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A genome scan for candidate genes involved in


the adaptation of turbot (Scophthalmus
maximus)
Article in Marine Genomics May 2015
DOI: 10.1016/j.margen.2015.04.011 Source: PubMed

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Retrieved on: 24 October 2016

MARGEN-00322; No of Pages 10
Marine Genomics xxx (2015) xxxxxx

Contents lists available at ScienceDirect

Marine Genomics
journal homepage: www.elsevier.com/locate/margen

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a r t i c l e

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Article history:
Received 16 February 2015
Received in revised form 28 April 2015
Accepted 28 April 2015
Available online xxxx

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Keywords:
Adaptation
Atlantic Ocean
Fish
Population genomics

Departamento de Gentica, Universidad de Santiago de Compostela, Facultad de Biologa, Santiago de Compostela E-15706, Spain
University of Leuven, Laboratory of Biodiversity and Evolutionary Genomics, Charles Deberiotstraat 32, B-3000 Leuven, Belgium
Departamento de Gentica, Universidad de Santiago de Compostela, Facultad de Veterinaria, Lugo E-27002, Spain

i n f o

a b s t r a c t

Partitioning phenotypic variance in genotypic and environmental variance may benet from the population
genomic assignment of genes putatively involved in adaptation. We analyzed a total of 256 markers (120
microsatellites and 136 Single Nucleotide Polymorphisms SNPs), several of them associated to Quantitative
Trait Loci (QTL) for growth and resistance to pathologies, with the aim to identify potential adaptive variation
in turbot Scophthalmus maximus L. The study area in the Northeastern Atlantic Ocean, from Iberian Peninsula
to the Baltic Sea, involves a gradual change in temperature and an abrupt change in salinity conditions. We
detected 27 candidate loci putatively under selection. At least four of the ve SNPs identied as outliers are
located within genes coding for ribosomal proteins or directly related with the production of cellular proteins.
One of the detected outliers, previously identied as part of a QTL for growth, is a microsatellite linked to a
gene coding for a growth factor receptor. A similar set of outliers was detected when natural populations were
compared with a sample subjected to strong articial selection for growth along four generations. The observed
association between FST outliers and growth-related QTL supports the hypothesis of changes in growth as an
adaptation to differences in temperature and salinity conditions. However, further work is needed to conrm
this hypothesis.
2015 Published by Elsevier B.V.

Romn Vilas a,, Sara G. Vandamme b,1, Manuel Vera c,2, Carmen Bouza c, Gregory E. Maes b,3,
Filip A.M. Volckaert b, Paulino Martnez c

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3Q4

A genome scan for candidate genes involved in the adaptation of turbot


(Scophthalmus maximus)

1Q3

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1. Introduction

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Partitioning the phenotypic variance into genetic and environmental components can be challenging due to statistical correlations
between genetic and environmental variation and the constitutive
interaction between both effects. However, phenotypic differences
may reect differences in the underlying genetic variation caused
by natural selection acting over generations in a process of adaptation to particular environmental conditions. This effect has been
demonstrated through genetic variation of candidate genes within

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Corresponding author at: Departamento de Gentica, Universidad de Santiago de


Compostela, Facultad de Biologa, Santiago de Compostela E-15706, Spain. Tel/fax: +34
982 822428.
E-mail addresses: roman.vilas@usc.es (R. Vilas), sara.vandamme@ilvo.vlaanderen.be
(S.G. Vandamme), manuel.verar@udg.edu (M. Vera), mcarmen.bouza@usc.es (C. Bouza),
Gregory.Maes@bio.kuleuven.be (G.E. Maes), Filip.Volckaert@bio.kuleuven.be
(F.A.M. Volckaert), paulino.martinez@usc.es (P. Martnez).
1
Present address: Institute for Agricultural and Fisheries Research (ILVO), Animal
Sciences UnitFisheries, Ankerstraat 1, B 8400 Ostend, Belgium.
2
Present address: Laboratori d'Ictiologia Gentica, Departamento de Biologa, Facultad
de Ciencias, Universidad de Girona, Campus de Montilivi s/n, E-17071 Girona, Spain.
3
Present address: Centre for Sustainable Tropical Fisheries and Aquaculture, School of
Marine and Tropical Biology, James Cook University, Townsville QLD 4811, Australia.

populations along an environmental gradient (Colosimo et al.,


2005; Orsini et al., 2012; Alberto et al., 2013; Hemmer-Hansen
et al., 2014). The choice of the genes putatively subjected to natural
selection can be made on the basis of functional knowledge
(Hoffman and Willi, 2008) or by identifying Quantitative Trait Loci
(QTL) involved in the development of phenotypes directly related
to survival and reproduction (Slate, 2005; Storz, 2005). However,
these methods require a detailed characterization of gene function
or performing experimental crosses, which is not always feasible.
The population genomics approach can also be used for detecting
candidate genes. This involves the screening of a large set of markers
scattered across the genome in order to distinguish the effects of
natural selection inuencing specic loci from the effects of processes that act on the whole genome, such as gene ow, genetic drift and
inbreeding (Luikart et al., 2003). In contrast to the latter, loci
targeted by selection or closely linked to a locus under selection
(known as selective sweeps) should display three characteristics
which can be statistically tested: (1) a skewed allele frequency
distribution, (2) low variation within populations and (3) a higher
differentiation between locally adapted populations (e.g. Lewontin
and Krakauer, 1973; Schltterer, 2002). Therefore, scanning the
patterns of variation at the genomic level in order to identify loci

http://dx.doi.org/10.1016/j.margen.2015.04.011
1874-7787/ 2015 Published by Elsevier B.V.

Please cite this article as: Vilas, R., et al., A genome scan for candidate genes involved in the adaptation of turbot (Scophthalmus maximus), Mar.
Genomics (2015), http://dx.doi.org/10.1016/j.margen.2015.04.011

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Q5
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Fin clip samples were obtained from 286 specimens of turbot


(S. maximus) captured at ve locations across the Northeastern Atlantic
Ocean (Fig. 1): the English Channel (EC, 50.85 N; 1.1 E); the Baltic Sea in
the vicinity of the Island of Bornholm, Denmark (BS, 55.07 N; 14.55 E);
the German Bight (NS, 55.60 N; 8.0 E); the Cantabric Sea (CS, 43.41 N;
7.30 E); and the Atlantic Galician coast of the Iberian Peninsula (AG,
42.14 N; 8.43 W). An additional farmed population (FAR) native to the
Northeastern Atlantic Ocean was analyzed. This population is the result
of four generations of strong articial selection for growth carried out in
aquaculture facilities in Galicia, Northwestern Spain. A total of 48
individuals per site (except 46 at CS) were analyzed. No specic permission for sampling was required for this study since all wild individuals
sampled were obtained from commercial shing and those from the
farm were broods previously stored from an ongoing genetic breeding
program. No protected species was sampled.

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2.2. Genetic markers

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Whole genomic DNA was extracted by using standard phenol


chloroform procedures. Samples were genotyped for a total of 256
codominant markers: 120 microsatellites and 136 single nucleotide
polymorphisms (SNPs). All microsatellite loci had been characterized (Pardo et al., 2006, 2007; Bouza et al., 2008) and mapped
(Bouza et al., 2007, 2012) previously. Because their linkage relationships could be used as a selection criterion, the 24 linkage groups
that constitute the turbot genetic map were included. Furthermore,
linkage equilibrium between markers could be assumed as loci
assigned to the same linkage group were generally separated by
large genetic distances. A second criterion for selection of
microsatellites was their signicant association with previously
reported growth-and-resistance related QTL ( Snchez-Molano
et al., 2011; Rodrguez-Ramilo et al., 2011, 2013, 2014). Among the
120 microsatellites analyzed, 92 were linked to expressed sequence
tags (ESTs) (Bouza et al., 2008, 2012) and 28 were anonymous
(Pardo et al., 2007). Thirty of these 92 EST-linked microsatellite
markers and the 28 anonymous ones had been previously studied
in a population genomics screen using some of the samples of the
present study (Vilas et al., 2010). Therefore, we extend that study
by analyzing new samples, and the variation at 62 additional ESTlinked microsatellites and 136 SNPs. Fourteen of the EST-linked
microsatellites described in Bouza et al. (2008) were also used by
Vandamme et al. (2014). The anonymous microsatellites Sma149,
2/5TG14 and Sma22 and the EST-linked (E code) SmaE7, SmaE14
and SmaE40, showed signicant association with growth-related
traits (Snchez-Molano et al., 2011). One of them, Sma149, was also
associated with resistance and survival time to the infection with
the parasite Philasterides dicentrarchi (Rodrguez-Ramilo et al.,
2013). In addition, we used another seven microsatellites associated
with QTL for resistance to pathologies: Sma168, Sma147, Sma144,
Sma77, SmaE41, SmaE30 and SmaE36 (Rodrguez-Ramilo et al.,
2011, 2013, 2014). Microsatellites were genotyped on an ABI
PRISM 3730 automatic sequencer (Applied Biosystems) and allele
scoring was performed with GENEMAPPER 4.0 software (Applied
Biosystems). All the 29 SNPs from Vera et al. (2011) included in the
turbot map as framework markers (Bouza et al., 2012) were incorporated to the study. In addition, another 107 recently developed SNPs
from the last turbot EST database enriched with two 454 runs (Vera
et al., 2013; Ribas et al., 2013; Pereiro et al., 2012) were analyzed. The
mapping position of these 107 SNPs was unknown. However, we
thought it was appropriate to check either for the proximity of the
outlier SNPs to other outliers based on their estimated position or
for their location within the condence interval of previously

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2.1. Sampling

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2. Materials and methods

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that behave differently is useful for detecting adaptive polymorphisms, particularly if combined with complementary strategies
such as the use of functional information and appropriate QTL
(Vasemgi and Primmer, 2005; Stinchcombe and Hoekstra, 2007;
Hansen et al., 2012; Schoville et al., 2012). However, it is important
to emphasize the limited status of candidates for outlier loci, which
begs for additional experimental conrmation because of important
limitations of the population genomics approach (Bierne et al.,
2013). Correlations across populations and variation in effective
population size can substantially increase the FST variance among
loci and therefore make it difcult to separate historical or demographic effects from natural selection in highly subdivided species
(Nei and Maruyama, 1975; Robertson, 1975). Furthermore, the
effects of selection and genetic drift are frequently intertwined. For
example, when selection is involved in the reinforcement of
reproductive barriers, this may facilitate genome-wide neutral
divergence via genetic drift (Nosil et al., 2009). In combination
with population subdivision, the hitchhiking effect and the presence
of intrinsic genetic incompatibilities in hybrid zones, may cause a
correlation between genetic and environmental variation (Bierne,
2011; Bierne et al., 2011). Furthermore, other factors besides a
complex demography and cryptic genetic structure, can also
determine the appearance of selective effects on particular loci
(Barrett and Hoekstra, 2011; Roesti et al., 2012).
The turbot (Scophthalmus maximus L.; Scopththalmidae) is a
marine sh with high fecundity and vagility, which probably
explains high levels of gene ow between large panmictic populations. This hypothesis is concordant with the evidence of low genetic
structure along its geographic range (Blanquer et al., 1992; Bouza
et al., 1997, 2002; Coughlan et al., 1998; Vandamme et al., 2014).
The observed spatial genetic homogeneity of neutral markers is an
advantage in relating FST-outlier loci with signatures of divergent
selection because it suggests a low genetic drift scenario
(Beaumont, 2005; Nielsen et al., 2009). Vandamme et al. (2014)
found evidence for putative divergent selection at both sides of a
hydrodynamic front in the North Sea. However, the very wide
distribution range across several environmental gradients implies
the capacity of the sh to survive and reproduce in very different
conditions. The Atlantic area involves a gradient in light, temperature and salinity. Temperature differences range between 16 C in
the Iberian Peninsula to 8 C in the southern part of the Baltic Sea
and differences in salinity range from 35 in the Iberian Peninsula
to 8 in the Baltic (HELCOM, 2003; lvarez et al., 2005). Whereas
temperature changes gradually with latitude, an abrupt salinity
drop is observed between the Baltic and the North Sea. The transition
zone between the Baltic and the North Sea is recognized as a
biogeographical barrier for many marine species (Johanneson and
Andr, 2006). Differences in temperature and photoperiod are
expected to particularly affect the survival and reproduction of a
sh such as turbot that lives in relatively shallow waters near the
coast. Although populations living across these gradients have
usually shown low genetic structure for presumably neutral
markers, differentiation between them is statistically signicant
(Nielsen et al., 2004; Suzuki et al., 2004; Florin and Hglund, 2007;
Vilas et al., 2010; Vandamme et al., 2014). An explanation for this
result is that turbot consist of several demes locally adapted to
environmental differences in a background of relatively high levels
of gene ow (Nielsen et al., 2004; Vilas et al., 2010).
With the aim to identify genes with an adaptive genetic pattern we
perform a genome scan analysis of ve natural populations living in
different environmental conditions by using a total of 256 codominant
markers, most of them closely linked to functionally annotated genes
(Bouza et al., 2012). The analysis included 13 microsatellites located at
QTL related to growth and resistance to pathologies and the genetic
variation in natural populations was also compared with an additional
sample subjected to strong growth selection during several generations.

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R. Vilas et al. / Marine Genomics xxx (2015) xxxxxx

Please cite this article as: Vilas, R., et al., A genome scan for candidate genes involved in the adaptation of turbot (Scophthalmus maximus), Mar.
Genomics (2015), http://dx.doi.org/10.1016/j.margen.2015.04.011

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Fig. 1. Map of Europe showing the location of collecting sites: Atlantic Galician coast (AG), Cantabric Sea (CS), English Channel (EC), North Sea (NS) and Baltic Sea (BS).

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2.3. Genetic variation within and among populations

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Allele frequencies, average number of alleles per locus (A) and


gene diversity (He ) were calculated using FSTAT v2.9.3 (Goudet,
2001). This program was also used to estimate FST values among populations (Weir and Cockerham, 1984). Conformance to Hardy
Weinberg proportions was tested using exact tests as implemented
in GENEPOP v3.4 (Raymond and Rousset, 1995). Critical signicance
levels were adjusted for multiple tests using the Bonferroni
correction.

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2.4. Outlier tests for selection

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To search for putative signatures of selection we applied a test


based on estimates of FST (Beaumont and Balding, 2004). The basic
idea is that the inuence of divergent selection on a locus will
increase F ST compared to that expected at neutral loci. Thus,
evidence for divergent selection is obtained by looking for outliers
with higher FST values than expected under neutrality. Conversely,
unexpectedly low values would be indicative of balancing selection.
The method is based on the work by Lewontin and Krakauer (1973),

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reported QTL. For this purpose, we used the recently assembled


turbot genome (A. Figueras, Spanish National Research Council,
Vigo, unpublished) anchored to the linkage map of turbot using
mapped markers. Scaffolds anchored to the turbot map with a
signicant homology of marker sequences (mostly full identity but
always N 98%) and including more than two markers (frequently
tens of markers) were considered for positioning the SNPs. In this
way, non-mapped SNPs were located between the two closest
adjacent loci of turbot linkage groups anchored to specic scaffolds
(Hermida et al., 2013). SNPs were genotyped by using the
MassARRAY technology from SEQUENOM, Inc. San Diego, CA, USA,
in the University of Santiago de Compostela Genomics Platform.

improved by using stochastic simulations to obtain the expected


neutral distribution of the statistic (Beaumont and Nichols, 1996)
and inserted in a Bayesian framework (Beaumont and Balding,
2004). A logistic-regression model is used to estimate population
and locus specic F ST values in combination with a hierarchical
Bayesian approach. The posterior probability of including a locus
specic effect (), presumably caused by selection, is estimated by
calculating a Bayes factor (BF), which is the ratio of the posterior
probabilities of selective and neutral models given the data (Foll
and Gaggiotti, 2008). A log10BF = 1.52 is considered very strong
evidence of different statistical support for both models and
corresponds to a posterior probability (P) between 0.97 and 0.99.
For values above 2 evidence is interpreted as decisive (P = 0.99
1). The Beaumont and Balding (2004) method implemented in
BayeScan v2.01 (Foll and Gaggiotti, 2008) has some advantages
over the approach implemented in Fdist (Beaumont and Nichols,
1996). For example, it does not assume a symmetrical model of
gene ow and therefore allows particularly distinct populations. It
also avoids the problems associated with the simulation of the
neutral distribution of FST by using the empirical estimate, a value
calculated with a sample of loci which also includes the outlier ones.
We analyzed markers in all populations to reveal loci with a major
overall effect. Because we included one population (BS) living in a
different natural environment and one sample (FAR) originating from
an articially reared stock, we performed a triple approach: (1) analysis
of the ve natural populations including BS; (2) analysis of FAR and all
natural populations except BS; and (3) analysis of all natural
populations except BS. In addition, we conducted pairwise comparisons,
as recommended by Tsakas and Krimbas (1976) and Vitalis et al.
(2001), in order to detect local selective effects driven by the different
conditions represented by the BS and FAR samples. Because
microsatellites and SNPs differ in the mutation process, which affects
their levels of variability within and among populations, data
corresponding to both types of markers were analyzed separately.

Please cite this article as: Vilas, R., et al., A genome scan for candidate genes involved in the adaptation of turbot (Scophthalmus maximus), Mar.
Genomics (2015), http://dx.doi.org/10.1016/j.margen.2015.04.011

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t1:1
t1:2
t1:3
t1:4

R. Vilas et al. / Marine Genomics xxx (2015) xxxxxx

Table 1
Gene diversity (He), HardyWeinberg equilibrium departures (FIS) and test for conformance to expected values by locus and population for 58 microsatellite loci of turbot, analyzed by
Vilas et al. (2010), of which 28 are anonymous (left) and 30 EST-linked (right) in two new populations: the English Channel (EC) and the farmed population (FAR). Signicance level
*: P b 0.0008.

t1:5

EC

FAR

EC

FAR

He

FIS

He

FIS

Locus

He

FIS

He

FIS

Sma284
Sma42
Sma168
Sma247
2/5TG14
Sma135
Sma22
Sma147
Sma14
Sma137
Sma142
Sma149
F8I11/8/17
Sma113
Sma117
Sma34
Sma18
Sma184
Sma185
Sma38
Sma100
Sma144
Sma175
Sma205
Sma19
Sma146
Sma282
Sma77

0.862
0.938
0.785
0.759
0.867
0.910
0.867
0.804
0.903
0.320
0.936
0.889
0.865
0.808
0.859
0.868
0.882
0.666
0.723
0.833
0.594
0.962
0.418
0.510
0.920
0.829
0.691
0.831

0.084
0.007
0.197*
0.033
0.061
0.053
0.067
0.053*
0.081
0.037
0.304*
0.043
0.153
0.193
0.095
0.053
0.019
0.217*
0.047*
0.119
0.214*
0.026
0.021
0.183
0.005
0.079
0.076
0.064

0.867
0.906
0.631
0.646
0.825
0.821
0.878
0.691
0.765
0.377
0.873
0.801
0.497
0.699
0.731
0.802
0.868
0.257
0.525
0.807
0.408
0.876
0.341
0.459
0.895
0.770
0.551
0.759

0.183
0.058
0.057
0.054
0.041
0.015
0.075
0.085
0.007
0.051
0.269
0.037
0.112
0.043
0.083
0.142
0.069
0.028
0.213
0.123
0.081
0.001
0.024
0.053
0.022
0.101
0.112
0.018

SmaE1
SmaE2
SmaE3
SmaE7
SmaE4
SmaE29
SmaE8
SmaE12
SmaE14
SmaE16
SmaE25
SmaE28
SmaE38
SmaE20
SmaE35
SmaE40
SmaE41
SmaE42
SmaE19
SmaE22
SmaE26
SmaE32
SmaE30
SmaE43
SmaE10
SmaE13
SmaE31
SmaE33
SmaE36
SmaE39

0.869
0.284
0.586
0.617
0.510
0.367
0.610
0.879
0.862
0.379
0.789
0.430
0.887
0.666
0.347
0.854
0.693
0.432
0.703
0.799
0.645
0.706
0.527
0.116
0.383
0.858
0.846
0.814
0.766
0.856

0.250
0.617*
0.066
0.139*
0.184
0.074
0.080
0.052
0.226
0.598*
0.076
0.135
0.026
0.086
0.624*
0.029
0.066
0.169
0.005
0.040
0.360*
0.085
0.115
0.053
0.104
0.027
0.133*
0.189
0.051
0.111

0.770
0.172
0.505
0.516
0.469
0.308
0.662
0.797
0.870
0.043
0.656
0.245
0.727
0.427
0.000
0.793
0.660
0.538
0.508
0.262
0.461
0.574
0.576
0.354
0.164
0.781
0.745
0.680
0.583
0.647

0.080
0.093
0.010
0.111
0.066
0.084
0.119
0.046
0.101
0.011
0.027
0.154
0.024
0.287
0.000
0.051
0.177
0.071
0.024
0.032
0.123
0.160
0.121
0.057
0.063
0.048
0.225
0.073
0.014
0.130

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3. Results

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3.1. Genetic variation within and among populations

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In general, both microsatellites and SNP loci showed genotypic


frequencies in accordance with the expected values under Hardy
Weinberg equilibrium (Tables 1 and 2). Four loci showed positive and signicant FIS values across several populations suggesting the presence of
null alleles (SmaE139, SmaE120, SmaE61 and SmaE96). HardyWeinberg
deviations were particularly frequent in the sample from the English
Channel (EC; Tables 1 and 2), which is consistent with the population genetic structure observed in that geographic area (Vandamme et al., 2014).
Microsatellite analysis revealed similar levels of genetic variability in natural populations (He ranged from 0.577 in BS to 0.614 in EC; A ranged
from 6.6 in the Baltic Sea (BS) to 7.4 in Atlantic Galician coast (AG). The
farmed population (FAR) showed the lowest variability (He = 0.555
and A = 5.2), which is consistent with the presumed loss of diversity
associated with the founder effect and the successive generations of selection. This effect was not detected with SNPs, which is consistent with the
loss of rare alleles due to genetic drift. Natural population structure quantied as FST revealed that 3.7% of the variation at microsatellites and 2.9%
of the variation at SNPs were due to differences between populations.
When analyzed pairwise, the highest differentiation between natural
populations was detected between BS and AG (FST = 0.031 and 0.043
for microsatellites and SNPs, respectively; P = 0.003) and between BS
and EC (FST = 0.034 for microsatellites and SNPs; P = 0.003), while the
lowest one between AG and Cantabric Sea (CS) for microsatellites
(FST = 0.004; P = 0.003) and between German Bight (NS) and CS for
SNPs (FST = 0.000; P = 0.173). The farmed population showed, as expected due to drift, the highest divergence (the pairwise FST ranged from
0.047 to 0.075 for microsatellites and from 0.037 to 0.085 for SNPs;
P b 0.003). FST for SNPs was statistically signicant only when natural
populations were compared with BS or FAR samples (Table 3).

273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291

R
O
P

T
C
E

271
272

269
270

267
268

265
266

Locus

t1:7
t1:8
t1:9
t1:10
t1:11
t1:12
t1:13
t1:14
t1:15
t1:16
t1:17
t1:18
t1:19
t1:20
t1:21
t1:22
t1:23
t1:24
t1:25
t1:26
t1:27
t1:28
t1:29
t1:30
t1:31
t1:32
t1:33
t1:34
t1:35
t1:36

t1:6

3.2. Tests for outliers in natural populations

292

The global genome scan analysis using microsatellites in all natural


populations identied 15 outliers with a degree of evidence interpreted
as decisive (log10BF N 2; P N 0.99; Table 4). Three other markers were
identied as outliers with very strong evidence (log10BF = 1.52;
P = 0.970.99; Table 4). This means that genomic regions closely linked
to 15% of the microsatellite loci analyzed are likely under selection. For
microsatellites Sma42, Sma22, Sma144 and SmaE112 populations were
more similar than expected under neutrality, thus suggesting stabilizing
selection. The remaining 14 outliers revealed a pattern of variation
consistent with divergent selection. The outlier status of two markers
(SmaE7 and Sma146) conrmed previous results by Vilas et al. (2010).
The other two previously reported (SmaE4 and SmaE12) showed some
statistical support (log10BF = 1; P = 0.910.97). The markers SmaE4
and SmaE7 were also identied as outliers by Vandamme et al. (2014).
The only marker subjected to balancing selection (Sma144), which was
consistently identied by Vilas et al. (2010), was conrmed in the present
study (Table 4). When the BS population was excluded from the analysis,
an additional locus showed a decisive signicance (Sma149); 12 loci did
not change their signicance status while eight changed, particularly
SmaE167, now being non-signicant. Seven out of 13 microsatellites at
QTL were outliers in any of the two global comparisons performed
representing more than 50% tested.
Global analysis of SNPs in natural populations identied ve outlier
loci (6.8%), three of them (SmaSNP247, SmaSNP181 and SmaSNP314)
highly signicant (Table 5, Fig. 2). These three markers are located within
genes coding for ribosomal proteins: L18a, L13 and L1, respectively (Vera
et al., 2011, 2013). The posterior probability of the other two SNPs
(SmaSNP298 and SmaSNP281) was between 0.91 and 0.97, which
corresponds with a log10BF of 1 (strong evidence). SmaSNP298
and SmaSNP281 are located at the coding region of the queuine tRNAribosyltransferase locus and the 5UTR of the gene coding for a

293
294

Please cite this article as: Vilas, R., et al., A genome scan for candidate genes involved in the adaptation of turbot (Scophthalmus maximus), Mar.
Genomics (2015), http://dx.doi.org/10.1016/j.margen.2015.04.011

295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323

R. Vilas et al. / Marine Genomics xxx (2015) xxxxxx

t2:1
t2:2
t2:3

Table 2
Gene diversity (He), HardyWeinberg equilibrium departures (FIS) and test for conformance to expected values by locus and population of 62 new EST-linked microsatellites of turbot. EC,
BS, NS, CS, AG, and FAR are samples from the English Channel, Baltic Sea, North Sea, Cantabric Sea, Atlantic Galician coast, and the farmed population, respectively. Signicance level *: P b

t2:4

0.0008.

t2:5

EC

BS

NS

CS

AG

FAR

He

FIS

He

FIS

He

FIS

He

FIS

He

FIS

He

FIS

t2:7
t2:8
t2:9
t2:10
t2:11
t2:12
t2:13
t2:14
t2:15
t2:16
t2:17
t2:18
t2:19
t2:20
t2:21
t2:22
t2:23
t2:24
t2:25
t2:26
t2:27
t2:28
t2:29
t2:30
t2:31
t2:32
t2:33
t2:34
t2:35
t2:36
t2:37
t2:38
t2:39
t2:40
t2:41
t2:42
t2:43
t2:44
t2:45
t2:46
t2:47
t2:48
t2:49
t2:50
t2:51
t2:52
t2:53
t2:54
t2:55
t2:56
t2:57
t2:58
t2:59
t2:60
t2:61
t2:62
t2:63
t2:64
t2:65
t2:66
t2:67
t2:68

Smax144
Smax272
SmaE139
Smax187
Smax218
Smax261
SmaE84
SmaE86
Smax194
Smax315
SmaE120
SmaE96
Smax154
Smax159
SmaE127
SmaE61
SmaE99
Smax244
SmaE100
Smax205
Smax283
Smax290
Smax170
Smax220
Smax254
Smax310
Smax158
Smax255
SmaE118
SmaE128
SmaE52
Smax164
Smax189
Smax215
Smax225
SmaE97
SmaE316
Smax231
SmaE117
SmaE82
Smax270
Smax302
SmaE137
Smax183
Smax184
Smax277
SmaE79
Smax180
Smax248
Smax284
SmaE78
Smax191
Smax197
Smax227
SmaE105
SmaE112
SmaE50
SmaE71
Smax276
SmaE72
SmaE167
Smax168

0.823
0.149
0.776
0.069
0.450
0.439
0.781
0.902
0.693
0.368
0.531
0.855
0.942
0.640
0.716
0.707
0.499
0.710
0.549
0.691
0.752
0.725
0.739
0.190
0.779
0.747
0.553
0.506
0.578
0.908
0.045
0.477
0.586
0.333
0.591
0.605
0.736
0.429
0.148
0.638
0.454
0.554
0.790
0.142
0.839
0.734
0.881
0.595
0.853
0.748
0.343
0.506
0.210
0.342
0.632
0.819
0.542
0.695
0.568
0.823
0.290
0.504

0.132
0.040
0.520*
0.016
0.260*
0.111
0.072
0.081
0.050
0.038
0.559*
0.644*
0.0590
0.201*
0.130
0.186
0.043
0.405*
0.240
0.035
0.058*
0.096
0.065
0.094
0.117
0.026
0.162
0.038
0.046
0.028
0.012
0.048
0.001
0.085
0.257
0.008
0.046
0.108
0.250
0.103
0.032
0.279
0.240
0.071
0.153
0.022
0.069
0.030
0.053
0.318*
0.444
0.227
0.290
0.058
0.031
0.065
0.088
0.166
0.043
0.077
0.200
0.009

0.758
0.155
0.802
0.021
0.409
0.475
0.595
0.841
0.724
0.118
0.411
0.762
0.914
0.441
0.676
0.757
0.327
0.587
0.501
0.691
0.610
0.687
0.650
0.061
0.701
0.680
0.575
0.505
0.707
0.870
0.022
0.264
0.615
0.410
0.564
0.626
0.754
0.298
0.021
0.593
0.494
0.648
0.784
0.245
0.685
0.788
0.843
0.533
0.781
0.731
0.340
0.697
0.121
0.230
0.586
0.746
0.585
0.664
0.504
0.832
0.000
0.538

0.032
0.192
0.376
0.000
0.068
0.096
0.001
0.007
0.177
0.056
0.482*
0.497*
0.015
0.009
0.140
0.295*
0.053
0.218
0.040
0.045
0.116
0.114
0.025
0.021
0.129
0.049
0.047
0.032
0.182
0.193
0.000
0.195
0.175
0.116
0.245
0.054
0.181
0.428
0.000
0.076
0.033
0.410
0.279
0.391
0.365*
0.054
0.074
0.059
0.081
0.064
0.248
0.405
0.057
0.243
0.076
0.060
0.109
0.098
0.132
0.028
0.000
0.225

0.847
0.041
0.712
0.062
0.406
0.473
0.631
0.897
0.709
0.345
0.505
0.705
0.935
0.448
0.724
0.716
0.328
0.401
0.463
0.626
0.621
0.724
0.663
0.155
0.677
0.576
0.514
0.491
0.635
0.922
0.118
0.558
0.589
0.287
0.591
0.611
0.769
0.462
0.138
0.595
0.345
0.581
0.781
0.154
0.825
0.691
0.859
0.584
0.829
0.752
0.339
0.623
0.271
0.342
0.606
0.789
0.606
0.665
0.621
0.771
0.349
0.504

0.008
0.010
0.648*
0.014
0.126
0.056
0.022
0.094
0.029
0.025
0.440*
0.471*
0.048
0.066
0.059
0.284
0.200
0.099
0.099
0.098
0.039
0.035
0.101
0.192
0.133
0.093
0.012
0.103
0.147
0.119
0.056
0.028
0.115
0.258
0.154
0.174
0.011
0.217
0.057
0.124
0.025
0.398
0.136
0.080
0.267
0.054
0.040
0.212
0.020
0.002
0.005
0.388*
0.020
0.181
0.138
0.019
0.037
0.064
0.211
0.134
0.512*
0.048

0.813
0.160
0.754
0.085
0.442
0.482
0.751
0.911
0.713
0.427
0.293
0.778
0.913
0.425
0.794
0.758
0.229
0.630
0.493
0.629
0.643
0.678
0.747
0.160
0.597
0.562
0.616
0.505
0.488
0.899
0.065
0.287
0.583
0.520
0.628
0.553
0.787
0.441
0.321
0.614
0.428
0.588
0.683
0.022
0.815
0.730
0.847
0.573
0.838
0.766
0.368
0.677
0.196
0.334
0.639
0.755
0.609
0.662
0.574
0.814
0.374
0.443

0.117
0.084
0.557*
0.025
0.065
0.323
0.044
0.171
0.055
0.325
0.667*
0.608*
0.070
0.023
0.014
0.283*
0.052
0.275
0.179
0.002
0.019
0.049
0.041
0.084
0.020
0.033
0.144
0.100
0.070
0.085
0.023
0.060
0.029
0.060
0.074
0.115
0.181
0.013
0.053
0.114
0.118
0.331
0.051
0.000
0.146
0.071
0.152
0.289
0.118
0.092
0.064
0.261
0.111
0.042
0.191
0.021
0.124
0.093
0.109
0.014
0.048
0.129

0.817
0.100
0.776
0.082
0.260
0.499
0.876
0.876
0.698
0.426
0.332
0.767
0.921
0.475
0.762
0.711
0.281
0.611
0.451
0.675
0.672
0.705
0.731
0.172
0.669
0.596
0.593
0.499
0.671
0.920
0.082
0.361
0.605
0.524
0.603
0.675
0.780
0.439
0.195
0.621
0.334
0.598
0.716
0.137
0.819
0.742
0.846
0.530
0.871
0.745
0.320
0.660
0.154
0.192
0.623
0.792
0.609
0.674
0.554
0.807
0.339
0.461

0.081
0.044
0.592*
0.021
0.117
0.127
0.023
0.023
0.194
0.027
0.579*
0.429*
0.029
0.051
0.012
0.296
0.039
0.522*
0.108
0.018
0.023
0.086
0.053
0.093
0.034
0.091
0.175
0.194
0.287*
0.017
0.033
0.180
0.173
0.046
0.070
0.043
0.041
0.288
0.069
0.060
0.252
0.359
0.093
0.068
0.124
0.032
0.040
0.099
0.051
0.049
0.105
0.084
0.080
0.085
0.063
0.033
0.145
0.050
0.210
0.098
0.121
0.031

0.725
0.395
0.711
0.000
0.448
0.496
0.737
0.729
0.592
0.313
0.510
0.641
0.893
0.490
0.722
0.769
0.607
0.605
0.192
0.657
0.724
0.726
0.563
0.333
0.700
0.267
0.313
0.498
0.460
0.806
0.082
0.259
0.525
0.157
0.408
0.491
0.658
0.631
0.126
0.591
0.504
0.410
0.677
0.157
0.765
0.702
0.771
0.505
0.788
0.729
0.282
0.556
0.061
0.543
0.500
0.772
0.397
0.558
0.444
0.657
0.193
0.299

0.190
0.084
0.330
0.000
0.138
0.139
0.086
0.169
0.162
0.088
0.433
0.701*
0.182
0.172
0.116
0.190*
0.111
0.402
0.108
0.029
0.035
0.053
0.171
0.000
0.012
0.034
0.011
0.102
0.138
0.181
0.033
0.035
0.070
0.060
0.084
0.151
0.113
0.207
0.296
0.127
0.115
0.220
0.690*
0.082
0.101
0.098
0.034
0.010
0.022
0.028
0.408
0.250
0.021
0.036
0.167
0.136
0.055
0.082
0.329
0.059
0.338
0.066

324
325
326
327
328
329
330
331

N
C
O

phosphatidyl inositol transfer protein, respectively (Vera et al., 2013). The


ve outliers displayed patterns of differentiation consistent with a model
of divergent selection. Pairwise population analysis of SNPs conrmed the
apparent association of the outlier pattern with the BS sample (Table 6).
Analysis of the relationship between the recently assembled turbot genome with the genetic map (Bouza et al., 2012) located the highly significant outliers SmaSNP247 and SmaSNP181 in QTLs for resistance to
Aeromonas salmonicida and P. dicentrarchi, respectively (Table 7).

R
O

Locus

t2:6

3.3. Comparison between natural and farmed populations

332

The global outlier microsatellite analysis including FAR instead of BS


revealed 14 highly consistent outliers (log10BF N 2; P N 0.99). Two of
them (Sma42 and Sma22) were signicant for balancing selection. The
remaining 12 were signicant for divergent selection (Table 4). Other
four loci (SmaE112, Sma168, SmaE12 and Sma142) were also signicant
(P = 0.970.99) for divergent selection. In total, only ve loci (Sma42,

333

Please cite this article as: Vilas, R., et al., A genome scan for candidate genes involved in the adaptation of turbot (Scophthalmus maximus), Mar.
Genomics (2015), http://dx.doi.org/10.1016/j.margen.2015.04.011

334
Q8
335
336
337
338

6
t3:1
t3:2
t3:3
t3:4

R. Vilas et al. / Marine Genomics xxx (2015) xxxxxx

Table 3
Pairwise FST values from SNPs (above diagonal) and microsatellites (below diagonal). EC,
BS, NS, CS, AG and FAR are samples for the English Channel, Baltic Sea, North Sea, Cantabric
Sea and Atlantic Galician coast, respectively. P b 0.0033.

t3:5
t3:6
Q1
t3:7
t3:8
t3:9
t3:10
t3:11

339

EC
BS
NS
CS
AG
FAR

Table 5
Outlier analysis based on BayeScan of SNPs in natural populations of turbot. is the locus
specic effect; P is the posterior probability that the locus is under selection and FST is the
xation index.

EC

BS

NS

CS

AG

FAR

Outlier

FST

t5:5

0.034*
0.020*
0.022*
0.025*
0.047*

0.034

0.026*
0.031*
0.030*
0.075*

0.001
0.032*

0.009*
0.015*
0.064*

0.003
0.041*
0.000

0.004
0.067*

0.008
0.043*
0.002
0.003

0.071*

0.046*
0.085*
0.037*
0.041*
0.043*

SmaSNP247
SmaSNP281
SmaSNP314
SmaSNP298
SmaSNP181

2.114
1.432
1.485
1.346
1.316

1.000
0.967
0.985
0.914
0.993

0.0765
0.0469
0.0491
0.0441
0.0412

t5:6
t5:7
t5:8
t5:9
t5:10

generalized pattern consistent with balancing selection. Two of these


loci, Sma22 and Sma144, are located at QTL for growth and resistance,
respectively (Rodrguez-Ramilo et al., 2013, 2014). The observation
that more than half of the outlier microsatellites are located in QTL
seems to conrm the adaptive signicance of the associated traits. A statistical association between outlier loci identied by the population genomics approach and QTL for putatively adaptive traits has previously
been observed in sh (Rogers and Bernatchez, 2005, 2007).

365
366

4.1. Microsatellite based outliers along a temperate and salinity gradient

373

Because the outlier analysis was performed with samples from populations living along a temperature cline, it is likely that the presumed
adaptive process is related to differences for this variable or any other
correlated with it. Many environmental variables are dependent on
temperature, including differences in metabolism, the distribution of
food, predators, parasites and competitors. Differences in temperature
as an explanation for the atypical behavior of several markers is consistent with the result that most outliers remained signicant when the
analysis did not include the two atypical samples (BS because of the
low salinity and FAR because of farming), but only those populations living along a temperature gradient (AG, CS, EC, and NS). Although temperature is gradually changing within the Atlantic Ocean, population
genetic structure of turbot appears to be mostly associated with a relatively strong break within the North Sea (Vandamme et al., 2014). Four
of these outliers (Sma22, SmaE7, 2/5TG14 and Sma149) are located at a
growth-related QTL (Snchez-Molano et al., 2011; Rodrguez-Ramilo
et al., 2014). Locus SmaE7 is linked to a gene coding for the receptor
for a broblast growth factor, which is likely involved in adaptation

374
375

355

4. Discussion

356

364

This study identied several candidate genes for local adaptation in


natural populations of turbot living across a temperature and salinity
gradient in the North Atlantic. Candidate loci were associated with 20
different linkage groups (Table 7). Fifteen microsatellites (12.5%), ve
of them located in QTL related to growth and pathogen resistance,
showed very high statistical support as outliers. Another ve loci were
putative outliers (Table 4). Most outliers showed evidence of divergent
selection suggesting that they are involved in local adaptation. However, four markers (Sma42, Sma22, Sma144 and SmaE112) revealed a

t4:1
t4:2
t4:3
Q2
t4:4

Table 4
Outlier analysis based on BayeScan of the microsatellite loci in all ve turbot populations including the Baltic Sea and the farmed population (5p/BS and 5p/FAR, respectively) and in four
populations excluding the Baltic and the farmed population (4p/noBS-FAR). is the locus specic effect; P is the posterior probability that the locus is under selection and FST is the xation
index. Outliers identied by Vilas et al. (2010) and Vandamme et al. (2014) are indicated as V10 and V14, respectively. Signicance level ***: P N 0.99; **: P = 0.970.99; *: P = 0.910.97.

357
358
359
360
361
362
363

R
O

352
353

350
351

348
349

346
347

344
345

t4:5

342
343

354

Sma22, Sma144, SmaE112 and SmaE12) displayed patterns of variation


consistent with a model of balancing selection (Table 4). However,
SmaE12 was identied to be subjected to divergent selection when
FAR was replaced by BS. In addition, this locus was identied as outlier
only when the analysis involved BS or FAR. Nonetheless, most outliers
were common to the three global tests (Table 4).
Only one SNP (SmaSNP273) was identied as outlier (P = 0.995)
after replacing BS by FAR, showing signals consistent with divergent selection. Furthermore, this marker was only signicantly identied when
FAR was compared with either of the Iberian samples (P = 0.991 and
0.946 for comparisons with CS and AG, respectively). This marker is located at the 3UTR of the ubiquitin-protein ligase locus. The global analysis of SNPs excluding both BS and FAR did not reveal any outliers.
Pairwise comparisons between natural populations and FAR using
both microsatellites and SNPs basically conrmed the results of the
global analysis (results not shown).

340
341

5p/BS

Sma42
Sma117
Sma22
Sma144
Sma147
Sma146
SmaE7
SmaE22
F8I11/8/17
SmaE33
2/5TG14
SmaE84
SmaE137
SmaE112
SmaE167
Sma168
SmaE99
SmaE117
SmaE12
SmaE4
Sma149
Sma142

t4:7
t4:8
t4:9
t4:10
t4:11
t4:12
t4:13
t4:14
t4:15
t4:16
t4:17
t4:18
t4:19
t4:20
t4:21
t4:22
t4:23
t4:24
t4:25
t4:26
t4:27
t4:28

V10

Outlier

t4:6

t5:1
t5:2
t5:3
t5:4

V10
V10V14

V10
V10V14

1.198
1.240
1.541
1.113
1.552
1.333
2.487
0.974
1.071
1.099
1.257
3.016
1.751
1.842
1.911
1.021
1.328
1.636
0.751
1.096
0.546
0.055

5p/FAR

4p/noBS-FAR

FST

FST

FST

***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
**
**
**
*
*
ns
ns

0.0064
0.0649
0.0048
0.0068
0.0887
0.0700
0.1883
0.0520
0.0553
0.0582
0.0656
0.2737
0.1051
0.0038
0.1313
0.0555
0.0774
0.1061
0.0415
0.0584
0.0335
0.0188

1.097
1.508
1.192
0.528
1.854
1.317
2.694
1.384
1.328
1.502
1.401
2.961
1.855
1.140
0.033
1.120
1.773
1.250
1.228
0.127
0.971
0.616

***
***
***
*
***
***
***
***
***
***
***
***
***
**
ns
**
***
ns
**
ns
***
**

0.0122
0.1184
0.0114
0.0203
0.1539
0.1022
0.2611
0.1091
0.1032
0.1187
0.1091
0.3023
0.1552
0.0123
0.0407
0.0904
0.1504
0.1084
0.0115
0.0415
0.0779
0.0573

1.170
1.659
1.354
1.075
2.060
1.552
2.752
1.380
1.410
1.656
1.605
3.227
2.073
1.494
0.047
1.400
1.540
1.459
0.946
0.245
0.994
0.500

*
***
***
***
***
***
***
***
***
***
***
***
***
**
ns
***
***
ns
ns
ns
***
ns

0.0044
0.0631
0.0037
0.0046
0.0940
0.0566
0.1680
0.0500
0.0494
0.0644
0.0594
0.2355
0.0965
0.0034
0.0188
0.0531
0.0627
0.0667
0.0057
0.0210
0.0340
0.0201

Please cite this article as: Vilas, R., et al., A genome scan for candidate genes involved in the adaptation of turbot (Scophthalmus maximus), Mar.
Genomics (2015), http://dx.doi.org/10.1016/j.margen.2015.04.011

367
368
369
370
371
372

376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391

R
O

R. Vilas et al. / Marine Genomics xxx (2015) xxxxxx

Fig. 2. FST outliers identied by BayeScan analysis of the global dataset with ve natural samples (136 SNPs). A log10 Bayes factor (BF) of 1.0, 1.5 and 2.0 corresponds to posterior probabilities that a locus is under selection of 0.91, 0.97 and 0.99, respectively. For a log10BF N 2 the evidence is commonly interpreted as decisive.

409
410
411
412
413
414
415
416
417
418
419
420
421

407
408

405
406

403
404

N
C
O

401
402

399
400

t6:7

Outlier

BS/EC

BS/NS

BS/CS

BS/AG

t6:8
t6:9
t6:10
t6:11
t6:12

SmaSNP247
SmaSNP281
SmaSNP314
SmaSNP298
SmaSNP181

1.000
0.846
0.830
0.963
0.986

0.998
0.711
0.860
0.779
0.995

1.000
0.579
0.967
0.679
0.949

1.000
0.488
0.972
0.859
0.963

423

Table 7
Markers linked to loci likely under selection in turbot. LG is the linkage group in the genetic
map of Bouza et al. (2012). Outliers within the condence interval of QTL for growth
(G) and resistance (R) are indicated.

t7:1
t7:2
t7:3
t7:4

Table 6
Outlier analysis of SNPs between pairs of natural populations of turbot. EC, BS, NS, CS, AG,
and FAR are samples from the English Channel, Baltic Sea, North Sea, Cantabric Sea,
Atlantic Galician coast, and the farmed population, respectively. Posterior probability of
being under selection of ve SNPs identied as outliers in the global analysis is shown.
The remaining six pairwise comparisons (which do not involve BS) revealed no outliers.

397
398

also explain differences between populations in the availability of


resources used for growth. Both the identication of several growthrelated loci in natural populations living along a gradient of temperature
and the comparison with a sample subjected to strong articial
selection for growth, support the hypothesis of changes in body size as
a by-product of the adaptation to differences in temperature or any
other variable correlated with it.
Four microsatellites linked to genes (SmaE167, SmaE117, SmaE12
and SmaE4) were identied as outliers only when BS was included in
the analysis, which suggests that this sample affects most the outlier
pattern. Two of them (SmaE12 and SmaE4) were previously identied
as outliers; they are most likely related to the salinity cline at the
entrance of the Baltic Sea (Vilas et al., 2010; Vandamme et al., 2014).
In contrast to SmaE4, very high statistical support is found for

t6:1
t6:2
t6:3
t6:4
t6:5
t6:6

395
396

422

(Vilas et al., 2010; Vandamme et al., 2014). Most markers identied as


outliers remained highly signicant when the Baltic sample (BS) was
replaced for a farmed population which had been strongly selected for
growth during four generations (FAR). One marker (Sma149), located
at a QTL for both growth and resistance to the infection with
P. dicentrarchi (Rodrguez-Ramilo et al., 2013), was identied as an
outlier only when the analysis included the FAR sample (Table 4). This
could be related to the opportunistic infection of farmed populations
originating from the study area by this parasite (Iglesias et al., 2001).
However, Sma149 also seems to be an outlier after excluding FAR and
BS samples.
Taken together these observations suggest (1) that the anomalous
behavior of most markers does not solely depend on the peculiar
characteristics of the Baltic Sea (e.g. low salinity) and (2) an adaptive
relationship between temperature and growth rate. The latter
hypothesis is supported by the detection of several outliers along a
cline of temperature which are located in QTLs for growth. However,
when the growth selected farm sample is excluded from the outlier
analysis, such markers are still identied as outliers. This result suggests
a strong effect on wild populations. The presumed relationship between
temperature and growth rate can be direct or mediated by other traits.
For example, the optimal temperature for the growth of turbot declines
with increasing size (Imsland et al., 1996), an observation consistent
with the hypothesis of differences in body size as an adaptation to
cold. Another possibility is that adaptation to differences in temperature
entails changes in allele frequency by selection of genes involved in
growth due to the effect of temperature on reproduction and the need
of the species to nd a balance between the amount of resources used
in reproduction and somatic growth. In fact, as commonly observed in
sh, the highest growth rate occurs before sexual maturity. Thus,
differences in fecundity may be due to local adaptation, which could

393
394

392

Outlier

LG QTL Annotation

Sma42
Sma117
Sma22
Sma144
Sma147
Sma146
SmaE7
SmaE22
F8I11/8/17
SmaE33
2/5TG14
SmaE84
SmaE137
SmaE112
SmaE167
Sma168
SmaE99
SmaE117

1
21
11
3
6
14
6
12
24
23
19
7
16

22
2
9
9

G
R
R

SmaE12
SmaE4
Sma149
Sma142
SmaSNP247
SmaSNP281
SmaSNP314
SmaSNP298
SmaSNP181

17
8
15
17

13
21
4

G/R

Fibroblast growth factor receptor

Trifunctional enzyme subunit beta, mitochondrial-like

Cathepsin O
Fibrinogen alpha chain-like
Villin-1
T-complex protein 1 subunit gamma-like

Cytosolic malate dehydrogenase thermostable form


Microtubule-associated tumor suppressor 1 homolog
A-like
Trap alpha-translocon protein
Beta-microglobuline

Ribosomal protein L18a


Phosphatidyl inositol transfer protein
Ribosomal protein L1
Queuine t-RNA ribosyltransferase
Ribosomal protein L13

Please cite this article as: Vilas, R., et al., A genome scan for candidate genes involved in the adaptation of turbot (Scophthalmus maximus), Mar.
Genomics (2015), http://dx.doi.org/10.1016/j.margen.2015.04.011

424
425
426
427
428
429
430
431
432
433
434
435
436

t7:5
t7:6
t7:7
t7:8
t7:9
t7:10
t7:11
t7:12
t7:13
t7:14
t7:15
t7:16
t7:17
t7:18
t7:19
t7:20
t7:21
t7:22
t7:23
t7:24
t7:25
t7:26
t7:27
t7:28
t7:29
t7:30
t7:31
t7:32

444
445
446
447
448
449
450
451
Q9
452
453
454
455

458

Unlike the microsatellite markers, the outlier analysis based on SNPs


revealed a much lower number of signicant markers. This might be attributed to different mutational dynamics on the functional constraints
461
experienced (DeFaveri et al., 2013), but also that these markers had not
462
been a priori selected by their association to previously detected QTLs.
463
Five gene-associated SNPs showed evidence for divergent selection,
464
but only when BS was included in the pairwise analyses. The outlier pat465
tern was always consistent with a model assuming divergent selection
466
between BS and the remaining populations. These results suggest local
467
adaptation of turbot populations to salinity (although there are other
468
potential drivers than salinity such as differences in environmental pol469
lution or in any other selective pressure). However, it is striking that at
470
least four of the ve outliers are either linked to genes coding for ribo471
somal proteins or directly related to the production of cellular proteins.
472
In addition, the two most signicant SNPs are within ribosomal genes
473
located in two QTL for resistance to pathologies, which could be related
474
to the known inuence of salinity on the immune system of sh
475
(Bowden, 2008). It is interesting that SNP outliers seem to be related
476
to the BS sample while microsatellite outliers are mainly related to the
477
Atlantic samples. This difference could be related to the close functional
478
relationship of the SNP outliers. The relatively low number of SNP out479
liers detected in Atlantic populations suggests an effect on SNPs specif480
ically linked to the Baltic Sea and it is likely that the functional
481
relationship is not a coincidence.
482
The Baltic Sea, almost fully surrounded by land and heavily inu483
enced by freshwater runoff, has a distinct salinity gradient from south
484
to north (HELCOM, 2003). The limited exchange rate explains the very
485
high level of pollution (HELCOM, 2011; Hong et al., 2012). Therefore,
486
at least two hypotheses could explain putative signatures of divergent
487
selection on ribosomal genes associated to the comparisons between
488
the Baltic with any other population: (1) they are the outcome of local
489
adaptation to salinity or (2) they are related to environmental pollution.
490
It is known that the expression of ribosomal proteins is induced by ex491
Q10 posure of marine sh to pollutants (Campbell and Devlin, 1997;
492
Handley-Goldstone et al., 2005; Oh et al., 2009). Although differences
493
in gene expression do not necessarily imply changes in allele frequen494
cies, eventually ribosomal genes may record the effects of natural selec495
tion leading to adaptation to a more polluted environment; particularly,
496
as in this case, when markers are located within the coding region or the
497
5UTR (Hoffman and Willi, 2008; Roelofs et al., 2010). On the other
498
hand, if turbot populations from the Baltic Sea have adapted to low sa499
linity conditions by increased fecundity, a trait correlated with differ500
ences in female size, this adaptation may involve changes in genes

Acknowledgments

538

We thank Luca Insua for the technical assistance and Carlos


Fernndez for the help with the turbot database. SNP genotyping
services were provided by the University of Santiago de Compostela
node of the Spanish National Centre of Genotyping (CeGen-ISCIII). S.V.
acknowledges a PhD scholarship of ILVO-Vlaanderen and G.E.M.
acknowledges a fellowship of the Research Foundation-Flanders. This
study was supported by the Consolider Ingenio Aquagenomics
(CSD200700002) and the Science and Education Spanish Ministry
(AGL2009-11782).

539

References

548

Alberto, F.J., Derory, J., Boury, C., Frigerio, J.M., Zimmermann, N.E., Kremer, A., 2013.
Imprints of natural selection along environmental gradients in phenology-related
genes of Quercus petraea. Genetics 195, 495512.
lvarez, I., de Castro, M., Gmez-Gesteira, M., Prego, R., 2005. Inter- and intra-annual
analysis of the salinity and temperature evolution in the Galician Ras Baixas-ocean
boundary (northwest Spain). J. Geophys. Res. 110 (C04008).
Barrett, R.D.H., Hoekstra, H.E., 2011. Molecular spandrels: test of adaptation at the genetic
level. Nat. Rev. Genet. 12, 767780.
Beaumont, M.A., 2005. Adaptation and speciation: what can FST tell us? Trends Ecol. Evol.
20, 435440.
Beaumont, M.A., Balding, D.J., 2004. Identifying adaptive genetic divergence among
populations from genome scans. Mol. Ecol. 13, 969980.
Beaumont, M.A., Nichols, R.A., 1996. Evaluating loci for the use in the genetic analysis of
population structure. Proc. Biol. Sci. 263, 16191626.
Bierne, N., 2011. The distinctive footprints of local hitchhiking in a varied environment
and global hitchhiking in a subdivided population. Evolution 64, 32543272.
Bierne, N., Welch, J., Loire, E., Bonhomme, F., David, P., 2011. The coupling hypothesis:
why genome scans may fail to map local adaptation genes. Mol. Ecol. 20, 20442072.

549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566

459
460

443

4.2. SNP based outliers and the unique position of the Baltic Sea

441
442

501
502

R
O

457

439
440

related to somatic growth and reproduction (Nissling et al., 2013).


Genes encoding ribosomal proteins may show the effects of natural selection for growth because the biosynthesis of these proteins is related
to the rate of ribosomal formation, an essential process in actively dividing tissues. However, both hypotheses are not mutually exclusive because ribosomal proteins also show extra-ribosomal functions
probably relevant in response to environmental stress. Hence, the unexpected behavior of these SNPs could be simply the result of adaptation
to harsher environmental conditions.
All explanations considered so far assumed that selection plays a
causal role in the atypical behavior of these loci. However, outliers
could be evidencing other phenomena such as differences associated
with the hybridization of distinct populations at the study area, not necessarily related to adaptation to different environmental conditions
(Bierne et al., 2013). The possibility of hybrid zones of turbot from the
transition zone between the Baltic Sea and the North Sea has been suggested by Nielsen et al. (2004). The effect of different selective pressures
acting on both sides of the transition zone reduces gene ow in this area.
In such case, outlier analyses would identify genomic regions with reduced gene ow due to its putative contribution to local adaptation.
However, outliers in cryptic hybrid zones could also be revealing genetic
incompatibilities or differences in mate choice between diverging populations (Bierne et al., 2011). Actually, there are many other caveats that
make it advisable to consider FST outliers just as candidate loci, requiring
further investigation (Bierne et al., 2013). Here, we identied several
loci that deserve the status of candidates to be involved in the adaptation of turbot populations most likely related to differences in temperature and salinity (and maybe pollution). Furthermore, results suggest
that the presumed adaptive process entails changes in growth rate as
a side effect, which is consistent with studies suggesting that turbot in
the Baltic Sea grows slower and reaches a smaller size in comparison
with other populations from the North Atlantic Ocean and the Mediterranean Sea (Stankus, 2003; Nissling et al., 2013). Finding genetic
markers associated with growth whose variation in natural populations
may be showing the effects of adaptation to different environmental
conditions has implications for aquaculture of this species because
they may serve to improve conditions for cultivation.

456

SmaE117 and SmaE167. Locus SmaE12 was an outlier only when the
analysis included BS or FAR. Nonetheless, this locus showed higher FST
than expected under neutrality when the analysis includes BS and the
opposite pattern when this sample was replaced by FAR. This suggests
that the variation at SmaE12 is driven in opposite directions by these
two populations under different evolutionary pressures, including
those potentially imposed by differences in salinity and growth rate.
This marker is closely linked to the gene coding for the trap alphatranslocon protein, which is related to membrane processes (Vilas
et al., 2010). An explanation for these contrasting patterns is the adaptation to varying conditions of salinity, negatively affecting the reproductive success. Consistent with this hypothesis, it has been observed
that eggs of turbot from the Baltic populations have high survival rates
in salinities between 10 and 15, conditions under which no or
only low survival occurs among the turbot from the North Sea (Karas
and Klingsheim, 1997; Nissling et al., 2006). Recent observations suggest that selection for a high fecundity result in lower somatic growth
in the Baltic Sea (Nissling et al., 2013). However, as in the case of temperature, it is likely that other environmental factors correlated with
differences in salinity may be operating (Vandamme et al., 2014).

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R. Vilas et al. / Marine Genomics xxx (2015) xxxxxx

Please cite this article as: Vilas, R., et al., A genome scan for candidate genes involved in the adaptation of turbot (Scophthalmus maximus), Mar.
Genomics (2015), http://dx.doi.org/10.1016/j.margen.2015.04.011

503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
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535
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537

540
541
542
543
544
545
546
547

R. Vilas et al. / Marine Genomics xxx (2015) xxxxxx

N
C
O

R
O

Nielsen, E.E., Hemmer-Hansen, J., Larsen, P.F., Bekkevold, D., 2009. Population genomics of
marine shes: identifying adaptive variation in space and time. Mol. Ecol. 18,
31283150.
Nissling, A., Johansson, U., Jacobsson, M., 2006. Effects of salinity and temperature conditions on the reproductive success of turbot (Scophthalmus maximus) in the Baltic Sea.
Fish. Res. 80, 230238.
Nissling, A., Florin, A.B., Thorsen, A., Bergstrm, U., 2013. Egg production of turbot,
Scophthalmus maximus, in the Baltic Sea. J. Sea Res. 84, 7786.
Nosil, P., Funk, D.J., Ortiz-Barrientos, D., 2009. Divergent selection and heterogeneous genomic divergence. Mol. Ecol. 18, 375402.
Oh, J.H., Moon, H.B., Choe, E.S., 2009. Changes in differentially expressed genes in the liver
of Oryzias latipes by binary exposure to carcinogenic polycyclic aromatic hydrocarbons. Kor. J. Environ. Biol. 27, 391396.
Orsini, L., Spanier, K.I., De Meester, L., 2012. Genomic signature of natural and anthropogenic stress in wild populations of the waterea Daphnia magna: validation in space,
time and experimental evolution. Mol. Ecol. 21, 21602175.
Pardo, B.G., Hermida, M., Fernndez, C., Bouza, C., Prez, M., Llavona, A., Snchez, L.,
Martnez, P., 2006. A set of highly polymorphic microsatellites useful for kinship
and population analysis in turbot (Scophthalmus maximus L.). Aquac. Res. 37,
15781582.
Pardo, B.G., Fernndez, C., Hermida, M., Vzquez-Lpez, A., Prez, M., Presa, P., Calaza, M.,
lvarez-Dios, J.A., Comesaa, A.S., Raposo-Guilln, J., Bouza, C., Martnez, P., 2007. Development and characterization of 248 novel microsatellites markers in turbot
(Scophthalmus maximus). Genome 50, 329332.
Pereiro, P., Balseiro, P., Romero, A., Dios, S., Forn-Cuni, G., Fuste, B., Planas, J.V., Beltrn, S.,
Novoa, B., Figueras, A., 2012. High throughput sequence analysis of turbot
(Scophthalmus maximus) transcriptome using 454-pyrosequencing for the discovery
of antiviral immune genes. Plos One 7 (e35369).
Raymond, M., Rousset, F., 1995. GENEPOP (version 1.2): population genetics software for
exact test and ecumenicism. J. Hered. 86, 248249.
Ribas, L., Pardo, B.G., Fernndez, C., lvarez-Dios, J.A., Gmez-Tato, A., Quiroga, M.I., Planas,
J.V., Sitja-Bobadilla, A., Martnez, P., Piferrer, F., 2013. A combined strategy involving
Sanger and 454 pyrosequencing increases genomic resources to aid in the management of reproduction, disease control and genetic selection in the turbot
(Scophthalmus maximus). BMC Genomics 14, 180.
Robertson, A., 1975. Remarks on the LewontinKrakauer test. Genetics 80, 396.
Rodrguez-Ramilo, S.T., Toro, M.A., Bouza, C., Hermida, M., Pardo, B.G., Cabaleiro, S.,
Martnez, P., Fernndez, C., 2011. QTL detection for Aeromonas salmonicida resistance
related traits in turbot (Scophthalmus maximus). BMC Genomics 12, 541.
Rodrguez-Ramilo, S.T., Fernndez, J., Toro, M.A., Bouza, C., Hermida, M., Fernndez, C.,
Pardo, B.G., Cabaleiro, S., Martnez, P., 2013. Uncovering QTL for resistance and survival time to Philasterides dicentrarchi in turbot (Scophthalmus maximus). Anim. Genet.
44, 149157.
Rodrguez-Ramilo, S.T., De la Herrn, R., Ruiz-Rejn, C., Hermida, M., Fernndez, C.,
Pereiro, P., Figueras, A., Bouza, C., Toro, M.A., Martnez, P., Fernndez, J., 2014. Identication of quantitative trait loci associated to resistance to viral haemorrhagic
septicaemia (VHS) in turbot (Scophthalmus maximus): a comparison between bacterium, parasite and virus diseases. Mar. Biotechnol. http://dx.doi.org/10.1007/s10126013-9544-x.
Roelofs, D., Morgan, J., Sturzenbaun, S., 2010. The signicance of genome-wide transcriptional regulation in the evolution of stress tolerance. Evol. Ecol. 24, 527539.
Roesti, M., Hendry, A.P., Salzburger, W., Berner, D., 2012. Genome divergence during evolutionary diversication as revealed in replicate lake-stream stickleback population
pairs. Mol. Ecol. 21, 28522862.
Rogers, S.M., Bernatchez, L., 2005. Integrating QTL mapping and genome scans towards
the characterization of candidate loci under parallel selection in the lake whitesh
(Coregonus clupeaformis). Mol. Ecol. 14, 351361.
Rogers, S.M., Bernatchez, L., 2007. The genetic architecture of ecological speciation and
the association with signatures of selection in natural lake whitesh (Coregonus sp.,
Salmonidae) species pairs. Mol. Biol. Evol. 24, 14231438.
Snchez-Molano, E., Cerna, A., Toro, M.A., Bouza, C., Hermida, M., Pardo, B.G., Cabaleiro, S.,
Fernndez, C., Martnez, P., 2011. Detection of growth-related QTL in turbot
(Scophthalmus maximus). BMC Genomics 12, 473.
Schltterer, C., 2002. A microsatellite-based multilocus screen for the identication of
local selective sweeps. Genetics 160, 753763.
Schoville, S.D., Bonin, A., Franois, O., Lobreaux, S., Melodelima, C., Manel, S., 2012. Adaptive genetic variation on the landscape: methods and cases. Annu. Rev. Ecol. Evol.
Syst. 43, 2343.
Slate, J., 2005. Quantitative trait locus mapping in natural populations: progress, caveats
and future directions. Mol. Ecol. 14, 363379.
Stankus, S., 2003. The peculiarities of turbot (Psetta maxima L.) biology and their role in
the ecosystem of the Baltic Sea coastal zone of Lithuania. Acta Zool. Lit. 13, 217238.
Stinchcombe, J.R., Hoekstra, H.E., 2007. Combining population genomics and quantitative
genetics: nding the genes underlying ecologically important traits. Heredity 100,
158170.
Storz, J.F., 2005. Using genome scans of DNA polymorphism to infer adaptive population
divergence. Mol. Ecol. 14, 671688.
Suzuki, N., Nishida, M., Yoseda, K., stnda, C., ahin, T., Amaoka, K., 2004. Phylogeographic relationships within the Mediterranean turbot inferred by mitochondrial
DNA haplotype variation. J. Fish Biol. 65, 580585.
Tsakas, S., Krimbas, C.B., 1976. Testing heterogeneity of F-values: suggestion and a correction. Genetics 84, 399401.
Vandamme, S.G., Maes, G., Raeymaekers, J., Cottenie, K., Imsland, A.K., Hellemans, B.,
Lacroix, G., Mac Aoidh, E., Martinsohn, J., Martnez, P., Robbens, J., Vilas, R.,
Volckaert, F., 2014. Environmental selective pressure reinforces historical differentiation in turbot (Scophthalmus maximus). Mol. Ecol. 23, 618636.

Bierne, N., Roze, D., Welch, J., 2013. Pervasive selection or is it? Why are FST outliers
sometimes so frequent? Mol. Ecol. 22, 261264.
Blanquer, A., Alayse, J.P., Berrada-Rkhami, O., Berrebi, P., 1992. Allozyme variation in
turbot (Psetta maxima) and brill (Scophthalmus rhombus) (Osteichtyes,
Pleuronectiformes, Scophthalmidae) throughout their range in Europe. J. Fish Biol.
41, 725736.
Bouza, C., Snchez, L., Martnez, P., 1997. Gene diversity analysis in natural populations
and cultured stocks of turbot (Scophthalmus maximus L.). Anim. Genet. 28, 2836.
Bouza, C., Presa, P., Castro, J., Snchez, L., Martnez, P., 2002. Allozyme and microsatellite
diversity in natural and domestic populations of turbot (Scophthalmus maximus) in
comparison with other Pleuronectiformes. Can. J. Fish. Aquat. Sci. 59, 14601473.
Bouza, C., Hermida, M., Pardo, B.G., Fernndez, C., Fortes, G.G., Castro, J., Snchez, L., Presa,
P., Prez, M., Sanjun, A., de Carlos, A., lvarez-Dios, J.A., Ezcurra, S., Cal, R.M., Piferrer,
F., Martnez, P., 2007. A microsatellite genetic map of turbot (Scophthalmus maximus).
Genetics 177, 24572467.
Bouza, C., Hermida, M., Milln, A., Vilas, R., Vera, M., Fernndez, C., Calaza, M., Pardo, B.G.,
Martnez, P., 2008. Characterization of EST-derived microsatellites for gene mapping
and evolutionary genomics in turbot. Anim. Genet. 39, 666670.
Bouza, C., Hermida, M., Pardo, B.G., Vera, M., Fernndez, C., de la Herrn, R., Navajas-Prez,
R., lvarez-Dios, J.A., Gmez-Tato, A., Martnez, P., 2012. An Expressed Sequence Tag
(EST)-enriched genetic map of turbot (Scophthalmus maximus): a useful framework
for comparative genomics across model and farmed teleosts. BMC Genet. 13, 54.
Bowden, T.J., 2008. Modulation of the immune system of sh by their environment. Fish
Shellsh Immunol. 25, 373383.
Campbell, P.M., Devlin, R.H., 1997. Increased CYP1A1 and ribosomal protein L5 gene
expression in a teleost: the response of juvenile Chinook salmon to coal dust
exposure. Aquat. Toxicol. 38, 115.
Colosimo, P.F., Hosemann, K.E., Balabhadra, S., Villareal, G., Dickson, M., Grimwood, J.,
Schmutz, J., Myers, R.M., Schluter, D., Kingsley, D.M., 2005. Widespread parallel
evolution in sticklebacks by repeat xation of ectodysplasin alleles. Science 307,
19281933.
Coughlan, J.P., Imsland, A.K., Galvin, P.T., Fitzgerald, R.D., Naevdal, G., Cross, T.F., 1998.
Microsatellite DNA variation in wild populations and farmed strains of turbot from
Ireland and Norway: a preliminary study. J. Fish Biol. 52, 916922.
DeFaveri, J., Viitaniemi, H., Leder, E., Meril, J., 2013. Characterizing genic and nongenic
molecular markers: comparison of microsatellites and SNPs. Mol. Ecol. Resour. 13,
377392.
Florin, A.B., Hglund, J., 2007. Absence of population structure of turbot (Psetta maxima)
in the Baltic Sea. Mol. Ecol. 16, 115126.
Foll, M., Gaggiotti, O., 2008. Estimating selection with different markers and varying
demographic scenarios: a Bayesian perspective. Genetics 180, 977993.
Goudet, J., 2001. FSTAT, a program to estimate and test gene diversities and xation
indices (version 2.9.3). Available from. http://www.unil.ch/izea/softwares/fstat.html.
Handley-Goldstone, H.M., Grow, M.W., Stegeman, J.J., 2005. Cardiovascular gene
expression proles of dioxin exposure in zebrash embryos. Toxicol. Sci. 85,
683693.
Hansen, M.M., Olivieri, I., Waller, D.M., Nielsen, E.E., GeM Working Group, 2012.
Monitoring adaptive genetic responses to environmental change. Mol. Ecol. 21,
13111329.
HELCOM, 2003. The Baltic marine environment 19992002. Balt. Sea Environ. Proc. N 87.
HELCOM, 2011. The fth Baltic Sea pollution load compilation (PLC-5). Balt. Sea Environ.
Proc. No. 128.
Hemmer-Hansen, J., Therkildsen, N.O., Meldrup, D., Nielsen, E.E., 2014. Conserving marine
biodiversity: insights from life-history trait candidate genes in Atlantic cod (Gadus
morhua). Conserv. Genet. 15, 213228.
Hermida, M., Bouza, C., Fernndez, C., Sciara, A.A., Rodrguez-Ramilo, S.T., Fernndez, J.,
Martnez, P., 2013. Compilation of mapping resources in turbot (Scophthalmus
maximus): a new integrated consensus genetic map. Aquaculture 414, 1925.
Hoffman, A.A., Willi, Y., 2008. Detecting genetic responses to environmental change. Nat.
Rev. Genet. 9, 421432.
Hong, B., Swaney, D.P., Mrth, C.M., Smedberg, E., Hgg, H.E., Humborg, C., Howarth, R.W.,
Bouraoui, F., 2012. Evaluating regional variation of net anthropogenic nitrogen and
phosphorus inputs (NANI/NAPI), major drivers, nutrient retention pattern and
management implications in the multinational areas of Baltic Sea basin. Ecol.
Model. 227, 17135.
Iglesias, R., Param, A., lvarez, M.F., Leiro, J., Fernndez, J., Sanmartn, M.L., 2001.
Philasterides dicentrarchi (Ciliophora: Scuticociliatida) as the causative agent of the
scuticociliatosis in farmed turbot Scophthalmus maximus in Galicia (NW Spain). Dis.
Aquat. Org. 46, 4755.
Imsland, A.K., Sunde, L.M., Folkvord, A., Stefansson, S.O., 1996. The interaction of
temperature and sh size on growth of juvenile turbot. J. Fish Biol. 49, 926940.
Johanneson, K., Andre, C., 2006. Life on the margin: genetic isolation and diversity loss in a
peripheral marine ecosystem, the Baltic Sea. Mol. Ecol. 15, 20132029.
Karas, P., Klingsheim, V., 1997. Effects of temperature and salinity on embryonic
development of turbot (Scophthalmus maximus L.) from the North Sea, and
comparisons with Baltic populations. Helgol. Mar. Res. 51, 241247.
Lewontin, R., Krakauer, J., 1973. Distribution of gene frequency as a test of the theory of
the selective neutrality of polymorphisms. Genetics 74, 175195.
Luikart, G., England, P.R., Tallmon, D., Jordon, S., Taberlet, P., 2003. The power and promise
of population genomics: from genotyping to genome typing. Nat. Rev. Genet. 4,
981994.
Nei, M., Maruyama, T., 1975. LewontinKrakauer test for neutral genes. Genetics 80, 395.
Nielsen, E.E., Nielsen, P.H., Meldrup, D., Hansen, M.M., 2004. Genetic population structure
of turbot (Scophthalmus maximus L.) supports the presence of multiple hybrid zones
for marine shes in the transition zone between the Baltic Sea and the North Sea.
Mol. Ecol. 13, 585595.

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R. Vilas et al. / Marine Genomics xxx (2015) xxxxxx

Vasemgi, A., Primmer, C.R., 2005. Challenges for identifying functionally important genetic variation: the promise of combining complementary research strategies. Mol.
Ecol. 14, 36233642.
Vera, M., lvarez-Dios, J.A., Milln, A., Pardo, B.G., Bouza, C., Hermida, M., Fernndez, C., de
la Herrn, R., Molina-Luzn, M.J., Martnez, P., 2011. Validation of single nucleotide
polymorphism (SNP) markers from an immune Expressed Sequenced Tag (EST)
turbot (Scophthalmus maximus) data base. Aquaculture 313, 3141.
Vera, M., lvarez-Dios, J.A., Fernndez, C., Bouza, C., Vilas, R., Martnez, P., 2013.
Development and validation of single nucleotide polymorphisms (SNPs) markers
from two transcriptomic 454-run of turbot (Scophthalmus maximus) using high
throughput genotyping. Int. J. Mol. Sci. 14, 56945711.

Vilas, R., Bouza, C., Vera, M., Milln, A., Martnez, P., 2010. Variation in anonymous and
EST-microsatellites suggests adaptive population divergence in turbot. Mar. Ecol.
Prog. Ser. 420, 231239.
Vitalis, R., Dawson, K., Boursot, P., 2001. Interpretation of variation across marker loci as
evidence of selection. Genetics 158, 18111823.
Weir, B.S., Cockerham, C.C., 1984. Estimating F-statistics for the analysis of population
structure. Evolution 38, 13581370.

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Genomics (2015), http://dx.doi.org/10.1016/j.margen.2015.04.011

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