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ICES Journal of Marine Science: Journal du Conseil 2005 62(8):1699-1704; doi:10.1016/j.icesjms.2005.06.001
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© 2005 International Council for the Exploration of the Sea

Population structure of Merluccius merluccius along the Iberian Peninsula coast

Ana G.F. Castilloa, Paula Alvarezb and Eva Garcia-Vazqueza,*

a Departamento de Biologia Funcional, Universidad de Oviedo C/Julian Claveria s/n, 33006 Oviedo, Spain
b AZTI-Technological Institute for Fisheries and Food, Herrera Kaia Portualdea z/g, 20110 Pasaia (Gipuzkoa), Basque Country, Spain

*tel: +34 943004800; tel: +34 985102726; fax: +34 985103534. e-mail: egv{at}fq.uniovi.es; palvarez{at}pas.azti.es.

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.

Keywords: Iberian Peninsula, Merluccius merluccius, microsatellites, population model, stock structure

Received 22 April 2004; accepted 10 June 2005.


   Introduction
Top
 Introduction
Material and methods
 Results
 Discussion
 References
 
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 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
Top
 Introduction
 Material and methods
Results
 Discussion
 References
 
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). 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 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.


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  		Table 1 Samples analysed. Origin: ICES Area where the samples were caught. n: number of samples analysed.

 


Figure 1
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  		Figure 1 Map showing the locations where the samples were caught.

 
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 [GenBank] ), Mmer Hk9b (AF136628 [GenBank] ), Mmer Hk20 (AF136695 [GenBank] ) (Morán et al., 1999); Mmer UEAW01 (X87461 [GenBank] , Rico et al., 1997); Mm 110-8 (AF121788 [GenBank] , D'Amato et al., 1999). PCR reactions were carried out in a total volume of 20 µl containing: 10–20 ng of DNA; 20 pmol of each primer; 250 µM of each deoxynucleotide; MgCl2 (1.5 µM for Mmer Hk9b, Mmer Hk3b and Mmer Hk20; 2 µM for Mmer UEAW01; 2.5 µM for Mm110-8); 10 mM of Tris–HCl (pH 9.0 at 25°C); 50 mM of KCl; 0.1% of Triton®X-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 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 Hardy–Weinberg 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 ({delta}µ)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
Top
 Introduction
 Material and methods
 Results
Discussion
 References
 
High genetic variation was found for all samples at all loci, with some differences between loci (Table 2). In all, 65 alleles (size ranges 109–208 bp) were found at the locus Mmer Hk9b, whereas the locus Mm 110-8 was less variable, with 14 alleles (size ranges 132–180). 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 Hardy–Weinberg 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 populations, significant deviations from Hardy–Weinberg were found at one or two loci. These deviations were always due to a significant deficit of heterozygotes with respect to those expected under Hardy–Weinberg 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.


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  		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 Hardy–Weinberg conditions. Ho, mean heterozygosity observed. NA, mean number of alleles per locus.

 
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 = 0.111). The same occurred for the population pairs VIIIc-E and VIIIc-C (p = 0.199), VIIIc-C and VIIIc-W (p = 0.111), VIIIc-W and IX-N (p = 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.


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  		Table 3 Statistical significance (p values) of pairwise genetic differentiation between samples.

 
Although most genetic variation was due to within-sample variation, FST (0.0189); statistically significant (p = 0.003) was 20.2% of total FIT (0.0935). Estimated gene flow among the whole eight populations was Nem = 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 = 0.026).

Table 4 presents a matrix with two genetic distances between populations, ({delta}µ)2 (below diagonal) and Nei (1978) (above diagonal). The tree constructed based on ({delta}µ)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). ({delta}µ)2 genetic distances were also significantly associated with geographic distances between samples (r = 0.786, p < 0.05).


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  		Table 4 Matrix of genetic distances between the populations analysed. Nei (1978) distances, above diagonal. ({delta}µ)2 distances, below diagonal.

 


Figure 2
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  		Figure 2 Neighbor-Joining tree constructed on the basis of ({delta}µ)2 distances between samples.

 

   Discussion
Top
 Introduction
 Material and methods
 Results
 Discussion
References
 
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 ({delta}µ)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 isolation-by-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 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.


   References
Top
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 Material and methods
 Results
 Discussion
 References
 

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