Zoological Journal of the Linnean Society (2001), 132: 487–499.

With 3 figures
doi:10.1006/zjls.2000.0282, available online at http://www.idealibrary.com on

Allozyme variation and genetic inter-relationships

between seven flatfish species (Pleuronectiformes)
ATHANASIOS EXADACTYLOS∗ and JOHN P. THORPE
School of Biological Sciences, The University of Liverpool, Port Erin Marine Laboratory, Port Erin,
Isle of Man, IM9 6JA

Received September 2000; accepted for publication October 2000

A total of 11 enzymes coded by 14 loci were assayed for each of 17 populations (from the north east Atlantic and
Mediterranean) of seven flatfish species, including representatives of three of the four European families of the
order Pleuronectiformes. Diagnostic alleles were observed for each species and there were fixed differences between
the species at many loci. Thus all species were genetically distinct, although there were some common alleles. Hobs
was higher than the average of a range of fish species and was also higher than that of vertebrate species as a
whole. It seems that flatfish as a group may show higher levels of genetic variation than other fish. Values of
genetic identity for all pairwise comparisons fell comfortably within the ranges expected. The data support the
grouping of plaice and flounder into a single genus, Pleuronectes, but it is concluded that the retention of the dab
in a separate genus, Limanda, is justified. A high level of genetic divergence was found between Dover and thickback
soles. Genetic divergence data support the hypothesis that Pleuronectes flesus luscus in the Aegean Sea is a distinct
subspecies of Pleuronectes flesus (flounder). The data show a clear separation of the Mediterranean Dover sole
population from those in the Atlantic. Low genetic divergence was observed between the Aegean Sea and Atlantic
brill populations. It is speculated that about 5 Mya the families Pleuronectidae and later Soleidae evolved from the
ancestral Scophthalmidae.  2001 The Linnean Society of London

ADDITIONAL KEY WORDS: allozyme electrophoresis – systematics – genetic identity – phylogeny.

INTRODUCTION Scophthalmidae are distinguished from the Pleur-

The Order Pleuronectiformes (flatfish) is well rep-
resented throughout the seas of the World and includes
many species spread over six families (review by
Hensley, 1997). Flatfish vary in adult size from about
10 g (e.g. solenette, Buglossidium luteum) to >200 kg
(halibut, Hippoglossus hippoglossus). The Pleuro-
nectiformes are generally visual predators, many of
them specializing in invertebrate prey on sandy or
muddy substrata. Some of the larger species eat fish.
Almost all species are benthic and many have the
ability to hide partly buried in sand or mud. Four
families occur in northern Europe; Scophthalmidae
(turbot and similar species), Bothidae (scaldfishes),
Pleuronectidae (plaice family) and Soleidae (soles) (for
summary of British species see Wheeler, 1992). The
∗ Corresponding author. Current address: Biodiversity Con-
servation Laboratory, Department of Environment Studies,
University of the Aegean, Mytilene, GR 81100, Greece. E-mail:
thexad@env.aegean.gr
487
0024–4082/01/080487+13 $35.00/0
onectidae because the former have both eyes on the
left side of the head and the latter on the right, although
in various species of each some reversed specimens
can be found (Quero et al., 1986). Species from both
families feature a well developed terminal mouth with
a prominent lower jaw. The Bothidae differ in lacking a
preopercular margin. The Soleidae are morphologically
quite distinct from other flatfishes with char-
acteristically slender bodies and a smoothly rounded
margin of the head projecting forward of the mouth
(Cunningham, 1890).
Conventional studies of the relationships of the
Pleuronectiformes, based on anatomical and mor-
phological data (Hubbs, 1945; Lauder & Liem, 1983)
are not entirely compatible with more recent work
using molecular methods (Verneau et al., 1994). The
molecular data suggest that the Pleuronectidae and
Scophthalmidae are monophyletic, but that the Pleuro-
nectiformes as a whole are considerably less closely
related (i.e. less recently evolved) than previously be-
lieved. Verneau et al. (1994) used mainly samples from
the Mediterranean area.
 2001 The Linnean Society of London
488 A. EXADACTYLOS and J. P. THORPE
The aim of our study was to repeat part of the work
of Verneau et al. (1994) using enzyme electrophoresis,
with samples of mostly the same species, but largely
from Northern Europe, to reexamine genetic di-
vergence in the order Pleuronectiformes and hence
gain a further insight into the phylogeny. We used
17 populations from seven flatfish species including
representatives of each of the three main families
(Pleuronectidae, Scophthalmidae and Soleidae) found
in European waters.
Apart from the work of Verneau et al. (1994), enzyme
electrophoresis has been used in a number of studies
in flatfish, primarily species of Pleuronectidae (e.g.
Purdom et al., 1976; Ward & Beardmore, 1977; Ward
& Galleguillos, 1978, 1983; Galleguillos & Ward, 1982;
McAndrew et al., 1982; Berrebi et al., 1983, 1985, 1990;
Quignard et al., 1984; She et al., 1987; Brule, 1989;
Blanquer et al., 1992). To date other molecular methods
have been little used to elucidate genetic relationships
in flatfish (see e.g. Coughlan et al., 1996, 1998;
McGowan & Reith, 1999), although they are being used
increasingly in fish and fisheries research generally
(reviews by e.g. Park & Moran, 1995; Ward & Grewe,
1995; Thorpe et al., 2000).
MATERIAL AND METHODS
SAMPLE COLLECTION SITES
All fish samples were collected by trawl, during 1995
and 1996 and processed fresh. Extraction of liver and
skeletal muscle was performed as described by Exa-
dactylos (1997). Specimens were kept at −75°C until
required. The populations sampled and the sampling
sites are described in Table 1 and Figure 1, respectively;
overall seven species (17 populations) were studied.
The species were Solea solea (Dover sole) and Micro-
chirus variegatus (thickback sole) (Soleidae); Scoph-
thalmus maximus (turbot) and Scophthalmus rhombus
(brill) (Scophthalmidae) and Pleuronectes platessa
(plaice), Pleuronectes flesus (flounder) and Limanda
limanda (dab) (Pleuronectidae).
ANALYSIS OF ELECTROPHORETIC VARIATION
Allozyme electrophoresis was carried out using stand-
ard horizontal starch gels (12.5%, Sigma Chemicals)
and staining systems (e.g. Murphy et al., 1990). The
homogenates were centrifuged at 3000 g for 15 min for
skeletal muscle and 5 min for liver tissue. Eye tissue
gave poor resolution. Loci and alleles are designated
following Allendorf & Utter (1979) and Shaklee et al.
(1990). Solea solea, Dover sole, was used as a standard
for the measurement of the relative mobilities of alleles
for all species. The buffer systems used are shown in
Table 2.
The programme Biosys-2 (Swofford & Selander,
1989) was used to estimate the variability and genetic
differentiation between species and between popu-
lations within species. Hunbias was chosen as a better
measure of genetic variation in a sample than a direct
count of heterozygosity. Hunbias values were calculated
for each locus and each of the seven species (popu-
lations within species were pooled).
Contingency chi-square analyses between popu-
lations within species and between species within
families were used for testing the level of heterogeneity
of allele frequencies. All results from chi-square and
exact tests were corrected under the sequential Bon-
ferroni procedure (Holm, 1979; Rice, 1989). F-statistics
were calculated on three levels of hierarchy: popu-
lations, species, families.
Matrices of Nei’s (1972, 1978) Genetic Identity, I,
and Distance, D, were calculated for each pair of popu-
lations and averaged for each pair of species and
families. A cluster analysis was performed on these
matrices of genetic identities, using the unweighted
pair group arithmetic means algorithm (UPGMA)
(Sneath & Sokal, 1973). Prevosti’s distance (Wright,
1978) was also employed to estimate the phylogenetic
relationship through the Wagner procedure (Farris,
1972). The goodness of fit statistics in both cases were
the criteria for evaluating which clustering method
produced the best tree.
RESULTS
ALLELE FREQUENCIES
A total of 11 enzymes coded by 14 loci were assayed
for all species. Allele frequencies are shown in Table
3. Thirteen of these loci were polymorphic (P<0.99
criterion) in one or more species, but this high level of
genetic variation was mainly a result of the marked
polymorphism observed within the S. solea popu-
lations. Ck-1 was the only locus consistently mono-
morphic in all the species studied. The loci Pgm and
G3pdh-1 were consistently polymorphic for all species.
Several loci showed diagnostic alleles (sensu Ayala,
1983). Mean number of alleles per locus and percentage
of loci polymorphic were calculated as an indication of
genetic variability within species (Table 4).
Contingency chi-square analyses for heterogeneity of
allele frequencies between populations within species
showed significant differentiation only in Solea solea
and only at three loci (Aat-4, Ldh-2, Pgdh). Dif-
ferentiation between the S. solea populations is dis-
cussed in detail by Exadactylos et al. (1998, 1999). A
contingency chi-square analysis between species
within families detected significant differences be-
tween the species for most of the loci, except those
shown in Table 5.
 SYSTEMATICS IN PLEURONECTIFORMES 489

Table 1. Sampling information protocol for the seven flatfish species studied. N=number of fish in sample

Species Location Sample site Code Date N Mean

standard
length in
cm [SE]

1 Solea solea King William’s Bank/Irish Sea 54°28N-3°58W S/CUM 05/11/95 50 34.52 [3.65]
2 Solea solea Chicken Rock/Isle of Man 53°58N-4°58W S/IOM 07/06/94 and 14/ 73 29.82 [5.12]
02/96
3 Solea solea German Bight/North Sea 53°55N-6°40E S/GER 29/09/95 18 25.3 [5.90]
4 Solea solea Kish Bank/Irish Sea 53°18N-5°47W S/IRL 12/09/94 and 22/ 18 23 [4.92]
09/95
5 Solea solea Newsombe Bank/North Sea 52°26N-1°15E S/EAN 07/09/94 43 19.7 [1.60]
6 Solea solea L’ı̂le d’Oleron/Bay of Biscay 45°37N-1°46W S/FRA 22/12/94 48 26.99 [2.40]
7 Solea solea Thermaikos Bay/Aegean Sea 40°13N-22°86E S/GRE 15/07/94 and 12/ 54 22.40 [2.69]
05/94
8 Microchirus variegatus Kish Bank/Irish Sea 53°18N-5°47W TS/IRL 22/09/95 20 12.43 [1.40]
9 Microchirus variegatus Chicken Rock/Isle of Man 53°58N-4°58W TS/IOM 14/02/96 4 12.00 [0.82]
10 Scophthalmus Chicken Rock/Isle of Man 53°58N-4°58W T/IOM 29/09/95 8 28.38 [1.32]
maximus
11 Scophthalmus Chicken Rock/Isle of Man 53°58N-4°58W B/IOM 07/09/96 11 36.80 [6.98]
rhombus
12 Scophthalmus Kish Bank/Irish Sea 53°18N-5°47W B/IRL 22/09/95 5 34.50 [5.45]
rhombus
13 Scophthalmus Thermaikos Bay/Aegean Sea 40°13N-22°86E B/GRE 24/12/95 10 19.71 [5.53]
rhombus
14 Pleuronectes platessa Chicken Rock/Isle of Man 53°58N-4°58W P/IOM 07/09/96 15 24.85 [2.59]
15 Pleuronectes flesus Chicken Rock/Isle of Man 53°58N-4°58W F/IOM 14/02/96 9 38.22 [5.40]
16 Pleuronectes flesus Thermaikos Bay/Aegean Sea 40°13N-22°86E F/GRE 24/12/95 7 27.24 [2.85]
17 Limanda limanda Chicken Rock/Isle of Man 53°58N-4°58W D/IOM 14/02/96 11 25.20 [4.86]

PANMIXIA of genetic differentiation between populations within

After pooling of rare alleles, exact tests of significance
(analogous to Fisher’s exact test for 2×2 contingency
tables; see Sokal & Rohlf, 1995) were performed on
the divergence from Hardy–Weinberg equilibrium. In
all the samples no significant deviation from the ex-
pected proportions of genotypes was observed. Sig-
nificant values were observed from the Li & Horvitz
(1953) chi-square analysis for deficiency of hetero-
zygotes using the FIS index, but only in the Irish Coast
S. solea population at the locus Pgdh and the Cumbrian
Coast S. solea population at the locus Mdhp-2. No
significant divergences were detected in any popu-
lations of the other flatfish species.
GENETIC DIVERGENCE
Observed (Hobs) and expected (Hunbias) heterozygosities
for the 13 variable loci are shown in Table 4. Hunbias
values ranged from 0.019 for the Isle of Man Scoph-
thalmus rhombus (brill) population to 0.196 for the
Greek S. solea population. There was a low mean level
species (FST=0.029). The matrices of Nei’s (1978) Gen-
etic Identity, I, between populations, species and famil-
ies are shown in Tables 6 and 7 respectively. In the
interspecies comparisons I ranged from 0.73 between
Pleuronectes platessa (plaice) and Pleuronectes flesus
(flounder) to zero in all comparisons between species
from different families. Within species all pairs of
population were very similar (I>0.99).
The UPGMA dendrogram using Nei’s (1978) Genetic
Identity (I) (Fig. 2), suggests that P. platessa and P.
flesus were the most closely related species pair and
that within a single family S. solea and Microchirus
variegatus (thickback sole) were the most divergent
species pair. There was a clear separation between the
three families and in each case the species within a
family clustered together. According to the goodness of
fit statistics for species divergence, the UPGMA de-
ndrogram (Fig. 2) gave a marginally better fit than the
Wagner procedure dendrogram using Prevosti’s distance
(Fig. 3), but the trees obtained from the two procedures
showed essentially the same branching pattern.
490 A. EXADACTYLOS and J. P. THORPE

S/IOM (72), TS/IOM (4),

P/IOM (15), F/IOM (9),
D/IOM (11), T/IOM (8), S/CUM (50)
B/IOM (11)

S/IRL (18),
TS/IRL (20),
B/IRL (5) S/GER (18)

S/FRA (48) S/EAN (43)

S/GRE (54),
F/GRE (7),
B/GRE (10)

Figure 1. Outline map of Europe to show the sampling sites of the 17 populations of seven species of flatfish. Figures
in brackets indicate the total number of fish in each sample. Abbreviations used for each sample follow
Table 1.

Table 2. Summary of the 14 enzyme loci studied. Details of buffers are given by Murphy et al. (1990)

Abbreviation of EC Number Tissue No. of loci Buffer system

enzyme

AAT 2.6.1.1 Muscle 2 Tris-Citrate, pH 8.0

CK 2.7.3.2 Muscle 1 Tris-Citrate, pH 8.0
GPI 5.3.1.9 Muscle/Liver 2 Discontinuous Tris-Borate-Citrate, pH 8.2–8.7
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 2 Discontinuous Tris-Borate-Citrate, pH 8.2–8.7
MDH 1.1.1.37 Muscle 1 Tris-Citrate, pH 8.0
MDHP 1.1.1.40 Muscle 1 Tris-Versene-Borate, pH 8.0
PGM 2.7.5.1 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

DISCUSSION (e.g. Ward & Beardmore, 1977; Ward & Galleguillos,

LEVELS OF VARIATION
The mean levels of heterozygosity (0.064) and poly-
morphism (0.46) over all the populations studied (Table
4) were somewhat higher than the means quoted for
fish species in various reviews (Powell, 1975; Nevo,
1978; Fujio & Kato, 1979; Smith & Fujio, 1982). How-
ever, values observed in this study fell comfortably
within the range observed in previous studies of flatfish
1978; Galleguillos & Ward, 1982; Berrebi et al., 1985;
She et al., 1987; Brule, 1989; Blanquer et al., 1992),
indicating that flatfishes as a group may show higher
levels of genetic variation than most other fish species.
GENETIC RELATIONSHIPS BETWEEN SPECIES
All 14 loci were diagnostic (with fixed allele differences)
for one or more species and overall complete dis-
 SYSTEMATICS IN PLEURONECTIFORMES 491

Table 3. Allele frequencies for all seven flatfish species. Samples 1–7: Solea solea, 8, 9: Microchirus variegatus, 10:
Scophthalmus maximus, 11–13: Scophthalmus rhombus, 14: Pleuronectes platessa, 15, 16: Pleuronectes flesus, 17:
Limanda limanda. See Table 1 for further details

Locus Samples
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Aat-2
(N) 54 48 72 50 18 43 18 20 4 8 11 5 10 15 9 7 11
80 0.000 0.010 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
100 1.000 0.958 0.972 0.990 1.000 1.000 0.972 1.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
110 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 0.045 0.000 0.000 0.000 0.000 0.000 0.000
115 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.955 1.000 1.000 0.000 0.000 0.000 0.000
120 0.000 0.031 0.028 0.010 0.000 0.000 0.028 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
125 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.067 0.000 0.000 0.000
140 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.933 1.000 1.000 1.000
Aat-4
(N) 54 48 72 50 18 43 18 20 4 8 11 5 10 15 9 7 11
80 0.130 0.031 0.014 0.030 0.028 0.023 0.056 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
85 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.045 0.000 0.000 0.000 0.000 0.000 0.000
100 0.796 0.938 0.958 0.970 0.917 0.953 0.861 1.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
105 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.955 0.900 0.950 0.000 0.000 0.000 0.000
110 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
120 0.074 0.031 0.028 0.000 0.056 0.023 0.083 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
125 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.100 0.050 0.000 0.000 0.000 0.000
140 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 1.000 1.000 1.000
Ck-1
(N) 54 48 72 50 18 43 18 20 4 8 11 5 10 15 9 7 11
55 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 1.000 1.000
60 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000
70 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 1.000 1.000 1.000 0.000 0.000 0.000 0.000
100 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
Gpi-1
(N) 54 48 72 50 18 43 18 20 4 8 11 5 10 15 9 7 11
70 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000
75 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
80 0.046 0.010 0.007 0.010 0.028 0.035 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
100 0.843 0.917 0.944 0.970 0.944 0.965 0.917 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
110 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 1.000 1.000 1.000 0.000 0.000 0.000 0.000
115 0.046 0.042 0.035 0.000 0.028 0.000 0.083 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
120 0.065 0.031 0.014 0.020 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
140 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 1.000 1.000 1.000
Gpi-3
(N) 54 48 72 50 18 43 18 20 4 8 11 5 10 15 9 7 11
60 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.900 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
70 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.100 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
90 0.028 0.042 0.049 0.040 0.056 0.023 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
100 0.880 0.885 0.826 0.930 0.806 0.930 0.778 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
110 0.056 0.052 0.083 0.010 0.056 0.000 0.028 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
115 0.037 0.021 0.042 0.020 0.083 0.047 0.194 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
120 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
125 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 1.000 1.000 0.000 0.000 0.000 0.000
140 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 1.000 1.000 1.000
G3pdh-1
(N) 54 48 72 50 18 43 18 20 4 8 11 5 10 15 9 7 11
70 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.100 0.000 0.000 0.000 0.000 0.000
75 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.111 0.000 0.182
80 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.063 0.000 0.000 0.000 0.000 0.000 0.000 0.000

continued
492 A. EXADACTYLOS and J. P. THORPE

Table 3 – continued

Locus Samples
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

85 0.037 0.031 0.007 0.000 0.028 0.070 0.028 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
90 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.875 1.000 0.900 0.950 0.000 0.000 0.000 0.000
95 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.889 1.000 0.818
100 0.889 0.906 0.889 0.960 0.889 0.884 0.861 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
105 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.975 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
110 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.063 0.000 0.000 0.000 0.000 0.000 0.000 0.000
115 0.074 0.063 0.104 0.040 0.083 0.047 0.111 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
120 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.025 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
125 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.050 0.000 0.000 0.000 0.000
140 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000
Idh-2
(N) 54 48 72 50 18 43 18 20 4 8 11 5 10 15 9 7 11
80 0.065 0.052 0.035 0.010 0.028 0.023 0.083 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
95 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.063 0.000 0.000 0.000 0.000 0.000 0.000 0.000
100 0.907 0.948 0.965 0.990 0.972 0.977 0.917 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
110 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
115 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.875 1.000 1.000 1.000 0.000 0.000 0.000 0.000
120 0.028 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
125 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.063 0.000 0.000 0.000 0.000 0.000 0.000 0.000
140 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 1.000 1.000 1.000
Ldh-1
(N) 54 48 72 50 18 43 18 20 4 8 11 5 10 15 9 7 11
80 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
100 0.963 1.000 0.938 0.990 0.972 0.930 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
110 0.037 0.000 0.063 0.010 0.028 0.070 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
115 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.071 0.000
120 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 1.000 1.000 1.000 0.000 0.000 0.000 0.000
125 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 1.000 0.929 0.000
140 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000
Ldh-2
(N) 54 48 72 50 18 43 18 20 4 8 11 5 10 15 9 7 11
70 0.028 0.021 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
90 0.019 0.000 0.007 0.000 0.000 0.035 0.000 1.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
95 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 1.000 1.000 0.000 0.000 0.000 0.000
100 0.787 0.948 0.979 1.000 0.972 0.895 0.917 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
105 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.067 0.111 0.143 1.000
110 0.167 0.031 0.014 0.000 0.028 0.070 0.083 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
120 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
140 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.933 0.889 0.857 0.000
Mdh-2
(N) 54 48 72 50 18 43 18 20 4 8 11 5 10 15 9 7 11
50 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 1.000 1.000 1.000
55 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.100 0.000 0.000 0.000 0.000
60 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
65 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 1.000 0.900 0.900 0.000 0.000 0.000 0.000
80 0.028 0.010 0.090 0.020 0.056 0.035 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
90 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.100 0.000 0.000 0.000 0.000 0.000
100 0.898 0.938 0.882 0.970 0.944 0.907 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
120 0.074 0.052 0.028 0.010 0.000 0.058 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
Mdhp-2
(N) 54 48 72 50 18 43 18 20 4 8 11 5 10 15 9 7 11
50 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
70 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.100 0.000 0.000 0.000

continued
 SYSTEMATICS IN PLEURONECTIFORMES 493

Table 3 – continued

Locus Samples
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

75 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.143 0.000
80 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.900 0.833 0.857 1.000
90 0.056 0.083 0.063 0.020 0.083 0.047 0.056 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
95 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.167 0.000 0.000
100 0.898 0.896 0.917 0.980 0.917 0.919 0.944 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
105 0.046 0.021 0.021 0.000 0.000 0.035 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
120 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 1.000 1.000 1.000 0.000 0.000 0.000 0.000
Pgm
(N) 54 48 72 50 18 43 18 20 4 8 11 5 10 15 9 7 11
50 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.967 0.111 0.000 0.000
55 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.050 0.000 0.000 0.000 0.000
60 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.050 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
65 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.938 0.000 0.000 0.000 0.000 0.000 0.000 0.000
70 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.950 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
75 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.955 1.000 0.950 0.000 0.000 0.000 0.000
80 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.045 0.000 0.000 0.000 0.000 0.000 0.000
85 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.063 0.000 0.000 0.000 0.000 0.000 0.000 0.000
90 0.037 0.010 0.021 0.020 0.000 0.012 0.028 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
95 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.033 0.889 0.929 0.000
100 0.880 0.906 0.979 0.980 1.000 0.942 0.944 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
110 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.091
115 0.083 0.083 0.000 0.000 0.000 0.047 0.028 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
140 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.071 0.909
Pgdh
(N) 54 48 72 50 18 43 18 20 4 8 11 5 10 15 9 7 11
55 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.925 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
60 0.028 0.021 0.000 0.000 0.000 0.012 0.028 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
65 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.075 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
75 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 1.000 0.000
80 0.000 0.125 0.049 0.110 0.056 0.105 0.222 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
85 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.067 0.000 0.000 0.000
95 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.867 0.000 0.000 0.000
100 0.741 0.729 0.799 0.850 0.944 0.849 0.722 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
110 0.231 0.125 0.153 0.040 0.000 0.035 0.028 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
115 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.067 0.000 0.000 0.000
120 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 1.000 1.000 1.000 0.000 0.000 0.000 0.000
125 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000
Sod-1
(N) 54 48 72 50 18 43 18 20 4 8 11 5 10 15 9 7 11
55 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000
60 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 1.000 1.000 0.000
70 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.000 1.000 1.000 1.000 0.000 0.000 0.000 0.000
80 0.046 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
100 0.954 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

crimination was achieved for all individuals of each
of the seven species. The presence of diagnostic loci
provides a clear indication of lack of gene flow between
populations, thus indicating that they are re-
productively isolated (Ayala, 1983).
Overall, levels of genetic divergence between the
populations and species can be reduced to a single
value using any of the various published statistical
indices of genetic identity or distance (see e.g. Thorpe,
1982; Nei, 1987). One of the most commonly used
indices is the genetic identity (I) of Nei (1972, 1978),
which ranges from one (populations identical) to zero
(total dissimilarity). Thorpe (1982), using published
data, plotted the probability of populations, within
494 A. EXADACTYLOS and J. P. THORPE

Table 4. Mean heterozygosities (H), mean number of alleles per locus, and the percentage of polymorphic loci in 17
populations of seven flatfish species. A locus is considered polymorphic if the frequency of the most common allele
does not exceed 0.99. Expected heterozygosities are Nei’s (1978) unbiased estimate. Populations are numbered as in
Table 1

Species Population Mean sample Mean no. of Percentage of Mean heterozygosity (H)
size per locus alleles per loci
[SE] locus [SE] polymorphic Observed [SE] Expected [SE]

Solea solea 1. S/GRE 54 [0.0] 2.8 [0.3] 85.7 0.165 [0.027] 0.196 [0.033]
Solea solea 2. S/FRA 48 [0.0] 2.7 [0.3] 78.6 0.112 [0.021] 0.134 [0.030]
Solea solea 3. S/IOM 72 [0.0] 2.6 [0.3] 85.7 0.110 [0.024] 0.124 [0.029]
Solea solea 4. S/CUM 50 [0.0] 2.1 [0.2] 78.6 0.046 [0.014] 0.057 [0.019]
Solea solea 5. S/IRL 18 [0.0] 2.1 [0.2] 71.4 0.056 [0.014] 0.098 [0.026]
Solea solea 6. S/EAN 43 [0.0] 2.4 [0.3] 78.6 0.108 [0.020] 0.114 [0.023]
Solea solea 7. S/GER 18 [0.0] 2.1 [0.3] 71.4 0.119 [0.029] 0.147 [0.037]
Microchirus variegatus 8. TS/IRL 20 [0.0] 1.3 [0.1] 28.6 0.036 [0.018] 0.034 [0.017]
Microchirus variegatus 9. TS/IOM 4 [0.0] 1.0 [0.0] 0.0 0.000 [0.000] 0.000 [0.000]
Scophthalmus 10. T/IOM 8 [0.0] 1.4 [0.2] 21.4 0.045 [0.025] 0.043 [0.024]
maximus
Scophthalmus 11. B/IOM 11 [0.0] 1.2 [0.1] 21.4 0.019 [0.010] 0.019 [0.010]
rhombus
Scophthalmus 12. B/IRL 5 [0.0] 1.2 [0.1] 21.4 0.043 [0.023] 0.043 [0.023]
rhombus
Scophthalmus 13. B/GRE 10 [0.0] 1.3 [0.1] 28.6 0.036 [0.017] 0.035 [0.016]
rhombus
Pleuronectes platessa 14. P/IOM 15 [0.0] 1.4 [0.2] 35.7 0.057 [0.024] 0.054 [0.023]
Pleuronectes flesus 15. F/IOM 9 [0.0] 1.3 [0.1] 28.6 0.056 [0.025] 0.066 [0.029]
Pleuronectes flesus 16. F/GRE 7 [0.0] 1.3 [0.1] 28.6 0.061 [0.029] 0.058 [0.027]
Limanda limanda 17. D/IOM 11 [0.0] 1.1 [0.1] 14.3 0.026 [0.018] 0.035 [0.025]
Total 45.8 0.064 [0.020]

Table 5. Non-significant values from a contingency
2 table for heterogeneity

of allele frequencies between species within families, after the sequential
Bonferroni correction at a′=0.005. All the other loci differed significantly

Families Locus No. of
2 df P

alleles

Pleuronectidae Aat-2 2 3.688 3 0.297

Mdhp-2 4 26.758 9 0.015
Scophthalmidae G3pdh-1 5 14.875 12 0.248
Idh-2 3 0.697 6 0.350
Soleidae Aat-2 3 14.609 16 0.553
Sod-1 2 25.472 8 0.013

species, between congeneric species and between dif-
ferent genera, falling into certain values of genetic
identity (I) and distance (D). Between genera most I
values fall below about 0.4, for congeneric species
between about 0.35 and 0.85 and for conspecific popu-
lations above about 0.85.
For the 17 populations studied in the present work,
values of genetic identity for all conspecific com-
parisons for populations of Solea solea (Dover sole),
Microchirus variegatus (thickback sole), Scophthalmus
rhombus (brill) and Pleuronectes flesus (flounder) fell
comfortably within the expected range (all were above
0.9). Of the pairs of congeneric species the mean genetic
identity value between plaice and flounder was 0.72
and between turbot (Scophthalmus maximus) and brill
it was 0.57. Between confamiliar genera some of the I
values, which ranged from 0.29 to 0.59 (mean=0.38),
were surprisingly high. Between species in different
families all values were zero.
It should be noted that the sampling errors for
 SYSTEMATICS IN PLEURONECTIFORMES 495

Table 6. Estimates of Nei’s (1978) Unbiased Genetic Distance (below the diagonal) and Nei’s (1978) Unbiased Genetic
Identity (above the diagonal) between the 17 flatfish populations. Sample numbers are as in Table 1. Genetic distance
values left blank are infinity

Population 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

1. S/GRE 0.997 0.995 0.994 0.994 0.996 0.995 0.305 0.300 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
2. S/FRA 0.003 0.999 0.999 0.998 0.999 0.999 0.304 0.299 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
3. S/IOM 0.005 0.001 0.998 1.000 0.998 0.997 0.306 0.300 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
4. S/CUM 0.006 0.001 0.002 1.000 0.999 0.997 0.296 0.291 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
5. S/IRL 0.006 0.002 0.000 0.000 1.000 0.999 0.300 0.294 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
6. S/EAN 0.004 0.001 0.002 0.001 0.000 0.997 0.308 0.303 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
7. S/GER 0.005 0.001 0.003 0.003 0.001 0.003 0.302 0.296 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
8. TS/IRL 1.186 1.190 1.185 1.216 1.205 1.178 1.199 0.999 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
9. TS/IOM 1.203 1.207 1.203 1.234 1.223 1.195 1.216 0.001 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
10. T/IOM 0.575 0.572 0.573 0.000 0.000 0.000 0.000
11. B/IOM 0.553 1.000 1.000 0.000 0.000 0.000 0.000
12. B/IRL 0.559 0.000 1.000 0.000 0.000 0.000 0.000
13. B/GRE 0.557 0.000 0.000 0.000 0.000 0.000 0.000
14. P/IOM 0.734 0.717 0.441
15. F/IOM 0.309 1.000 0.579
16. F/GRE 0.333 0.000 0.590
17. D/IOM 0.819 0.547 0.527

Table 7. Matrix of Nei’s (1978) D and I (below and above diagonal respectively) between the flatfish species. Values
are means. The range between the least and most genetically divergent populations within the species is given in
parentheses. ‘–’ indicates no comparison within the species in question since only one population was sampled

Species No. of 1 2 3 4 5 6 7
populations

1. Solea solea 7 0.303 0.000 0.000 0.000 0.000 0.000

(0.291–0.308)
2. Microchirus 2 1.195 0.000 0.000 0.000 0.000 0.000
variegatus (1.178–1.234)
3. Scophthalmus 1 0.574 0.000 0.000 0.000
maximus (0.572–0.575)
4. Scophthalmus 3 0.556 0.000 0.000 0.000
rhombus (0.553–0.559)
5. Pleuronectes 1 0.726 0.441
platessa (0.717–0.734)–
6. Pleuronectes 2 0.320 0.584
flesus (0.309–0.333) (0.579–0.590)
7. Limanda 1 0.819 0.538
limanda – (0.527–0.547)

estimates of genetic divergence between species are
not affected by the number of individuals examined
and depend almost entirely on the number of loci
analysed (Nei & Roychoudhury, 1974). Even sample
sizes as small as one individual will give fairly robust
estimates of I and D (Nei, 1978; Gorman & Renzi,
1979) and so the small sample sizes for some of the
species we studied are not important.
The results shown in the UPGMA and Wagner pro-
cedure generated trees (Figs 2, 3) show the plaice,
Pleuronectes platessa, and the flounder, Pleuronectes
flesus (formerly Platichthys flesus), as the most closely
related species pair and thus provide support for their
regrouping into a single genus (see Wheeler, 1992).
This is in accordance with what is known of the ability
to hybridize; plaice and flounder hybridize quite freely
in the Danish Belt Sea and western parts of the Baltic
(Ward & Galleguillos, 1978; Galleguillos & Ward, 1982)
and hybrid males are fertile (Sick et al., 1963). Our
results also show I values between 0.44 and 0.59
496 A. EXADACTYLOS and J. P. THORPE

0.0 0.2 0.4 0.6 0.8 1.0

Sole GRE
Sole FRA
Sole IOM
Sole CUM
Sole IRL
Sole EAN
Sole GER
Thickback IRL
Thickback IOM
Turbot IOM
Brill IOM
Brill IRL
Brill GRE
Plaice IOM
Flounder IOM
Flounder GRE
Dab IOM

Figure 2. UPGMA dendrogram using Nei’s (1978) Unbiased Genetic Identity (I) between the 17 populations of seven
species of flatfish. Goodness of fit statistics: f (Farris, 1972)=0.275; F (Prager & Wilson, 1976)=0.786; % standard
deviation (Fitch & Margoliash, 1967)=2.178; Cophenetic correlation=1.000.

0 0.09 0.18 0.27 0.35 0.44 0.53

Sole GRE
Sole FRA
Sole GER
Sole IOM
Sole IRL
Sole CUM
Sole EAN
Thickback IRL
Thickback IOM
Turbot IOM
Brill IOM
Brill IRL
Brill GRE
Plaice IOM
Flounder IOM
Flounder GRE
Dab IOM

Figure 3. Wagner procedure dendrogram using Prevosti’s distance (Wright, 1978) between the 17 populations of seven
species of flatfish. Goodness of fit statistics: f (Farris, 1972)=1.592; F (Prager & Wilson, 1976)=1.549; % standard
deviation (Fitch & Margoliash, 1967)=6.668; Cophenetic correlation=0.998.

between dab, Limanda limanda, and plaice and floun-
der, suggesting that all three species could be con-
sidered congeneric. However, over a larger sample of
allozyme loci Ward & Galleguillos (1983), in their major
genetic study of Pleuronectidae, obtained rather lower
I values between these three species. Their values for
dab/plaice and dab/flounder are borderline at around
0.35. The two species of Scophthalmus studied, turbot,
S. maximus and brill, S. rhombus appear to be correctly
placed in the same genus, although some authors (e.g.
Blanquer et al., 1992) have separated the turbot as
Psetta maximus. The two are sympatric over most of
their current geographic range, except on the Moroccan
coast where only turbot occur (Blanquer et al., 1992)
and it could be of interest to compare them using
samples from a wider area. The high level of genetic

divergence between Dover and thickback sole (I=0.29–
0.31) supports their current systematic positions in
different genera.
Galleguillos & Ward (1982) and Berrebi et al. (1983,
1985) reported the occurrence of two subspecies of
flounder in the Mediterranean basin, Pleuronectes fle-
sus italicus in the Adriatic Sea and Pleuronectes flesus
luscus in the Aegean and Black Seas. Both genetic
and morphological analyses indicated that subspecies
flesus (from the Atlantic) and luscus were more closely
related to each other than either was to italicus, sug-
gesting that the italicus stock may have separated
from flesus before luscus. Our results confirm the low
genetic divergence between the subspecies luscus and
flesus.
A surprising result is the apparently low level of
genetic divergence observed between the Aegean and
Atlantic brill populations. This contrasts with a clear
separation of Mediterranean Dover sole from those in
the Atlantic. Flatfishes of the families Scophthalmidae
and Pleuronectidae are considered to be of northern
origin (Greenwood et al., 1973). Paleohistorical
hypotheses concerning flounder (Berrebi, 1988) and
turbot (Renaud et al., 1990) suggest that the
Mediterranean populations originated from an At-
lantic parent population during cold episodes. A very
rapid and more recent south–north post-glacial re-
colonization could explain the low genetic dif-
ferentiation between geographically distant Atlantic
and Mediterranean brill populations, since this would
have been too recent to allow much differentiation.
Besides the possible contribution of these historical
events, evolutionary processes such as continuing high
levels of gene flow through possible adults migration or
passive exchange of pelagic larvae, may have reduced
levels of genetic differentiation between the Atlantic
and Mediterranean basins.
Values of genetic distance have been claimed to
increase linearly with time of evolutionary divergence.
Although this relationship can be controversial and
depends upon the validity of various theoretical as-
sumptions, there is little doubt that isolated popu-
lations diverge genetically with time (Nei, 1987;
Thorpe, 1989). Nei’s Genetic Distance, D, values be-
tween plaice and flounder indicate a divergence time
of approximately 1.6 Myr, whilst between turbot and
brill a divergence time of 2.6 Myr was estimated, using
a conservative calibration factor of 1 D=5 Myr (Nei,
1987). Reproductive isolation between these species is
still not complete, since occasional hybridization still
occurs in some areas and artificially produced hybrids
exhibit normal growth rates (Heap & Thorpe, 1987).
If it is speculated that about 5 Mya the families
Pleuronectidae and later Soleidae evolved from the
ancestral Scophthalmidae, this would be in agreement
with the suggestion of Lauder & Liem (1983) that
SYSTEMATICS IN PLEURONECTIFORMES 497
the Citharidae are the most primitive pleuronectiform
family, being ancestral to the Scophthalmidae and then
to the three remaining families (Bothidae, Pleuro-
nectidae and Soleidae). However, Verneau et al. (1994),
following Hubbs (1945), regarded the family Soleidae
as the most primitive family. Their electrophoretic data
indicated that the Pleuronectidae and Scophthalmidae
were sister taxa, although they have never been de-
scribed as such by Hubbs (1945) nor by Lauder & Liem
(1983) on the basis of morpho-anatomical criteria. In
the future analyses of flatfish interrelationships using,
for example, sequence data for nuclear and mito-
chondrial DNA or other molecular methods, may pro-
vide additional data in the many areas where flatfish
phylogeny remains unclear (see Hensley, 1997).
ACKNOWLEDGEMENTS
We thank P. Newton and his colleagues at DANI,
Belfast and the crew of the University of Liverpool
research vessel ‘Roagan’ for providing samples. We
also thank R.D.M. Nash and A.J. Geffen for advice
and comments on this study. A. Exadactylos ac-
knowledges the financial support of the Greek Schol-
arship Foundation.
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