ELSEVIER Journal of Sea Research 40 (1998) 117–129

Population structure of the Dover sole, Solea solea L., in a background
of high gene flow
Athanasios Exadactylos, Audrey J. Geffen
Ł
, John P. Thorpe
Port Erin Marine Laboratory, School of Biological Sciences, The University of Liverpool, Port Erin, Isle of Man IM9 6JA, UK
Received 7 November 1996; accepted 3 June 1997
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 P
99
criterion. A
phenogram using Prevosti’s Distance generated by the Wagner method exhibited a geographic pattern in the clustering of
populations. Estimates of N
m
(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.
Keywords: allozyme electrophoresis; gene flow; genetic variability; isolation by distance
1. Introduction
The Dover sole, Solea solea L., is the most com-
mon member of the Soleidae in European waters. It is
common both inshore and offshore, extremely adapt-
able either in estuarine waters or at sea, although not
Ł
Corresponding author. E-mail: geffen@liverpool.ac.uk
found in deep waters (Quero et al., 1986). S. solea
commands a consistently high market price (Bond,
1979) sustained by relative scarcity, fine texture and
flavour (Wheeler, 1969) and good keeping qualities
(Baynes et al., 1993). In recent years, total landings in
the northeast Atlantic amounted to an annual average
of 35,000 t (Rijnsdorp et al., 1992). In the Mediter-
ranean the landings are about 5 t annually.
1385-1101/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved.
PII S1 3 8 5 - 1 1 0 1 ( 9 8 ) 0 0 0 1 5 - X
118 A. Exadactylos et al. / Journal of Sea Research 40 (1998) 117–129
The characterisation of genetic population struc-
ture is vital for the management of ecologically and
economically important fish, such as S. solea. In
the marine environment gene flow typically occurs
through the dispersal of larvae or migration of adults.
However, an assumption of high gene flow in species
with a high potential for dispersal is not always cor-
rect (e.g. Knowlton and Keller, 1986). Therefore, the
extent of gene flow will generally determine the ge-
netic heterogeneity of the species, in the absence of
localised selection (Altukhov, 1981). These genetic
distinctions are important for management plans for
harvested species and are used to predict whether
a locally depleted population will be successfully
repopulated by immigrants (Shaklee, 1983; Utter,
1991).
For many marine species, a geographically broad
genetic survey of populations provides an efficient
and reliable method of determining geographic pat-
terns of population structure (Avise et al., 1990).
Many authors report increased genetic differentia-
tion with greater geographical distance between pop-
ulations (e.g. Larcson et al., 1989; Slatkin, 1993);
therefore it is appropriate to consider geographical
distance itself when evaluating existing barriers to
gene flow. On the other hand, there are numerous
examples of population homogeneity among marine
fish species (e.g. Grant et al., 1984, 1987). Vari-
ous studies investigating genetic structure over the
geographic range in plaice (Pleuronectes platessa),
flounder (Pleuronectes flesus), brill (Scophthalmus
rhombus) and turbot (S. maximus) lead to differing
conclusions. A high degree of population differenti-
ation has been found in flounder (Galleguillos and
Ward, 1982; Berrebi et al., 1985) and plaice (Ward
and Beardmore, 1977; Simonsen et al., 1988), but
no apparent differentiation in brill and limited dif-
ferentiation in turbot (Blanquer et al., 1992). These
studies suggest that factors other than the dispersal
potential during pelagic stages of the species may be
important, or have been so in the past.
The present work considers all the above param-
eters and reports a spatial-scale study of the genetic
structure of the Dover sole. Since extensive infor-
mation has already been gathered about the biology,
ecology and behaviour of the species (e.g. Kout-
sikopoulos et al., 1991; Molinero and Flos, 1991;
Dorel et al., 1991; Rogers, 1992; Amara et al.,
1993), a study of genetic population structure can
now be considered in relation to these factors.
2. Material and methods
2.1. Sampling protocol
Seven populations of adult S. solea were sam-
pled during 1994 and 1995. Three populations were
from the Irish Sea, two from the North Sea, one
from the Bay of Biscay and one from the Mediter-
ranean (Fig. 1). Sample size varied from 18 to 73
individuals. All fish were collected by trawl and pro-
cessed fresh. The sample collection dates did not
include patterns which would systematically bias the
results, such as summer feeding migrations, spawn-
ing season or winter offshore migration of adults and
juveniles. Sex, standard lengths and body weights
were recorded (Table 1), prior to the extraction of
tissues. Skeletal muscle from both sides of the body,
liver and eyes were used for electrophoresis. To stan-
dardise the sampling procedure (Weir, 1990) tissue
was taken from close to the tail of the fish for ev-
ery specimen. All tissues were immediately frozen
at 30ºC to 190ºC depending on the facilities
available during the extraction and the transportation
(liquid nitrogen containers, Polystyrene boxes with
dry ice or freezer). Once returned to the laboratory
the tissue samples were stored at 75ºC.
Allozyme electrophoresis was carried out using
standard horizontal starch gel techniques (Richard-
son et al., 1986; Murphy et al., 1990). The ho-
mogenisation buffer used was a mixture of Tris–HCl
1.2 g l
1
, EDTA 0.37 g l
1
, NADP 0.04 g l
1
at pH
6.8 (Pasteur et al., 1985).
2.2. Statistical analysis
A total of 22 enzyme systems were assayed (Ta-
ble 2). Genotypic data were analysed using the com-
puter program BIOSYS-1, Release 1.7 (Swofford
and Selander, 1989). The mean number of alleles per
locus, the mean observed heterozygosity per locus,
the mean expected heterozygosity per locus under
random mating (H
unbias
) (Nei, 1978), plus the pro-
portion of polymorphic loci (Harris and Hopkinson,
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)
Sample location Code Source Date N Mean standard
length in cm (S.E.)
54º28N–3º58W,
King William Banks,Irish Sea
CUM Commercial trawler hired by
Port Erin Marine Lab.
11,5,95 50 34.52 (3.65)
53º58N–4º58W,
Chicken Rock,Irish Sea
IOM Sampling cruise by Port Erin,
Research Vessel Roagan
07,06,94 and
14,02,96
73 29.82 (5.12)
53º55N–6º40E,
German Bight,North Sea
GER Sampling cruise supervised by
RIVO-DLO, Netherlands
09,29,95 18 25.30 (5.90)
53º18N–5º47W,
Kish Bank,Irish Sea
IRL Sampling cruise supervised by
DANI, Belfast
12,09,94 and
22,09,95
18 23.00 (4.92)
52º26N–1º15E,
Newsombe Bank,North Sea
EAN Commercial trawler hired by
MAFF, Lowestoft
09,7,94 43 19.70 (1.60)
45º37N–1º46W,
L’Ile d’Oleron,Bay of Biscay
FRA Commercial trawler hired by
CNRS-IFREMER, L’Houmeau
22,12,94 48 26.99 (2.40)
40º13N–22º86E,
Thermaikos Bay,Aegean Sea
GRE Commercial trawler 15,07,94 and
12,05,94
54 22.40 (2.69)
variation. H
unbias
was chosen as a better measure of
genetic variation in a sample than a direct count of
heterozygosity. H
unbias
is considered to be indepen-
dent of sample size, natural selection and inbreeding
(Nei and Roychoudhury, 1974).
Departures of genotype frequencies from Hardy–
Weinberg expectations were tested using exact tests
(Lessios, 1992; Sokal and Rohlf, 1995) analogous
to Fisher’s exact test for 2 ð 2 contingency tables.
They were used rather than the more commonly
used goodness-of-fit chi-square tests since expected
genotype frequencies were often quite small even
after pooling of rare alleles. If more than two alleles
were present at a locus, all alleles, other than the
most common one were pooled. Deficiencies of het-
erozygotes in each population were estimated for all
polymorphic loci using the inbreeding index F
is
, and
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
and Horvitz (1953):
¡
2
D F
2
is
N(k 1)I d.f. D k(k 1),2
where N is the sample size for the populations and k
is the number of alleles at a locus.
For each locus the statistical significance of the
inter-population variation in allelic frequencies was
calculated by contingency chi-square analysis. All
results from chi-square and exact test analyses were
corrected for multiple simultaneous tests using the
sequential Bonferroni procedure (Rice, 1989).
Genetic differentiation among populations was
estimated using the F
st
estimates and their signif-
icance was tested using the equation by Workman
and Niswander (1970):
¡
2
D 2NF
st
(k 1)I d.f. D (k 1)(s 1)
where N is the total number of individuals sam-
pled across s populations and k is the number of
alleles at a locus. Unbiased estimates of F-statistics
were calculated using the computer program F-STAT
(Goudet, 1994). Individual multi-locus genotypes
were required by FSTAT while BIOSYS-1 accepted
genotype frequencies.
The amount of gene flow (N
m
) was estimated
from the estimates by Weir and Cockerham (1984)
of F
st
. The approximation of Slatkin (1993)
N
m
D ((1,F
st
) 1),4
was used to estimate the effective migration rate be-
tween all pairs of populations and the relationship
between N
m
and geographic distance between pop-
ulations was examined. The shortest geographical
distance between each pair of populations was esti-
mated by measuring the coastline within the bound-
aries of general zooplankton distribution in these
areas according to FAO maps.
Prevosti’s Distance (Wright, 1978) among the var-
ious samples was used to estimate genetic distances.
A cluster analysis was performed on the matrix of
genetic distances generated using the Wagner proce-
dure (Farris, 1972).
A. Exadactylos et al. / Journal of Sea Research 40 (1998) 117–129 121
3. Results
3.1. Allele frequencies
The allele frequencies of the 33 loci analysed for
all seven populations are shown in Appendix A. Nei
(1978) and Gorman and Renzi (1979) underlined the
need to examine a large number of loci because the
reliability (i.e. sampling errors) of summary statis-
tics such as heterozygosity, genetic distance and F
st
depends more on this than on the number of indi-
viduals screened. From a total of 33 loci, 27 were
polymorphic (frequency of the most common allele
Ä 0.99) in most of the populations. The statistical
analyses of genetic diversity were carried out for all
27 polymorphic loci (Table 3). Additional alleles at
five loci (AAT-2, LDH-2, SOD-1, IDH-2, EST-I-3)
were found only in populations from Greece (GRE)
and the Bay of Biscay (FRA), whereas the allele
90 at the locus G6PDH-2 was restricted to northern
populations, suggesting a greater range of alleles in
adult S. solea, in southern Europe. The populations
FRA and GRE were pooled to form a southern Eu-
ropean basin, which exhibited significantly more al-
leles than in the northern European basin (Wilcoxon
signed-ranked test, p - 0.001).
Contingency chi-square analysis for heterogene-
ity of allele frequencies between samples within
basins showed significant differentiation at loci
ACOH (¡
2
D 22.518, d.f. 4, p - 0.001), EST-
I-1 (¡
2
D 32.539, d.f. 4, p - 0.001) and PEP-I-2
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).

2
D 36.262, d.f. 8, p - 0.001), only within the
northern European basin. However, a contingency
chi-square analysis for independence of allele fre-
quencies, with pooling of rare alleles across all
seven populations indicated significant differences
at loci ACOH (¡
2
D 31.767, d.f. 6, p - 0.001),
AAT-1 (¡
2
D 41.919, d.f. 12, p - 0.001), EST-I-1

2
D 35.250, d.f. 6, p - 0.001), G6PDH-2 (¡
2
D
42.835, d.f. 12, p - 0.001), LDH-2 (¡
2
D 59.773,
d.f. 18, p - 0.001), PEP-I-2 (¡
2
D 41.291, d.f.
12, p - 0.001) and PGDH (¡
2
D 65.781, d.f. 18,
p - 0.001). Overall, among the seven populations
there was extensive heterogeneity.
There was no clear geographic trend in the fre-
quencies of the most common allele at most loci.
However, the frequency values of the most common
allele at the GPI-1 locus increased significantly with
latitude, but the cline became non-significant after
the sequential Bonferroni correction.
3.2. Conformity to Hardy–Weinberg expectations
From 156 exact tests with pooling of rare alleles
and sequential Bonferroni correction for multiple
tests there was no evidence of significant departure
from random mating within the populations studied.
However, significant values were observed from
the Li and Horvitz (1953) chi-square analysis for de-
ficiency of heterozygotes using the inbreeding index
F
is
in the Irish Coast population (IRL) at the loci
G6PDH-3 and PGDH, the German Bight population
122 A. Exadactylos et al. / Journal of Sea Research 40 (1998) 117–129
Fig. 2. Mean heterozygosity plotted against latitude for the seven
populations of Solea solea. Regression (H D 0.395 0.00627L)
was significant at o D 0.05 (r D 0.81, F
1.6
D 9.52, p D 0.027).
(GER) at the MDHP-1 locus and the Cumbrian Coast
population (CUM) at the MDHP-2 locus (F
is
D 1,
¡
2
> 50, p - 0.001 for up to 20 tests).
3.3. Heterozygosity
Estimates of mean heterozygosity (H) were in-
versely related to latitude (Fig. 2). About 65% of
the variation in heterozygosity could be explained
by the variation in latitude. Heterozygosity values at
loci AAT-1, GPI-1, PEP-II and PGM differed signif-
icantly between the northern and southern European
basins (ANOVA, F
2.5
> 6.7; p - 0.05).
3.4. Population structure
The mean value of Wright’s F
st
was not sta-
tistically significant, indicating little evidence of
significant genetic heterogeneity among the seven
populations. Wright’s F
st
values were significantly
greater than zero using the chi-square test of Work-
man and Niswander (1970) only for the loci LDH-2
and PGDH (Table 4).
Relatively high levels of gene flow were indicated
from estimates of Weir and Cockerham’s F
st
values
for each pair of populations (Table 5), despite the
significant differences among populations in allele
frequencies from the contingency chi-square analy-
ses. These gene flow estimates between pairs of pop-
ulations were not significantly related to geographic
distance (r
s
D 0.46015 - r
s
at o
0
D 0.005), in-
dicating little isolation by distance across the range
sampled. However, patterns of gene flow determined
stepwise by a strategy of progressively pooling sam-
ples to the south, showed that local gene flow values
are, in most cases, higher than values calculated over
long geographic distances. Gene flow was highest
between geographically proximal populations. As
more samples were added, estimates of gene flow
decreased and remained constant at comparatively
low levels. The mean N
m
value gave an estimate of
roughly nine migrants exchanged between popula-
tions per generation (Table 4).
3.5. Genetic distance
The Wagner procedure dendogram using Pre-
vosti’s Distance (Fig. 3) indicated that population
GRE was most divergent, the Irish Sea populations
were closely related, as are the North Sea popula-
tions. Population GER was clustered at a midpoint
of the tree with population FRA. The goodness of
fit statistics measure the degree to which the output
reflects the corresponding input distances (Swofford,
1981) and hence can be used to choose between
trees generated by different methods. In this case the
Wagner procedure tree gave a better fit.
4. Discussion
4.1. Genetic variability within populations
The observed heterozygote deficiencies may be
due to inbreeding or presence of sub-populations. In
both processes frequencies of homozygotes tend to
increase, by fixing the most common allele in the
first case, and when different populations are mixed
together, in the second. Other destabilising forces
could be the effect of selection on the loci in ques-
tion, or mutation. Further investigations would be
necessary to indicate which process is taking place.
For some of these loci significant F
is
values may
have resulted from the chance occurrence of single
very rare homozygotes for rare alleles. Nevertheless,
156 overall exact tests revealed no significant devia-
tion from Hardy–Weinberg equilibrium, hence, there
is little evidence to reject random mating within the
populations studied.
The latitudinal decline in heterozygosity was par-
alleled by a significant cline in allele frequencies of
A. Exadactylos et al. / Journal of Sea Research 40 (1998) 117–129 123
Table 4
Summary of estimates of Wright’s F-statistics and gene flow (N
m
) at all 27 polymorphic loci, over the seven populations of Solea solea
Locus F
is
F
it
F
st
¡
2
d.f. N
m
(Workman and Niswander, 1970)
ACOH 0.1028 0.1498 0.0524 31.754 6 4.521
AAT-1 0.4020 0.4180 0.0267 32.360 12 9.113
AAT-2 0.1766 0.1878 0.0136 16.483 12 18.132
AAT-4 0.0709 0.1090 0.0411 49.813 12 5.833
DDH-1 0.1578 0.1714 0.0162 19.634 12 15.182
EST-I-1 0.0337 0.0264 0.0582 35.269 6 4.046
EST-I-3 0.3012 0.3187 0.0251 30.421 12 9.710
EST-II-1 0.2003 0.2082 0.0099 5.999 6 25.003
EST-II-3 0.2467 0.2511 0.0059 7.151 12 42.123
GCDH-2 0.2288 0.2391 0.0134 16.241 12 18.407
G6PDH-2 0.1791 0.2047 0.0313 37.936 12 7.737
G6PDH-3 0.2380 0.2589 0.0275 33.330 12 8.841
GPI-1 0.0709 0.0921 0.0228 41.450 18 10.715
GPI-3 0.2311 0.2478 0.0217 39.451 18 11.271
GR-1 0.2720 0.2913 0.0264 31.786 12 9.220
G3PDH-1 0.1582 0.1670 0.0104 12.605 12 23.788
IDH-2 0.0588 0.0418 0.0160 19.392 12 15.375
LDH-1 0.0420 0.0628 0.0217 13.150 6 11.271
LDH-2 0.0706 0.1316 0.0656 119.261 18 3.561
MDH-2 0.0373 0.0166 0.0200 24.240 12 12.250
MDHP-1 0.4868 0.4946 0.0152 9.211 6 16.197
MDHP-2 0.2896 0.2968 0.0101 12.241 12 24.502
PEP-I-2 0.1897 0.2089 0.0237 28.724 12 10.299
PEP-II 0.2009 0.2190 0.0227 27.512 12 10.763
PGM 0.0767 0.0453 0.0292 35.390 12 8.312
PGDH 0.2962 0.3225 0.0373 67.811 18 6.452
SOD-1 0.3709 0.3950 0.0384 23.270 6 6.260
Mean 0.1802 0.2016 0.0262 30.572 11.556 9.292
Values of F
st
significantly greater than zero corrected by the sequential Bonferroni procedure at o
0
D 0.005 are bold.
Table 5
Estimates of Prevosti’s Distance (below the diagonal) and estimates of Weir and Cockerham’s N
m
(above the diagonal) among the seven
populations of Solea solea
Population GRE FRA IOM CUM IRL EAN GER
GRE 18.4320 10.6660 5.2330 12.0260 14.2250 14.3330
FRA 0.0460 26.8440 8.7720 29.6110 142.1300 50.7060
IOM 0.0490 0.0340 16.9250 ♦ 22.6130 9.6540
CUM 0.0630 0.0450 0.0300 20.8080 11.3730 5.1650
IRL 0.0590 0.0430 0.0280 0.0310 62.5760 23.8780
EAN 0.0490 0.0340 0.0350 0.0400 0.0430 22.0600
GER 0.0590 0.0460 0.0510 0.0480 0.0480 0.0500
♦ D negative F
st
value, equivalent to an N
m
value of infinity.
the most common allele at the locus GPI-1. Asso-
ciations of allele frequencies with a latitudinal cline
correlated, for example, with temperature may result
from natural selection acting on allozyme loci, or
secondary intergradation of populations previously
differentiated during allopatry (Endler, 1977), but in
this case may well result from sampling error, since
only 1 of 33 loci gave a significant cline. Never-
theless, the latter became non-significant after the
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.
4.2. Differentiation between populations
The chi-square contingency analyses indicated
a significant heterogeneity in allele frequencies
within basins, and between populations. Heterozy-
gosity (H) values also varied significantly between
basins, indicating the level of genetic diversity of
the species. Average heterozygosity in these S. solea
populations was higher than the average for ma-
rine teleosts (H D 0.052 š 0.036, Smith and Fujio,
1982) but within the range observed in other flatfish
species (Ward and Beardmore, 1977; Galleguillos
and Ward, 1982; Blanquer et al., 1992). These find-
ings contradict the assumption that marine organisms
capable of extensive dispersal (those that undergo
lengthy planktonic larval development) will neces-
sarily demonstrate widespread genetic homogeneity
(Scheltema, 1986). S. solea spawn large numbers of
pelagic eggs (e.g. Kotoulas et al., 1995); therefore,
high levels of gene flow and genetic uniformity could
be expected. However, other factors such as tempera-
ture and salinity gradients, wind and current patterns
or oceanographic fronts may restrict larval disper-
sal and promote geographic isolation. Furthermore,
recruitment of S. solea larvae into the adult popu-
lation that spawned them or restricted movement of
adults within the spawning grounds (Rijnsdorp et al.,
1992; Amara et al., 1993) could also serve to reduce
the homogenisation of population allele frequencies
between spawning stocks. The observed pattern of
genetic patchiness could be the result of localised
selection, genetic drift or single-generation sampling
effects.
Nevertheless, the results indicated a relatively
high level of gene flow between the S. solea popula-
tions examined. These estimates rely on several as-
sumptions, including random breeding, populations
at genetic equilibrium and neutral alleles (Slatkin,
1985a; Waples, 1987; Slatkin and Barton, 1989;
Cockerham and Weir, 1993). However, these esti-
mates of gene flow based on F-statistics are aver-
ages over a number of populations, so there may
be no gene flow at present between the popula-
tions. The apparent absence of isolation-by-distance
suggests that S. solea may not be at genetic equilib-
rium. If populations have not reached equilibrium the
F-statistics will underestimate the degree of differen-
tiation expected at equilibrium and the estimates of
gene flow will overestimate the true levels (Slatkin,
1985b). Waples (1987) also points out that the accu-
racy of gene flow estimates depends on the stability
of the patterns of gene flow. Gene flow values were
sufficiently high to imply near-panmixia between the
two North Sea populations and FRA and IRL, in-
dicating the possibility of a probable movement of
migrants through the English Channel. This result is
A. Exadactylos et al. / Journal of Sea Research 40 (1998) 117–129 125
in agreement with tagging experiments carried out
by Greer Walker and Emerson (1990).
Kotoulas et al. (1995) studied the genetic structure
of S. solea in mixed juvenile and adult populations
over several spatial scales, and concluded that the
strongest result was an east to west pattern of popu-
lation differentiation, while a north to south pattern
was also significant. Biological data indicate near-
panmixia, for example, transport of eggs and larvae
of S. solea from the offshore spawning areas to
the inshore estuarine nurseries by diffusive mecha-
nisms (Koutsikopoulos et al., 1991), offshore move-
ments towards spawning grounds (Greer Walker and
Emerson, 1990), mixing in spawning grounds of
adults originating from adjacent nursery grounds
(Koutsikopoulos and Lacroix, 1992; Rijnsdorp et al.,
1992) or random dispersion mechanisms resulting in
nursery grounds containing juveniles from different
spawning grounds (Marchand, 1991; Koutsikopoulos
and Lacroix, 1992).
4.3. Differentiation within basins
The geographic clustering of the populations in
the Wagner procedure dendogram agree with the
conclusions of many Mediterranean biogeographers.
McCullach and De Deckker (1989) have hypothe-
sised that the history of the Mediterranean, com-
bined with the present hydrographic patterns, might
have promoted and maintained the differentiation of
Mediterranean populations. Since the Pleistocene the
history of the Mediterranean can be seen as a suc-
cession of glacial and interglacial periods with asso-
ciated regressions and transgressions (Blanc, 1968).
It is possible that during one of the regressions the
Mediterranean and Atlantic populations of S. solea
separated. It would be interesting to be able to date
the time of separation and relate it to geological
events. Unfortunately, because of the small number
of populations sampled across the total distribution
of the species, especially in the western Mediter-
ranean, we were unable to calibrate the molecular
clock and thus relate the genetic distance to evolu-
tionary time (Thorpe, 1982).
Besides the possible contribution of the history
and hydrographic barriers, evolutionary processes
such as genetic drift and founder effect, and,or se-
lection, may have produced the observed genetic
differentiation between the northern and southern
European basins. Various physical parameters, es-
pecially water temperature during the reproductive
period, vary within the range of the species and may
produce or maintain genetic differentiation. A pat-
tern emerged from the comparison between basins
and their estimates of genetic variability, revealing a
slight reduction of the latter in the northern European
basin, possibly resulting from, for example, a pop-
ulation bottleneck, or local reduction in population
size. The absence of isolation by distance provided
backing for a model of geographic isolation. The
analysis of more samples would help to complete the
picture and possibly give a better understanding of
the local and wide-range structuring of S. solea.
Acknowledgements
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
B. Turner from the CEFAS laboratory at Lowestoft,
England, P. Newton and his colleagues at DANI,
Belfast, and the crew of our research vessel Roagan
for providing us with the samples. We also thank
R.D.M. Nash, A. Hill, S.M. Lynch, G. Kotoulas,
P. Panagiotaki, E.R. Daka for advice and support.
A. Exadactylos acknowledges the financial support
of the Greek Scholarship Foundation throughout the
course of the research programme.
Appendix A. Allele frequencies for Solea solea at
seven locations
Locus Population
CUM IOM GER IRL EAN FRA GRE
ACP:
(N) 50 72 18 18 43 48 54
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000
ACOH:
(N) 50 72 18 18 43 48 54
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
AAT-1:
(N) 50 72 18 18 43 48 54
100 1.000 0.965 1.000 1.000 0.953 0.948 0.917
110 0.000 0.000 0.000 0.000 0.012 0.000 0.074
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
Appendix A (continued)
Locus Population
CUM IOM GER IRL EAN FRA GRE
AAT-2:
(N) 50 72 18 18 43 48 54
80 0.000 0.000 0.000 0.000 0.000 0.010 0.000
100 0.990 0.972 0.972 1.000 1.000 0.958 1.000
120 0.010 0.028 0.028 0.000 0.000 0.031 0.000
AAT-4:
(N) 50 72 18 18 43 48 54
80 0.030 0.014 0.056 0.028 0.023 0.031 0.130
100 0.970 0.958 0.861 0.917 0.953 0.938 0.796
120 0.000 0.028 0.083 0.056 0.023 0.031 0.074
DDH-1:
(N) 50 72 18 18 43 48 54
90 0.000 0.042 0.000 0.028 0.070 0.063 0.065
100 0.960 0.917 0.833 0.944 0.872 0.865 0.889
110 0.040 0.042 0.167 0.028 0.058 0.073 0.046
EST-I-1:
(N) 50 72 18 18 43 48 54
98 0.000 0.000 0.139 0.000 0.093 0.115 0.037
100 1.000 1.000 0.861 1.000 0.907 0.885 0.963
EST-I-3:
(N) 50 72 18 18 43 48 54
90 0.000 0.000 0.000 0.000 0.000 0.021 0.056
100 0.980 0.965 1.000 0.917 0.977 0.958 0.880
110 0.020 0.035 0.000 0.083 0.023 0.021 0.065
EST-II-1:
(N) 50 72 18 18 43 48 54
95 0.040 0.042 0.083 0.000 0.081 0.042 0.074
100 0.960 0.958 0.917 1.000 0.919 0.958 0.926
EST-II-3:
(N) 50 72 18 18 43 48 54
95 0.010 0.028 0.000 0.000 0.012 0.031 0.028
100 0.990 0.972 0.972 1.000 0.977 0.969 0.963
105 0.000 0.000 0.028 0.000 0.012 0.000 0.009
FBA-1:
(N) 50 72 18 18 43 48 54
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000
GCDH-2:
(N) 50 72 18 18 43 48 54
80 0.000 0.028 0.028 0.000 0.012 0.042 0.000
100 0.990 0.951 0.972 1.000 0.930 0.948 0.972
120 0.010 0.021 0.000 0.000 0.058 0.010 0.028
G6PDH-2:
(N) 50 72 18 18 43 48 54
90 0.000 0.035 0.000 0.000 0.058 0.000 0.000
100 1.000 0.965 1.000 1.000 0.919 0.948 0.917
110 0.000 0.000 0.000 0.000 0.023 0.052 0.083
Appendix A (continued)
Locus Population
CUM IOM GER IRL EAN FRA GRE
G6PDH-3:
(N) 50 72 18 18 43 48 54
80 0.130 0.028 0.083 0.000 0.047 0.000 0.074
100 0.870 0.944 0.917 0.944 0.953 0.990 0.917
120 0.000 0.028 0.000 0.056 0.000 0.010 0.009
GPI-1:
(N) 50 72 18 18 43 48 54
80 0.010 0.007 0.000 0.028 0.035 0.010 0.046
100 0.970 0.944 0.917 0.944 0.965 0.917 0.843
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
GPI-3:
(N) 50 72 18 18 43 48 54
90 0.010 0.083 0.028 0.056 0.000 0.052 0.056
100 0.930 0.826 0.778 0.806 0.930 0.885 0.880
110 0.040 0.049 0.000 0.056 0.023 0.042 0.028
120 0.020 0.042 0.194 0.083 0.047 0.021 0.037
GR-1:
(N) 50 72 16 18 43 48 54
90 0.010 0.021 0.000 0.000 0.000 0.031 0.074
100 0.980 0.965 0.969 1.000 0.907 0.896 0.898
110 0.010 0.014 0.031 0.000 0.093 0.073 0.028
GAPDH-1:
(N) 50 72 18 18 43 48 54
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000
G3PDH-1:
(N) 50 72 18 18 43 48 54
85 0.000 0.007 0.028 0.028 0.070 0.031 0.037
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
IDH-2:
(N) 50 72 18 18 43 48 54
80 0.010 0.035 0.083 0.028 0.023 0.052 0.065
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
LDH-1:
(N) 50 72 18 18 43 48 54
100 0.990 0.938 1.000 0.972 0.930 1.000 0.963
110 0.010 0.063 0.000 0.028 0.070 0.000 0.037
LDH-2:
(N) 50 72 18 18 43 48 54
70 0.000 0.000 0.000 0.000 0.000 0.021 0.028
90 0.000 0.007 0.000 0.000 0.035 0.000 0.019
100 1.000 0.979 0.917 0.972 0.895 0.948 0.787
110 0.000 0.014 0.083 0.028 0.070 0.031 0.167
LDH-4:
(N) 50 72 18 18 43 48 54
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
Appendix A (continued)
Locus Population
CUM IOM GER IRL EAN FRA GRE
MDH-1:
(N) 50 72 18 18 43 48 54
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000
MDH-2:
(N) 50 72 18 18 43 48 54
80 0.020 0.090 0.000 0.056 0.035 0.010 0.028
100 0.970 0.882 1.000 0.944 0.907 0.938 0.898
120 0.010 0.028 0.000 0.000 0.058 0.052 0.074
MDHP-1:
(N) 50 72 18 18 43 48 54
90 0.040 0.042 0.056 0.083 0.116 0.094 0.037
100 0.960 0.958 0.944 0.917 0.884 0.906 0.963
MDHP-2:
(N) 50 72 18 18 43 48 54
90 0.020 0.063 0.056 0.083 0.047 0.083 0.056
100 0.980 0.917 0.944 0.917 0.919 0.896 0.898
105 0.000 0.021 0.000 0.000 0.035 0.021 0.046
PEP-I-1:
(N) 50 72 18 18 43 48 54
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000
PEP-I-2:
(N) 50 72 18 18 43 48 54
95 0.010 0.021 0.000 0.028 0.081 0.031 0.065
100 0.990 0.972 0.889 0.917 0.919 0.927 0.935
105 0.000 0.007 0.111 0.056 0.000 0.042 0.000
PEP-II:
(N) 50 72 18 18 43 48 54
95 0.000 0.035 0.000 0.000 0.047 0.010 0.046
100 0.990 0.944 1.000 0.972 0.953 0.948 0.926
105 0.010 0.021 0.000 0.028 0.000 0.094 0.028
PGM:
(N) 50 72 18 18 43 48 54
90 0.020 0.021 0.028 0.000 0.012 0.010 0.037
100 0.980 0.979 0.944 1.000 0.942 0.906 0.880
115 0.000 0.000 0.028 0.000 0.047 0.083 0.083
PGDH:
(N) 50 72 18 18 43 48 54
60 0.000 0.000 0.028 0.000 0.012 0.021 0.028
80 0.110 0.049 0.222 0.056 0.105 0.125 0.000
100 0.850 0.799 0.722 0.944 0.849 0.729 0.741
110 0.040 0.153 0.028 0.000 0.035 0.125 0.231
SOD-1:
(N) 50 72 18 18 43 48 54
80 0.000 0.000 0.000 0.000 0.000 0.000 0.046
100 1.000 1.000 1.000 1.000 1.000 1.000 0.954
CUM D Cumbria; IOM D Isle of Man; GER D German Bight;
IRL D Ireland; EAN D East Anglia; FRA D France; GRE D
Greece.
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