ICES Journal of Marine Science, 62: 1699e1704 (2005)
doi:10.1016/j.icesjms.2005.06.001
Population structure of Merluccius merluccius
along the Iberian Peninsula coast
Ana G. F. Castillo, Paula Alvarez, and Eva Garcia-Vazquez
Castillo, A. G. F., Alvarez, P., and Garcia-Vazquez, E. 2005. Population structure of
Merluccius merluccius along the Iberian Peninsula coast. e ICES Journal of Marine
Science, 62: 1699e1704.
Prespawning hake caught in eight locations around the Iberian Peninsula were genetically
analysed. The distribution of variation at five microsatellite loci suggests that the species
follows a population model of isolation by distance in this geographical area. Three
different areas can be identified: the Mediterranean Sea, the Portuguese coast, and the
Cantabric Sea. The lack of differentiation between samples caught in the VIIIa,b,d and in
the VIIIc ICES Areas suggests that, based on genetic information, the boundary between
northern and southern stocks of European hake should be reconsidered.
Ó 2005 International Council for the Exploration of the Sea. Published by Elsevier Ltd. All rights reserved.
Keywords: Iberian Peninsula, Merluccius merluccius, microsatellites, population model,
stock structure.
Received 22 April 2004; accepted 10 June 2005.
A. G. F. Castillo and E. Garcia-Vazquez: Departamento de Biologia Funcional,
Universidad de Oviedo, C/Julian Claveria s/n, 33006 Oviedo, Spain. P. Alvarez: AZTITechnological Institute for Fisheries and Food, Herrera Kaia, Portualdea z/g, 20110
Pasaia (Gipuzkoa), Basque Country, Spain; tel: C34 943004800; e-mail: palvarez@
pas.azti.es; tel: C34 985102726; fax: C34 985103534; e-mail: [email protected].
Introduction
The European hake (Merluccius merluccius) is distributed
in the Northeast Atlantic, from the coast of Mauritania at
about 21(N to 62(N off the western coast of Norway and
the waters of Iceland (Pitcher and Alheit, 1995). The
economic value of hake in Europe is relatively high
compared with other markets, so hake in this area are
characterized by low catches and high market prices.
Management of the species is based on the assumption of
the existence of three different stocks: Mediterranean,
northern and southern. Differentiation of these stocks is not
very clear (Roldan et al., 1998). The boundary of the two
Atlantic stocks, Cap Breton Canyon, was defined mainly
based on management considerations (Anon., 2004).
Significant spatial differentiation within each stock was
described employing highly variable loci as genetic
markers (Lundy et al., 2000; Castillo et al., 2004), but
the biological basis of the separation between the two
Atlantic stocks, as recognized by ICES (International
Council for the Exploration of the Sea) remains unclear
(Lundy et al., 1999).
The relevance of genetic differentiation is particularly
important for developing hake stock management plans.
Annual catches of both northern and southern hake stocks
1054-3139/$30.00
decreased sharply in the past few decades. Recovery plans
are currently being considered, treating each stock
separately. A complete description of the genetic variation
of the species, at small oceanographic scales, will help to
precisely define stock boundaries. The next step would be
to develop protection plans or other management measures
on the basis of the indentified population structure.
There are three objectives of this study: (i) to examine
the population genetic structure of European hake along the
coast of the Iberian Peninsula, (ii) to evaluate the boundary
of the two Atlantic stocks, as well as the boundary of
Mediterranean and Atlantic hake located in Spanish waters,
and (iii) to analyse the genetic variation of prespawning
adult hake at five hypervariable microsatellite loci.
Material and methods
Sample collection
Prespawning adult hake were collected from eight locations
along the Iberian Peninsula coast (Figure 1) in September
2001. Table 1 shows the number of individuals analysed
per location. Four sampling locations were considered for
the northern stock (ICES Area VIIIa,b,d) and three for the
southern (ICES Area VIIIc-E, VIIIc-C, and VIIIc-W).
Ó 2005 International Council for the Exploration of the Sea. Published by Elsevier Ltd. All rights reserved.
1700
A. G. F. Castillo et al.
VIIIa,b,d
VIIIc-W
VIIIc-C
VIIIc-E
IX-N
IBERIAN
PENINSULA
IX-C
Med
IX-S
Figure 1. Map showing the locations where the samples were caught.
Within ICES Area IX (southern stock), we considered three
different locations at different latitudes along the Portuguese
and Spanish Atlantic coast, labelled from north to south as
IX-N, IX-C, and IX-S. Finally, we considered one sample
from the Spanish Mediterranean coast (Med, Mediterranean
hake stock). Two samples (VIIIa,b,d and Med) and some
Table 1. Samples analysed. Origin: ICES Area where the samples
were caught. n: number of samples analysed.
Sample
Sea
VIIIa,b,d* Atlantic OceaneBay
of Biscay
VIIIc-E
Atlantic OceaneBay
of Biscay
VIIIc-C
Atlantic OceaneBay
of Biscay
VIIIc-W Atlantic OceaneBay
of Biscay
IX-N
Atlantic Ocean
IX-C
Atlantic Ocean
IX-S
Atlantic Ocean
Med*
Mediterranean Sea
*From Castillo et al. (2004).
Origin
n
ICES VIIIa,b,d
60
ICES VIIIc East
19
ICES VIIIc Middle
43
ICES VIIIc West
44
ICES IX North
ICES IX Middle
ICES IX South
Southeast Spanish coast
20
38
49
55
individuals from the samples VIIIc-C and VIIIc-W had
been analysed previously (Castillo et al., 2004). As they
had been collected the same year, they were included in the
present analysis for a detailed study of local genetic
differentiation around the Iberian Peninsula.
A small portion (approximately 1 cm2) of gill or muscle
was removed from freshly caught fish and stored in 97%
ethanol at room temperature.
DNA extraction and microsatellite analysis
DNA was extracted following Estoup et al. (1996). The
following microsatellite loci were amplified by PCR: Mmer
Hk3b (GenBank accession number AF136627), Mmer
Hk9b (AF136628), Mmer Hk20 (AF136695) (Morán
et al., 1999); Mmer UEAW01 (X87461, Rico et al.,
1997); Mm 110-8 (AF121788, D’Amato et al., 1999). PCR
reactions were carried out in a total volume of 20 ml
containing: 10e20 ng of DNA; 20 pmol of each primer;
250 mM of each deoxynucleotide; MgCl2 (1.5 mM for
Mmer Hk9b, Mmer Hk3b and Mmer Hk20; 2 mM for
Mmer UEAW01; 2.5 mM for Mm110-8); 10 mM of
TriseHCl (pH 9.0 at 25(C); 50 mM of KCl; 0.1% of
TritonÒX-100; and 0.5 units of Taq DNA polymerase
(Promega). PCR consisted of an initial denaturing step at
95(C for 5 min; 35 cycles of 95(C for 20 s; annealing at
Population structure of M. merluccius
50(C, 53(C or 55(C for 20 s (Mmer Hk3b, Mmer Hk20,
and Mm 110-8, Mmer Hk9b, and Mmer UEAW01,
respectively); extension at 72(C for 20 s and a final
extension at 72(C for 7 min. For the new samples, the
size of the PCR products was determined by automated
fluorescent scanning detection employing an ABI 3100
automated sequencer (Perkin Elmer) and the GENESCAN
analysis software (ABI). Genotyping of the samples
previously analysed was carried out in acrylamide gels
(Castillo et al., 2004).
Data analysis
Allele frequencies, parameters of genetic variation within
samples (heterozygosities, mean number of alleles per
locus), and Nei (1978) genetic distances among samples
were obtained with the program Genetix 4.01 (Belkhir
et al., 1999). Conformity to the HardyeWeinberg equilibrium and statistical significance of heterozygote excess or
defect were determined with the program GENEPOP 3.3
(Raymond and Rousset, 1995) by the Markov chain method
with 1000 replicates. A global FST analysis and estimates of
gene flow among samples as the number of migrants per
generation Nem were also performed with the program
GENEPOP 3.3. Statistical significance of genetic differences between paired samples and hierarchical analysis of
the molecular variance (AMOVA) were obtained using
the program ARLEQUIN (Schneider et al., 2000). The
significance of the differences of genetic parameters (allele
richness, He, Ho, FST, FIS) between groups of samples was
tested with the computer package FSTAT (2001 updated
version of Goudet, 1995). Values of (dm)2 genetic distances
between samples (Goldstein et al., 1995) were estimated
employing the program Rst-CALC (Goodman, 1997). The
dendrograms based on genetic distances were constructed
by the method Neighbor-Joining (Saitou and Nei, 1987)
employing the program Neighbor of the computer package
Phylip 3.6.a.2 (Felsenstein, 1993) and were visualized with
the program TreeView 1.5 (Page, 1996). Mantel tests to
examine association between genetic and geographic
distances were performed with the program Genetix 4.01
(Belkhir et al., 1999).
Results
High genetic variation was found for all samples at all loci,
with some differences between loci (Table 2). In all, 65
alleles (size ranges 109e208 bp) were found at the locus
Mmer Hk9b, whereas the locus Mm 110-8 was less variable,
with 14 alleles (size ranges 132e180). The sample caught in
ICES Area VIIIa,b,d (northern Atlantic stock) was the most
variable (Table 2), with an average of 21 alleles per locus.
With respect to conformity to HardyeWeinberg genetic
equilibrium, the samples VIIIa,b,d, VIIIc-C, and
IX-N did not show significant deviations from the
equilibrium genotypic frequencies. For the other five
1701
Table 2. Parameters of genetic variability in the populations
studied. Private alleles, number of alleles exclusive of a population.
He, mean heterozygosity expected across loci under Hardye
Weinberg conditions. Ho, mean heterozygosity observed. NA,
mean number of alleles per locus.
Populations
Private
alleles
8
1
9
7
4
3
2
7
VIIIa,b,d
VIIIc-E
VIIIc-C
VIIIc-W
IX-N
IX-C
IX-S
Med
He
0.9038
0.8695
0.8936
0.8961
0.8698
0.8765
0.8766
0.7858
Ho
(0.0511)
(0.0441)
(0.0614)
(0.0476)
(0.0678)
(0.0726)
(0.0610)
(0.2075)
0.8948
0.7210
0.8747
0.8275
0.8125
0.8229
0.7784
0.7061
(0.0642)
(0.2018)
(0.0373)
(0.1583)
(0.1250)
(0.0846)
(0.0691)
(0.1887)
NA
21.4
12.8
18.6
18.4
14.2
18.0
17.2
19.4
populations, significant deviations from HardyeWeinberg
were found at one or two loci. These deviations were always
due to a significant deficit of heterozygotes with respect to
those expected under HardyeWeinberg conditions. The
Mediterranean sample had a lower mean heterozygosity,
both observed (0.7061) and expected (0.7858), than the
Atlantic samples at these five microsatellite loci. However,
there were no significant differences at any parameter of
genetic variability (allele richness, heterozygosity values)
between Bay of Biscay, Atlantic, and Mediterranean
samples.
With respect to the genitic differentiation between
samples, p values for each population pair across all loci
were statistically significant in most cases (Table 3).
However, they were not significant for most neighbour
samples. For example, VIIIa,b,d and VIIIc-E were not
statistically different (p Z 0.111). The same occurred for
the population pairs VIIIc-E and VIIIc-C (p Z 0.199),
VIIIc-C and VIIIc-W (p Z 0.111), VIIIc-W and IX-N
(p Z 0.158), and so on. The statistically different neighbour
samples were IX-S and Med (p ! 0.001). These results
suggest a population model of isolation by distance.
Table 3. Statistical significance (p values) of pairwise genetic
differentiation between samples.
VIII
a,b,d
VIII
c-E
VIII
c-C
VIII
c-W
IX-N
IX-C
IX-S
VIIIc-E 0.111
e
e
e
e
e
e
VIIIc-C 0.049 0.199
e
e
e
e
e
VIIIc-W!0.001 0.008 0.111
e
e
e
e
IX-N
0.081 0.123 0.154 0.158
e
e
e
IX-C
0.355 0.012 0.602!0.001 0.101
e
e
IX-S
!0.001!0.001 0.004!0.001!0.001 0.056
e
Med
!0.001!0.001!0.001!0.001!0.001!0.001!0.001
1702
A. G. F. Castillo et al.
Although most genetic variation was due to withinsample variation, FST (0.0189); statistically significant
(p Z 0.003) was 20.2% of total FIT (0.0935). Estimated
gene flow among the whole eight populations was
Nem Z 8.39 migrants per generation, after correction for
size (mean sample size 35.4). Genetic variance was due to
not only differences between the Mediterranean sample and
the rest, but also significant differences between Bay of
Biscay and Atlantic samples (Area VIII vs. Area IX).
Molecular variance due to differences between the two
groups of samples, although very low (0.75% total
variance), was statistically significant (p Z 0.026).
Table 4 presents a matrix with two genetic distances
between populations, (dm)2 (below diagonal) and Nei (1978)
(above diagonal). The tree constructed based on (dm)2
genetic distances is given in Figure 2. It is remarkable that
samples clustered by (macro)geographic distance, the
Mediterranean one apart of the rest. Samples from Area
IX clustered, closer to the Mediterranean sample than
samples from Area VIII. The apparent association between
geographic and genetic distances was confirmed with
Mantel tests. The coefficient of Pearson r between Nei
(1978) genetic and geographic distances was 0.810
(p ! 0.01). (dm)2 genetic distances were also significantly
associated with geographic distances between samples
(r Z 0.786, p ! 0.05).
Discussion
Genetic variation described in this work for the hake
populations around the Iberian Peninsula was similar to that
previously reported for these regions (Lundy et al., 1999;
Castillo et al., 2004). A high number of alleles per locus are
expected for these hypervariable microsatellites, permitting
description of fine population structure, if it exists. It seems
to be the case for European hake stocks around the Iberian
Peninsula. These stocks follow a population model of
isolation by distance. Significant Mantel tests were
obtained for both (dm)2 and Nei (1978) genetic distances
and geographic distances. Prespawning adult hakes sampled around the Iberian Peninsula seem to be a continuum;
neighbouring samples are genetically similar, but genetic
differences arise between samples caught apart. Genetic
differentiation seems to be a function of geographical
distance. The discontinuity between the Mediterranean and
the Atlantic hake stocks (Roldan et al., 1998; Lo Brutto
et al., 2004; Castillo et al., 2004) is again supported by
these results, as the Strait of Gibraltar may be considered
a barrier to gene flow for this and other marine fish such as
swordfish (Kotoulas et al., 1995) and sparids (Bargelloni
et al., 2003).
Lundy et al. (1999) and Castillo et al. (2004) also
reported (macro)geographic population differentiation in
Atlantic Merluccius merluccius. Other marine fish with
high dispersal potential also exhibit genetic structuring
among Atlantic stocks, for example the Atlantic mackerel
Scomber scombrus (Nesbø et al., 2000) and the Atlantic
cod Gadus morhua (Nielsen et al., 2001). Present results,
obtained on a smaller oceanographic scale, demonstrate
a clear differentiation between prespawning adults caught in
quite proximal oceanographic areas without an apparent
geographical discontinuity. Hake spawning in the Bay of
Biscay, as a whole, seem to have subtle but significant
genetic differences compared with hake spawning along the
Atlantic coast of the Iberian Peninsula. This suggests the
existence of a sort of homing. Adults could return to spawn
near or adjacent to the area where they were born. A fine
genetic structuring has also been reported for cod, with
highly significant differences observed among populations
at DNA markers in a pattern consistent with an isolationby-distance model of population structure (Pogson et al.,
1995). It is very difficult to know at present if this model is
also suitable for hake, because the adults studied in this
work were not spawning but prespawning adults. A more
accurate definition of the spawning stocks could be
obtained from analysis of young embryos at the spawning
sites before their drift.
These results are relevant for management purposes. The
boundary thought to exist between northern and southern
hake stocks, which should be between ICES Area VIIIa,b,d
Table 4. Matrix of genetic distances between the populations analysed. Nei (1978) distances, above diagonal. (dm)2 distances, below
diagonal.
VIIIa,b,d
VIIIc-E
VIIIc-C
VIIIc-W
IX-N
IX-C
IX-S
Med
VIIIa,b,d
VIIIc-E
VIIIc-C
VIIIc-W
IX-N
IX-C
IX-S
Med
e
0.4114
0.7143
0.7702
0.4083
1.1524
1.2436
3.7716
0.040
e
0.2267
0.1911
0.1607
0.3320
0.2637
3.5488
ÿ0.015
0.003
e
0.6492
0.4553
0.4352
0.4737
4.7857
0.084
0.091
0.029
e
0.2510
0.3357
0.3311
3.3204
ÿ0.001
0.042
0.015
0.036
e
0.2326
0.3751
2.6462
0.000
0.057
ÿ0.040
0.073
ÿ0.004
e
0.1374
3.1447
0.070
0.126
0.040
0.073
0.078
0.022
e
3.0864
0.359
0.377
0.323
0.311
0.289
0.258
0.238
e
Population structure of M. merluccius
1703
VIIIa,b,d
VIIIc-E
IX-N
VIIIc-W
VIIIc-C
IX-S
IX-C
Med
0.1
Figure 2. Neighbor-Joining tree constructed on the basis of (dm)2 distances between samples.
and VIIIc, is not supported by our results of microsatellite
variation. The sample caught in Area VIIIa,b,d was not
statistically different from that caught in Area VIIIc-E
(East). Taking into account the high variability found at the
markers employed in this study, significant genetic differences, if they exist, should have been revealed as they
appear between other sample pairs. Thus, the boundary
between both stocks should be reconsidered based on
biological evidence, as suggested by Lundy et al. (1999)
and the ICES Working Group on the Assessment of
Southern Shelf Stocks of Hake, Monk, and Megrim (Anon.,
2004).
The existence of subtle genetic differentiation between
samples caught in different geographical locations around
the Iberian Peninsula is also important for future plans for
population restoration. These results suggest the existence
of several spawning areas in which the breeders are
genetically different. All efforts at protecting hake spawning
areas should then be concentrated in more than one or two
points around the Peninsula, given the apparently complex
genetic structure of the Iberian hake.
Acknowledgements
We are indebted to Ivan Gonzalez Pola for his collaboration
in laboratory tasks. This work has been supported by EU
Contract MARINEGGS QLK5-CT1999-01157.
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