Genetic structure of Octopus vulgaris around the Iberian Peninsula and Canary Islands as indicated by microsatellite DNA variation
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12
Genetic structure of Octopus vulgaris around the Iberian
Peninsula and Canary Islands as indicated by
microsatellite DNA variation
C. Cabranes, P. Fernandez-Rueda, and J. L. Martı́nez
Cabranes, C., Fernandez-Rueda, P., and Martı́nez, 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 ¼ 18.3, mean He ¼ 0.874]. Analysis of the micro-
satellites allowed significant subpopulation structure to be identified, consistent with an isolation-by-distance model for Atlantic
Downloaded from http://icesjms.oxfordjournals.org/ by guest on October 26, 2015
populations. Differences between pairs of samples separated by ,200 km were not significant. From a fisheries management perspec-
tive, 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.
C. Cabranes and P. Fernandez-Rueda: Centro de Experimentación Pesquera, Consejerı́a de Medio Rural y Pesca, Avenida Principe de Asturias s/n,
33212 Gijón, Asturias, Spain. J. L. Martı́nez: Unidad de Secuenciación, Servicios Cientı́fico-Técnicos, Universidad de Oviedo, Campus “El Cristo”,
33006 Oviedo, Asturias, Spain. Correspondence to C. Cabranes: tel: þ34 985 319711; fax: þ34 985 312899; e-mail: carmecb@princast.es
Introduction (ICES, 2006). Despite such commercial interest, studies on the
Octopus vulgaris is a benthic cephalopod, distributed broadly on identity and distribution of Iberian stocks are scarce.
rocky, sandy, and muddy substrata from the coast to the edge of Molecular genetic approaches have been applied successfully to
the continental shelf at depths up to 200 m and in diverse habitats. stock discrimination studies in fisheries (Murphy et al., 2002).
The species has been long considered a cosmopolitan resident of Knowledge of the genetic structure of a species can be a useful
temperate and tropical seas (Roper et al., 1984), but the possible tool for management and can be used to determine whether a
occurrence of cryptic species among O. vulgaris-like octopods locally collapsed stock can be repopulated by immigrants. Such
has been reported (Guerra et al., 1999; Söller et al., 2000). The information may assist in the identification of different stocks of
species has a lifespan of 1 year, and juvenile recruitment is sen- exploited species, characterized by different population par-
sitive to unpredictable environmental fluctuations. In some cases, ameters such as recruitment and mortality patterns (Maltagliati
uncontrolled harvesting in certain areas makes it essential to have et al., 2002). Large stocks of exploited species may be impacted
a clear picture of population substructuring, to allow rational by harvesting even when they show just modest population
management of the resource. decline. This may occur through genetic erosion resulting from
Octopus vulgaris fixes its eggs in rocky caves or to an appropri- genetic drift, inbreeding, prolonged bottlenecks, or through les-
ate substratum, and there is a paralarval phase, but adults have sened fitness resulting from chance fixation of detrimental alleles
limited migratory capacity (Guerra, 1992). It is thought that (Ryman et al., 1995).
adult O. vulgaris forage around a “home-range” of 15 m Genetic markers can produce evidence of stock separation, so
(Mather, 1993), but may make inshore –offshore migrations providing the basis for better management of whole populations
related to spawning (Mangold, 1983). The currents that transport and thence sustainable fisheries. Microsatellite DNA loci have
fish larvae may also transport octopus paralarvae, so there is scope been extensively used in population studies, because they
for wider octopus dispersal during the early stages of life. provide highly polymorphic loci that can identify fine-scale struc-
The species is of great interest as a commercial resource for turing (Shaw et al., 1999; Casu et al., 2002; Perez-Losada et al.,
Spain and Portugal and is therefore subjected to heavy fishing, 2002; Murphy et al., 2002; Castillo et al., 2005). Recently, micro-
carried out by trawling and by various small-scale gears, such as satellite markers have been isolated for O. vulgaris (Greatorex
traps, pots, fykenets, and setnets. Total annual cephalopod land- et al., 2000).
ings in the Iberian Peninsula have ranged between 11 151 and The aim of the present study was to use microsatellite DNA
17 514 t for the past 9 years; the catches represent 97 –99% markers to enhance knowledge of genetic structuring in the O. vul-
of the total catch of the species in the whole ICES area garis population around the Iberian Peninsula and Canary Islands.
# 2007 International Council for the Exploration of the Sea. Published by Oxford Journals. All rights reserved.
For Permissions, please email: journals.permissions@oxfordjournals.orgGenetic structure of Octopus vulgaris around the Iberian Peninsula and Canary Islands 13
Material and methods equilibrium and the statistical significance of heterozygote excess
Sampling or deficit were tested using the Fisher’s exact test, with the level
of significance determined by a Markov chain method using
Six samples of O. vulgaris were collected at five Atlantic sites and
GENEPOP 3.3 software (Raymond and Rousset, 1995). The
one in the Mediterranean Sea, around the Iberian Peninsula and
same software was used to test for genotypic linkage disequili-
Canary Islands, between March 2005 and March 2006. One
brium for each pair of loci in each population.
sample was from the Cantabrian Sea, north of the Iberian
To estimate genetic differentiation among samples, two
Peninsula (Asturias, n ¼ 34), three from the west coast of the
methods were used. First, we tested for simple frequency differen-
Iberian Peninsula (Galicia, n ¼ 48; Portugal, n ¼ 48; and Cádiz,
tiation between pairs of samples with Fisher’s exact test
n ¼ 35), another from the Canary Islands (n ¼ 33), and the last
implemented in the GENEPOP 3.3 software package. Second, we
from the Mediterranean Sea, east of the Iberian Peninsula
estimated the pairwise genetic differentiation among samples
(Murcia, n ¼ 48) (Figure 1). All samples were from adult octo-
using FST (Weir, 1996) with the FreeNA software package
puses. From each animal, a small piece of muscular tissue from
(Chapuis and Estoup, 2007).
the tip of the arm was excised and preserved in absolute ethanol.
To quantify genetic affinities among samples, pairwise Chord
distances, Dchord, (Cavalli-Sforza and Edwards, 1967) were calcu-
DNA extraction and microsatellite analysis lated from a dataset corrected for null alleles using the FreeNA
DNA was extracted following the Chelex-based method described software package (Chapuis and Estoup, 2007). The correlation
by Estoup et al. (1996). All six samples were screened for variation coefficient between the matrix of Chord genetic distances and geo-
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at five polymorphic microsatellite loci (Oct3, Oct8, Ov6, Ov10, graphical distances was calculated and its probability estimated by
and Ov12) previously isolated and characterized for O. vulgaris a Mantel test. Both genetic distances and the Mantel test were
by Greatorex et al. (2000). PCR reactions were carried out under calculated using the GENETIX 4.02 software package (Belkhir
conditions set out in Greatorex et al. (2000) in a total volume of et al., 1996).
20 ml. Amplification products were resolved on an ABI PRISM A dendrogram based on Chord genetic distances
3100 Genetic Analyser, and analysed using GeneMapper v.3.5 (Cavalli-Sforza and Edwards, 1967) was constructed with boot-
software (Applied Biosystems). strap support for branches (2000 replicates) using the UPGMA
method of clustering, employing the program NEIGHBOR of
Data analysis the PHYLIP 3.6 computer package (Felsenstein, 1993). Bootstrap
The software Micro-Checker 2.2.3 (Van Oosterhout et al. 2004) support on branches is computed by resampling loci with the
was used to identify possible genotyping errors (i.e. stuttering, program SEQBOOT of the PHYLIP 3.6 computer package
large allele dropout, and null alleles) within the microsatellite (Felsenstein, 1993). The dendrogram was visualized with the
dataset by performing 1000 randomizations. Microsatellite poly- program MEGA.4 (Tamura et al., 2007).
morphism within samples was measured as the mean number A sequential Bonferroni technique (Rice, 1989) was used to
of alleles (Na) per locus, and observed and unbiased expected adjust significance levels for multiple simultaneous comparisons.
heterozygosity was calculated using the GENETIX 4.02 software
package (Belkhir et al., 1996). Deviations from Hardy –Weinberg Results
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 out-
crossing 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).
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 con-
formity 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.
Figure 1. Geographic locations of Octopus vulgaris samples taken in The presence of null alleles was detected for locus Oct03 in all
the Atlantic and Mediterranean. populations and for locus Ov12 in all populations except the14 C. Cabranes et al.
Table 1. Levels of genetic variations observed at five microsatellite DNA loci within six Iberian Peninsula Octopus vulgaris samples: allele size (in
base pairs), number of alleles (Na), observed heterozygosity (Ho) and unbiased expected heterozygosity (He), and means across all samples and
loci.
Locus and parameter Asturias Galicia Cádiz Portugal Canary Islands Murcia Mean
Oct03
.....................................................................................................................................................................................................................................................................
Number of alleles 25 26 21 21 22 27 23.67
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .
Allele size 126–187 117–195 117 – 177 117 – 177 117 –180 117 –187 117 –195
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .
H o 0.652 0.593 0.529 0.541 0.387 0.586 0.548
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .
He 0.957** 0.960*** 0.954*** 0.915*** 0.948*** 0.958*** 0.949
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .
Ov06
.....................................................................................................................................................................................................................................................................
Number of alleles 8 14 12 15 13 17 13.20
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .
Allele size 129–170 119–161 117 – 154 126 – 179 117 –154 117 –170 117 –179
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .
H o 0.735 0.707 0.914 0.867 0.807 0.902 0.822
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .
He 0.724 0.739 0.880 0.887 0.855 0.891 0.829
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .
Oct08
.....................................................................................................................................................................................................................................................................
Number of alleles 17 17 18 17 17 19 17.50
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .
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Allele size 130–165 111–167 128 – 165 128 – 169 115 –163 115 –165 111 –169
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .
H o 0.969 0.881 0.849 0.738 0.655 0.907 0.833
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .
He 0.921 0.903 0.915 0.915* 0.905** 0.919 0.913
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .
Ov10
.....................................................................................................................................................................................................................................................................
Number of alleles 17 16 19 17 13 12 15.70
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .
Allele size 115–150 115–145 109 – 148 111 – 152 117 –143 107 –137 107 –152
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .
H o 0.875 0.900 0.914 0.870 0.719 0.911 0.865
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .
He 0.918 0.909 0.932 0.914 0.815 0.836 0.887
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .
Ov12
.....................................................................................................................................................................................................................................................................
Number of alleles 18 26 17 27 16 25 21.50
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .
Allele size 175–371 170–375 162 – 330 167 – 399 175 –375 167 –338 162 –399
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .
H o 0.632 0.650 0.484 0.711 0.750 0.879 0.684
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .
He 0.936*** 0.939*** 0.903*** 0.913*** 0.895* 0.945 0.922
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .
N 34 48 35 48 33 48 –
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .
Mean N a 17.0 19.8 17.4 19.4 16.2 20.0 18.31
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .
Mean Ho 0.773 0.746 0.738 0.745 0.664 0.837 0.750
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .
Mean He 0.854 0.850 0.909 0.900 0.835 0.898 0.874
Deviations from Hardy– Weinberg expectations within loci: *p , 0.05; **p , 0.01; ***p , 0.001. Emboldened values are not significant at p , 0.05 after
sequential Bonferroni correction.
Canary Islands and Murcia. Locus Oct08 showed null alleles only The fact that samples clustered by geographic distance is remark-
for Portugal and the Canary Islands, and for loci Ov06 and Ov10, able. Neighbouring populations such as Portugal and Cádiz,
null alleles were not detected for any population. and Asturias and Galicia grouped together, whereas the
In terms of genetic differentiation between samples, p-values Mediterranean sample (Murcia) was separate from the rest.
estimated by pairwise differentiation were statistically significant
in all cases except Portugal –Cádiz (Table 2). However, after Table 2. Statistical significance (p-values) of pairwise genic
differentiation test between samples (above diagonal). Pairwise
sequential Bonferroni adjustment, Asturias –Galicia was not stat-
estimates of multilocus FST between samples of Octopus vulgaris
istically significant (p , 0.05). Estimation of FST indicated signifi- (below diagonal).
cant levels of intersample genetic variance in all pairwise
comparisons except Portugal– Cádiz (p , 0.05), as shown in Asturias Galicia Cádiz Portugal Canary Murcia
Table 2. Chord genetic distances (Cavalli-Sforza and Edwards, Islands
1967) were lowest between the geographically closest pairs of Asturias – 0.034 ,0.001 ,0.001 ,0.001 ,0.001
. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . .
samples (Portugal–Cádiz, 0.018; Table 3). Galicia 0.014* – ,0.001 ,0.001 ,0.001 ,0.001
. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . .
There was a pattern of increasing genetic differences with Cádiz 0.035* 0.020* – 0.240 ,0.001 ,0.001
. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . .
increasing geographic distance between samples for Atlantic Portugal 0.025* 0.012* 0.005 – ,0.001 ,0.001
. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . .
material, shown by the Mantel test for Chord genetic distance Canary 0.054* 0.036* 0.013* 0.015* – ,0.001
(Cavalli-Sforza and Edwards, 1967; p ¼ 0.03). Otherwise, the Islands
. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . .
Mantel test failed to reveal isolation-by-distance if Mediterranean Murcia 0.038* 0.033* 0.026* 0.021* 0.042* –
and Atlantic populations were considered.
Emboldened values are not significant at p , 0.05 after sequential
The UPGMA dendrogram constructed from Chord genetic dis- Bonferroni correction.
tances (Cavalli-Sforza and Edwards, 1967) is shown in Figure 2. *p , 0.05.Genetic structure of Octopus vulgaris around the Iberian Peninsula and Canary Islands 15
Table 3. Chord genetic distances among samples from the (Casu et al., 2002). If deviations were the result of the presence
different regions studied. of null alleles, they would probably be found in all samples, but
in the present study, the Ov06 locus was in Hardy– Weinberg equi-
Asturias Galicia Cádiz Portugal Canary
Islands librium for all populations. The Atlantic sample (Vig) analysed by
Casu et al. (2002) and the sample for Galicia analysed here were
Galicia 0.021 – – – –
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .
from the same population, so differences found with regard to
Cádiz 0.021 0.020 – – –
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . Hardy –Weinberg equilibrium could be caused by temporal vari-
Portugal 0.023 0.019 0.018 – –
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . ation in the population as well as differences in the techniques
Canary 0.032 0.032 0.020 0.021 – employed.
Islands
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . Microsatellite loci Ov10 and Ov12 were also employed in an
Murcia 0.039 0.038 0.033 0.033 0.041 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 sig-
nificant deviations for the Ov12 microsatellite, so supporting the
Discussion results of the present study for these microsatellite loci.
The utility of microsatellite DNA markers for examining subtle Two estimators of genetic divergence (exact test of sample
population genetic structuring within the Cephalopoda has been differentiation and FST) were used to test the extent of genetic
demonstrated previously (Shaw et al., 1999; Perez-Losada et al., difference within populations of O. vulgaris. The exact test and
2002). Our study has shown a high degree of genetic variability
Downloaded from http://icesjms.oxfordjournals.org/ by guest on October 26, 2015
FST gave similar results for most samples and significant levels of
within and between populations of O. vulgaris at all microsatellite inter-sample differentiation for O. vulgaris around the Iberian
loci, a finding particularly notable for Ov12 with its 56 alleles. In Peninsula and Canary Islands, except for Portugal– Cádiz.
previous studies, Casu et al. (2002) found a similar Na (21) for Asturias –Galicia revealed no significant difference with an exact
the locus Ov06, and Murphy et al. (2002) found a high level of test after sequential Bonferroni correction.
polymorphism in O. vulgaris for the loci Ov10 and Ov12. For A notable result of our study is the existence of a fine spatial
locus Ov12, the mean Na we determined was lower than obtained substructure in O. vulgaris populations in the Atlantic which is a
by Murphy et al. (2002), whereas for locus Ov10, the polymorph- function of geographical distance. Significant Mantel tests were
ism was similar in both studies. The differences between the results obtained for Chord genetic distances (Cavalli-Sforza and
of the two studies could be a consequence of the different tech- Edwards, 1967), and those results showed a population model of
niques used to resolve PCR products and/or the different isolation-by-distance for the Atlantic populations.
sampling area. Previous studies of the genetic structure of O. vulgaris from the
Observed and expected heterozygosity values were high (mean Mediterranean Sea using allozymes (Maltagliati et al., 2002) and
Ho 0.750, mean He 0.874), in accord with earlier studies on cepha- microsatellite loci (Casu et al., 2002) excluded isolation-
lopods (Shaw et al., 1999; Perez-Losada et al., 2002; Garoia et al., by-distance in O. vulgaris Mediterranean populations.
2004) and on O. vulgaris specifically (Casu et al., 2002; Murphy Maltagliati et al. (2002) suggested that O. vulgaris in the
et al., 2002). Mediterranean followed a basic island model in a background of
Deviations from Hardy –Weinberg equilibrium were observed high gene flow. One explanation for the different results could
for some populations, basically for loci Oct03 and Ov12, owing be the difference in geographical area studied.
to a deficit of heterozygotes in terms of those expected. The pre- Additionally, in the case of the Maltagliati et al. (2002) result,
sence of null alleles was confirmed as the cause of the deviation. we can explain divergence between results because they used allo-
Deficiencies in heterozygous genotypes have been found before zyme electrophoresis to investigate genetic variability. Previous
in cephalopod populations and suggest the presence of non- studies (Shaw et al., 1999; Perez-Losada et al., 2002) found that
amplified alleles (null alleles) as the cause of the observed depar- microsatellites have a greater power than allozymes to resolve
tures from Hardy –Weinberg equilibrium (Shaw et al., 1999; genetic relationships among closely related subpopulations of
Perez-Losada et al., 2002). aquatic species, with a facility for gene flow on a small geographical
A previous study of the genetic structure of the Ov06 O. vulgaris scale, and are more suitable for resolving historical relationships at
microsatellite locus from the Mediterranean Sea, but which an intraspecific level. In the case of the analysis of Casu et al.
included an Atlantic Ocean sample (Vig), detected deficiencies (2002), the use of just one microsatellite (Ov6) may not have
of heterozygous genotypes for most of the populations analysed, been powerful enough to find associations of genetic differen-
and the presence of null alleles was proposed as a possible cause tiation with geographic distribution of samples.
In this study, divergence was observed with the distance iso-
lation 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
Figure 2. UPGMA tree constructed on the basis of Chord genetic fisheries management perspective, the results may be considered
distances between samples. Bootstrap support from 2000 replications as supporting coordinated management of neighbouring stocks
is indicated on the branches. around the Iberian Peninsula.16 C. Cabranes et al.
Acknowledgements ICES. 2006. Report of the Working Group on Cephalopod Fisheries
and Life History. ICES Document CM 2006/LRC: 14. 43 pp.
We thank several people at the “Instituto Canario Ciencias
Maldonado, A. 1985. Evolution of the Mediterranean basins and a
Marinas”, CIFAP, IMIDA, “D. Xeneral Recursos Mariños”
reconstruction of the Cenozoic palaeoceanography. In Western
(Spain), and IPIMAR (Portugal) who collected the tissue Mediterranean, pp. 18 – 61. Ed. by R. Margalef. Pergamon Press,
samples for this work, especially J. Ro, J. L. Muñoz, J. Cerezo, London. 374 pp.
R. Arnaiz, and M. Gaspar. We also thank the Luarca and Puerto Maltagliati, F., Belcari, P., Cau, D., Casu, M., Sartor, P., Vargiu, G., and
de Vega Fishermen’s Guilds staff for their collaboration in the col- Castelli, A. 2002. Allozyme genetic variability and gene flow in
lection of samples in Asturias. Eva Garcı́a Vazquez, Gonzalo O. vulgaris (Cephalopoda, Octopodidae) from the Mediterranean
Machado, and Daniel Campo Falgueras collaborated in the statisti- Sea. Bulletin of Marine Science, 71: 473– 486.
cal analysis, and M. Casu and an anonymous referee made valuable Mangold, K. 1983. Octopus vulgaris. In Cephalopod Life Cycles,
1, pp. 335– 364. Ed. by P. R. Boyle. Academic Press, New York.
comments on the submitted manuscript.
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Mather, J. A. 1993. Octopuses as predators: implications for manage-
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