Aquaculture 460 (2016) 124–135

Contents lists available at ScienceDirect

Aquaculture

journal homepage: www.elsevier.com/locate/aquaculture

Genetic decoupling of spat origin from hatchery to harvest of Mytilus

galloprovincialis cultured in suspension
B. Díaz-Puente a,b, M. Miñambres b, G. Rosón a,c, A. Aghzar d, P. Presa a,b,⁎
a
ECIMAT Marine Station of Toralla (University of Vigo), Illa de Toralla s/n, E-36331, Vigo, Spain
b
Department of Biochemistry, Genetics and Immunology, Faculty of Biology, University of Vigo, 36310, Vigo, Spain
c
Department of Applied Physics, Faculty of Marine Sciences, University of Vigo, Campus Lagoas-Marcosende s/n, Vigo, Spain
d
Université Moulay Ismail, École Supérieure de Technologie (EST), Département de Génie de l'environnement et agro-biotechnologie, B.P. 170, 54000 Khénifra, Morocco

a r t i c l e i n f o a b s t r a c t

Article history: Productivity of Mediterranean mussel aquaculture in suspension could be enhanced with selected hatchery
Received 25 October 2015 strains for a variety of candidate traits. While selective breeding and reproduction are well established
Received in revised form 5 April 2016 technologies, several key parameters required to grow selected strains outdoors still remain elusive. For instance,
Accepted 15 April 2016
external recruitment dynamics on suspended devices is poorly known and therefore it is uncertain whether a
Available online 19 April 2016
harvested strain comes from the one initially seeded or has been partially replaced by wild recruits during the
Keywords:
pre-fattening period. The incorporation of genetic technologies to the management of mussel stocks makes
Genetic assignment feasible to disentangle the genetic contribution of different seed sources, provided that an appropriate type of
Hatchery mussel seed marker and a suitable experimental design are properly combined. Using multiplexed microsatellites we have
Microsatellites traced back the individual origin of a batch of harvested mussels, which grew in suspension ropes that were
Mytilus galloprovincialis initially seeded with full-sib juveniles. We show that mean heterozygosity decreased by 28% between months
Recruitment rate 8 and 13 in suspension, a phenomenon thought to be caused by fall-offs of the more exposed heterozygous
individuals of the cohort. Bayesian tests allowed to ascertaining that 98.3% of the adult harvest came from
the original hatchery full-sib family while only 1.7% mussels were recruited from the wild. Such low external
recruitment on unprotected ropes allows to being confident on the feasibility of culturing selected strains,
provided that both, seed density and retention timing are adequately managed.
Statement of relevance: Genetic assessment of mussel recruitment in suspension.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction
Worldwide production of mussels from aquaculture has been
steadily growing in the last decades, from 70,000 tons in early 1950s
to 1,755,694 tons in 2013 (FAO, Fishstat). In 2013, Spain produced
188,944 tons of mollusks and its 96.94% (74,513,064 €) corresponded
to the Mediterranean mussel (Mytilus galloprovincialis) cultivated in
Galician Rías (MAGRAMA, 2014). Most local production is consumed
in Spain, either in fresh (1.2 kg/year per capita; 50–70 cents/kg in
origin) or canned (0.3 kg/year; 40 cents/kg in origin) (González-Laxe
and Martín-Palmero, 2014). Such high production is due to the high
primary productivity of Rías Gallegas, a phenomenon provoked by the
combination of nutrient-rich sea water entering local estuaries during
upwelling events and local circulation patterns (Bode et al., 1996).
That phytoplankton productivity sustains a high density of planktonic
⁎ Corresponding author at: Department of Biochemistry, Genetics and Immunology,
Faculty of Biology, University of Vigo, 36310 Vigo, Spain.
E-mail addresses: borjadp@uvigo.es (B. Díaz-Puente), minhambres@gmail.com
(M. Miñambres), groson@uvigo.es (G. Rosón), aaghzar@gmail.com (A. Aghzar),
pressa@uvigo.es (P. Presa).
mussel larvae during the spawning season, i.e. ≈ 20 × 103 larvae/m3
surrounding cultivation rafts (Cáceres-Martínez and Figueras, 1998a).
Mussel seed required for suspended cultivation in rafts is obtained by
farmers either by scrapping juveniles from intertidal beds of exposed
rocks or from short spat-collectors suspended from rafts (Fuentes
et al., 1998). Therefore, the availability of wild spat has played a decisive
role in the development of mussel farming and the growth of the whole
mussel industry (Cáceres-Martínez and Figueras, 1998b). Growing
competitiveness in shellfish aquaculture is now fueling research
in many fields, such as coastal and estuary management strategies
(e.g. Brigolin et al., 2009), new spat collectors and cultivation devices
(e.g. Aghzar et al., 2012), nutritional optimization in the nursery
(Nevejan et al., 2007; Miñambres et al., 2011) or physiologic and behav-
ioral related processes (Liu et al., 2011). Nevertheless, several problems
jeopardize a higher mussel productivity in suspended devices, such as
predators (e.g. Beadman et al., 2003), storm episodes (Harger and
Landenberger, 1971), industrial contamination (Lado-Ínsua et al.,
2011) and HABs (Harmful microalgae blooming episodes) now
worsening because of global warming (e.g. Anderson et al., 2012).
An innovative approach to overcome the lowering productivity of
mussel cultivation in suspension caused by those handicaps consists

http://dx.doi.org/10.1016/j.aquaculture.2016.04.016
0044-8486/© 2016 Elsevier B.V. All rights reserved.
 B. Díaz-Puente et al. / Aquaculture 460 (2016) 124–135
on the combination of environmental prediction with hatchery-selected
mussel strains. Such approach should allow to shortening or re-
mastering the production cycle which nowadays consists of four main
steps i.e. (1) Seeding: juveniles (≈ 4.5 mm in length) from collector
ropes or from rocky scrapping are wrapped on raft-suspended ropes,
(2) Prefattening: juveniles grow for 3–6 months in suspension,
(3) Thinning-out: juveniles from the initial rope are unfolded into 2–3
growing ropes holding ≈50 kg mussels each and allowed to grow for
12 additional months, (4) Harvesting: mussels of commercial size
(70–100 mm) are removed from the ≈ 200 ropes per raft generally
holding ≈150 kg mussels each (30–35 individuals per kg) (e.g. Figueras,
2007). Reproductive expertise in the Mediterranean mussel
(e.g. Miñambres et al., 2011) and recent estimates of heritability for
diverse traits (e.g. 0.230 for shell length and 0.392 for body weight in
Mytilus edulis (Alcapán et al., 2007), or 0.640 for shell length and 0.350
for body weight in M. galloprovincialis (Nguyen et al., 2014) suggest
that selection could be performed in hatchery and juveniles grew in sus-
pension afterwards, for instance to attain a shorter production cycle.
However, it is uncertain whether a full harvest comes from the selected
hatchery strain seeded 20–24 months before or if naturally recruited
free spat have been recruited to the suspended rope or the extent to
which these later have out-competed the selected hatchery juveniles.
Since the first characterization of microsatellites in fishes two
decades ago (Estoup et al., 1993) these markers have been progressively
incorporated into population genetics (e.g. Presa et al., 1994) and then
into breeding programs of several species (e.g. Vandeputte et al.,
2011) as well as in stock assignment, genealogical traceability and
individual identification (e.g. Chistiakov et al., 2006). The incorporation
of multilocus genotyping as supporting technology in mussel produc-
tion is particularly relevant because it can help the genetic management
of natural source populations (e.g. Diz and Presa, 2009) as well as the
increment of productivity of hatchery stocks through marker-assisted
selection (e.g. Moen et al., 2009). Using a full-sib hatchery progeny of
mussels and multiplexed microsatellite markers we aimed to clarify
the time, place, rate and percentage of wild spat that is recruited to
cultivation ropes originally seeded with selected juveniles from the
hatchery. Knowledge of those variables would allow to assessing the
final identity of the selected stock, to increase cultivation efficiency,
and to re-engineering the production cycle according to the mussel
growth characteristics. We specifically aim to know 1) when and
where recruitment takes place along the rope sleeve and its impact on
protected and unprotected ropes, 2) the proportion of immigrants
recruited to pre-seeded cultivation ropes that is finally harvested,
3) the practical implications and viability of growing selected hatchery
strains in suspension systems.
2. Materials and methods
2.1. Control strain and experimental design
In October 2007 several full-sib progenies of M. galloprovincialis
were produced at ECIMAT Marine Station to assist experimental
designs, e.g. Miñambres et al., (2011). Briefly, thirty mature mussels
from Ría de Vigo (NW Spain) were kept at 4 °C overnight, then rinsed
with seawater and individually immersed in a beaker containing
seawater previously filtered (1 μm) and sterilized using a UV lamp.
Spawning was induced by immersing the beakers in a thermostatic
bath containing current water at 23 °C. Individuals were allowed to
complete their spawning and then each oocyte batch was filtered
through a 40 μm sieve and immersed in a beaker containing 1 μm
filtered and sterilized seawater. Oocytes were counted and their quality
(roundness) assessed under a microscope. A similar procedure was
followed for the male sperm and the volume required to achieve an
8:1 spermatozoid:oocyte proportion was enforced after checking its
motility. After fecundation, fertilized oocytes were counted through in-
spection of the second polar body and placed into a fiberglass tank of
125
150 L volume at an initial density of 20 embryos/ml. Tanks containing
independent full-sib offspring were aerated with individual pumps
and kept in closed circuit with water renewal and tank cleaning on a
48 h basis. Larvae were fed every other day with 1 L microalgae culture
of average density 1.68 × 104 cells/μl diet containing 50% Isochrysis
galbana & 50% Pavlova luthery, to reach a final concentration of
110 cell/μl in a 150 L tank. For maintenance tasks, larvae were collected
from tanks using progressive sieves adjusted to the advancement of
developmental stages.
When larvae started the pediveliger phase (≅28 days) and became
competent for substrate settlement (Widdows, 1991), three polyester
4.5 m length cultivation ropes used as settlement substrate were
introduced in the respective tanks containing 13,500 post larvae from
a single family (3000 individuals/m). Post-larvae were fed every 48 h
with a 10 L microalgae diet of average density 7.3 × 103 cells/μl designed
for mussel post-larvae (Miñambres et al., 2011) which contained 50%
Isochrysis galbana (Class Prymnesiophyceae; cell strain # CCMP1323),
25% Tetraselmis suecica (Class Prasinophyceae; cell strain # CCMP904),
and 25% Chaetoceros gracilis (Class Coscinodiscophyceae; cell strain #
CCMP1317).
In January 2008 (90 day-old juveniles averaging 3224 ± 1045 μm in
length) one replicate rope was kept indoors as a control group while
another two replicates were transferred to the ECIMAT jetty for grow-
out in suspension (Fig. 1). One replicate was protected by a tubular
plastic mesh (18 mm mesh size, 20 cm wide and closed on the edges)
and the other replicate was suspended unprotected (free predation
allowed). Three PVC sticks were placed at the upper, middle and bottom
end of the seeded ropes to minimize mussel detachment and anchored
to the plastic mesh so that predators could not feed inside (Fig. 2).
Between April 2008 (6 month-old juveniles) and June 2008 (8 month-
old juveniles), protected (P) and unprotected (U) ropes were periodi-
cally sampled and mussels were frozen until DNA extraction. One-
hundred and forty individuals were sampled per month on 3 April, 8
May and 6 June and consisted of 20 individuals from a 5 cm rope section
at three depths per rope, i.e. 60 individuals from the upper (U), middle
(M), and bottom (B) part of each replica rope. Since the hatchery control
progeny (HCP) was kept indoors and no external recruitment was
possible, a single batch of 20 individuals was taken per sampling. After
the third sampling performed in June 2008, the protected replicate
was removed because mussel out-growth was physically constrained
by the protective mesh, while its unprotected replica was allowed to
grow for five additional months. On 14 November 2008 (13 month-
old juveniles) five additional samples were taken as two samples from
niches where mussels were previously scrapped in spring (UUSS,
Unprotected Upper Sampled Site; UBSS, Unprotected Bottom Sampled
Site), one sample from a bottom weight attached to the rope tip
(ERCG, External Recruit Control Group), one sample from rope surfaces
which were initially seeded with the hatchery progeny (EHCP,
Experimental Hatchery Control Progeny) and a fifth sample scrapped
from the sea rocks neighboring the jetty and used as external control
from Ría de Vigo (ECRV).
2.2. Microsatellite genotyping
DNA from 457 mussels was extracted and purified from 10 mg of
mantle tissue following the FENOSALT protocol for marine taxa (Pérez
and Presa, 2011) and preserved frozen in ultrapure water until bio-
chemical analyses. All individuals were screened by PCR amplification
for six microsatellite markers developed for this species, four of them
(Mgμ1, Mgμ3, Mgμ4, Mgμ5) described by Presa et al., (2002), and two
additional ones (Mch8-D10 and Mgμ8-D5) reported by Ouagajjou
et al., (2011) and Ouagajjou and Presa (2015), respectively. About
50 ng per individual DNA were used in a PCR reaction of 15 μL consisting
of 1 U Taq DNA Polymerase (BIOTAQ™) in 1 × NH4 reaction buffer
(Bioline), 1 μm of each primer, 200 μM of each dNTP and the MgCl2 con-
centration reported for each locus. The forward primer of each
126 B. Díaz-Puente et al. / Aquaculture 460 (2016) 124–135

Fig. 1. Atlantic façade of Ría de Vigo showing the location of ECIMAT Marine Station (Black circle) where hatchery and outdoors culture experiments were performed.

Fig. 2. Schematic diagram of the experimental design implemented to test mussel spat recruitment on hatchery-seeded culture ropes. Treatments: HCP, hatchery control progeny; UUR,
Upper unprotected rope; MUR, medium unprotected rope; BUR, bottom unprotected rope; UPR, upper protected rope; MPR, medium protected rope; BPR, bottom protected rope; EHCP,
experimental hatchery control progeny; UUSS, unprotected upper sampled site; UBSS, unprotected bottom sampled site; ERCG, external recruit control group, ECRV, external control from
Ría de Vigo.
 B. Díaz-Puente et al. / Aquaculture 460 (2016) 124–135
locus was fluorescently labeled with Cy5 (5-N-N-diethyl-tetramethyl-
indodicarbocyanine) for laser detection. PCR reactions were performed
in a Mastercycler Gradient Thermocycler (Eppendorf) following the
amplification profile of one cycle at 95 °C for 5 min, 35 cycles at the
annealing temperature for 40 s, 72 °C for 1 min and 94 °C for 1 min,
and ending by an extension step at 72 °C for 30 min. PCR products
were visualized in 2% agarose gels and electrophoresed in acrylamide
gels using an ALFexpressII automatic fragment analyzer (GE
Healthcare). Allele calling was performed using internal size standards
of 80 bp, 114 bp, 180 bp, 230 bp and 402 bp as well as external size
controls from initial allele scorings of each marker. Raw genotypic
data and allelic frequencies were plotted in an Excel spreadsheet to
detect odd distributions, allelic misinterpretations and ambiguities
and then re-checked on electrophoretic gels or re-genotyped when
allele call doubts remained.
2.3. Statistical analyses
Adjustment of observed genotype distributions to those expected
under simple Mendelian segregation rules was checked with chi-
square contingency tests using SPSS 17.0. Allele frequencies, observed
heterozygosity and expected heterozygosity were estimated with
FSTAT 2.9.3.2. (Goudet, 1995) and compared among the first three
samplings within treatment (HCP, hatchery control progeny; UUR,
upper unprotected rope; MUR, middle unprotected rope; BUR, bottom
unprotected rope; UPR, upper protected rope; MPR, middle protected
rope; BPR, bottom protected rope) and among treatments within
sampling. Heterozygosity of the control progeny was assessed by
comparing that of the three spring samplings with the heterozygosity
in the 13 month-old adult batch harvested in autumn (EHCP, experi-
mental hatchery control progeny grown unprotected) using the permu-
tation approach indicated in FSTAT 2.9.3.2 (Goudet, 1995). Bayesian
probabilities of multilocus genotypes were explored to detect individ-
uals not belonging to the experimental hatchery progeny, i.e. aimed to
detect free swimming mussel spat recruits from Ría de Vigo. Population
genotype data from Ría de Vigo was taken as external reference to the
control progeny. Assignment/exclusion probabilities and first genera-
tion immigrants were calculated by the bayesian method of Rannala
and Mountain (1997) as implemented in GENECLASS 2.0. (Piry et al.,
2004). Such bayesian inference on genotype frequencies of the sampled
groups was performed using the Monte-Carlo resampling and simula-
tion algorithm of Rannala and Mountain (1997). This method better
fitted present data since other alternatives such as Paetkau et al.
(2004) require that samples be in HWE equilibrium. The distance
method of Cornuet et al. (1999) does not assume HWE but assigned
individuals to the hatchery progeny that showed incompatibilities
with the experimental control progeny in two loci (data not shown).
The minimum amount of recruitment was estimated empirically as
the number of atypical multilocus genotypes incompatible with the
hatchery progeny as well as mathematically, as the proportion of
individuals statistically excluded from the hatchery progeny. Assign-
ment probabilities computed were: L-Home, assignment probability to
the source group (a genotype is miss-assigned if it has a low multilocus
probability in the group it was sampled); Pim, probability of being
immigrant (p b 0.01) within its sampling group; Pfs, probability of
belonging to the full-sib family; Ni/Ng, No. of incompatible loci between
offspring and genitors per genotyped loci. Those statistics were applied
to all treatments, including the external control of recruits (ERCG)
settled on the rope tip weight as well as to the external control group
from Ría de Vigo (ECRV) consisting of mussels scrapped from rocky
shores neighboring the jetty.
3. Results and discussion
Selection programs in the aquaculture industry have been imple-
mented only in a few species and many of them grown for commercial
127
purposes are still essentially wild (e.g. Gjedrem, 2000). Therefore, the
determination of pedigree during the establishment of new broodstocks
(e.g. Matusse et al., 2016) as well as the traceability of their offspring at
harvesting (this study) will likely be a growing application of current
genetic technologies. The experimental design used in this study,
where mussel breeders and their offspring are physically and genetically
identifiable at the individual level, from the beginning to the end of the
production cycle, allows to assessing the feasibility of growing selected
hatchery strains of Mediterranean mussels in suspension.
3.1. Polymorphism of microsatellite markers
The six loci analyzed were highly polymorphic in natural popula-
tions (Ouagajjou et al., 2010) and exhibited 13 alleles in the present
full-sib offspring, i.e. two alleles per locus and one null allele inferred
in locus Mgμ4. Both parents of that hatchery progeny were opposite
homozygotes for locus Mgμ1, the father homozygote and the mother
heterozygote for loci Mgμ5 and Mch8, the father heterozygote and the
mother homozygote for locus Mgμ8 and both heterozygous for loci
Mgμ3 and Mgμ4 (Table 1). The wild population of Ría de Vigo (ECRV)
used as external control, showed that locus Mgμ3 and locus Mgμ4
were the less (5 alleles) and the most (15 alleles) polymorphic ones,
respectively. Loci Mgμ8, Mgμ1, Mgμ5 and Mch8 exhibited 14, 10, 10
and 6 alleles respectively (Table 1).
3.2. Segregation patterns per locus and treatment
In general, the segregation of most loci across treatments fitted
expected genotypic proportions at equilibrium. Unexpected genotypic
proportions were observed in 2 out of 126 tests performed on the first
three samplings within treatment and locus, i.e. 1.58% of cases for
α = 0.01 (Table 1). Those two deviations to HWE expectations were
due to significant excess of heterozygotes in locus Mgμ8 of sampling II
and treatment BPR (Table 1) or to a significant deficit of heterozygotes
in locus Mch8 of sampling II and treatment MPR (Table 1). Those two
cases of unexpected genotypic proportions fit expected Type I error
and therefore are assumable to a random refusal of the null hypothesis.
Pooled data across treatments showed heterozygote deficits in loci
Mgμ3 and Mgμ4 of sampling I, in loci Mgμ8 and Mgμ4 of sampling II
and in locus Mgμ3 of sampling III. The heterozygote deficit of locus
Mgμ4 across samplings was due to co-segregation of null alleles,
which were not described before in this locus. Loci Mgμ3, Mch8 and
Mgμ4 also showed a highly significant heterozygote deficit in sampling
IV on 13 month-old adults from the experimental hatchery control
progeny (EHCP) harvested from suspension ropes, (Table 1), while
loci Mgμ8 and Mgμ5 segregated as expected. Some deficits are likely
due to null alleles, as described above for locus Mgμ4, but when such
deficit is systematic across loci it has also been ascribed to other causes
such as selection against heterozygotes in early stages of mollusk
development (e.g. Zouros and Foltz, 1984). That latter possibility has
been hypothesized by earlier investigations in Mediterranean mussels
where the loss of more exposed heterozygote individuals would entail
the final rise of the global population homozygosity as a consequence
of such fall-off losses (Myrand et al., 2002) (see also Section 3.3.3).
3.3. Changes in heterozygosity
3.3.1. Heterozygote differences within treatment
There were no differences of heterozygosity among samplings with-
in treatment (protected rope/unprotected rope) except a significant
fluctuation of heterozygosity (from sampling I to sampling III) of locus
Mgμ8 in treatments MUR and MPR, and of locus Mgμ3 in treatments
MPR and BPR (Table 2). Those four tests represented 9.52% of cases
and were scattered among treatments and locus so they do not support
any specific shift trend of this parameter.
 128
Table 1
Chi-square contingency tests on genotypic segregations of six microsatellite loci in seven treatments within sampling: HCP (hatchery control progeny); UUR (upper unprotected rope); MUR (middle unprotected rope); BUR (bottom unprotected
rope); UPR (upper protected rope); MPR (middle protected rope); BPR (bottom protected rope); EHCP (Experimental hatchery control progeny grew unprotected). Sampling I — 6 month old juveniles (03/04/2008), Sampling II — 7 month old
juveniles (08/05/2008), Sampling III — 8 month old juveniles (06/06/2008), Sampling IV — 13 month old juveniles (14/11/2008). Sampling size is given in parentheses; figures in the first four rows correspond to parental and offspring genotype
per locus. Asterisk indicates a significant deviation to Mendelian expected segregations (*p b 0.01).

Locus Mgμ5 Mgμ8 Mgμ3 Mch8 Mgμ1 Mgμ4

Father 132–132 194–200 138–140 203–203 142–142 173–191

Mother 126–132 200–200 138–140 203–205 156–156 173–null

Expected 1/2126– 1/2132– p(χ2) 1/2194– 1/2200– p(χ2) 1/4138– 1/2138– 1/4140– p(χ2) 1/2203– 1/2203– p(χ2) 142–156 p(χ2) ¼ 173–173 1/4173– 1/4191- p(χ2)

B. Díaz-Puente et al. / Aquaculture 460 (2016) 124–135

genotypic 132 132 200 200 138 140 140 203 205 + ¼ 173-null 191 null
ratio

Sampling I HCP (20) 10 10 1.000 12 7 0.511 7 6 7 0.435 10 10 1.000 20 – 15 3 2 0.248

UUR (20) 8 12 0.525 7 13 0.337 4 8 8 0.599 9 11 0.752 18 – 9 4 7 0.780
MUR 20) 9 11 0.752 13 5 0.171 14 5 1 0.014 11 9 0.752 17 – 15 3 2 0.248
BUR (20) 13 7 0.337 5 10 0.269 6 7 7 0.621 15 5 0.102 19 – 11 2 5 0.415
UPR (20) 7 13 0.337 4 16 0.047 11 8 1 0.077 12 8 0.525 20 – 17 2 1 0.056
MPR (20) 10 10 1.000 3 17 0.018 12 5 3 0.080 13 7 0.337 20 – 13 1 1 0.067
BPR (20) 9 11 0.752 9 9 1.000 7 7 4 0.747 8 12 0.525 19 – 15 1 2 0.048
Total (140) 66 74 0.632 53 77 0.135 61 46 31 0.003* 78 62 0.338 133 – 95 16 20 0.001*
Sampling II HCP (19) 11 8 0.744 10 9 1.000 5 3 11 0.072 10 9 1.000 19 – 10 6 3 0.725
UUR (20) 7 13 0.337 7 13 0.337 7 6 7 0.435 11 9 1.000 20 – 11 3 6 0.727
MUR (20) 10 8 0.738 17 3 0.018 9 6 5 0.343 9 11 0.752 20 – 10 5 5 1.000
BUR (40) 18 17 0.598 29 9 0.017 13 17 10 0.728 30 10 0.021 40 – 21 8 8 0.897
UPR (20) 7 10 0.492 5 11 0.280 6 5 9 0.235 14 6 0.197 20 – 10 5 4 0.921
MPR (20) 7 10 0.492 17 2 0.012 3 9 8 0.372 18 2 0.006* 20 – 14 2 2 0.122
BPR (20) 9 5 0.445 19 1 0.001* 3 10 7 0.659 10 10 1.000 20 – 14 3 1 0.091
Total (159) 69 71 0.905 104 48 0.001* 46 56 57 0.026 102 57 0.011 159 – 90 32 29 0.001*
Sampling III HCP (20) 12 6 0.310 7 12 0.328 2 5 13 0.039 10 10 1.000 20 – 12 2 5 0.424
UUR (20) 6 8 0.705 5 8 0.431 7 6 7 0.435 12 8 0.525 18 – 11 6 2 0.455
MUR (20) 7 9 0.723 2 11 0.039 3 8 9 0.394 14 6 0.197 20 – 6 6 6 0.792
BUR (20) 10 8 0.738 10 9 1.000 4 9 7 0.780 11 9 0.752 20 – 12 7 1 0.204
UPR (20) 8 9 0.732 9 10 0.746 5 6 9 0.343 15 5 0.102 20 – 9 6 3 0.723
MPR (20) 6 9 0.464 12 8 0.525 4 3 13 0.024 14 6 0.197 20 – 12 3 5 0.711
BPR (18) 8 10 0.738 10 8 1.000 8 3 7 0.105 12 6 0.310 18 – 8 5 3 0.881
Total (138) 65 59 0.703 55 66 0.440 33 40 65 0.001* 88 50 0.021 136 – 70 35 25 0.349
Sampling IV EHCP (20) 7 10 0.502 14 6 0.197 0 0 20 0.001* 19 1 0.001* 20 – 19 0 1 0.005*
 B. Díaz-Puente et al. / Aquaculture 460 (2016) 124–135 129

Table 2
Statistical comparison of heterozygosity per locus and treatment (*p b 0.01). HCP (hatchery control progeny); EHCP (External hatchery control progeny grew unprotected); UUR (upper
unprotected rope site); MUR (middle unprotected rope site); BUR (bottom unprotected rope site); UPR (upper protected rope site); MPR (middle protected rope site); BPR (bottom
protected rope site) and among treatments within samples. Mean heterozygosity was compared among spring samples (SI, SII, SIII) as well as between spring samples and the autumn
sample (SIV).

Treatment Samples (I, II, III) Mgμ5 Mgμ8 Mgμ3 Mch8 Mgμ1 Mgμ4 Six loci

HCP SI 0.500 0.600 0.300 0.500 1.000 0.200 0.520

SII 0.600 0.500 0.200 0.500 1.000 0.300 0.520
SIII 0.700 0.400 0.300 0.500 1.000 0.100 0.500
p (SI-SII-SIII) 0.180 0.520 0.340 0.990 1.000 0.100 0.900
Average (SI-SII-SIII) 0.579 0.509 0.237 0.492 1.000 0.190 0.501
EHCP SIV 0.444 0.700 0.000 0.050 1.000 0.000 0.359
p (SI-II-III vs SIV) 0.003* 0.001* 0.001* 0.001* 1.000 0.001* 0.003*
UUR SI 0.400 0.400 0.400 0.600 1.000 0.200 0.500
SII 0.400 0.400 0.300 0.500 1.000 0.200 0.470
SIII 0.500 0.400 0.300 0.400 1.000 0.300 0.480
p (SI-SII-SIII) 0.740 0.990 0.510 0.520 1.000 0.250 0.820
Average (SI-SII-SIII) 0.389 0.358 0.333 0.483 1.000 0.220 0.461
MUR S1 0.450 0.720 0.250 0.450 1.000 0.150 0.500
SII 0.560 0.850 0.300 0.550 1.000 0.250 0.590
SIII 0.440 0.150 0.400 0.300 1.000 0.330 0.440
p (SI-SII-SIII) 0.400 0.010* 0.410 0.070 1.000 0.230 0.020
Average (SI-SII-SIII) 0.481 0.627 0.317 0.433 1.000 0.241 0.517
BUR S1 0.650 0.380 0.350 0.250 1.000 0.110 0.460
SII 0.530 0.760 0.430 0.250 1.000 0.220 0.530
SIII 0.560 0.530 0.450 0.450 1.000 0.350 0.560
p (SI-SII-SIII) 0.110 0.180 0.630 0.250 1.000 0.020 0.120
Average (SI-SII-SIII) 0.568 0.611 0.413 0.300 1.000 0.227 0.520
UPR S1 0.350 0.200 0.400 0.400 1.000 0.100 0.408
SII 0.410 0.310 0.250 0.300 1.000 0.260 0.423
SIII 0.470 0.470 0.300 0.250 1.000 0.330 0.471
p (SI-SII-SIII) 0.500 0.540 0.470 0.560 1.000 0.040 0.440
Average (SI-SII-SIII) 0.407 0.327 0.317 0.317 1.000 0.171 0.433
MPR S1 0.500 0.400 0.300 0.400 1.000 0.100 0.450
SII 0.400 0.900 0.500 0.100 1.000 0.100 0.500
SIII 0.400 0.600 0.200 0.300 1.000 0.200 0.450
p (SI-SII-SIII) 0.410 0.010* 0.010* 0.090 1.000 0.820 0.110
Average (SI-SII-SIII) 0.442 0.542 0.300 0.250 1.000 0.113 0.441
BPR S1 0.450 0.500 0.390 0.600 1.000 0.060 0.500
SII 0.640 0.950 0.500 0.500 1.000 0.170 0.630
SIII 0.440 0.560 0.170 0.330 1.000 0.310 0.470
p (SI-SII-SIII) 0.050 0.100 0.010* 0.110 1.000 0.030 0.020
Average (SI-SII-SIII) 0.500 0.679 0.357 0.483 1.000 0.173 0.532
Among treatments 0.100 0.490 0.840 0.340 1.000 0.970 0.410
across samples (p)
Among treatments SI 0.060 0.120 0.640 0.050 1.000 0.750 0.170
per sample (p) SII 0.010* 0.020 0.010* 0.010* 1.000 0.360 0.010*
SIII 0.070 0.590 0.030 0.490 1.000 0.020 0.450

3.3.2. Heterozygote differences among treatments
No differences in heterozygosity were apparent among treatments
across the first three samplings I, II and III (Table 2), indicating that
neither the treatment nor the position along the suspended rope had
any effect on heterozygosity. Sampling II showed significant fluctua-
tions of heterozygosity across treatments for loci Mgμ5, Mgμ3 and
Mch8 as well as globally across loci (Table 2). Since the heterozygosity
change among treatments within sampling II did not show any trend
among loci, it can be reasonably ascribed to random.
3.3.3. Heterozygote differences between samplings of the hatchery control
progeny (HCP)
No differences of heterozygosity were observed among the first
three samplings of the experimental hatchery control progeny (HCP).
However, a significant decrease of heterozygosity was observed in
sampling IV in four out of the five segregating loci and a heterozygote
increment in locus Mgμ8. The global trend across loci was significantly
skewed towards heterozygote deficit (Table 2). While heterozygosity
was steady during the first three samplings (Juveniles aged 6-, 7-
and 8-months) it decreased afterwards by 28% in sampling IV on
13 month-old adults. A progressive reduction of heterozygosity of
juveniles along the pre-fattening period is a phenomenon described in
mollusks and particularly in mussels (e.g. Raymond et al., 1997). Present
data are congruent with a multilocus heterozygosity (MLH) drop)
reported in M. edulis cultivated on suspended sleeves (Myrand et al.,
2009). In such study, a low heterozygosity at harvest was causally
ascribed to the selective loss of highly heterozygous individuals which
moved to the best-oxygenated and exposed niches of the sleeve and
therefore which would suffer higher mortalities through fall-offs
(Myrand et al., 2009). Noteworthy, such hypothesis is also congruent
with the significant correlation found between heterozygosity and
growth rate at high cultivation densities in M. edulis (Gentili and
Beaumont, 1988) and is also congruent with the heterozygosity
drop observed in the present full-sib offspring of M. galloprovincialis
cultivated outdoors.
3.4. Genetic assignments of 13 month-old harvested adults
3.4.1. EHCP (experimental hatchery control progeny)
All adult mussels, which grew unprotected were assigned to the
hatchery progeny (EHCP) (Sampling IV, Appendix A). Individual
EHCP-11 was statistically excluded from its sampling group because of
its infrequent genotype (191/191) for locus Mgμ4. Since that locus
showed evidence of null alleles (see Table 1 and subsection 3.2) and
the rest of loci matched belonging criteria to the EHCP, its real genotype
likely was 191/null, and therefore this individual cannot be classified as
an immigrant. Individual EHCP-17 was not excluded from its sampled
130 B. Díaz-Puente et al. / Aquaculture 460 (2016) 124–135
group, but was significantly classed as immigrant to that group. That
was caused by its infrequent genotype 203/205 in locus Mch8 of group
EHCP. Noteworthy, the significant Pim probability of individual EHCP-
17 to group EHCP clearly misclassified it as non-immigrant to its true
source. Those two above misclassifications due to null alleles or to
infrequent genotypes were fully compatible with the EHCP hatchery
progeny. Therefore, no recruitment of wild larvae or post-larvae was
observed on the cultivation rope that had been pre-seeded with a
hatchery progeny 13 months before.
3.4.2. ECRV (external control from Ría de Vigo) and ERCG (external recruit
control group)
All individuals that were scrapped on rocks and used as a refer-
ence from the mussel population of Ría de Vigo (ECRV) as well as
all individuals from the external control or recruitment group
(ERCG) showed genotype incompatibilities in at least 2 loci regard-
ing the hatchery progeny (Appendix A). Noteworthy, eight out of
48 ERCG mussels were not assigned to their sampling group (e.g.
ERCG-7, P(L-Home b 0.002) and seven additional ones were classified
as immigrants (e.g. ERCG-2, Pim = 0.001). The cause of such high 15/
48 missassignment ratio was their low multilocus probability in their
sampling group, a phenomenon facilitated by a high locus polymor-
phism, a high population size in this area and the limited sample size
of the reference home group for assignment. The multilocus geno-
typic divergence observed between the hatchery progeny (EHCP)
and both, the external recruit control (ERCG) and the external con-
trol from Ría de Vigo (ECRV), indicate that a low probability exists
Fig. 3. Log-likelihood distribution of assignment probabilities (X-axis) of all multilocus genotypes (Y-axis; absolute frequency) to the experimental hatchery control progeny (EHCP). The
arrow indicates the lowest probability threshold for multilocus genotypes from EHCP.
of finding redundant multilocus genotypes between wild recruits
and the well characterized hatchery strain. This result allows being
confident in the recruitment figures estimated with the current
genotyping tool.
3.4.3. UBSS and UUSS (unprotected bottom/unprotected upper, spring-
sampled free-niche sites)
Two individuals of the unprotected upper sampling site (UUSS-14
and UUSS-19) and none of the UBSS sampling site were compatible
with the hatchery progeny (Appendix A). Therefore, all UBSS mussels
and 46 out 48 UUSS mussels were wild recruits into free niches left by
early spring samplings. The compatibility of two UUSS mussels (UUSS-
14 and UUSS-19) with EHCP multilocus genotypes suggests that
flanking EHCP juveniles moved to free niches of the rope. Alternate
explanations for that phenomenon are the unadverted incomplete
removal of EHCP juveniles during spring samplings (I, II and III) or a
genotypic redundancy between the hatchery progeny and the wild
population (see Section 3.4.2). If those two latter explanations were
reasonably less probable than the former, the percentage of mussels
that shifted over the rope was ≈ 4%, what is congruent with the well
know displacement of juvenile mussels in suspension (e.g. Sénéchal
et al., 2008). Noteworthy, ten individuals were excluded from sampling
groups UUSS and UBSS, i.e. P(L-Home b 0.05) as well as from the
hatchery progeny (EHCP) (Pfs b 0.001) and 23 individuals, including
the former ones were classified as putative immigrants (Pim b 0.01) to
those sampling groups (17 of them at the 1% probability). Due to the
high polymorphism of present microsatellite markers (Presa et al.,
 B. Díaz-Puente et al. / Aquaculture 460 (2016) 124–135
Table 3
Recruitment impact per sampling and treatment between April 2008 and November 2008 on experimental culture ropes grew in suspension. R/G (%), ratio number of recruits/number of
individuals genotyped. PR (niches sampled on protected ropes); UUR (upper niche sampled on unprotected ropes); EHCP (External hatchery control progeny grew unprotected); ERCG
(external recruit control group) composed by recruits from Ría de Vigo settled to the bottom weight attached to the ropes; UBSS and UUSS (unprotected bottom or upper sampled site)
were sibs were sampled in spring.
Sampling I Sampling II
(03/04/2008 6 months) (08/05/2008 7 months)
PR UR PR UR
R/G (%) 0/60 (0) 0/60 (0) 0/60 (0) 0/80 (0)
2002) and to the high fecundity of mussels, it is not improbable that
unique wild multilocus genotypes from independent fecundations in
the wild population or Ría de Vigo recruit randomly to free niches of
the rope.
3.4.4. Log-likelihood distributions of assignment probabilities
Distributions of multilocus genotypes versus their maximum
likelihood of assignment to the hatchery progeny showed two dis-
joint scenarios, i.e. a normal distribution of highly frequent
multilocus genotypes from the hatchery progeny (left side of
Fig. 3) and a scattered distribution of less frequent genotypes from
external sources (immigrants to the rope, right side of Fig. 3).
While some overlapping exists between the two distributions, the
global likelihood assignment to the home group was correct in 90%
of cases, i.e. the two mismatches observed in the experimental con-
trol group (EHCP, see Section 3.4.1) were fully compatible with a
hatchery progeny multilocus composition. In this sense, the efficien-
cy of current assignment programs to identify immigrants fully relies
on the composition and size of the sample. In most cases where sam-
ple sizes are limited, assignment programs find more immigrants
than real, e.g. current ERCG group. This is not a problem with con-
trolled aquaculture crosses since one can tell after visual inspection
if odd multilocus genotypes can belong to a given family, even if
they have a low probability of occurrence.
3.4.5. Estimation of mussel recruitment in the period April 2008 –
November 2008
No predation was observed on the protected rope from January
2008 to June 2008 (Table 3) as estimated by seed counting on a
10 cm stretch at three depths of the rope (data not shown). That ob-
servation indicates that the initial rope protection impeded the ac-
tion of putative predators. The absence of predation also on the
unprotected rope (data not shown) suggests that its impact on cur-
rent suspended cultures was very low during that period in that cul-
turing area. External spat fixation was also fairly null during the first
three months in suspension (April 2008 to June 2008) in all sampling
sites of the unprotected rope (Table 3). Only one individual from
sampling III (UURS-13, June 2008) showed a diploid genotype
(146/146) in locus Mgμ1 that was incompatible with the parental ge-
notype of the experimental progeny (Male 142/142-female 156/
156) (Table 3). Since two simultaneous meiotic mutations in both
parents are unlikely (≈ 10− 6 –10 − 8 ), most probably individual
UUR-13 is an external recruit from Ría de Vigo, thus amounting
1.7% of external recruitment from April 2008 to June 2008. Notewor-
thy, null external recruitment was observed in sampling IV (Novem-
ber 2008) on the 13 month-old adult culture from the experimental
hatchery control progeny (EHCP, Table 3), opposite to free niches
where spring samplings allowed for a full re-settlement of external
spat (UBSS and UUSS, Section 3.4.3, Appendix A and Table 3). In sum-
mary, a full harvest of a hatchery strain is achievable provided that
the appropriate juvenile density has been settled on cultivation
sleeves in the previous winter.
131
Sampling III Sampling IV
(06/06/2008 8 months) (14/11/2008 13 months)
PR UUR EHCP ERCG UBSS UUSS
0/58 (0) 1/60 (1.7) 0/20 (0) 48/48 (100) 24/24 (100) 22/24 (91.6)
4. Conclusion
A handicap exists to cultivate selected mussel strains in marine areas
where fish predation has been identified as a major cause of juvenile
loss in mussel farms (e.g. Hayden, 1995), especially on small mussels
(b20 mm) (Rilov and Schiel, 2006). High predation requires sleeve
protection of selected juveniles at the cultivation take-off, such as that
used in the present pilot experiment and in order to maximize the
probability of harvesting a full hatchery strain of mussel grown in
suspension, it is recommendable to prevent external recruitment by
both, optimizing juvenile density on ropes and assuring asynchrony
between natural spawning and seeding-out timing. Theoretically,
upcoming selective breeding programs for mussels would allow to
obtaining high-growth selected seeds in the hatchery (e.g. Nguyen
et al., 2014) which could be used for attaining the commercial size
earlier than current wild seed in classic production cycles. Besides,
hatchery-produced mussel strains could become a solution to actual
needs of producers and markets, such as a) to increase the number of
production sleeves in place of spat collectors, b) to provide seed in
places where its natural recruitment to collectors is scarce or in places
where several mussel species cohabit and difficulties exist to extract
the desirable one (Kamermans et al., 2013), e.g. Andalucía coast
(Southern Spain) where M. galloprovincialis, Mytilaster minimus and
Perna picta share rocky substrates (Tirado et al., 2005), c) to ignore
coastline dumping which disallows seed gathering from rocky shores,
d) to avoid conflict among artisanal fishing sectors, e.g. colliding
extractive activities of the barnacle Pollicipes pollicipes and blue mussel
M. galloprovincialis which can be found co-distributed on the same
rocky shores in Galicia (Molares and Freire, 2003), and e) to reduce
the ecological impact because of the accidental removal of collateral
species when scrapping mussel juveniles on rocky shores.
Nevertheless, more research on the necessary know-how for mussel
seed production and transfer methodologies should predate the
implementation of large-scale cultivation of selected hatchery strains.
Impact of hatchery-selected mussels on the genetic variability of natural
populations as well as on the loading capacity of the ecosystems where
massive cultivation occurs, are additional demanded priors. Also,
accurate estimates of the economic viability of the main variables in
play are needed, e.g. the production cost of selected seed in hatchery
and its return in labor costs as compared to free traditional seed sources.
Finally, the control of selected seed from hatchery should be adequately
legislated since actual local regulations do not foresee any massive cul-
tivation and harvesting of hatchery mussels (e.g. Fishing Law 11/2008).
Acknowledgements
This study has been feasible thanks to grant #AE01-131H-641.11
“Agrupación Estratégica Oceanografía-ECIMAT, AEOE” from Consellería
de Educación e Ordenación Universitaria da Xunta de Galicia (Galician
Regional Government) cofounded by the European Regional Develop-
ment Fund (ERDF). We thank Damián Costas, Pablo Álvarez, Montse
Pérez and Alfonso Pita for their experimental advice and management
expertise.
132 B. Díaz-Puente et al. / Aquaculture 460 (2016) 124–135

Appendix A
Genotypes of genitors and offspring from five treatments in sampling IV (November 2008, 13 month-old mussels). L-Home, assignment probability to
the source group (A genotype is misassigned if it has a low multilocus probability in the group it was sampled); Pim, probability of being immigrant
(p b 0.01) within its sampling group; Pfs, probability of belonging to the full-sib family; Ni/Ng, per locus number of incompatible loci between off-
spring and genitors per genotyped; Nominal levels: *, p b 0.05; **, p b 0.01; ***, p b 0.001. UUR (upper unprotected rope site from spring); EHCP (Ex-
ternal hatchery control progeny grew unprotected); ERCG (external control of recruitment composed by recruits settled to the bottom weight
attached to tip of the ropes); UBSS (bottom unprotected sampled site were sibs were previously sampled); UUSS (upper unprotected upper sampled
site) were sibs were previously sampled); na (genotype not available); ECRV (External Control group from Ría de Vigo).

Locus Mgμ5 Mgμ8 Mgμ3 Mch8 Mgμ1 Mgμ4 P (L-Home) Pim Pfs Ni/Ng

Father 132–132 194–200 138–140 203–203 142–142 173–191

Mother 126–132 200–200 138–140 203–205 156–156 173–null

Sampling I – – – – – – – – –
Sampling II – – – – – – – – –
Sampling III UUR–13 126–132 200–200 140–140 203–203 146–146 173–173 0.020* b0.001*** b0.001*** 1/6
Sampling IV
EHCP (Experimental hatchery control progeny) EHCP–1 132–132 194–200 140–140 203–203 142–156 173–173 0.812 0.799 0.812 0/6
EHCP–2 126–132 194–200 140–140 203–203 142–156 173–173 0.530 0.508 0.530 0/6
EHCP–3 na 194–200 140–140 203–203 142–156 173–173 0.796 0.788 0.796 0/5
EHCP–4 132–132 194–200 140–140 203–203 142–156 173–173 0.812 0.818 0.812 0/6
EHCP–5 126–132 194–200 140–140 203–203 142–156 na 0.447 0.448 0.447 0/6
EHCP–6 132–132 na 140–140 203–203 142–156 173–173 0.818 0.830 0.818 0/5
EHCP–7 132–132 na 140–140 203–203 142–156 173–173 0.832 0.841 0.832 0/5
EHCP–8 132–132 194–200 140–140 203–203 142–156 173–173 0.812 0.807 0.812 0/6
EHCP–9 na 194–200 140–140 203–203 142–156 173–173 0.807 0.787 0.807 0/5
EHCP–10 126–132 194–200 140–140 203–203 142–156 173–173 0.530 0.527 0.530 0/6
EHCP–11 126–132 194–200 140–140 203–203 142–156 191–191 0.007** b0.001*** 0.007** 0/6
EHCP–12 132–132 194–200 140–140 203–203 142–156 173–173 0.812 0.801 0.812 0/6
EHCP–13 132–132 194–200 140–140 203–203 142–156 173–173 0.812 0.819 0.812 0/6
EHCP–14 126–132 194–200 140–140 203–203 142–156 173–173 0.530 0.493 0.530 0/6
EHCP–15 126–132 194–200 140–140 203–203 142–156 173–173 0.530 0.501 0.530 0/6
EHCP–16 126–132 200–200 140–140 203–203 142–156 173–173 0.406 0.387 0.406 0/6
EHCP–17 132–132 200–200 140–140 203–205 142–156 173–173 0.097 0.007** 0.097 0/6
EHCP–18 132–132 200–200 140–140 203–203 142–156 173–173 0.719 0.712 0.719 0/6
EHCP–19 132–132 na 140–140 203–203 142–156 173–173 0.843 0.838 0.843 0/5
EHCP–20 na 194–200 140–140 203–203 142–156 173–173 0.795 0.799 0.795 0/5
ERCG (External recruitment control group) ERCG–1 128–132 196–202 140–140 203–203 138–138 191–191 0.041* 0.001** b0.001*** 3/6
ERCG–2 130–130 190–214 140–140 203–203 164–164 183–183 0.059 0.001** b0.001*** 4/6
ERCG–3 130–130 na 140–140 203–203 138–138 177–185 0.127 0.037** b0.001*** 3/5
ERCG–4 132–142 200–208 140–140 203–205 134–146 183–183 0.082 0.005** b0.001*** 4/6
ERCG–5 126–134 200–200 138–140 203–203 144–156 173–183 0.389 0.358 b0.001*** 3/6
ERCG–6 130–130 214–214 140–140 203–203 138–166 167–173 0.202 0.023* b0.001*** 4/6
ERCG–7 126–136 192–192 138–138 205–205 140–140 167–167 0.002** b0.001*** b0.001*** 5/6
ERCG–8 132–132 200–200 138–138 203–203 144–154 173–183 0.215 0.034* b0.001*** 2/6
ERCG–9 126–132 192–198 140–140 203–205 142–142 167–185 0.173 0.094 b0.001*** 3/6
ERCG–10 126–132 192–198 140–140 203–205 144–146 173–173 0.552 0.523 b0.001*** 2/6
ERCG–11 na 212–212 140–140 203–203 146–146 191–191 0.534 0.414 b0.001*** 2/5
ERCG–12 124–130 202–206 140–140 203–203 138–138 191–191 0.623 0.506 b0.001*** 3/6
ERCG–13 130–138 190–200 140–140 203–203 144–156 na 0.592 0.477 b0.001*** 3/5
ERCG–14 124–142 192–192 140–140 203–205 132–146 165–173 0.118 0.044* b0.001*** 4/6
ERCG–15 128–134 194–194 138–140 203–203 144–156 175–185 0.385 0.320 b0.001*** 3/6
ERCG–16 132–134 196–214 140–140 203–203 158–158 175–179 0.158 0.086 b0.001*** 4/6
ERCG–17 134–134 196–208 140–140 203–203 138–138 171–171 0.272 0.138 b0.001*** 4/6
ERCG–18 124–134 194–198 140–140 203–205 158–158 173–185 0.028* 0.003** b0.001*** 4/6
ERCG–19 124–134 na 138–140 203–203 140–140 191–191 0.195 0.128 b0.001*** 2/5
ERCG–20 134–140 na 140–140 203–203 144–158 167–183 0.493 0.422 b0.001*** 3/5
ERCG–21 140–140 194–202 140–140 203–203 138–142 171–173 0.175 0.086 b0.001*** 4/6
ERCG–22 126–132 192–200 140–140 203–203 140–144 183–183 0.717 0.683 b0.001*** 3/6
ERCG–23 132–132 200–200 140–140 201–211 138–138 175–179 0.013* b0.001*** b0.001*** 3/6
ERCG–24 132–138 200–200 138–140 203–203 144–150 167–175 0.151 0.022* b0.001*** 3/6
ERCG–25 132–132 190–198 140–140 203–203 138–146 173–173 0.665 0.595 b0.001*** 2/6
ERCG–26 132–132 200–200 140–140 203–203 132–140 175–179 0.302 0.191 b0.001*** 2/6
ERCG–27 132–132 192–198 140–140 203–203 146–156 183–183 0.544 0.503 b0.001*** 3/6
ERCG–28 126–132 192–192 140–140 203–203 138–144 169–177 0.181 0.083 b0.001*** 3/6
ERCG–29 132–134 190–198 140–140 205–205 144–156 169–175 0.035* 0.010* b0.001*** 5/6
ERCG–30 126–134 na 140–140 203–203 140–142 167–167 0.441 0.339 b0.001*** 3/5
ERCG–31 132–132 200–208 140–140 203–203 140–146 167–171 0.406 0.290 b0.001*** 3/6
ERCG–32 126–132 192–198 140–140 203–203 142–144 175–185 0.438 0.383 b0.001*** 3/6
ERCG–33 132–132 190–198 140–140 203–203 140–146 167–175 0.548 0.499 b0.001*** 3/6
ERCG–34 124–132 190–190 140–140 203–209 142–146 167–167 0.026* 0.002** b0.001*** 5/6
ERCG–35 122–132 198–200 140–140 203–203 144–144 183–183 0.437 0.158 b0.001*** 4/6
ERCG–36 134–140 212–214 140–140 203–203 144–144 167–183 0.604 0.515 b0.001*** 4/6
ERCG–37 124–132 192–204 140–140 203–203 144–144 185–185 0.506 0.361 b0.001*** 4/6
ERCG–38 na 214–214 140–140 203–203 134–146 173–173 0.693 0.554 b0.001*** 2/5
ERCG–39 na 194–202 140–140 203–203 144–146 165–183 0.629 0.464 b0.001*** 3/5
 B. Díaz-Puente et al. / Aquaculture 460 (2016) 124–135 133

(continued)
Appendix A (continued)

Locus Mgμ5 Mgμ8 Mgμ3 Mch8 Mgμ1 Mgμ4 P (L-Home) Pim Pfs Ni/Ng

Father 132–132 194–200 138–140 203–203 142–142 173–191

Mother 126–132 200–200 138–140 203–205 156–156 173–null

ERCG–40 132–132 198–206 140–140 203–203 134–134 173–173 0.541 0.356 0.001** 2/6
ERCG–41 120–132 198–202 138–138 203–203 140–140 173–183 0.046* 0.004** b0.001*** 4/6
ERCG–42 132–132 198–198 138–138 203–203 144–144 173–173 0.321 0.243 b0.001*** 2/6
ERCG–43 132–138 200–200 140–140 203–203 140–140 167–173 0.482 0.366 b0.001*** 3/6
ERCG–44 134–134 200–200 140–140 203–205 138–144 173–173 0.386 0.333 b0.001*** 2/6
ERCG–45 132–136 192–202 140–140 203–203 140–144 173–183 0.766 0.677 b0.001*** 4/6
ERCG–46 132–132 192–208 132–140 203–203 138–146 191–191 0.297 0.103 b0.001*** 3/6
ERCG–47 132–134 194–214 138–138 203–205 162–168 175–185 0.005** b0.001*** b0.001*** 4/6
ERCG–48 126–132 192–212 140–140 203–203 144–158 173–173 0.804 0.734 b0.001*** 2/6
UBSS (Unprotected bottom sampled site) UBSS–1 134–134 200–200 138–140 203–203 138–146 183–183 0.830 0.695 b0.001*** 3/6
UBSS–2 na 202–208 140–140 203–203 136–136 177–177 0.026* b0.001*** b0.001*** 3/5
UBSS–3 132–132 198–198 138–142 203–203 138–138 173–173 0.129 0.047* b0.001*** 3/6
UBSS–4 122–132 200–200 140–140 201–201 150–158 181–181 0.004** b0.001*** b0.001*** 4/6
UBSS–5 126–132 192–198 140–142 205–205 138–138 165–173 0.016* b0.001*** b0.001*** 5/6
UBSS–6 132–132 194–194 140–140 203–203 138–146 167–167 0.437 0.040* b0.001*** 4/6
UBSS–7 132–132 200–200 138–140 195–195 140–140 179–181 0.041* b0.001*** b0.001*** 3/6
UBSS–8 132–132 198–198 140–140 203–203 144–160 167–177 0.229 0.013* b0.001*** 3/6
UBSS–9 132–132 200–200 140–140 203–205 144–156 155–183 0.827 0.608 b0.001*** 2/6
UBSS–10 124–134 200–200 140–140 203–203 146–146 181–189 0.305 0.044* b0.001*** 2/6
UBSS–11 134–134 198–200 138–140 203–205 134–146 173–183 0.327 0.056 b0.001*** 4/6
UBSS–12 134–134 202–208 132–138 203–203 138–152 173–173 0.016* b0.001*** b0.001*** 4/6
UBSS–13 132–132 200–200 138–140 203–203 144–156 167–167 0.873 0.770 b0.001*** 2/6
UBSS–14 134–134 200–200 140–140 203–203 142–156 173–173 0.880 0.782 b0.001*** 1/6
UBSS–15 134–140 208–208 140–140 203–203 156–156 173–189 0.152 0.011* b0.001*** 4/6
UBSS–16 132–132 200–200 140–140 203–203 156–156 167–167 0.987 0.957 b0.001*** 2/6
UBSS–17 132–132 192–208 140–140 203–203 142–156 167–167 0.662 0.509 b0.001*** 2/6
UBSS–18 126–132 192–200 140–140 203–203 140–140 167–169 0.350 0.040* b0.001*** 3/6
UBSS–19 126–132 192–200 140–140 205–205 142–156 171–171 0.075 0.001** b0.001*** 3/6
UBSS–20 132–132 200–200 140–140 203–205 142–156 175–175 0.561 0.063 b0.001*** 1/6
UBSS–21 134–134 200–200 140–140 203–203 146–156 183–183 0.951 0.899 b0.001*** 3/6
UBSS–22 132–132 200–200 140–140 203–203 138–138 183–183 0.975 0.941 b0.001*** 2/6
UBSS–23 132–132 200–200 140–140 203–203 140–140 155–183 0.847 0.637 b0.001*** 2/6
UBSS–24 134–134 200–200 140–140 203–205 146–156 183–183 0.890 0.820 b0.001*** 3/6
UUSS (Upper unprotected sampled site) UUSS–1 134–140 202–208 140–140 205–205 140–140 183–183 0.002** b0.001*** b0.001*** 5/6
UUSS–2 124–132 190–190 138–138 203–203 142–156 171–183 0.035* 0.004** b0.001*** 3/6
UUSS–3 132–132 200–200 140–140 203–203 142–156 173–181 0.945 0.896 0.007** 1/6
UUSS–4 132–132 200–200 138–138 203–203 140–146 167–173 0.905 0.850 b0.001*** 2/6
UUSS–5 132–132 200–200 138–138 203–203 138–146 167–173 0.841 0.790 b0.001*** 2/6
UUSS–6 124–132 192–198 138–140 203–203 144–146 167–173 0.063 b0.001*** b0.001*** 4/6
UUSS–7 132–132 200–200 140–140 203–203 144–156 173–173 0.974 0.969 0.009** 1/6
UUSS–8 132–132 196–206 140–140 203–203 140–140 185–185 0.767 0.551 b0.001*** 3/6
UUSS–9 na 200–204 138–140 203–205 160–160 185–185 0.135 b0.001*** b0.001*** 3/5
UUSS–10 126–134 200–200 140–140 205–205 140–140 185–185 0.106 0.030* b0.001*** 4/6
UUSS–11 132–132 200–200 140–140 201–201 142–156 173–181 0.403 0.226 b0.001*** 2/6
UUSS–12 132–132 200–200 138–140 205–205 142–166 173–187 0.223 0.002** b0.001*** 3/6
UUSS–13 134–134 200–200 138–142 203–205 170–170 157–157 0.006** b0.001*** b0.001*** 4/6
UUSS–14 126–132 194–200 138–140 203–203 142–156 173–173 0.540 0.175 0.016* 0/6
UUSS–15 132–132 200–200 140–140 203–203 146–170 173–179 0.818 0.366 b0.001*** 2/6
UUSS–16 132–132 212–214 138–140 203–205 134–138 173–173 0.694 0.156 b0.001*** 2/6
UUSS–17 132–132 200–208 140–140 203–203 138–138 175–183 0.471 0.161 b0.001*** 3/6
UUSS–18 130–140 192–204 140–140 203–203 146–146 171–175 0.041* 0.001** b0.001*** 4/6
UUSS–19 126–132 200–200 140–140 203–203 142–156 173–173 0.905 0.851 0.406 0/6
UUSS–20 134–140 192–200 140–140 203–203 132–144 173–173 0.189 0.012* b0.001*** 3/6
UUSS–21 132–132 200–200 138–140 203–203 150–156 169–181 0.330 0.007** b0.001*** 2/6
UUSS–22 132–140 190–200 138–140 201–201 148–148 167–167 0.014* b0.001*** b0.001*** 5/6
UUSS–23 132–132 200–200 140–140 203–203 144–146 167–167 0.838 0.723 b0.001*** 2/6
UUSS–24 124–132 200–200 138–140 203–203 170–170 167–173 0.727 0.588 b0.001*** 3/6
ECRV (External Control Ría de Vigo) ECRV–1 132–132 196–196 140–140 203–203 144–144 171–183 0.577 0.091 b0.001*** 3/6
ECRV–2 130–130 198–198 138–138 201–203 134–144 167–175 0.330 0.116 b0.001*** 5/6
ECRV–3 130–134 194–206 142–138 205–211 140–140 173–173 0.228 0.024* b0.001*** 5/6
ECRV–4 128–134 194–204 142–140 203–205 138–144 177–177 0.444 0.241 b0.001*** 5/6
ECRV–5 126–132 192–192 140–138 201–207 144–144 167–177 0.542 0.297 b0.001*** 4/6
ECRV–6 132–132 196–208 140–140 201–203 144–144 169–199 0.441 0.038* b0.001*** 4/6
ECRV–7 128–140 194–194 140–136 203–203 138–138 173–173 0.094 0.001** b0.001*** 4/6
ECRV–8 132–136 190–200 140–138 201–205 144–144 173–195 0.338 0.005** b0.001*** 5/6
ECRV–9 126–130 194–200 140–138 203–203 140–156 173–173 0.535 0.099 b0.001*** 2/6
ECRV–10 130–132 198–206 142–140 203–203 146–146 171–173 0.391 0.163 b0.001*** 5/6
ECRV–11 126–130 198–204 140–138 201–203 140–144 163–167 0.478 0.098 b0.001*** 5/6
ECRV–12 132–136 210–220 140–138 209–203 132–144 167–173 0.306 0.020* b0.001*** 5/6
ECRV–13 132–132 192–212 140–138 203–203 142–142 171–177 0.569 0.290 b0.001*** 3/6
ECRV–14 128–134 200–210 140–138 203–205 134–148 167–167 0.093 b0.001*** b0.001*** 4/6
ECRV–15 134–138 192–204 140–140 201–207 144–144 173–187 0.242 0.050 b0.001*** 5/6

(continued on next page)

134 B. Díaz-Puente et al. / Aquaculture 460 (2016) 124–135

(continued)
Appendix A (continued)

Locus Mgμ5 Mgμ8 Mgμ3 Mch8 Mgμ1 Mgμ4 P (L-Home) Pim Pfs Ni/Ng

Father 132–132 194–200 138–140 203–203 142–142 173–191

Mother 126–132 200–200 138–140 203–205 156–156 173–null

ECRV–16 132–132 214–214 140–138 203–205 144–144 167–167 0.931 0.806 b0.001*** 3/6
ECRV–17 126–132 194–202 138–138 203–203 140–144 177–177 0.875 0.760 b0.001*** 3/6
ECRV–18 130–132 198–206 140–138 201–205 142–142 173–177 0.768 0.571 b0.001*** 5/6
ECRV–19 124–132 194–204 140–140 203–203 144–144 169–173 0.956 0.927 b0.001*** 4/6
ECRV–20 132–134 212–214 140–132 201–205 144–144 165–173 0.319 0.024* b0.001*** 6/6
ECRV–21 132–132 202–202 140–138 205–205 140–142 173–173 0.869 0.760 b0.001*** 3/6
ECRV–22 124–142 194–198 140–138 203–203 146–146 179–179 0.079 b0.001*** b0.001*** 4/6
ECRV–23 124–140 196–202 140–138 205–205 132–132 155–155 0.036* b0.001*** b0.001*** 5/6
ECRV–24 124–132 192–202 138–138 209–209 144–150 155–167 0.094 0.002** b0.001*** 5/6
ECRV–25 126–134 202–206 138–132 205–205 142–144 173–177 0.503 0.297 b0.001*** 6/6
ECRV–26 124–132 204–214 138–138 203–205 144–144 169–175 0.740 0.529 b0.001*** 4/6
ECRV–27 132–132 194–206 142–138 203–205 140–146 185–185 0.311 0.007** b0.001*** 4/6
ECRV–28 134–134 194–194 138–138 203–205 134–144 173–173 0.882 0.739 b0.001*** 2/6
ECRV–29 124–138 194–204 140–140 203–203 142–142 173–173 0.617 0.357 b0.001*** 3/6
ECRV–30 122–132 196–208 138–138 205–205 144–144 175–187 0.211 0.010* b0.001*** 5/6

References
Aghzar, A., Talbaoui, M., Benajiba, M.H., Presa, P., 2012. Influence of depth and diameter of
rope collectors on settlement density of Mytilus galloprovincialis spat in Baie de M'diq
(Alboran Sea). Mar. Freshw. Behav. Physiol. 45, 51–61.
Alcapán, A.C., Nespolo, R.F., Toro, J.E., 2007. Heritability of body size in the Chilean blue
mussel (Mytilus chilensis Hupe 1854): effects of environment and ageing. Aquac.
Res. 38, 313–320.
Anderson, D.M., Cembella, A.D., Hallegraeff, G.M., 2012. Progress in understanding harm-
ful algal blooms: paradigm shifts and new technologies for research, monitoring, and
management. Ann. Rev. Mar. Sci. 4, 143–176.
Beadman, H., Caldow, R., Kaiser, M., Willows, R., 2003. How to toughen up your mussels:
using mussel shell morphology plasticity to reduce predation losses. Mar. Biol. 142,
487–494.
Bode, A., Casas, B., Férnandez, E., Marañón, E., Serret, P., Varela, M., 1996. Phytoplankton
biomass and production in shelf waters off NW Spain: spatial and seasonal variability
in relation to upwelling. Hydrobiologia 341, 225–234.
Brigolin, D., Dal Maschio, G., Rampazzo, F., Giani, M., Pastres, R., 2009. An individual-based
population dynamic model for estimating biomass yield and nutrient fluxes through
an off-shore mussel (Mytilus galloprovincialis) farm. Estuar. Coast. Shelf Sci. 82,
365–376.
Cáceres-Martínez, J., Figueras, A., 1998a. Distribution and abundance of mussel (Mytilus
galloprovincialis Lmk) larvae and post-larvae in the Ría de Vigo (NW Spain). J. Exp.
Mar. Biol. Ecol. 229, 277–287.
Cáceres-Martínez, J., Figueras, A., 1998b. Mussel (Mytilus galloprovincialis Lamarck) colo-
nization on artificial substrates in the Ría de Vigo of NW Spain. J. Shellfish Res. 17,
153–157.
Chistiakov, D., Hellemans, B., Volckaert, F., 2006. Microsatellites and their genomic distri-
bution, evolution, function and applications: a review with special reference to fish
genetics. Aquaculture 255, 1–29.
Cornuet, J.M., Piry, S., Luikart, G., Estoup, A., Solignac, M., 1999. New methods employing
multilocus genotypes to select or exclude populations as origins of individuals. Ge-
netics 153, 1989–2000.
Diz, A., Presa, P., 2009. The genetic diversity pattern of Mytilus galloprovincialis in Galician
Rías (NW Iberian estuaries). Aquaculture 287, 278–285.
Estoup, A., Presa, P., Krieg, F., Vaiman, D., Guyomard, R., 1993. CT)n and (GT)n
microsatellites: a new class of genetic markers for Salmo trutta L. (brown trout. He-
redity 71, 488–496.
FAO, Food and Agriculture Organization of the United Nations. FIGIS. FishStat (database).
URL: http://data.fao.org/ref/babf3346-ff2d-4e6c-9a40-ef6a50fcd422.html?version=1.0
Figueras, A., 2007. Biología y cultivo del mejillón (Mytilus galloprovincialis) en Galicia.
Consejo Superior de Investigaciones Científicas, Madrid.
Fishing Law 11/2008. Galician government (Xunta de Galicia). URL: http://www.xunta.
es/dog/Publicados/2008/20081216/Anuncio4C1EE_es.html
Fuentes, J., Molares, J., Villalba, A., 1998. Growth, mortality and parasitization of mussels
cultivated in the Ría de Arousa (NW Spain) from two sources of seed: intertidal
rocky shore vs. collector ropes. Aquaculture 162, 231–240.
Gentili, M.R., Beaumont, A.R., 1988. Environmental stress, heterozygosity, and growth rate
in Mytilus edulis L. J. Exp. Mar. Biol. Ecol. 120, 145–153.
Gjedrem, T., 2000. Genetic improvement of cold-water fish species. Aquac. Res. 31, 25–33.
González-Laxe, F., Martín-Palmero, F., 2014. El mercado de mejillón en España. Distrib.
Consum. 3, 102–111http://www.mercasa.es/files/multimedios/1406496600_
DISTRIBUCION_Y_CONSUMO_133_articulo_mejillon.pdf.
Goudet, J., 1995. FSTAT (version 1.2): a computer program to calculate F-statistics.
J. Hered. 86, 485–486.
Harger, J.R.E., Landenberger, D.L., 1971. The effect of storms as a density dependent mor-
tality factor on populations of sea mussels. Veliger 14, 195–201.
Hayden, B., 1995. Factors affecting recruitment of farmed greenshell mussels, Perna canalic-
ulus (Gmelin) 1791, the Malborough sounds. University of Otago, New Zealand, p. 168.
Kamermans, P., Galley, T., Boudry, P., Fuentes, J., McCombie, H., Batista, F.M., Blanco, A.,
Dominguez, L., Cornette, F., Pincot, P., Beaumont, A., 2013. Blue mussel hatchery tech-
nology in Europe. In: Allan, G., Burnell, G. (Eds.), Advances in Aquaculture Hatchery
Technology. Woodhead Publishing Limited, Cambridge, U.K, pp. 339–373.
Lado-Ínsua, T., Pérez, M., Diz, A.P., Presa, P., 2011. Temporal estimates of genetic diversity
in some Mytilus galloprovincialis populations impacted by the prestige oil-spill. Cont.
Shelf Res. 31, 466–475.
Liu, G., Stapleton, E., Innes, D., Thompson, R., 2011. Aggregational behavior of the blue
mussels Mytilus edulis and Mytilus trossulus: a potential pre-zygotic reproductive iso-
lation mechanism. Mar. Ecol. Evol. Persp. 32, 480–487.
MAGRAMA, 2014. Ministerio de Agricultura. Alimentación Y Medio Ambiente. Datos de
producción de Acuicultura (Accessed on 01 July 2014) http://www.magrama.gob.
es/es/pesca/temas/acuicultura/produccion_engorde_2012_tcm7-305691.pdf.
Matusse, N.R.D., Pita, A., Pérez, M., Trucco, M.I., Peleteiro, J.B., Presa, P., 2016. First-
generation genetic drift and inbreeding risk in hatchery stocks of the wreckfish
Polyprion americanus. Aquaculture 451, 125–136.
Miñambres, M., Pérez, M., Alvarez, P., Presa, P., 2011. In: García-Estévez, J.M., Olabarría, C.,
Pérez, S., Rolán-Álvarez, E., Rosón, G. (Eds.), Cálculo de parámetros elementales para
el cultivo de microalgas en criadero y su aplicación en el diseño de raciones
alimentarias. Métodos y Técnicas en Investigación Marina, Vigo, pp. 159–172 In
Spanish.
Moen, T., Baranski, M., Sonesson, A.K., Kjøglum, S., 2009. Confirmation and fine-mapping
of a major QTL for resistance to infectious pancreatic necrosis in Atlantic salmon
(Salmo salar): population-level associations between markers and trait. BMC Geno-
mics 10, 1–14 doi:10.1186/1471-2164-10-368.
Molares, J., Freire, J.M., 2003. Development and perspectives for community-based man-
agement of the goose barnacle (Pollicipes pollicipes) fisheries in Galicia (NW Spain).
Fish. Res. 65, 485–492.
Myrand, B., Tremblay, R., Sevigny, J.M., 2002. Selection against blue mussels (Mytilus
edulis L.) homozygotes under various stressful conditions. J. Hered. 93, 238–248.
Myrand, B., Tremblay, R., Sevigny, J.M., 2009. Impact of suspension culture using mesh
sleeves on genetic characteristics of Mytilus edulis L. in Canada. Aquaculture 291,
147–153.
Nevejan, N., Davis, J., Little, K., Kiliona, A., 2007. Use of a formulated diet for mussel spat
Mytilus galloprovincialis (Lamarck 1819) in a commercial hatchery. J. Shellfish Res.
26, 357–363.
Nguyen, T.T.T., Hayes, B.J., Ingram, B.A., 2014. Genetic parameters and response to
selection in blue mussel (Mytilus galloprovincialis) using a SNP-based pedigree.
Aquaculture 420–421, 295–301.
Ouagajjou, Y., Presa, P., 2015. The connectivity of Mytilus galloprovincialis in Northern
Morocco: a gene flow crossroads between continents. Estuar. Coast. Shelf Sci. 152, 1–10.
Ouagajjou, Y., Aghzar, A., Miñambres, M., Presa, P., Pérez, M., 2010. Differential gene flow
between populations of Mytilus galloprovincialis distributed along Iberian and north
African coasts. Thalassas 26, 75–78.
Ouagajjou, Y., Presa, P., Astorga, M., Pérez, M., 2011. Microsatellites of Mytilus chilensis: a
genomic print of its taxonomic status within Mytilus sp. J. Shellfish Res. 30, 325–330.
Paetkau, D., Slade, R., Burden, M., Estoup, A., 2004. Direct, real-time estimation of migra-
tion rate using assignment methods: a simulation-based exploration of accuracy and
power. Mol. Ecol. 13, 55–65.
Pérez, M., Presa, P., 2011. FENOSALT: Un método sintético para la extracción de ADN de
peces y moluscos. In: García-Estévez, J.M., Olabarría, C., Pérez, S., Rolán-Álvarez, E.,
Rosón, G. (Eds.), Métodos Y Técnicas en Investigación Marina, Vigo, pp. 81–89 In
Spanish.
Piry, S., Alapetite, A., Cornuet, J.-M., Paetkau, D., Baudouin, L., Estoup, A., 2004. GeneClass2:
a software for genetic assignment and first-generation migrant detection. J. Hered.
95, 536–539.
Presa, P., Krieg, F., Estoup, A., Guyomard, R., 1994. Diversité et gestion génétique de la
truite commune: Apport de l'étude du polymorphisme des locus protéiques et
microsatellites. Genet. Sel. Evol. 26, 183–202.
 B. Díaz-Puente et al. / Aquaculture 460 (2016) 124–135
Presa, P., Pérez, M., Diz, A.P., 2002. Polymorphic microsatellite markers for blue mussels
(Mytilus spp.). Conserv. Genet. 3, 441–443.
Rannala, B., Mountain, J.L., 1997. Detecting immigration by using multilocus genotypes.
Proc. Natl. Acad. Sci. U. S. A. 94, 9197–9201.
Raymond, M., Vääntä, R.L., Thomas, F., Rousset, F., De Meeüs, T., Renaud, F., 1997. Hetero-
zygote deficiency in the mussel Mytilus edulis species complex revisited. Mar. Ecol.
Prog. Ser. 156, 225–237.
Rilov, G., Schiel, D.R., 2006. Trophic linkages across seascapes: subtidal predators limit effec-
tive mussel recruitment in rocky intertidal communities. Mar. Ecol. Prog. Ser. 327, 83–93.
Sénéchal, J., Grant, J., Archambault, M.-C., 2008. Experimental manipulation of suspended
culture stocks: growth and behavior juvenile mussels (Mytilus spp.). J. Shellfish Res.
27, 811–826.
135
Tirado, C., Macías, J.C., Villarías, R.M., Gaiteiro, J.M., Gómez, D., Martín, M.J., Rueda, J.L.,
Álamo, C., Manchado, M., Infante, C., 2005. Conclusiones derivadas del estudio sobre
el potencial del cultivo de mejillón Mytilus galloprovincialis Lamarck, 1819 en
Andalucía. Bol. Inst. Esp. Oceanogr. 21, 455–464 In Spanish.
Vandeputte, M., Rossignol, M.N., Pincent, C., 2011. From theory to practice: empirical eval-
uation of the assignment power of marker sets for pedigree analysis in fish breeding.
Aquaculture 314, 80–86.
Widdows, J., 1991. Physiological ecology of mussel larvae. Aquaculture 94, 147–163.
Zouros, E., Foltz, D.W., 1984. Possible explanations of heterozygote deficiency in bivalve
mollusks. Malacologia 25, 583–591.