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Abstract
To investigate the genetic population structure of the Dover sole, Solea solea L., allozyme electrophoresis was
performed on 303 fish collected from seven locations ranging from Cumbria, Great Britain, to Greece. A total of
22 enzyme systems were analysed, coded by 33 loci. Of these, 27 loci were polymorphic using the P99 criterion. A
phenogram using Prevosti’s Distance generated by the Wagner method exhibited a geographic pattern in the clustering of
populations. Estimates of Nm (effective number of migrants per generation between populations) were sufficiently high
to imply near-panmixia between the North Sea, Bay of Biscay and the Irish Coast populations, indicating a probable
movement of migrants through the English Channel. However, despite this high level of gene flow, striking patterns of
geographic differentiation were observed at a few loci. Allele frequencies at loci ACOH, EST-I-1, PEP-I-2 exhibited
genetic patchiness on both local and range-wide (within the northern and southern European basins) scales. This pattern of
genetic patchiness could be the result of localised selection, genetic drift or single-generation sampling effects. Estimates
of mean heterozygosity .H/ were inversely related to latitude. Evolutionary processes such as genetic drift and founder
effect, and=or selection, may have produced the observed difference in the number of alleles between the basins. Moreover,
the absence of isolation by distance provides support for a model of geographic isolation. Such a pattern of genetic
patchiness, revealing a slight reduction of genetic variability in the northern European basin, may suggest a population
bottleneck, or local reduction in population size. Various physical parameters, especially water temperature during the
reproductive period, vary within the range of the species, and may produce or maintain this genetic differentiation. These
results indicate the role of both ecological and evolutionary structuring mechanisms in determining the genetic population
structure of S. solea. 1998 Elsevier Science B.V. All rights reserved.
The characterisation of genetic population struc- 1993), a study of genetic population structure can
ture is vital for the management of ecologically and now be considered in relation to these factors.
economically important fish, such as S. solea. In
the marine environment gene flow typically occurs
through the dispersal of larvae or migration of adults. 2. Material and methods
However, an assumption of high gene flow in species
with a high potential for dispersal is not always cor- 2.1. Sampling protocol
rect (e.g. Knowlton and Keller, 1986). Therefore, the
extent of gene flow will generally determine the ge- Seven populations of adult S. solea were sam-
netic heterogeneity of the species, in the absence of pled during 1994 and 1995. Three populations were
localised selection (Altukhov, 1981). These genetic from the Irish Sea, two from the North Sea, one
distinctions are important for management plans for from the Bay of Biscay and one from the Mediter-
harvested species and are used to predict whether ranean (Fig. 1). Sample size varied from 18 to 73
a locally depleted population will be successfully individuals. All fish were collected by trawl and pro-
repopulated by immigrants (Shaklee, 1983; Utter, cessed fresh. The sample collection dates did not
1991). include patterns which would systematically bias the
For many marine species, a geographically broad results, such as summer feeding migrations, spawn-
genetic survey of populations provides an efficient ing season or winter offshore migration of adults and
and reliable method of determining geographic pat- juveniles. Sex, standard lengths and body weights
terns of population structure (Avise et al., 1990). were recorded (Table 1), prior to the extraction of
Many authors report increased genetic differentia- tissues. Skeletal muscle from both sides of the body,
tion with greater geographical distance between pop- liver and eyes were used for electrophoresis. To stan-
ulations (e.g. Larcson et al., 1989; Slatkin, 1993); dardise the sampling procedure (Weir, 1990) tissue
therefore it is appropriate to consider geographical was taken from close to the tail of the fish for ev-
distance itself when evaluating existing barriers to ery specimen. All tissues were immediately frozen
gene flow. On the other hand, there are numerous at 30ºC to 190ºC depending on the facilities
examples of population homogeneity among marine available during the extraction and the transportation
fish species (e.g. Grant et al., 1984, 1987). Vari- (liquid nitrogen containers, Polystyrene boxes with
ous studies investigating genetic structure over the dry ice or freezer). Once returned to the laboratory
geographic range in plaice (Pleuronectes platessa), the tissue samples were stored at 75ºC:
flounder (Pleuronectes flesus), brill (Scophthalmus Allozyme electrophoresis was carried out using
rhombus) and turbot (S. maximus) lead to differing standard horizontal starch gel techniques (Richard-
conclusions. A high degree of population differenti- son et al., 1986; Murphy et al., 1990). The ho-
ation has been found in flounder (Galleguillos and mogenisation buffer used was a mixture of Tris–HCl
Ward, 1982; Berrebi et al., 1985) and plaice (Ward 1.2 g l 1 , EDTA 0.37 g l 1 , NADP 0.04 g l 1 at pH
and Beardmore, 1977; Simonsen et al., 1988), but 6.8 (Pasteur et al., 1985).
no apparent differentiation in brill and limited dif-
ferentiation in turbot (Blanquer et al., 1992). These 2.2. Statistical analysis
studies suggest that factors other than the dispersal
potential during pelagic stages of the species may be A total of 22 enzyme systems were assayed (Ta-
important, or have been so in the past. ble 2). Genotypic data were analysed using the com-
The present work considers all the above param- puter program BIOSYS-1, Release 1.7 (Swofford
eters and reports a spatial-scale study of the genetic and Selander, 1989). The mean number of alleles per
structure of the Dover sole. Since extensive infor- locus, the mean observed heterozygosity per locus,
mation has already been gathered about the biology, the mean expected heterozygosity per locus under
ecology and behaviour of the species (e.g. Kout- random mating .Hunbias / (Nei, 1978), plus the pro-
sikopoulos et al., 1991; Molinero and Flos, 1991; portion of polymorphic loci (Harris and Hopkinson,
Dorel et al., 1991; Rogers, 1992; Amara et al., 1976), were calculated to assess intra-population
A. Exadactylos et al. / Journal of Sea Research 40 (1998) 117–129 119
Fig. 1. Map showing the sample locations of Solea solea adults. See Table 1 for population codes. The total number of individuals
sampled are given in parentheses.
Table 1
Sampling information protocol for population genetics of Solea solea (standard errors in brackets)
variation. Hunbias was chosen as a better measure of They were used rather than the more commonly
genetic variation in a sample than a direct count of used goodness-of-fit chi-square tests since expected
heterozygosity. Hunbias is considered to be indepen- genotype frequencies were often quite small even
dent of sample size, natural selection and inbreeding after pooling of rare alleles. If more than two alleles
(Nei and Roychoudhury, 1974). were present at a locus, all alleles, other than the
Departures of genotype frequencies from Hardy– most common one were pooled. Deficiencies of het-
Weinberg expectations were tested using exact tests erozygotes in each population were estimated for all
(Lessios, 1992; Sokal and Rohlf, 1995) analogous polymorphic loci using the inbreeding index Fis , and
to Fisher’s exact test for 2 ð 2 contingency tables. its significance was tested using the equation of Li
120 A. Exadactylos et al. / Journal of Sea Research 40 (1998) 117–129
Table 2
Enzyme systems, buffers and number of loci studied
Abbreviation of enzyme (substrate) E.C Number Tissue No. of loci Buffer system
ACP 3.1.3.2 Liver 1 Tris–Citrate–EDTA, pH 7.0
ACOH 4.2.1.3 Liver 1 Tris–Citrate–EDTA, pH 7.0
AAT 2.6.1.1 Muscle=liver 3 Tris–Citrate, pH 8.0
DDH 1.8.1.4 Liver 1 Tris–Citrate, pH 8.0
EST-I 3.1.–.– Liver 2 Discontinuous Tris–HCl–Borate, pH 8.2–8.5
(α and β naphthyl acetate)
EST-II 3.1.–.– Liver 2 Tris–Citrate–EDTA, pH 7.0
(4-methylumbelliferyl acetate)
FBA 4.1.2.13 Muscle 1 Tris–Citrate, pH 8.0
GCDH 1.1.1.118 Liver 1 Tris–Citrate–EDTA, pH 7.0
G6PDH 1.1.1.49 Muscle=liver 2 Tris–Versene–Borate, pH 8.0
GPI 5.3.1.9 Muscle=liver 2 Discontinuous Tris–Borate–Citrate, pH 8.2–8.7
GR 1.6.4.2 Liver 1 Tris–Citrate–EDTA, pH 7.0
GAPDH 1.2.1.12 Muscle 1 Tris–Versene–Borate, pH 8.0
G3PDH 1.1.1.8 Muscle 1 Tris–Citrate, pH 8.0
IDH 1.1.1.42 Liver 1 Tris–Versene–Borate, pH 8.0
LDH 1.1.1.27 Muscle=eyes 3 Discontinuous Tris–Borate–Citrate, pH 8.2–8.7
MDH 1.1.1.37 Muscle 2 Tris–Citrate, pH 8.0
MDHP 1.1.1.40 Muscle 2 Tris–Versene–Borate, pH 8.0
PEP-I 3.4.–.– Liver 2 Discontinuous Tris–HCl–Borate, pH 8.2–8.5
(u)
PEP-II 3.4.–.– Liver 1 Discontinuous Tris–HCl–Borate, pH 8.2–8.5
(Gly-Leu)
PGM 5.4.2.2 Muscle 1 Discontinuous Tris–Borate–Citrate, pH 8.2–8.7
PGDH 1.1.1.44 Muscle 1 Tris–Versene–Borate, pH 8.0
SOD 1.15.1.1 Liver 1 Tris–Citrate, pH 8.0
Table 3
Genetic variability at 33 loci in seven populations of adult Solea solea (standard errors in brackets)
Population a Mean sample size Mean no. of alleles Percentage of loci Mean heterozygosity
per locus (S.E.) per locus (S.E.) polymorphic b
Observed (S.E.) Expected c under Hardy–Weinberg
equilibrium (S.E.)
CUM 50 (0.0) 1.8 (0.1) 63.6 0.035 (0.008) 0.043 (0.011)
IOM 72 (0.0) 2.2 (0.2) 72.7 0.071 (0.013) 0.083 (0.015)
GER 17.9 (0.1) 1.8 (0.1) 60.6 0.083 (0.017) 0.107 (0.021)
IRL 18 (0.0) 1.7 (0.1) 48.5 0.034 (0.008) 0.064 (0.015)
EAN 43 (0.0) 2.2 (0.1) 75.8 0.085 (0.012) 0.101 (0.014)
FRA 48 (0.0) 2.3 (0.2) 72.7 0.088 (0.013) 0.108 (0.017)
GRE 54 (0.0) 2.4 (0.2) 78.8 0.114 (0.016) 0.141 (0.019)
Total 0.073 (0.012)
a See Table 1.
b A locus is considered polymorphic if the frequency of the most common allele does not exceed 0.99.
c Unbiased estimate (Nei, 1978).
122 A. Exadactylos et al. / Journal of Sea Research 40 (1998) 117–129
Table 4
Summary of estimates of Wright’s F-statistics and gene flow .Nm / at all 27 polymorphic loci, over the seven populations of Solea solea
Values of Fst significantly greater than zero corrected by the sequential Bonferroni procedure at Þ 0 D 0:005 are bold.
Table 5
Estimates of Prevosti’s Distance (below the diagonal) and estimates of Weir and Cockerham’s Nm (above the diagonal) among the seven
populations of Solea solea
the most common allele at the locus GPI-1. Asso- differentiated during allopatry (Endler, 1977), but in
ciations of allele frequencies with a latitudinal cline this case may well result from sampling error, since
correlated, for example, with temperature may result only 1 of 33 loci gave a significant cline. Never-
from natural selection acting on allozyme loci, or theless, the latter became non-significant after the
secondary intergradation of populations previously sequential Bonferroni correction for multiple tests.
124 A. Exadactylos et al. / Journal of Sea Research 40 (1998) 117–129
Fig. 3. Wagner procedure dendogram using Prevosti’s Distance (Wright, 1978) and goodness of fit statistics between the seven
populations of Solea solea. Goodness of fit statistics: Farris (1972) f D 0:045; Prager and Wilson (1976) F D 4:834; % standard
deviation (Fitch and Margoliash, 1967)D 5:61; cophenetic correlationD 0:96:
in agreement with tagging experiments carried out differentiation between the northern and southern
by Greer Walker and Emerson (1990). European basins. Various physical parameters, es-
Kotoulas et al. (1995) studied the genetic structure pecially water temperature during the reproductive
of S. solea in mixed juvenile and adult populations period, vary within the range of the species and may
over several spatial scales, and concluded that the produce or maintain genetic differentiation. A pat-
strongest result was an east to west pattern of popu- tern emerged from the comparison between basins
lation differentiation, while a north to south pattern and their estimates of genetic variability, revealing a
was also significant. Biological data indicate near- slight reduction of the latter in the northern European
panmixia, for example, transport of eggs and larvae basin, possibly resulting from, for example, a pop-
of S. solea from the offshore spawning areas to ulation bottleneck, or local reduction in population
the inshore estuarine nurseries by diffusive mecha- size. The absence of isolation by distance provided
nisms (Koutsikopoulos et al., 1991), offshore move- backing for a model of geographic isolation. The
ments towards spawning grounds (Greer Walker and analysis of more samples would help to complete the
Emerson, 1990), mixing in spawning grounds of picture and possibly give a better understanding of
adults originating from adjacent nursery grounds the local and wide-range structuring of S. solea.
(Koutsikopoulos and Lacroix, 1992; Rijnsdorp et al.,
1992) or random dispersion mechanisms resulting in
nursery grounds containing juveniles from different Acknowledgements
spawning grounds (Marchand, 1991; Koutsikopoulos
and Lacroix, 1992). We thank A.D. Rijnsdorp and his colleagues at
RIVO-DLO, F. Lagardere, J.P. Lagardere and crew
at La Rochelle, France, D.J. Symonds, B. Harley and
4.3. Differentiation within basins B. Turner from the CEFAS laboratory at Lowestoft,
The geographic clustering of the populations in England, P. Newton and his colleagues at DANI,
the Wagner procedure dendogram agree with the Belfast, and the crew of our research vessel Roagan
conclusions of many Mediterranean biogeographers. for providing us with the samples. We also thank
McCullach and De Deckker (1989) have hypothe- R.D.M. Nash, A. Hill, S.M. Lynch, G. Kotoulas,
sised that the history of the Mediterranean, com- P. Panagiotaki, E.R. Daka for advice and support.
bined with the present hydrographic patterns, might A. Exadactylos acknowledges the financial support
have promoted and maintained the differentiation of of the Greek Scholarship Foundation throughout the
Mediterranean populations. Since the Pleistocene the course of the research programme.
history of the Mediterranean can be seen as a suc-
cession of glacial and interglacial periods with asso- Appendix A. Allele frequencies for Solea solea at
ciated regressions and transgressions (Blanc, 1968). seven locations
It is possible that during one of the regressions the
Mediterranean and Atlantic populations of S. solea Locus Population
separated. It would be interesting to be able to date CUM IOM GER IRL EAN FRA GRE
the time of separation and relate it to geological ACP:
events. Unfortunately, because of the small number .N/ 50 72 18 18 43 48 54
of populations sampled across the total distribution 100 1.000 1.000 1.000 1.000 1.000 1.000 1.000
of the species, especially in the western Mediter- ACOH:
ranean, we were unable to calibrate the molecular .N/ 50 72 18 18 43 48 54
clock and thus relate the genetic distance to evolu- 100 1.000 1.000 0.917 1.000 0.988 1.000 0.926
120 0.000 0.000 0.083 0.000 0.012 0.000 0.074
tionary time (Thorpe, 1982).
Besides the possible contribution of the history AAT-1:
.N/ 50 72 18 18 43 48 54
and hydrographic barriers, evolutionary processes 100 1.000 0.965 1.000 1.000 0.953 0.948 0.917
such as genetic drift and founder effect, and=or se- 110 0.000 0.000 0.000 0.000 0.012 0.000 0.074
lection, may have produced the observed genetic 120 0.000 0.035 0.000 0.000 0.035 0.052 0.009
126 A. Exadactylos et al. / Journal of Sea Research 40 (1998) 117–129
AAT-4: GPI-1:
.N/ 50 72 18 18 43 48 54 .N/ 50 72 18 18 43 48 54
80 0.030 0.014 0.056 0.028 0.023 0.031 0.130 80 0.010 0.007 0.000 0.028 0.035 0.010 0.046
100 0.970 0.958 0.861 0.917 0.953 0.938 0.796 100 0.970 0.944 0.917 0.944 0.965 0.917 0.843
120 0.000 0.028 0.083 0.056 0.023 0.031 0.074 115 0.020 0.014 0.000 0.000 0.000 0.031 0.065
120 0.000 0.035 0.083 0.028 0.000 0.042 0.046
DDH-1:
GPI-3:
.N/ 50 72 18 18 43 48 54
.N/ 50 72 18 18 43 48 54
90 0.000 0.042 0.000 0.028 0.070 0.063 0.065
90 0.010 0.083 0.028 0.056 0.000 0.052 0.056
100 0.960 0.917 0.833 0.944 0.872 0.865 0.889
100 0.930 0.826 0.778 0.806 0.930 0.885 0.880
110 0.040 0.042 0.167 0.028 0.058 0.073 0.046
110 0.040 0.049 0.000 0.056 0.023 0.042 0.028
EST-I-1: 120 0.020 0.042 0.194 0.083 0.047 0.021 0.037
.N/ 50 72 18 18 43 48 54 GR-1:
98 0.000 0.000 0.139 0.000 0.093 0.115 0.037 .N/ 50 72 16 18 43 48 54
100 1.000 1.000 0.861 1.000 0.907 0.885 0.963 90 0.010 0.021 0.000 0.000 0.000 0.031 0.074
EST-I-3: 100 0.980 0.965 0.969 1.000 0.907 0.896 0.898
.N/ 50 72 18 18 43 48 54 110 0.010 0.014 0.031 0.000 0.093 0.073 0.028
90 0.000 0.000 0.000 0.000 0.000 0.021 0.056 GAPDH-1:
100 0.980 0.965 1.000 0.917 0.977 0.958 0.880 .N/ 50 72 18 18 43 48 54
110 0.020 0.035 0.000 0.083 0.023 0.021 0.065 100 1.000 1.000 1.000 1.000 1.000 1.000 1.000
EST-II-1: G3PDH-1:
.N/ 50 72 18 18 43 48 54 .N/ 50 72 18 18 43 48 54
95 0.040 0.042 0.083 0.000 0.081 0.042 0.074 85 0.000 0.007 0.028 0.028 0.070 0.031 0.037
100 0.960 0.958 0.917 1.000 0.919 0.958 0.926 100 0.960 0.889 0.861 0.889 0.884 0.906 0.889
115 0.040 0.104 0.111 0.083 0.047 0.063 0.074
EST-II-3:
.N/ 50 72 18 18 43 48 54 IDH-2:
95 0.010 0.028 0.000 0.000 0.012 0.031 0.028 .N/ 50 72 18 18 43 48 54
100 0.990 0.972 0.972 1.000 0.977 0.969 0.963 80 0.010 0.035 0.083 0.028 0.023 0.052 0.065
105 0.000 0.000 0.028 0.000 0.012 0.000 0.009 100 0.990 0.965 0.917 0.972 0.977 0.948 0.907
120 0.000 0.000 0.000 0.000 0.000 0.000 0.028
FBA-1:
LDH-1:
.N/ 50 72 18 18 43 48 54
.N/ 50 72 18 18 43 48 54
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000
100 0.990 0.938 1.000 0.972 0.930 1.000 0.963
GCDH-2: 110 0.010 0.063 0.000 0.028 0.070 0.000 0.037
.N/ 50 72 18 18 43 48 54 LDH-2:
80 0.000 0.028 0.028 0.000 0.012 0.042 0.000 .N/ 50 72 18 18 43 48 54
100 0.990 0.951 0.972 1.000 0.930 0.948 0.972 70 0.000 0.000 0.000 0.000 0.000 0.021 0.028
120 0.010 0.021 0.000 0.000 0.058 0.010 0.028 90 0.000 0.007 0.000 0.000 0.035 0.000 0.019
G6PDH-2: 100 1.000 0.979 0.917 0.972 0.895 0.948 0.787
.N/ 50 72 18 18 43 48 54 110 0.000 0.014 0.083 0.028 0.070 0.031 0.167
90 0.000 0.035 0.000 0.000 0.058 0.000 0.000 LDH-4:
100 1.000 0.965 1.000 1.000 0.919 0.948 0.917 .N/ 50 72 18 18 43 48 54
110 0.000 0.000 0.000 0.000 0.023 0.052 0.083 100 1.000 1.000 1.000 1.000 1.000 1.000 1.000
A. Exadactylos et al. / Journal of Sea Research 40 (1998) 117–129 127
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