ICES Journal of Marine Science: Journal du Conseil Advance Access originally published online on December 17, 2007
ICES Journal of Marine Science: Journal du Conseil 2008 65(1):12-16; doi:10.1093/icesjms/fsm178
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Genetic structure of Octopus vulgaris around the Iberian Peninsula and Canary Islands as indicated by microsatellite DNA variation
1 Centro de Experimentación Pesquera, Consejería de Medio Rural y Pesca, Avenida Principe de Asturias s/n, 33212 Gijón, Asturias, Spain
2 Unidad de Secuenciación, Servicios Científico-Técnicos, Universidad de Oviedo, Campus "El Cristo", 33006 Oviedo, Asturias, Spain
Correspondence to C. Cabranes: tel: +34 985 319711; fax: +34 985 312899; e-mail: carmecb{at}princast.es
Cabranes, C., Fernandez-Rueda, P., and Martínez, J. L. 2008. Genetic structure of Octopus vulgaris around the Iberian Peninsula and Canary Islands as indicated by microsatellite DNA variation. – ICES Journal of Marine Science, 65: 12–16.Microsatellite DNA markers were used for a genetic study of Octopus vulgaris, a cephalopod species of great commercial interest to Spain and Portugal, and therefore subjected to intensive fishing. Improving the demographic knowledge of marine resources supports more-responsible management and conservation. Genetic variation at five microsatellite loci screened in six samples from NE Atlantic and Mediterranean coasts of the Iberian Peninsula was high [mean number of alleles = 18.3, mean He = 0.874]. Analysis of the microsatellites allowed significant subpopulation structure to be identified, consistent with an isolation-by-distance model for Atlantic populations. Differences between pairs of samples separated by <200 km were not significant. From a fisheries management perspective, the results support coordinated management of neighbouring stocks of O. vulgaris around the Iberian Peninsula.
Keywords: genetic structure, microsatellite DNA, Octopus vulgaris, population differentiation
Received 6 August 2007; accepted 6 November 2007; advance access publication 17 December 2007.
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Introduction |
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 Top Introduction Material and methodsResultsDiscussionReferences |
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Octopus vulgaris is a benthic cephalopod, distributed broadly on rocky, sandy, and muddy substrata from the coast to the edge of the continental shelf at depths up to 200 m and in diverse habitats. The species has been long considered a cosmopolitan resident of temperate and tropical seas (Roper et al., 1984), but the possible occurrence of cryptic species among O. vulgaris-like octopods has been reported (Guerra et al., 1999; Söller et al., 2000). The species has a lifespan of
1 year, and juvenile recruitment is sensitive to unpredictable environmental fluctuations. In some cases, uncontrolled harvesting in certain areas makes it essential to have a clear picture of population substructuring, to allow rational management of the resource.
Octopus vulgaris fixes its eggs in rocky caves or to an appropriate substratum, and there is a paralarval phase, but adults have limited migratory capacity (Guerra, 1992). It is thought that adult O. vulgaris forage around a "home-range" of 15 m (Mather, 1993), but may make inshore–offshore migrations related to spawning (Mangold, 1983). The currents that transport fish larvae may also transport octopus paralarvae, so there is scope for wider octopus dispersal during the early stages of life.
The species is of great interest as a commercial resource for Spain and Portugal and is therefore subjected to heavy fishing, carried out by trawling and by various small-scale gears, such as traps, pots, fykenets, and setnets. Total annual cephalopod landings in the Iberian Peninsula have ranged between 11 151 and 17 514 t for the past 9 years; the catches represent 97–99% of the total catch of the species in the whole ICES area (ICES, 2006). Despite such commercial interest, studies on the identity and distribution of Iberian stocks are scarce.
Molecular genetic approaches have been applied successfully to stock discrimination studies in fisheries (Murphy et al., 2002). Knowledge of the genetic structure of a species can be a useful tool for management and can be used to determine whether a locally collapsed stock can be repopulated by immigrants. Such information may assist in the identification of different stocks of exploited species, characterized by different population parameters such as recruitment and mortality patterns (Maltagliati et al., 2002). Large stocks of exploited species may be impacted by harvesting even when they show just modest population decline. This may occur through genetic erosion resulting from genetic drift, inbreeding, prolonged bottlenecks, or through lessened fitness resulting from chance fixation of detrimental alleles (Ryman et al., 1995).
Genetic markers can produce evidence of stock separation, so providing the basis for better management of whole populations and thence sustainable fisheries. Microsatellite DNA loci have been extensively used in population studies, because they provide highly polymorphic loci that can identify fine-scale structuring (Shaw et al., 1999; Casu et al., 2002; Perez-Losada et al., 2002; Murphy et al., 2002; Castillo et al., 2005). Recently, microsatellite markers have been isolated for O. vulgaris (Greatorex et al., 2000).
The aim of the present study was to use microsatellite DNA markers to enhance knowledge of genetic structuring in the O. vulgaris population around the Iberian Peninsula and Canary Islands.
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Material and methods |
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TopIntroductionMaterial and methodsResultsDiscussionReferences |
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Sampling
Six samples of O. vulgaris were collected at five Atlantic sites and one in the Mediterranean Sea, around the Iberian Peninsula and Canary Islands, between March 2005 and March 2006. One sample was from the Cantabrian Sea, north of the Iberian Peninsula (Asturias, n = 34), three from the west coast of the Iberian Peninsula (Galicia, n = 48; Portugal, n = 48; and Cádiz, n = 35), another from the Canary Islands (n = 33), and the last from the Mediterranean Sea, east of the Iberian Peninsula (Murcia, n = 48) (Figure 1). All samples were from adult octopuses. From each animal, a small piece of muscular tissue from the tip of the arm was excised and preserved in absolute ethanol.
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DNA extraction and microsatellite analysis
DNA was extracted following the Chelex-based method described by Estoup et al. (1996). All six samples were screened for variation at five polymorphic microsatellite loci (Oct3, Oct8, Ov6, Ov10, and Ov12) previously isolated and characterized for O. vulgaris by Greatorex et al. (2000). PCR reactions were carried out under conditions set out in Greatorex et al. (2000) in a total volume of 20 µl. Amplification products were resolved on an ABI PRISM 3100 Genetic Analyser, and analysed using GeneMapper v.3.5 software (Applied Biosystems).
Data analysis
The software Micro-Checker 2.2.3 (Van Oosterhout et al. 2004) was used to identify possible genotyping errors (i.e. stuttering, large allele dropout, and null alleles) within the microsatellite dataset by performing 1000 randomizations. Microsatellite polymorphism within samples was measured as the mean number of alleles (Na) per locus, and observed and unbiased expected heterozygosity was calculated using the GENETIX 4.02 software package (Belkhir et al., 1996). Deviations from Hardy–Weinberg equilibrium and the statistical significance of heterozygote excess or deficit were tested using the Fisher’s exact test, with the level of significance determined by a Markov chain method using GENEPOP 3.3 software (Raymond and Rousset, 1995). The same software was used to test for genotypic linkage disequilibrium for each pair of loci in each population.
To estimate genetic differentiation among samples, two methods were used. First, we tested for simple frequency differentiation between pairs of samples with Fisher’s exact test implemented in the GENEPOP 3.3 software package. Second, we estimated the pairwise genetic differentiation among samples using FST (Weir, 1996) with the FreeNA software package (Chapuis and Estoup, 2007).
To quantify genetic affinities among samples, pairwise Chord distances, Dchord, (Cavalli-Sforza and Edwards, 1967) were calculated from a dataset corrected for null alleles using the FreeNA software package (Chapuis and Estoup, 2007). The correlation coefficient between the matrix of Chord genetic distances and geographical distances was calculated and its probability estimated by a Mantel test. Both genetic distances and the Mantel test were calculated using the GENETIX 4.02 software package (Belkhir et al., 1996).
A dendrogram based on Chord genetic distances (Cavalli-Sforza and Edwards, 1967) was constructed with bootstrap support for branches (2000 replicates) using the UPGMA method of clustering, employing the program NEIGHBOR of the PHYLIP 3.6 computer package (Felsenstein, 1993). Bootstrap support on branches is computed by resampling loci with the program SEQBOOT of the PHYLIP 3.6 computer package (Felsenstein, 1993). The dendrogram was visualized with the program MEGA.4 (Tamura et al., 2007).
A sequential Bonferroni technique (Rice, 1989) was used to adjust significance levels for multiple simultaneous comparisons.
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Results |
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TopIntroductionMaterial and methodsResultsDiscussionReferences |
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Estimates of variability at the five microsatellite DNA loci within all population samples, heterozygosity (Ho and He) within and means across loci and samples, size distributions and mean Na across loci, and tests for deviation from Hardy–Weinberg outcrossing expectations within loci are listed in Table 1. All samples revealed a high level of genetic variability. The locus Ov12 had the most alleles (56), and the locus Ov06 the least (24).
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Within-sample variability was uniformly high across all samples: the mean Na ranged between 16.2 from the Canary Islands and 20.0 from Murcia. The mean observed (Ho) and unbiased expected heterozygosity (He) ranged between 0.664 and 0.837, and between 0.835 and 0.909, respectively. In terms of conformity to Hardy–Weinberg genetic equilibrium, 13 of 30 single-locus tests for deviations from outcrossing predictions yielded significant results. All populations showed significant deviations from Hardy–Weinberg equilibrium for locus Oct03; the Canary Islands and Portugal showed significant deviations from Hardy–Weinberg equilibrium for locus Oct08, and all populations except Murcia showed significant deviations from equilibrium for locus Ov12. All tests except one (Oct08 from Portugal) remained significant when adjusted for table-wide significance by a sequential Bonferroni procedure. These deviations were always attributable to a significant deficit of heterozygotes with respect to those expected under Hardy–Weinberg conditions.
The presence of null alleles was detected for locus Oct03 in all populations and for locus Ov12 in all populations except the Canary Islands and Murcia. Locus Oct08 showed null alleles only for Portugal and the Canary Islands, and for loci Ov06 and Ov10, null alleles were not detected for any population.
In terms of genetic differentiation between samples, p-values estimated by pairwise differentiation were statistically significant in all cases except Portugal–Cádiz (Table 2). However, after sequential Bonferroni adjustment, Asturias–Galicia was not statistically significant (p < 0.05). Estimation of FST indicated significant levels of intersample genetic variance in all pairwise comparisons except Portugal–Cádiz (p < 0.05), as shown in Table 2. Chord genetic distances (Cavalli-Sforza and Edwards, 1967) were lowest between the geographically closest pairs of samples (Portugal–Cádiz, 0.018; Table 3).
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There was a pattern of increasing genetic differences with increasing geographic distance between samples for Atlantic material, shown by the Mantel test for Chord genetic distance (Cavalli-Sforza and Edwards, 1967; p = 0.03). Otherwise, the Mantel test failed to reveal isolation-by-distance if Mediterranean and Atlantic populations were considered.
The UPGMA dendrogram constructed from Chord genetic distances (Cavalli-Sforza and Edwards, 1967) is shown in Figure 2. The fact that samples clustered by geographic distance is remarkable. Neighbouring populations such as Portugal and Cádiz, and Asturias and Galicia grouped together, whereas the Mediterranean sample (Murcia) was separate from the rest.
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Discussion |
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TopIntroductionMaterial and methodsResultsDiscussionReferences |
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The utility of microsatellite DNA markers for examining subtle population genetic structuring within the Cephalopoda has been demonstrated previously (Shaw et al., 1999; Perez-Losada et al., 2002). Our study has shown a high degree of genetic variability within and between populations of O. vulgaris at all microsatellite loci, a finding particularly notable for Ov12 with its 56 alleles. In previous studies, Casu et al. (2002) found a similar Na (21) for the locus Ov06, and Murphy et al. (2002) found a high level of polymorphism in O. vulgaris for the loci Ov10 and Ov12. For locus Ov12, the mean Na we determined was lower than obtained by Murphy et al. (2002), whereas for locus Ov10, the polymorphism was similar in both studies. The differences between the results of the two studies could be a consequence of the different techniques used to resolve PCR products and/or the different sampling area.
Observed and expected heterozygosity values were high (mean Ho 0.750, mean He 0.874), in accord with earlier studies on cephalopods (Shaw et al., 1999; Perez-Losada et al., 2002; Garoia et al., 2004) and on O. vulgaris specifically (Casu et al., 2002; Murphy et al., 2002).
Deviations from Hardy–Weinberg equilibrium were observed for some populations, basically for loci Oct03 and Ov12, owing to a deficit of heterozygotes in terms of those expected. The presence of null alleles was confirmed as the cause of the deviation. Deficiencies in heterozygous genotypes have been found before in cephalopod populations and suggest the presence of non-amplified alleles (null alleles) as the cause of the observed departures from Hardy–Weinberg equilibrium (Shaw et al., 1999; Perez-Losada et al., 2002).
A previous study of the genetic structure of the Ov06 O. vulgaris microsatellite locus from the Mediterranean Sea, but which included an Atlantic Ocean sample (Vig), detected deficiencies of heterozygous genotypes for most of the populations analysed, and the presence of null alleles was proposed as a possible cause (Casu et al., 2002). If deviations were the result of the presence of null alleles, they would probably be found in all samples, but in the present study, the Ov06 locus was in Hardy–Weinberg equilibrium for all populations. The Atlantic sample (Vig) analysed by Casu et al. (2002) and the sample for Galicia analysed here were from the same population, so differences found with regard to Hardy–Weinberg equilibrium could be caused by temporal variation in the population as well as differences in the techniques employed.
Microsatellite loci Ov10 and Ov12 were also employed in an earlier genetic study of O. vulgaris (Murphy et al., 2002). In that study, most populations showed close conformity to Hardy–Weinberg equilibrium for the Ov10 microsatellite locus and significant deviations for the Ov12 microsatellite, so supporting the results of the present study for these microsatellite loci.
Two estimators of genetic divergence (exact test of sample differentiation and FST) were used to test the extent of genetic difference within populations of O. vulgaris. The exact test and FST gave similar results for most samples and significant levels of inter-sample differentiation for O. vulgaris around the Iberian Peninsula and Canary Islands, except for Portugal–Cádiz. Asturias–Galicia revealed no significant difference with an exact test after sequential Bonferroni correction.
A notable result of our study is the existence of a fine spatial substructure in O. vulgaris populations in the Atlantic which is a function of geographical distance. Significant Mantel tests were obtained for Chord genetic distances (Cavalli-Sforza and Edwards, 1967), and those results showed a population model of isolation-by-distance for the Atlantic populations.
Previous studies of the genetic structure of O. vulgaris from the Mediterranean Sea using allozymes (Maltagliati et al., 2002) and microsatellite loci (Casu et al., 2002) excluded isolation-by-distance in O. vulgaris Mediterranean populations. Maltagliati et al. (2002) suggested that O. vulgaris in the Mediterranean followed a basic island model in a background of high gene flow. One explanation for the different results could be the difference in geographical area studied.
Additionally, in the case of the Maltagliati et al. (2002) result, we can explain divergence between results because they used allozyme electrophoresis to investigate genetic variability. Previous studies (Shaw et al., 1999; Perez-Losada et al., 2002) found that microsatellites have a greater power than allozymes to resolve genetic relationships among closely related subpopulations of aquatic species, with a facility for gene flow on a small geographical scale, and are more suitable for resolving historical relationships at an intraspecific level. In the case of the analysis of Casu et al. (2002), the use of just one microsatellite (Ov6) may not have been powerful enough to find associations of genetic differentiation with geographic distribution of samples.
In this study, divergence was observed with the distance isolation model when all populations (Atlantic and Mediterranean) were included in the analysis. A possible explanation could be that the Atlantic and the Mediterranean have been isolated several times through the course of history, perhaps associated with substantial environmental changes in the latter (Maldonado, 1985; Bianco, 1990).
Our results failed to show significant differences between pairs of samples separated by <200 km (Portugal–Cádiz), so from a fisheries management perspective, the results may be considered as supporting coordinated management of neighbouring stocks around the Iberian Peninsula.
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Acknowledgements |
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We thank several people at the "Instituto Canario Ciencias Marinas", CIFAP, IMIDA, "D. Xeneral Recursos Mariños" (Spain), and IPIMAR (Portugal) who collected the tissue samples for this work, especially J. Ro, J. L. Muñoz, J. Cerezo, R. Arnaiz, and M. Gaspar. We also thank the Luarca and Puerto de Vega Fishermen’s Guilds staff for their collaboration in the collection of samples in Asturias. Eva García Vazquez, Gonzalo Machado, and Daniel Campo Falgueras collaborated in the statistical analysis, and M. Casu and an anonymous referee made valuable comments on the submitted manuscript.
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