- Email: [email protected]
Population genetic studies of the roughhead grenadier, Macrourus berglax L., in the North Atlantic Ocean
Fisheries Research 51 (2001) 207±215
Population genetic studies of the roughhead grenadier, Macrourus berglax L., in the North Atlantic Ocean Euthemia Katsarou, Gunnar Nñvdal* Department of Fisheries and Marine Biology, University of Bergen, Bergen High Technology Center, HIB Ð Thormohlensgt. 55, N-5020 Bergen, Norway
Abstract Samples of the roughhead grenadier, Macrourus berglax L., were collected from several locations off East and West Greenland and in the Norwegian Sea. A total of 651 individuals were screened using starch-gel electrophoresis to reveal polymorphic systems to be applied in studies of the genetic structure of the species. Ten enzymes were stained for and seven enzyme loci (G3PDH-1, GPI-1, GPI-2, LDH-1, LDH-2, MDH and PGM) displayed suf®cient activity and allele variation to be used in the routine work. The GPI-1 and PGM loci displayed high heterozygosities. Tests of accordance between observed distributions of phenotypes and expected Hardy±Weinberg distributions as well as tests of heterogeneity among sampling sites, were performed using w2 tests. No genetic differences within the three main areas (West Greenland, East Greenland and the Norwegian Sea) were revealed, while signi®cant differences were found among those areas indicating that the roughhead grenadier in the North Atlantic is composed of different stock units with their own gene pools rather than belonging to one panmictic population. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Polymorphism; Isozymes; Genetic structure; Macrourus berglax; Grenadier; Heterogeneity
1. Introduction Roughhead grenadier, Macrourus berglax L., is an abundant, mostly demersal species widely distributed in moderate to great depths (Parsons, 1976; Moyle and Cech, 1996) across the North Atlantic. It is found in the Labrador Sea (Mombeck, 1984; Sahrhage, 1982; Savvatimsky, 1984), Baf®n Bay (Forest et al., 1978; Savvatimsky, 1984), between Greenland and the Azores on the mid-Atlantic ridge (Sahrhage, 1986), from Denmark Strait to Jan Mayen (Magnusson, 1978; Sahrhage, 1986 and the references cited therein), to the west and north-west of Iceland (Magnusson, 1978, *
Corresponding author. Tel.: 47-55584401; fax: 47-55584450. E-mail address: [email protected] (G. Nñvdal).
1979), further east in the waters between Iceland and the Faeroe Islands (Sahrhage, 1986 and the references cited therein), and west of the British Isles, Ireland and Norway (Bakken et al., 1975; Hognestad and Vader, 1979; Eliassen and Lorentsen, 1982; Eliassen, 1983; Eliassen and Breiby, 1983). Occurrence of the species has also been reported in a number of the deep basins in the Barents Sea (Sahrhage, 1986 and the references cited therein), between Bear Island and Svalbard (Geistdoerfer, 1979), and as far as the Kola Peninsula (Savvatimsky, 1989). Investigations on roughhead grenadiers have been carried out since 1969, together with corresponding studies of other commercial ®sh species occurring in the same areas (P.I. Savvatimsky, personal communication). In the late 1970s the species was extensively studied in the North Atlantic (Magnusson, 1977;
0165-7836/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 7 8 3 6 ( 0 1 ) 0 0 2 4 6 - 6
208
E. Katsarou, G. Nñvdal / Fisheries Research 51 (2001) 207±215
Eliassen and Breiby, 1983; Savvatimsky, 1989). The growing interest in utilizing `new' resources has focused on the roughhead grenadier as a potential species for commercial harvest (Savvatimsky, 1989, 1992). Bottom trawls and particularly longline ®sheries are expected to be a promising method of utilizing the resource in the North Atlantic (Savvatimsky, 1992). The development of population genetic methods offers new tools for investigating the genetic structure of natural populations. The genetic structure of the roughhead grenadier was investigated by applying allozyme electrophoretic analysis of samples from different geographic areas in the North Atlantic (Fig. 1). The goals were to reveal genetic variation and to utilize the most useful traits for studies of possible population structure of the species. The investigation was based on the hypothesis that the roughhead grenadier in the North Atlantic consists of a single panmictic population. 2. Materials and methods During the period August 1991±June 1996, 651 individual samples were collected on board research vessels at a number of sampling locations at different ®shing grounds on the shelf and continental slopes off Greenland and Norway (Fig. 1). Four samples were
collected from Baf®n Bay and Davis Strait off the coast of West Greenland, 10 sample units were from the Irminger Sea and Dohrnbank off the coast of East Greenland and four samples were from Storegga and Lofoten in the Norwegian Sea. The sample size ranged from 2 to 260 specimens. For the statistical analyses, samples from adjacent localities were amalgamated. An overview of the material is given in Table 1 where also the samples are grouped according to the three main areas: West Greenland, East Greenland and the Norwegian Sea. Small pieces (approximately 1 cm3) of white muscle from close to the dorsal ®n were taken from each ®sh. Liver samples were taken from approximately one-third of the individuals (Table 1). All tissue samples were stored in Microwelles at ÿ208C on board the ship until they were transferred to an ultra freezer (ÿ808C) at the laboratory for further processing. From some localities the ®sh were frozen whole and tissue were sampled after thawing. The tissue enzymes were analysed by standard starch-gel electrophoresis (SGE) (Harris and Hopkinson, 1976; Murphy et al., 1990). Two buffer systems were assessed for their ability to accurately resolve polymorphic enzymes: (i) Histidine-citrate pH 7.0 (Harris and Hopkinson, 1976) and (ii) Tris-citrate-borate pH 8.6 (Selander et al., 1971). Each gel (8 mm thick) was sliced horizontally ®ve or six times and each slice was stained for a particular
Fig. 1. Sampling localities of roughhead grenadiers in the North Atlantic for population studies.
E. Katsarou, G. Nñvdal / Fisheries Research 51 (2001) 207±215
209
Table 1 Sampling sites, sampling data and fishing method (L: longline, BT: bottom trawl), depth range in metres (based on estimation from echo depth) and sample size (N) of muscle (M) and liver (L) tissues Location of sampling areasa
East Greenland Dohrnbank01 (dhr.b.01)
Dohrnbank02 (dhr.b.02)
Dohrnbank±Irminger (dhr.irm.) West Greenland Baffin Bay (bf.b.) Davis Strait (dv.st.) Norwegian Sea Storegga (nr.s.01) Lofoten (nr.s.02)
Position (latitude, longitude)
Collection data (date)
Fishing method
Echo depth (m)
658270 N 658270 N 658260 N 658290 N
318200 W 318240 W 318170 W 318260 W
10 10 21 21
September 1991 September 1991 June 1994 June 1994
L L L L
678080 N 678070 N 678090 N 678100 N
308350 W 308250 W 308280 W 308370 W
07 07 08 08
September September September September
1991 1991 1991 1991
M
L
628±389 600±394 594±446 519±559
260 44 23
95 ±b ±b
L L L L
750±535 548±371 540±375 341±512
33 5
±b ±b
648080 N 368020 W 628190 N 408250 W
29 August 1993 10 July 1994
L L
796±467 830±465
96 15
±b ±b
718350 N 608260 W 708280 N 608470 W
24 August 1993 27 August 1993
L L
651±563 439±388
19 43
19 43
698120 N 588400 W 698070 N 588410 W
29 August 1993 29 August 1993
L L
465±322 445±322
26 18
26 18
628200 N 018050 E 628210 N 018290 E
24 June 1995 24 June 1995
BT BT
585 530
43 12
12 ±b
678460 N 098470 E 678190 N 088480 E
28 June 1996 30 June 1996
BT BT
638 596
12 2
±b ±b
Total a b
N
651
213
For the statistical analyses, samples from adjacent localities were amalgamated. No liver tissue available.
enzyme system using procedures based on the protocols of Murphy et al. (1990). The enzyme patterns were immediately interpreted and photographed after staining and incubation. Non-speci®c esterase was screened by isoelectric focusing (IEF) on ampholine polyacrylamide gel, pH range 3.5±9.5, according to the instructions of Ampholine1 PAGplates, Pharmacia Biotechnology AB, Uppsala, Sweden (Anon., 1990). Enzyme nomenclature and allele designation followed Shaklee et al. (1990). The 10 enzymes (15 readable loci estimated) assayed, their abbreviations, the optimal buffer system used and the number of loci and alleles consistently scoreable are given in Table 2. Statistical analyses were carried out using the BIOSYS package program (Swofford and Selander, 1981), employing the Levene's (1949) correction for
small sample size. The exact signi®cance probabilities were calculated to test deviations from expected Hardy±Weinberg proportions. Contingency w2 analyses were performed to test the heterogeneity among samples. For multiple tests the signi®cance levels were adjusted according to the Bonferroni procedure (Weir, 1990). The genetic distance among samples was calculated using the unbiased genetic distance coef®cient of Nei (1978). 3. Results and discussion 3.1. Polymorphic variation Of the 10 enzymes examined, ®ve consistently produced variable isoenzyme patterns which are likely
210
E. Katsarou, G. Nñvdal / Fisheries Research 51 (2001) 207±215
Table 2 Enzymes and enzyme commission numbers (EC), quaternary structure of their functional proteins, number of loci scored, number of alleles, buffer systems used for electrophoresis, activity and technical quality of muscle (M) and liver (L) tissues and estimated numbers of loci Enzyme
Number of alleles
Buffer (pH)b
Activity and technical qualityc M
L
±
II
±
Loci descriptiona
Alcohol dehydrogenase (1.1.1.1)
ADH
Creatine phosphokinase (2.7.3.2)
CPK
1
I
Esterase (3.1.1.-)
EST-1 EST-2
1 2?
III
Glycerol-3-phosphate dehydrogenase (1.1.1.8)
G3PDH-1 G3PDH-2
2 1
I
Glucose-6-phosphate isomerase (5.3.1.9)
GPI-1 GPI-2
4 3
I, II
Isocitrate dehydrogenase (1.1.1.42)
IDHP-1 IDHP-2
1 1
I, II
L-Lactate
LDH-1 LDH-2
3 3
I, II
MDH
3
I
PGM
6
I, II
±
I
dehydrogenase (1.1.1.27)
Malate dehydrogenase (1.1.1.37) Phosphoglucomutase (5.4.2.2f) Sorbitol dehydrogenase (1.1.1.14)
SDH
a
The estimated numbers of loci are based only on muscle analyses. Buffers for horizontal SGE: I Ð discontinuous Histidine-citrate, pH 7.0; II Ð discontinuous Tris-citrate-borate, pH 8.6; III Ð indicates isoelectric focusing (IEF), pH 3.5±9.5. c Activity and technical quality code: : unclear, : faint, : intermediate, moderate, : abundant. b
to be controlled by seven polymorphic loci. An account of the results is given in Table 2 together with an overview of the results of the analyses of each enzyme. The number of presumptive loci which could be accurately discerned amounted to 15. The enzymes ADH and SDH were rejected because they showed no activity. Further CPK and EST were poorly resolved. However, seven polymorphic loci for the following enzymes were found to be consistently interpretable and were scored for all sample units; G3PDH (locus G3PDH-1), GPI (loci GPI-1 and GPI-2), LDH (loci LDH-1 and LDH-2), MDH (locus MDH) and PGM (locus PGM). Three monomorphic loci for the following proteins were found; G3PDH (locus G3PDH2) and IDHP (loci IDHP-1 and IDHP-2). Activity was only observed in skeletal muscle extracts while liver tissues extract failed to give adequate staining for any enzyme. The phenotype patterns for the ®ve variable enzymes are displayed in Fig. 2. For all polymorphic loci calculated, allele frequencies are given in Table 3.
For most samples the observed distributions of phenotypes were in accordance with expected Hardy±Weinberg distributions. Among those distributions which could be tested by a w2 goodness-of-®t test when pooling rare alleles as recommended (Swofford and Selander, 1981; Chakraborty and Leimar, 1987) (Table 4) and applying a Bonferroni correction of the level of signi®cance for multiple tests, none of the samples displayed signi®cant deviations for any loci. Accordingly, the assumed mode of inheritance is accepted for all the described polymorphic systems. However, although not testable, it was also noted that the genotype LDH-1-65/25 occurred once despite the expectance for this genotype being very low. The accordance between observed and expected distributions is further illustrated by the calculation of Selander's D-statistics for heterozygote de®ciency or excess, Table 5, and observed and expected heterozygosities (not shown). Both positive and negative Dcoef®cients occur, indicating no trend among the deviations. It is, however, noted that the most highest
E. Katsarou, G. Nñvdal / Fisheries Research 51 (2001) 207±215
211
Fig. 2. Outline of genotypes of isozyme polymorphic systems for roughhead grenadiers obtained by SGE. Designations of alleles are shown on the sides and of genotypes below each drawing. Arrows indicate anodic migration.
deviation which concerns PGM genotypes in sample dhr.b.02, represents a de®ciency of heterozygotes. Thus a Wahlund's effect cannot be excluded, but there was no other indication of this sample representing a mixture of different gene pools. Mean heterozygosity values within samples ranged from 0.06 to 0.1. This is well within the range for teleost ®shes: 0.00±0.12 (Nevo, 1978) and close to the average value, 0:060 0:038, calculated by Smith and Fujio (1982), based on enzymatic loci. This shows that
the level of genetic variation in isozyme markers in roughhead grenadier is reasonably high offering possibilities for population studies using isozyme markers as tool. However, novel methods of DNA analyses (Carvalho and Pitcher, 1995 and the references cited therein) probably would show more variation which may be used for throwing additional light upon the structure of the species. From the present data it is not possible to draw any conclusion about the possible dynamics of
212
E. Katsarou, G. Nñvdal / Fisheries Research 51 (2001) 207±215
Table 3 Allelic frequencies at seven polymorphic loci for the seven sampling locationsa Locus
Allele (N)
dhr.b.01 (327)
dhr.b.02 (38)
dhr.irm. (111)
bf.b. (62)
dv.st. (44)
nr.s.01 (55)
nr.s.02 (14)
G3PDH-1
100 120
0.992 0.008
1.000 ±
1.000 ±
1.000 ±
1.000 ±
1.000 ±
0.964 0.036
GPI-1
75 100 125 150
0.387 0.569 0.037 0.008
0.250 0.737 0.013 ±
0.347 0.595 0.050 0.009
0.460 0.500 0.040 ±
0.398 0.568 0.023 0.011
0.373 0.509 0.118 ±
0.286 0.607 0.071 0.036
GPI-2
85 100 115
0.009 0.910 0.081
± 0.947 0.053
0.014 0.887 0.099
± 0.984 0.016
± 0.920 0.080
0.009 0.900 0.091
± 1.000 ±
LDH-1
ÿ100 ÿ65 25
0.989 0.009 0.002
0.987 0.013 ±
0.995 0.005 ±
0.968 0.032 ±
0.966 0.034 ±
0.991 0.009 ±
0.964 0.036 ±
LDH-2
65 100 135
0.002 0.930 0.069
± 0.921 0.079
± 0.941 0.059
± 0.944 0.056
± 1.000 ±
± 0.927 0.073
± 0.893 0.107
MDH
100 115 130
0.994 0.003 0.003
1.000 ± ±
1.000 ± ±
1.000 ± ±
1.000 ± ±
1.000 ± ±
1.000 ± ±
PGM
± 0.670 0.008 0.242 0.070 0.011
± 0.776 ± 0.184 0.026 0.013
± 0.694 0.005 0.225 0.072 0.005
± 0.694 ± 0.145 0.153 0.008
0.011 0.705 ± 0.170 0.114 ±
± 0.664 ± 0.255 0.064 0.018
± 0.607 ± 0.321 0.071 ±
94 100 105 110 120 130
a The most common allele at each locus is arbitrarily assigned 100; mobilities of other alleles are relative to the common allele. For definition of sample station abbreviations, see Table 1. Allele designations based on mobilities are given in the left-hand column. Number of specimens are in parentheses.
Table 4 Test of accordance between observed and expected frequencies, with pooling of rare alleles (Swofford and Selander, 1981; Chakraborty and Leimar, 1987) and w2-test for deviation from Hardy±Weinberg equilibriuma Sampling sites Dohrnbank01 (dhr.b.01) Dohrnbank02 (dhr.b.02) Dohrnbank±Irminger (dhr.irm.) Baffin Bay (bf.b.) Davis Strait (dv.st.) Storegga (nr.s.01) Lofoten (nr.s.02) a
w2-test GPI-1
GPI-2
LDH-2
MDH
PGM
0.01 1.53 4.07 0.10 3.59 1.02 1.18
3.16 ± 1.701 ± ± 0.61 ±
1.83 ± ± ± ± ± ±
0.01 ± ± ± ± ± ±
2.69 9.08 2.73 6.59 2.72 0.00 1.18
w2: chi-squares value; and indicate significance level of <0.05 and 0.005, respectively. When applying a Bonferroni adjustment of significance level, none of the deviations were statistically significant. No value indicates too low numbers to be tested. Distributions of G3PDH-1 and LDH-1 phenotypes could not be tested for any sample.
E. Katsarou, G. Nñvdal / Fisheries Research 51 (2001) 207±215
213
Table 5 Coefficients for heterozygote deficiency or excess in the seven sampling locationsa Locus
Sampling area
Obs. Het.b
Exp. het.c
Dd
G3PDH-1 GPI-1 GPI-2 LDH-1 LDH-2 MDH PGM
Dohrnbank01 (dhr.b.01)
5 168 59 6 46 4 174
4.97 172.07 54.25 6.95 42.90 3.99 159.82
0.006 ÿ0.024 0.088 ÿ0.136 0.072 0.003 0.089
GPI-1 GPI-2 LDH-1 LDH-2 PGM
Dohrnbank02 (dhr.b.02)
12 4 1 6 8
15.19 3.84 1.00 5.60 13.96
ÿ0.210 0.042 0.000 0.071 ÿ0.427
GPI-1 GPI-2 LDH-1 LDH-2 PGM
Baffin Bay (bf.b.)
31 2 4 7 21
33.57 1.98 3.90 6.66 29.65
ÿ0.077 0.008 0.025 0.051 ÿ0.292
G3PDH-1 GPI-1 GPI-2 LDH-1 LDH-2 PGM
Storegga (nr.s.01)
30 11 1 8 28
32.63 10.08 1.00 7.49 27.22
ÿ0.081 0.091 0.000 0.069 0.029
GPI-1 GPI-2 LDH-1 LDH-2 PGM
Dohrnbank±Irminger (dhr.irm.)
67 25 1 13 46
58.39 22.58 1.00 12.29 51.61
0.148 0.107 0.000 0.057 ÿ0.109
GPI-1 GPI-2 LDH-1 LDH-2 PGM
Davis Strait (dv.st.)
29 7 3
23.07 6.52 2.93
0.257 0.074 0.024
16
20.54
ÿ0.221
1 6
1.00 7.89
0.000 ÿ0.239
1 3 6
1.00 2.78 7.593
0.000 0.080 ÿ0.210
G3PDH-1 GPI-1 GPI-2 LDH-1 LDH-2 PGM
Lofoten (nr.s.02)
a
Probability levels (0.99 criterion) resulting from tests for homogeneity among loci and localities are shown in the first two rows. Observed number of heterozygotes. c Expected number of heterozygotes. d Selander's D-statistic for the excess or deficiency of heterozygotes. b
the observed variation, i.e. whether the markers are selectively neutral or not. However, as pointed out by Ferguson (1995) selective traits may be valuable as markers for population studies with
respect to management application. However, the evolutionary signi®cance of interpopulation differences may be different for neutral and selective traits.
214
E. Katsarou, G. Nñvdal / Fisheries Research 51 (2001) 207±215
3.2. Variation among samples and areas Contingency w2 analysis of variation among all samples indicated heterogeneity in the total material of roughhead grenadier (Table 6) both for GPI-1 and for total distribution of alleles indicating that the samples have been drawn from more than one panmictic population. Splitting the material according to the three main areas: West Greenland, East Greenland and the Norwegian Sea and performing a similar test for the most variable enzyme systems, GPI-1 and PGM, no signi®cant differences were found within any of the areas. Further tests of each area versus the other ones showed signi®cant differences among all areas based upon one or the other of the two polymorphic systems, Table 7, and for both system combined (not shown in the table). The calculated values for genetic distances among samples were all low (0.000±0.004). This indicates that although two independent markers showed clear heterogeneity among samples collected from the different main areas, the total differentiation is low. A reasonable interpretation of these observations is that roughhead grenadier consists of at least one panmictic population within each of the sampled areas, and for management purposes they should be regarded as independent. However, the low overall genetic differentiation as indicated by the low values of the genetic distance coef®cients, indicates that the evolutionary signi®cance of the observed, between areas, differences is uncertain and possibly low. Further studies Table 6 Contingency w2 table for the inter-area results with respect to the seven loci for the nine sampling sitesa Locus
Number of alleles
G3PDH-1 GPI-1 GPI-2 LDH-1 LDH-2 MDH PGM
2 4 3 3 3 3 6
Total
w2
d.f.
P
10.092 36.195 15.536 10.828 8.889 3.975 42.998
6 18 12 12 12 12 30
0.121 0.006 0.213 0.544 0.712 0.984 0.059
128.513
102
0.039
a 2 w : chi-squares value test, d.f.: degrees of freedom and P: significant probability. When the level of significance is adjusted by a sequential Bonferroni correction (Weir, 1990) only GPI-1 and the total show significantly different distributions.
Table 7 Contingency w2 analysis for inter-area with respect to two most variable polymorphic loci, GPI-1 and PGMa Number w2 of alleles
d.f. P
GPI-1 East±West Greenland East Greenland±Norwegian Sea West Greenland±Norwegian Sea All areas
4 4 4 4
3.439 13.715 9.001 18.394
3 3 3 6
0.329 0.003 0.029 0.005
PGM East±West Greenland East Greenland±Norwegian Sea West Greenland±Norwegian Sea All areas
6 5 5 6
21.591 1.976 11.072 25.520
5 4 4 10
0.001 0.740 0.026 0.004
Area
a 2 w : chi-squares value, d.f.: degrees of freedom and P: significance probability.
using a denser sampling grid and applying additional markers would probably allow ®rmer conclusions about the structure of roughhead grenadiers in the North Atlantic. 4. Conclusion The contingency w2-test among the sampling sites revealed signi®cant heterogeneity at the polymorphic locus GPI-1 and for all polymorphic loci combined. No variation among samples were found within any of the three main areas: West Greenland, East Greenland and the Norwegian Sea. Pairwise comparisons using contingency w2 tests for the most variable polymorphic loci (GPI-1 and PGM) showed that the frequency distributions differed signi®cantly between the main areas. Thus, the present study provides strong evidence that the roughhead grenadier population in the North Atlantic does not form a single panmictic stock but is composed of at least three different stock units with their own gene pools. Acknowledgements The authors are greatly indebted to skippers and crew of different research and ®shing boats who have collected the material. Especially our thanks are directed to Nils-Roar Hareide who was in charge for most of the sampling in Greenland waters.
E. Katsarou, G. Nñvdal / Fisheries Research 51 (2001) 207±215
References Anon., 1990. Instruction for Ampholine1 PAGplate, precast polyacrylamide gels for analytical isoelectric focusing. Pharmacia Biotechnology AB, Uppsala, Sweden, 34 pp. Bakken, E., Lahn-Johannesen, J., Gjùsñter, J., 1975. Demersal fish on the continental slope off Norway. ICES CM/F: 29, 1±15. Carvalho, G.R., Pitcher, T.J. (Eds.), 1995. Molecular Genetics in Fisheries. Chapman & Hall, Padstow, Cornwall, UK, 141 pp. Chakraborty, R., Leimar, O., 1987. Genetic variation within a subdivided population. Genetic distance and molecular philogeny. In: Ryman, N., Utter, F. (Eds.), Population Genetics and Fishery Management. University of Washington, Washington, DC, pp. 89±120. Eliassen, J.-E., 1983. Distribution and abundance of roundnose grenadier (Coryphaenoides rupestris Gunnerus) (Gadiformes, Macrouridae) in northern and mid-Norway. ICES CM/G: 43, 1± 24 (mimeo). Eliassen, J.-E., Breiby, A., 1983. Forsùksfiske etter isgalt og blaÊkveite langs Trñnaegga, Nordland fylke i 1983. Rep. Ser. B: Ressurbiologi 1983, Vol. 6. Institutt for Fiskerifag, University of Tromsù, Norway, pp. 1±13 (in Norwegian). Eliassen, J.-E., Lorentsen, E., 1982. Toktrapport fra forsùksfiske etter isgalt og blaÊkveite utenfor Nordland og Tromsù i juni og juli 1982. Rep. Ser. B: Ressurbiologi 1982, Vol. 6. Institutt for Fiskerifag, University of Tromsù, Norway, pp. 1±9 (in Norwegian). Ferguson, A., 1995. Molecular genetics in fisheries: current and future perspectives. In: Carvalho, G.R., Pitcher, T.J. (Eds.), Molecular Genetics in Fisheries. Chapman & Hall, Padstow, Cornwall, UK, pp. 81±103. Forest, A., Minet, J.P., Perodou, J.B., 1978. Results of groundfish survey on Baffin Island shelf. ICNAF Research Document No. 78/VI/45, 10 pp. Geistdoerfer, P., 1979. New data on the reproduction of macrourids (Teleostei, Gadiformes). Sarsia 64, 109±112. Harris, H., Hopkinson, D.A., 1976. Handbook of Enzyme Electrophoresis in Human Genetics. North-Holland, Oxford. Hognestad, P.T., Vader, W., 1979. Saltvannsfiskene i Nord Norge, Vol. 6. Universitet i Tromsù, Tromura, 74 pp. (in Norwegian). Levene, H., 1949. On a matching problem arising in genetics. Ann. Math. Statist. 20, 91±94. Magnusson, J.V., 1977. Some notes on the spawning habits of Macrouridae at Iceland. ICES CM 1977/F: 49, 9 pp. Magnusson, J.V., 1978. On the distribution and spawning grounds of the roughhead grenadier (Macrourus berglax LacepeÂde) west of Iceland. ICES CM 1977/G: 36, 10 pp. Magnusson, J.V., 1979. Icelandic investigations on grenadier fish (Coryphaenoides rupestris and Macrourus berglax) in 1977. Ann. Biol., Copenhague 34, 223±225. Mombeck, F., 1984. Untersuchung der GrundfischbestaÈende vor Labrador (investigation of benthic fish stocks near Labrador). Inf. Fischw. Auslds. 31, 3±9 (in German).
215
Moyle, P.B., Cech Jr., J.J. (Eds.), 1996. Fishes: An Introduction to Ichthyology, 3rd Edition. Prentice-Hall, Englewood Cliffs, NJ, p. 590. Murphy, R.W., Sites Jr., J.W., Buth, D.G., Haufler, C.H., 1990. Proteins. I. Isozyme electrophoresis. In: Hills, D.M., Moritz, C. (Eds.), Molecular Systematics. Sinauer Associates, Inc., Sunderland, MA, pp. 45±126. Nei, M., 1978. Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89, 583± 590. Nevo, E., 1978. Genetic variation in natural populations: patterns and theory. Theoret. Pop. Biol. 13, 121±177. Parsons, L.S., 1976. Distribution and relative abundance of roundnose, roughhead and common grenadiers in the northwest Atlantic. ICNAF Select. Pap. 1, 73±88. Sahrhage, D., 1982. Erforschung der GrundfischbestaÈende vor Labrador (research of benthic fish stocks near Labrador). Inf. Fischw. Auslds. 29 (1), 4±13. Sahrhage, D., 1986. Mitteilungen aus dem Instistut fuÈr Seefischerei der Bundesforschungsanstalt fuÈr Fischerei. Commercially important grenadiers of the North Atlantic. Can. Transl. Fish. Aquat. Sci., Hamburg 37, 81. Savvatimsky, P.I., 1984. Biological aspects of roughhead grenadier (Macrourus berglax) from longline catches in the eastern Grand Bank area, 1982. NAFO Sci. Counc. Stud. 7, 45±51. Savvatimsky, P.I., 1989. Investigations of roughhead grenadier (Macrourus berglax L.) in the Northwest Atlantic, 1967±1983. NAFO Sci. Counc. Stud., 13, 59±75. Savvatimsky, P.I., 1992. Severnyj makrurus (Macrourus berglax L.) potentsial'nyj ob'ekt tralovogo i yarusnogo promysla v Severnoj Atlantike (the roughhead grenadier Macrourus berglax, a potential resource for trawl and longline fisheries). In: Savvatimskij, P.I. (Ed.), Studies of Biological Resources in the North Atlantic. #Issledovaniya Bioresursovsevernoj Atlantiki, Pinko, pp. 45±67 (in Russian). Selander, R.K., Smith, M.H., Yang, S.Y., Johnson, W.E., Gentry, J.R., 1971. Biochemical polymorphism and systematics in the genus Peromyscus. I. Variation in the old-field mouse (Peromyscus polionotus). Studies in Genetics, Vol. VI. The University of Texas Publication No. 103, pp. 49±90. Shaklee, J.B., Allendorf, F.W., Morizot, D.C., Writt, G.S., 1990. Gene nomenclature for protein-coding loci in fish. Trans. Am. Fish. Soc. 119, 2±15. Smith, P.J., Fujio, Y., 1982. Genetic variation in marine teleosts: high variability in habitat specialists and low variability in habitat generalists. Mar. Biol. 69, 7±20. Swofford, D.L., Selander, R.B., 1981. BIOSYS-1 (Release 1.7). A Computer Program for the Analysis of Allelic Variation in Genetics. Users Manual. Department of Genetics and Development, University of Illinois, IL, 63 pp: Appendices. Weir, B.S., 1990. Genetic Data Analysis. Methods for Discrete Population Genetic Data. Sinauer Association, Inc., Sunderland, MA, 377 pp.
Population genetic studies of the roughhead grenadier, Macrourus berglax L., in the North Atlantic Ocean Euthemia Katsarou, Gunnar Nñvdal* Department of Fisheries and Marine Biology, University of Bergen, Bergen High Technology Center, HIB Ð Thormohlensgt. 55, N-5020 Bergen, Norway
Abstract Samples of the roughhead grenadier, Macrourus berglax L., were collected from several locations off East and West Greenland and in the Norwegian Sea. A total of 651 individuals were screened using starch-gel electrophoresis to reveal polymorphic systems to be applied in studies of the genetic structure of the species. Ten enzymes were stained for and seven enzyme loci (G3PDH-1, GPI-1, GPI-2, LDH-1, LDH-2, MDH and PGM) displayed suf®cient activity and allele variation to be used in the routine work. The GPI-1 and PGM loci displayed high heterozygosities. Tests of accordance between observed distributions of phenotypes and expected Hardy±Weinberg distributions as well as tests of heterogeneity among sampling sites, were performed using w2 tests. No genetic differences within the three main areas (West Greenland, East Greenland and the Norwegian Sea) were revealed, while signi®cant differences were found among those areas indicating that the roughhead grenadier in the North Atlantic is composed of different stock units with their own gene pools rather than belonging to one panmictic population. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Polymorphism; Isozymes; Genetic structure; Macrourus berglax; Grenadier; Heterogeneity
1. Introduction Roughhead grenadier, Macrourus berglax L., is an abundant, mostly demersal species widely distributed in moderate to great depths (Parsons, 1976; Moyle and Cech, 1996) across the North Atlantic. It is found in the Labrador Sea (Mombeck, 1984; Sahrhage, 1982; Savvatimsky, 1984), Baf®n Bay (Forest et al., 1978; Savvatimsky, 1984), between Greenland and the Azores on the mid-Atlantic ridge (Sahrhage, 1986), from Denmark Strait to Jan Mayen (Magnusson, 1978; Sahrhage, 1986 and the references cited therein), to the west and north-west of Iceland (Magnusson, 1978, *
Corresponding author. Tel.: 47-55584401; fax: 47-55584450. E-mail address: [email protected] (G. Nñvdal).
1979), further east in the waters between Iceland and the Faeroe Islands (Sahrhage, 1986 and the references cited therein), and west of the British Isles, Ireland and Norway (Bakken et al., 1975; Hognestad and Vader, 1979; Eliassen and Lorentsen, 1982; Eliassen, 1983; Eliassen and Breiby, 1983). Occurrence of the species has also been reported in a number of the deep basins in the Barents Sea (Sahrhage, 1986 and the references cited therein), between Bear Island and Svalbard (Geistdoerfer, 1979), and as far as the Kola Peninsula (Savvatimsky, 1989). Investigations on roughhead grenadiers have been carried out since 1969, together with corresponding studies of other commercial ®sh species occurring in the same areas (P.I. Savvatimsky, personal communication). In the late 1970s the species was extensively studied in the North Atlantic (Magnusson, 1977;
0165-7836/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 7 8 3 6 ( 0 1 ) 0 0 2 4 6 - 6
208
E. Katsarou, G. Nñvdal / Fisheries Research 51 (2001) 207±215
Eliassen and Breiby, 1983; Savvatimsky, 1989). The growing interest in utilizing `new' resources has focused on the roughhead grenadier as a potential species for commercial harvest (Savvatimsky, 1989, 1992). Bottom trawls and particularly longline ®sheries are expected to be a promising method of utilizing the resource in the North Atlantic (Savvatimsky, 1992). The development of population genetic methods offers new tools for investigating the genetic structure of natural populations. The genetic structure of the roughhead grenadier was investigated by applying allozyme electrophoretic analysis of samples from different geographic areas in the North Atlantic (Fig. 1). The goals were to reveal genetic variation and to utilize the most useful traits for studies of possible population structure of the species. The investigation was based on the hypothesis that the roughhead grenadier in the North Atlantic consists of a single panmictic population. 2. Materials and methods During the period August 1991±June 1996, 651 individual samples were collected on board research vessels at a number of sampling locations at different ®shing grounds on the shelf and continental slopes off Greenland and Norway (Fig. 1). Four samples were
collected from Baf®n Bay and Davis Strait off the coast of West Greenland, 10 sample units were from the Irminger Sea and Dohrnbank off the coast of East Greenland and four samples were from Storegga and Lofoten in the Norwegian Sea. The sample size ranged from 2 to 260 specimens. For the statistical analyses, samples from adjacent localities were amalgamated. An overview of the material is given in Table 1 where also the samples are grouped according to the three main areas: West Greenland, East Greenland and the Norwegian Sea. Small pieces (approximately 1 cm3) of white muscle from close to the dorsal ®n were taken from each ®sh. Liver samples were taken from approximately one-third of the individuals (Table 1). All tissue samples were stored in Microwelles at ÿ208C on board the ship until they were transferred to an ultra freezer (ÿ808C) at the laboratory for further processing. From some localities the ®sh were frozen whole and tissue were sampled after thawing. The tissue enzymes were analysed by standard starch-gel electrophoresis (SGE) (Harris and Hopkinson, 1976; Murphy et al., 1990). Two buffer systems were assessed for their ability to accurately resolve polymorphic enzymes: (i) Histidine-citrate pH 7.0 (Harris and Hopkinson, 1976) and (ii) Tris-citrate-borate pH 8.6 (Selander et al., 1971). Each gel (8 mm thick) was sliced horizontally ®ve or six times and each slice was stained for a particular
Fig. 1. Sampling localities of roughhead grenadiers in the North Atlantic for population studies.
E. Katsarou, G. Nñvdal / Fisheries Research 51 (2001) 207±215
209
Table 1 Sampling sites, sampling data and fishing method (L: longline, BT: bottom trawl), depth range in metres (based on estimation from echo depth) and sample size (N) of muscle (M) and liver (L) tissues Location of sampling areasa
East Greenland Dohrnbank01 (dhr.b.01)
Dohrnbank02 (dhr.b.02)
Dohrnbank±Irminger (dhr.irm.) West Greenland Baffin Bay (bf.b.) Davis Strait (dv.st.) Norwegian Sea Storegga (nr.s.01) Lofoten (nr.s.02)
Position (latitude, longitude)
Collection data (date)
Fishing method
Echo depth (m)
658270 N 658270 N 658260 N 658290 N
318200 W 318240 W 318170 W 318260 W
10 10 21 21
September 1991 September 1991 June 1994 June 1994
L L L L
678080 N 678070 N 678090 N 678100 N
308350 W 308250 W 308280 W 308370 W
07 07 08 08
September September September September
1991 1991 1991 1991
M
L
628±389 600±394 594±446 519±559
260 44 23
95 ±b ±b
L L L L
750±535 548±371 540±375 341±512
33 5
±b ±b
648080 N 368020 W 628190 N 408250 W
29 August 1993 10 July 1994
L L
796±467 830±465
96 15
±b ±b
718350 N 608260 W 708280 N 608470 W
24 August 1993 27 August 1993
L L
651±563 439±388
19 43
19 43
698120 N 588400 W 698070 N 588410 W
29 August 1993 29 August 1993
L L
465±322 445±322
26 18
26 18
628200 N 018050 E 628210 N 018290 E
24 June 1995 24 June 1995
BT BT
585 530
43 12
12 ±b
678460 N 098470 E 678190 N 088480 E
28 June 1996 30 June 1996
BT BT
638 596
12 2
±b ±b
Total a b
N
651
213
For the statistical analyses, samples from adjacent localities were amalgamated. No liver tissue available.
enzyme system using procedures based on the protocols of Murphy et al. (1990). The enzyme patterns were immediately interpreted and photographed after staining and incubation. Non-speci®c esterase was screened by isoelectric focusing (IEF) on ampholine polyacrylamide gel, pH range 3.5±9.5, according to the instructions of Ampholine1 PAGplates, Pharmacia Biotechnology AB, Uppsala, Sweden (Anon., 1990). Enzyme nomenclature and allele designation followed Shaklee et al. (1990). The 10 enzymes (15 readable loci estimated) assayed, their abbreviations, the optimal buffer system used and the number of loci and alleles consistently scoreable are given in Table 2. Statistical analyses were carried out using the BIOSYS package program (Swofford and Selander, 1981), employing the Levene's (1949) correction for
small sample size. The exact signi®cance probabilities were calculated to test deviations from expected Hardy±Weinberg proportions. Contingency w2 analyses were performed to test the heterogeneity among samples. For multiple tests the signi®cance levels were adjusted according to the Bonferroni procedure (Weir, 1990). The genetic distance among samples was calculated using the unbiased genetic distance coef®cient of Nei (1978). 3. Results and discussion 3.1. Polymorphic variation Of the 10 enzymes examined, ®ve consistently produced variable isoenzyme patterns which are likely
210
E. Katsarou, G. Nñvdal / Fisheries Research 51 (2001) 207±215
Table 2 Enzymes and enzyme commission numbers (EC), quaternary structure of their functional proteins, number of loci scored, number of alleles, buffer systems used for electrophoresis, activity and technical quality of muscle (M) and liver (L) tissues and estimated numbers of loci Enzyme
Number of alleles
Buffer (pH)b
Activity and technical qualityc M
L
±
II
±
Loci descriptiona
Alcohol dehydrogenase (1.1.1.1)
ADH
Creatine phosphokinase (2.7.3.2)
CPK
1
I
Esterase (3.1.1.-)
EST-1 EST-2
1 2?
III
Glycerol-3-phosphate dehydrogenase (1.1.1.8)
G3PDH-1 G3PDH-2
2 1
I
Glucose-6-phosphate isomerase (5.3.1.9)
GPI-1 GPI-2
4 3
I, II
Isocitrate dehydrogenase (1.1.1.42)
IDHP-1 IDHP-2
1 1
I, II
L-Lactate
LDH-1 LDH-2
3 3
I, II
MDH
3
I
PGM
6
I, II
±
I
dehydrogenase (1.1.1.27)
Malate dehydrogenase (1.1.1.37) Phosphoglucomutase (5.4.2.2f) Sorbitol dehydrogenase (1.1.1.14)
SDH
a
The estimated numbers of loci are based only on muscle analyses. Buffers for horizontal SGE: I Ð discontinuous Histidine-citrate, pH 7.0; II Ð discontinuous Tris-citrate-borate, pH 8.6; III Ð indicates isoelectric focusing (IEF), pH 3.5±9.5. c Activity and technical quality code: : unclear, : faint, : intermediate, moderate, : abundant. b
to be controlled by seven polymorphic loci. An account of the results is given in Table 2 together with an overview of the results of the analyses of each enzyme. The number of presumptive loci which could be accurately discerned amounted to 15. The enzymes ADH and SDH were rejected because they showed no activity. Further CPK and EST were poorly resolved. However, seven polymorphic loci for the following enzymes were found to be consistently interpretable and were scored for all sample units; G3PDH (locus G3PDH-1), GPI (loci GPI-1 and GPI-2), LDH (loci LDH-1 and LDH-2), MDH (locus MDH) and PGM (locus PGM). Three monomorphic loci for the following proteins were found; G3PDH (locus G3PDH2) and IDHP (loci IDHP-1 and IDHP-2). Activity was only observed in skeletal muscle extracts while liver tissues extract failed to give adequate staining for any enzyme. The phenotype patterns for the ®ve variable enzymes are displayed in Fig. 2. For all polymorphic loci calculated, allele frequencies are given in Table 3.
For most samples the observed distributions of phenotypes were in accordance with expected Hardy±Weinberg distributions. Among those distributions which could be tested by a w2 goodness-of-®t test when pooling rare alleles as recommended (Swofford and Selander, 1981; Chakraborty and Leimar, 1987) (Table 4) and applying a Bonferroni correction of the level of signi®cance for multiple tests, none of the samples displayed signi®cant deviations for any loci. Accordingly, the assumed mode of inheritance is accepted for all the described polymorphic systems. However, although not testable, it was also noted that the genotype LDH-1-65/25 occurred once despite the expectance for this genotype being very low. The accordance between observed and expected distributions is further illustrated by the calculation of Selander's D-statistics for heterozygote de®ciency or excess, Table 5, and observed and expected heterozygosities (not shown). Both positive and negative Dcoef®cients occur, indicating no trend among the deviations. It is, however, noted that the most highest
E. Katsarou, G. Nñvdal / Fisheries Research 51 (2001) 207±215
211
Fig. 2. Outline of genotypes of isozyme polymorphic systems for roughhead grenadiers obtained by SGE. Designations of alleles are shown on the sides and of genotypes below each drawing. Arrows indicate anodic migration.
deviation which concerns PGM genotypes in sample dhr.b.02, represents a de®ciency of heterozygotes. Thus a Wahlund's effect cannot be excluded, but there was no other indication of this sample representing a mixture of different gene pools. Mean heterozygosity values within samples ranged from 0.06 to 0.1. This is well within the range for teleost ®shes: 0.00±0.12 (Nevo, 1978) and close to the average value, 0:060 0:038, calculated by Smith and Fujio (1982), based on enzymatic loci. This shows that
the level of genetic variation in isozyme markers in roughhead grenadier is reasonably high offering possibilities for population studies using isozyme markers as tool. However, novel methods of DNA analyses (Carvalho and Pitcher, 1995 and the references cited therein) probably would show more variation which may be used for throwing additional light upon the structure of the species. From the present data it is not possible to draw any conclusion about the possible dynamics of
212
E. Katsarou, G. Nñvdal / Fisheries Research 51 (2001) 207±215
Table 3 Allelic frequencies at seven polymorphic loci for the seven sampling locationsa Locus
Allele (N)
dhr.b.01 (327)
dhr.b.02 (38)
dhr.irm. (111)
bf.b. (62)
dv.st. (44)
nr.s.01 (55)
nr.s.02 (14)
G3PDH-1
100 120
0.992 0.008
1.000 ±
1.000 ±
1.000 ±
1.000 ±
1.000 ±
0.964 0.036
GPI-1
75 100 125 150
0.387 0.569 0.037 0.008
0.250 0.737 0.013 ±
0.347 0.595 0.050 0.009
0.460 0.500 0.040 ±
0.398 0.568 0.023 0.011
0.373 0.509 0.118 ±
0.286 0.607 0.071 0.036
GPI-2
85 100 115
0.009 0.910 0.081
± 0.947 0.053
0.014 0.887 0.099
± 0.984 0.016
± 0.920 0.080
0.009 0.900 0.091
± 1.000 ±
LDH-1
ÿ100 ÿ65 25
0.989 0.009 0.002
0.987 0.013 ±
0.995 0.005 ±
0.968 0.032 ±
0.966 0.034 ±
0.991 0.009 ±
0.964 0.036 ±
LDH-2
65 100 135
0.002 0.930 0.069
± 0.921 0.079
± 0.941 0.059
± 0.944 0.056
± 1.000 ±
± 0.927 0.073
± 0.893 0.107
MDH
100 115 130
0.994 0.003 0.003
1.000 ± ±
1.000 ± ±
1.000 ± ±
1.000 ± ±
1.000 ± ±
1.000 ± ±
PGM
± 0.670 0.008 0.242 0.070 0.011
± 0.776 ± 0.184 0.026 0.013
± 0.694 0.005 0.225 0.072 0.005
± 0.694 ± 0.145 0.153 0.008
0.011 0.705 ± 0.170 0.114 ±
± 0.664 ± 0.255 0.064 0.018
± 0.607 ± 0.321 0.071 ±
94 100 105 110 120 130
a The most common allele at each locus is arbitrarily assigned 100; mobilities of other alleles are relative to the common allele. For definition of sample station abbreviations, see Table 1. Allele designations based on mobilities are given in the left-hand column. Number of specimens are in parentheses.
Table 4 Test of accordance between observed and expected frequencies, with pooling of rare alleles (Swofford and Selander, 1981; Chakraborty and Leimar, 1987) and w2-test for deviation from Hardy±Weinberg equilibriuma Sampling sites Dohrnbank01 (dhr.b.01) Dohrnbank02 (dhr.b.02) Dohrnbank±Irminger (dhr.irm.) Baffin Bay (bf.b.) Davis Strait (dv.st.) Storegga (nr.s.01) Lofoten (nr.s.02) a
w2-test GPI-1
GPI-2
LDH-2
MDH
PGM
0.01 1.53 4.07 0.10 3.59 1.02 1.18
3.16 ± 1.701 ± ± 0.61 ±
1.83 ± ± ± ± ± ±
0.01 ± ± ± ± ± ±
2.69 9.08 2.73 6.59 2.72 0.00 1.18
w2: chi-squares value; and indicate significance level of <0.05 and 0.005, respectively. When applying a Bonferroni adjustment of significance level, none of the deviations were statistically significant. No value indicates too low numbers to be tested. Distributions of G3PDH-1 and LDH-1 phenotypes could not be tested for any sample.
E. Katsarou, G. Nñvdal / Fisheries Research 51 (2001) 207±215
213
Table 5 Coefficients for heterozygote deficiency or excess in the seven sampling locationsa Locus
Sampling area
Obs. Het.b
Exp. het.c
Dd
G3PDH-1 GPI-1 GPI-2 LDH-1 LDH-2 MDH PGM
Dohrnbank01 (dhr.b.01)
5 168 59 6 46 4 174
4.97 172.07 54.25 6.95 42.90 3.99 159.82
0.006 ÿ0.024 0.088 ÿ0.136 0.072 0.003 0.089
GPI-1 GPI-2 LDH-1 LDH-2 PGM
Dohrnbank02 (dhr.b.02)
12 4 1 6 8
15.19 3.84 1.00 5.60 13.96
ÿ0.210 0.042 0.000 0.071 ÿ0.427
GPI-1 GPI-2 LDH-1 LDH-2 PGM
Baffin Bay (bf.b.)
31 2 4 7 21
33.57 1.98 3.90 6.66 29.65
ÿ0.077 0.008 0.025 0.051 ÿ0.292
G3PDH-1 GPI-1 GPI-2 LDH-1 LDH-2 PGM
Storegga (nr.s.01)
30 11 1 8 28
32.63 10.08 1.00 7.49 27.22
ÿ0.081 0.091 0.000 0.069 0.029
GPI-1 GPI-2 LDH-1 LDH-2 PGM
Dohrnbank±Irminger (dhr.irm.)
67 25 1 13 46
58.39 22.58 1.00 12.29 51.61
0.148 0.107 0.000 0.057 ÿ0.109
GPI-1 GPI-2 LDH-1 LDH-2 PGM
Davis Strait (dv.st.)
29 7 3
23.07 6.52 2.93
0.257 0.074 0.024
16
20.54
ÿ0.221
1 6
1.00 7.89
0.000 ÿ0.239
1 3 6
1.00 2.78 7.593
0.000 0.080 ÿ0.210
G3PDH-1 GPI-1 GPI-2 LDH-1 LDH-2 PGM
Lofoten (nr.s.02)
a
Probability levels (0.99 criterion) resulting from tests for homogeneity among loci and localities are shown in the first two rows. Observed number of heterozygotes. c Expected number of heterozygotes. d Selander's D-statistic for the excess or deficiency of heterozygotes. b
the observed variation, i.e. whether the markers are selectively neutral or not. However, as pointed out by Ferguson (1995) selective traits may be valuable as markers for population studies with
respect to management application. However, the evolutionary signi®cance of interpopulation differences may be different for neutral and selective traits.
214
E. Katsarou, G. Nñvdal / Fisheries Research 51 (2001) 207±215
3.2. Variation among samples and areas Contingency w2 analysis of variation among all samples indicated heterogeneity in the total material of roughhead grenadier (Table 6) both for GPI-1 and for total distribution of alleles indicating that the samples have been drawn from more than one panmictic population. Splitting the material according to the three main areas: West Greenland, East Greenland and the Norwegian Sea and performing a similar test for the most variable enzyme systems, GPI-1 and PGM, no signi®cant differences were found within any of the areas. Further tests of each area versus the other ones showed signi®cant differences among all areas based upon one or the other of the two polymorphic systems, Table 7, and for both system combined (not shown in the table). The calculated values for genetic distances among samples were all low (0.000±0.004). This indicates that although two independent markers showed clear heterogeneity among samples collected from the different main areas, the total differentiation is low. A reasonable interpretation of these observations is that roughhead grenadier consists of at least one panmictic population within each of the sampled areas, and for management purposes they should be regarded as independent. However, the low overall genetic differentiation as indicated by the low values of the genetic distance coef®cients, indicates that the evolutionary signi®cance of the observed, between areas, differences is uncertain and possibly low. Further studies Table 6 Contingency w2 table for the inter-area results with respect to the seven loci for the nine sampling sitesa Locus
Number of alleles
G3PDH-1 GPI-1 GPI-2 LDH-1 LDH-2 MDH PGM
2 4 3 3 3 3 6
Total
w2
d.f.
P
10.092 36.195 15.536 10.828 8.889 3.975 42.998
6 18 12 12 12 12 30
0.121 0.006 0.213 0.544 0.712 0.984 0.059
128.513
102
0.039
a 2 w : chi-squares value test, d.f.: degrees of freedom and P: significant probability. When the level of significance is adjusted by a sequential Bonferroni correction (Weir, 1990) only GPI-1 and the total show significantly different distributions.
Table 7 Contingency w2 analysis for inter-area with respect to two most variable polymorphic loci, GPI-1 and PGMa Number w2 of alleles
d.f. P
GPI-1 East±West Greenland East Greenland±Norwegian Sea West Greenland±Norwegian Sea All areas
4 4 4 4
3.439 13.715 9.001 18.394
3 3 3 6
0.329 0.003 0.029 0.005
PGM East±West Greenland East Greenland±Norwegian Sea West Greenland±Norwegian Sea All areas
6 5 5 6
21.591 1.976 11.072 25.520
5 4 4 10
0.001 0.740 0.026 0.004
Area
a 2 w : chi-squares value, d.f.: degrees of freedom and P: significance probability.
using a denser sampling grid and applying additional markers would probably allow ®rmer conclusions about the structure of roughhead grenadiers in the North Atlantic. 4. Conclusion The contingency w2-test among the sampling sites revealed signi®cant heterogeneity at the polymorphic locus GPI-1 and for all polymorphic loci combined. No variation among samples were found within any of the three main areas: West Greenland, East Greenland and the Norwegian Sea. Pairwise comparisons using contingency w2 tests for the most variable polymorphic loci (GPI-1 and PGM) showed that the frequency distributions differed signi®cantly between the main areas. Thus, the present study provides strong evidence that the roughhead grenadier population in the North Atlantic does not form a single panmictic stock but is composed of at least three different stock units with their own gene pools. Acknowledgements The authors are greatly indebted to skippers and crew of different research and ®shing boats who have collected the material. Especially our thanks are directed to Nils-Roar Hareide who was in charge for most of the sampling in Greenland waters.
E. Katsarou, G. Nñvdal / Fisheries Research 51 (2001) 207±215
References Anon., 1990. Instruction for Ampholine1 PAGplate, precast polyacrylamide gels for analytical isoelectric focusing. Pharmacia Biotechnology AB, Uppsala, Sweden, 34 pp. Bakken, E., Lahn-Johannesen, J., Gjùsñter, J., 1975. Demersal fish on the continental slope off Norway. ICES CM/F: 29, 1±15. Carvalho, G.R., Pitcher, T.J. (Eds.), 1995. Molecular Genetics in Fisheries. Chapman & Hall, Padstow, Cornwall, UK, 141 pp. Chakraborty, R., Leimar, O., 1987. Genetic variation within a subdivided population. Genetic distance and molecular philogeny. In: Ryman, N., Utter, F. (Eds.), Population Genetics and Fishery Management. University of Washington, Washington, DC, pp. 89±120. Eliassen, J.-E., 1983. Distribution and abundance of roundnose grenadier (Coryphaenoides rupestris Gunnerus) (Gadiformes, Macrouridae) in northern and mid-Norway. ICES CM/G: 43, 1± 24 (mimeo). Eliassen, J.-E., Breiby, A., 1983. Forsùksfiske etter isgalt og blaÊkveite langs Trñnaegga, Nordland fylke i 1983. Rep. Ser. B: Ressurbiologi 1983, Vol. 6. Institutt for Fiskerifag, University of Tromsù, Norway, pp. 1±13 (in Norwegian). Eliassen, J.-E., Lorentsen, E., 1982. Toktrapport fra forsùksfiske etter isgalt og blaÊkveite utenfor Nordland og Tromsù i juni og juli 1982. Rep. Ser. B: Ressurbiologi 1982, Vol. 6. Institutt for Fiskerifag, University of Tromsù, Norway, pp. 1±9 (in Norwegian). Ferguson, A., 1995. Molecular genetics in fisheries: current and future perspectives. In: Carvalho, G.R., Pitcher, T.J. (Eds.), Molecular Genetics in Fisheries. Chapman & Hall, Padstow, Cornwall, UK, pp. 81±103. Forest, A., Minet, J.P., Perodou, J.B., 1978. Results of groundfish survey on Baffin Island shelf. ICNAF Research Document No. 78/VI/45, 10 pp. Geistdoerfer, P., 1979. New data on the reproduction of macrourids (Teleostei, Gadiformes). Sarsia 64, 109±112. Harris, H., Hopkinson, D.A., 1976. Handbook of Enzyme Electrophoresis in Human Genetics. North-Holland, Oxford. Hognestad, P.T., Vader, W., 1979. Saltvannsfiskene i Nord Norge, Vol. 6. Universitet i Tromsù, Tromura, 74 pp. (in Norwegian). Levene, H., 1949. On a matching problem arising in genetics. Ann. Math. Statist. 20, 91±94. Magnusson, J.V., 1977. Some notes on the spawning habits of Macrouridae at Iceland. ICES CM 1977/F: 49, 9 pp. Magnusson, J.V., 1978. On the distribution and spawning grounds of the roughhead grenadier (Macrourus berglax LacepeÂde) west of Iceland. ICES CM 1977/G: 36, 10 pp. Magnusson, J.V., 1979. Icelandic investigations on grenadier fish (Coryphaenoides rupestris and Macrourus berglax) in 1977. Ann. Biol., Copenhague 34, 223±225. Mombeck, F., 1984. Untersuchung der GrundfischbestaÈende vor Labrador (investigation of benthic fish stocks near Labrador). Inf. Fischw. Auslds. 31, 3±9 (in German).
215
Moyle, P.B., Cech Jr., J.J. (Eds.), 1996. Fishes: An Introduction to Ichthyology, 3rd Edition. Prentice-Hall, Englewood Cliffs, NJ, p. 590. Murphy, R.W., Sites Jr., J.W., Buth, D.G., Haufler, C.H., 1990. Proteins. I. Isozyme electrophoresis. In: Hills, D.M., Moritz, C. (Eds.), Molecular Systematics. Sinauer Associates, Inc., Sunderland, MA, pp. 45±126. Nei, M., 1978. Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89, 583± 590. Nevo, E., 1978. Genetic variation in natural populations: patterns and theory. Theoret. Pop. Biol. 13, 121±177. Parsons, L.S., 1976. Distribution and relative abundance of roundnose, roughhead and common grenadiers in the northwest Atlantic. ICNAF Select. Pap. 1, 73±88. Sahrhage, D., 1982. Erforschung der GrundfischbestaÈende vor Labrador (research of benthic fish stocks near Labrador). Inf. Fischw. Auslds. 29 (1), 4±13. Sahrhage, D., 1986. Mitteilungen aus dem Instistut fuÈr Seefischerei der Bundesforschungsanstalt fuÈr Fischerei. Commercially important grenadiers of the North Atlantic. Can. Transl. Fish. Aquat. Sci., Hamburg 37, 81. Savvatimsky, P.I., 1984. Biological aspects of roughhead grenadier (Macrourus berglax) from longline catches in the eastern Grand Bank area, 1982. NAFO Sci. Counc. Stud. 7, 45±51. Savvatimsky, P.I., 1989. Investigations of roughhead grenadier (Macrourus berglax L.) in the Northwest Atlantic, 1967±1983. NAFO Sci. Counc. Stud., 13, 59±75. Savvatimsky, P.I., 1992. Severnyj makrurus (Macrourus berglax L.) potentsial'nyj ob'ekt tralovogo i yarusnogo promysla v Severnoj Atlantike (the roughhead grenadier Macrourus berglax, a potential resource for trawl and longline fisheries). In: Savvatimskij, P.I. (Ed.), Studies of Biological Resources in the North Atlantic. #Issledovaniya Bioresursovsevernoj Atlantiki, Pinko, pp. 45±67 (in Russian). Selander, R.K., Smith, M.H., Yang, S.Y., Johnson, W.E., Gentry, J.R., 1971. Biochemical polymorphism and systematics in the genus Peromyscus. I. Variation in the old-field mouse (Peromyscus polionotus). Studies in Genetics, Vol. VI. The University of Texas Publication No. 103, pp. 49±90. Shaklee, J.B., Allendorf, F.W., Morizot, D.C., Writt, G.S., 1990. Gene nomenclature for protein-coding loci in fish. Trans. Am. Fish. Soc. 119, 2±15. Smith, P.J., Fujio, Y., 1982. Genetic variation in marine teleosts: high variability in habitat specialists and low variability in habitat generalists. Mar. Biol. 69, 7±20. Swofford, D.L., Selander, R.B., 1981. BIOSYS-1 (Release 1.7). A Computer Program for the Analysis of Allelic Variation in Genetics. Users Manual. Department of Genetics and Development, University of Illinois, IL, 63 pp: Appendices. Weir, B.S., 1990. Genetic Data Analysis. Methods for Discrete Population Genetic Data. Sinauer Association, Inc., Sunderland, MA, 377 pp.