossus hippoglossus (L.) (Foss et al., 1998), and long rough dab Hippoglossoides platessoides (Bloch) (Walsh, 1994). It should be noted that apart from the possible selection of the Syp I locus no indication of selection of the other RFLP markers have been found (Fevolden and Pogson, 1995). The sharp differences observed by Pogson et al. (1995) between populations calculated by the FST test were not seen in the present study as only the Barents Sea and the Scotian Shelf samples displayed significant genetic differences from other samples using the FST test. The lack of significant FST values in our study compared to the study of Pogson et al. (1995) might be explained by fewer nuclear RFLPs scored and less information gathered from each locus in our study. In conclusion the nuclear DNA RFLP analysis of Atlantic cod collected from six locations across the
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North Atlantic revealed a genetic substructure of the species across its range. In particular our results indicate a clear differentiation between west and east Atlantic population units and between Barents Sea population and other population in the east Atlantic.
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