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Phylogeographical history of the white seabream Diplodus sargus(Sparidae): Implications for insularityMercedes González-Wangüemertab; Elsa Froufec; Angel Pérez-Ruzafad; Paulo Alexandrinoa

a Centro de Investigação em Biodiversidade e Recursos Genéticos (CIBIO), Universidade do Porto,Porto, Portugal b Centro de Ciências do Mar (CCMAR), Universidade do Algarve, Faro, Portugal c

CIIMAR, Centro Interdisciplinar de Investigação Marinha e Ambiental, Porto, Portugal d Departamentode Ecología e Hidrología, Facultad de Biología, Universidad de Murcia, Murcia, Spain

First published on: 18 March 2011

To cite this Article González-Wangüemert, Mercedes , Froufe, Elsa , Pérez-Ruzafa, Angel and Alexandrino, Paulo(2011)'Phylogeographical history of the white seabream Diplodus sargus (Sparidae): Implications for insularity ', MarineBiology Research, 7: 3, 250 — 260, First published on: 18 March 2011 (iFirst)To link to this Article: DOI: 10.1080/17451000.2010.499438URL: http://dx.doi.org/10.1080/17451000.2010.499438

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ORIGINAL ARTICLE

Phylogeographical history of the white seabream Diplodus sargus

(Sparidae): Implications for insularity

MERCEDES GONZALEZ-WANGUEMERT1,2*, ELSA FROUFE3, ANGEL

PEREZ-RUZAFA4 & PAULO ALEXANDRINO1

1Centro de Investigacao em Biodiversidade e Recursos Geneticos (CIBIO), Universidade do Porto, Porto, Portugal, 2Centro de

Ciencias do Mar (CCMAR), Universidade do Algarve, Faro, Portugal, 3CIIMAR, Centro Interdisciplinar de Investigacao

Marinha e Ambiental, Porto, Portugal, and 4Departamento de Ecologıa e Hidrologıa, Facultad de Biologıa, Universidad de

Murcia, Murcia, Spain

Abstract

Partial sequences of the mitochondrial control region and its comparison with previously published cytochrome b (cyt-b)and microsatellite data were used to investigate the influence of island isolation and connectivity on white seabream geneticstructure. To achieve this, a total of 188 individuals from four island localities (Castellamare and Mallorca, MediterraneanSea; Azores and Canary Islands, Atlantic Ocean) and five coastal localities (Banyuls, Murcia and Tunisia, MediterraneanSea; Galicia and Faro, Atlantic Ocean) were analysed. Results showed high haplotype diversity and low to moderatenucleotide diversity in all populations (except for the Canary Islands). This pattern of genetic diversity is attributed to arecent population expansion which is corroborated by other results such as cyt-b network and demographic analyses. Lowdifferentiation among Mediterranean/Atlantic and coastal/island groups was shown by the AMOVA and FST values,although a weak phylogeographic break was detected using cyt-b data. However, we found a clear and significant island/distance effect with regard to the Azores islands. Significant genetic differentiation has been detected between the Azoresislands and all other populations. The large geographical distance between the European continental slope and the Azoresislands is a barrier to gene flow within this region and historic events such as glaciation could also explain this geneticdifferentiation.

Key words: Connectivity, fishes, islands, mtDNA, phylogeography, population structure

Introduction

In the marine realm, a high potential for dispersal

and/or the absence of strong barriers to migration

are believed to guarantee high connectivity between

distant populations, and to limit long-term popula-

tion subdivision. Conversely, species with low dis-

persal potential are expected to have clear patterns

of genetic structure (Duran et al. 2004). There are

examples showing that such expectations may be

unfounded (Uthicke & Benzie 2000; Lazoski et al.

2001), because a variety of biological, ecological and

historical factors might contribute to the struc-

turing of the populations through space and time

(Gonzalez-Wanguemert et al. 2004, 2007; Perez-Ruzafa

et al. 2006). In fact, marine fish species may exhibit

population structure owing to spawning behaviour

and self-recruitment (Gonzalez-Wanguemert et al.

2007), these being highly influenced by environ-

mental conditions and habitat characteristics (Gibson

1994; Cardinale & Arrhenius 2000; Pitchford et al.

2005). Also, ecological factors such as discontinuity

in suitable habitat may reduce gene flow among

populations of marine organisms. This loss of habitat

for species is very common between islands and

continental coasts, favouring the genetic divergence

between populations (Riginos & Nachman 2001;

Duran et al. 2004; Sa-Pinto et al. 2008; Zulliger et al.

2009). Some authors have found high genetic

*Correspondence: M. Gonzalez-Wanguemert, Centro de Ciencias do Mar (CCMAR), Universidade do Algarve, Campus de Gambelas,

8005-139 Faro, Portugal. E-mail: mercedes@mail.icav.up.pt; mwanguemert@ualg.pt.

Published in collaboration with the University of Bergen and the Institute of Marine Research, Norway, and the Marine Biological Laboratory,

University of Copenhagen, Denmark

Marine Biology Research, 2011; 7: 250�260

(Accepted 19 May 2010; Published online 21 March 2011)

ISSN 1745-1000 print/ISSN 1745-1019 online # 2011 Taylor & Francis

DOI: 10.1080/17451000.2010.499438

Downloaded By: [B-on Consortium - 2007] At: 15:41 18 March 2011

differentiation between oceanic island and contin-

ental populations from the North Atlantic Ocean

linked to isolation by current systems and geo-

graphic distances (Emerson 2002; Schonhuth et al.

2005; Domingues et al. 2007; Sa-Pinto et al. 2008).

Together with these features, we must also con-

sider historical factors that might contribute to the

genetic structuring of the populations. In our study

area (Mediterranean Sea and northeastern Atlantic

Ocean) we must consider events which occurred

during the Pliocene: the northeastern Atlantic ex-

perienced a progressive cooling and the sea surface

temperatures in the Azores islands experienced an

additional cooling (2�38C) (Crowley 1981; Briggs

1996; Adams et al. 1999; Domingues et al. 2006).

Several authors (e.g. Briggs 1974; Santos et al.

1995) suggested that this probably resulted in the

mass extinction of littoral fish from the Azores, and

that most of the organisms now present would have

come from southern regions. Santos et al. (1995)

suggested a post-glacial colonization of the Azores

from Madeira or the western coast of Africa.

However, other authors (e.g. Barton et al. 1998;

Domingues et al. 2007) maintained that sea surface

temperatures around the Azores did not decrease

meaningfully, so the elimination of littoral fish

populations by glaciation was unlikely. On the other

hand, the Mediterranean Sea was gradually sepa-

rated from the Atlantic Ocean during the Messinian

Salinity Crisis (5.59�5.33 Ma), although short

periods of separation between the Atlantic and

Mediterranean waters occurred during the Quatern-

ary in response to cyclical ice ages and the associated

sea level changes (Patarnello et al. 2007).

Diplodus sargus (Linnaeus, 1758) is a commercial

species which includes seven subspecies (Bauchot

& Hureau 1990) in the Atlantic and Indian Oceans,

the Mediterranean Sea and the Persian Gulf. The

life history of D. sargus shows a pattern consistent

with digynic hermaphroditism, changing from male

to female through a nonfunctional intersexual phase.

White seabream achieve sexual maturity during the

second or third year of life. Spawning occurs from

March to June and the onset and duration of

spawning season appear to be influenced by sea

water temperatures (Morato et al. 2003). Diplodus

sargus larvae spend 3�4 weeks in the open sea before

reaching a favourable environment for metamorpho-

sis and later recruitment into the adult population

(Gonzalez-Wanguemert et al. 2004). During this

phase of the life cycle, larvae are at the mercy of

prevailing currents. This species behaves as ‘cyclic

migrants’ and can migrate into lagoons after meta-

morphosis and spend the early stages of their life

cycle in these environments with very different tem-

peratures and salinities. Juveniles move to shallow

areas (B5 m) whilst adults are more abundant in

the surf zone (Pajuelo & Lorenzo 2004) at depths of

between 10 and 50 m depending upon substrate

availability (Harmelin-Vivien et al. 1995).

Several authors have studied the genetic struc-

ture of D. sargus using different molecular markers

(Lenfant & Planes 1996, 2002; Gonzalez-Wanguemert

et al. 2004, 2006, 2007, 2010; Bargelloni et al. 2005;

Perez-Ruzafa et al. 2006; Domingues et al. 2007). In

general, allozyme studies detected significant genetic

differentiation between populations at different spa-

tial (101�103 km) and temporal scales in the Med-

iterranean and Atlantic regions. However, some authors

did not find signs of genetic differentiation among

Atlantic and Mediterranean populations of D. sargus

using allozymes (Bargelloni et al. 2005) and mito-

chondrial control region and the first intron of the

S7 ribosomal protein gene (Domingues et al. 2007).

Gonzalez-Wanguemert et al. (2010) found signifi-

cant genetic differentiation between the Azores

population and other studied populations using

microsatellites and cyt-b region. These last authors

suggested that the breakdown of effective genetic

exchange between the Azores and the others samples

could be explained simultaneously by hydrographic

(deep water) and hydrodynamic (isolating current

regimes) factors acting as barriers to the free dis-

persal of white seabream (adults and larvae) and by

historical factors which could have favoured the

survival of Azorean white seabream population at

the last glaciation.

In this study, we want to analyse the influence of

isolation and connectivity on the white seabream

genetic structure considering a number of localities

and individuals distributed across the Western and

Central Mediterranean Sea and Northeastern Atlantic

Ocean. To achieve this aim, we analysed a highly

variable marker (control region) and the obtained

data were compared with previously published data

from cyt-b region and microsatellites (Gonzalez-

Wanguemert et al. 2010). Discussions considering

data from mtDNA (cyt-b and control region) and

nuclear (microsatellites) markers can finally try to

conclude which are the factors that can explain the

white seabream genetic structure.

Material and methods

Current patterns from study area

The Northeastern Atlantic Current system is domi-

nated by the Gulf Stream, which splits into two main

branches, the North Atlantic Current (North) and

the Azores Current (East) (Domingues et al. 2006).

Close to the Azores Islands, each of these currents

divides into two branches, one of which flows south,

Phylogeography of white seabream 251

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feeding the Canaries Current (Santos et al. 1995)

(Figure 1). Mediterranean physical oceanography is

forced by topography, wind stress and buoyancy flux

at the surface due to fresh water inputs and heat

fluxes (Robinson et al. 2001; Patarnello et al. 2007).

Atlantic water is less dense than Mediterranean

water, and enters at the surface through the Strait

of Gibraltar. This mass of water forms a semi-permanent

anticyclonic gyre in the Alboran Sea (SE Spain),

generating an oceanic front located from Almeria to

Oran (AOF, Figure 1). This surface water flows

to the East following the North African coast

(Algerian Current) with eddies near the Libyan

coast (Alhammoud et al. 2004) (Figure 1).

Sampling and DNA extractions

To achieve the defined aims, we analysed a total

of 188 individuals from four island localities

(Castellamare and Mallorca, Mediterranean Sea;

Azores and Canary Islands, Atlantic Ocean) and

five continental coastal localities (Banyuls, Murcia

and Tunisia, Mediterranean Sea; Galicia and Faro,

Atlantic Ocean) (Figure 1). Samples were collected

at local markets while some individuals were

sampled by SCUBA diving during 2004�2005.

Muscle samples were removed from fresh fish and

stored in 99% ethanol immediately after collection.

Total genomic DNA was extracted from small (3�5 mg) pieces of tissue following a protocol based on

Sambrook et al. (1989) with minor modifications

(increasing the time of Proteinase K digestion to

24 h and adding 7 ml Proteinase K per sample). The

extracted DNA was resuspensed in an elution

buffer and stored at �208C until further use.

PCR amplification and sequencing

We used sequences of non-coding mitochondrial,

control region, because it has been demonstrated

that this genetic marker shows adequate levels of

sequence variation in the sparid family for popula-

tion genetic and phylogeography studies (Jousson

et al. 2000; Bargelloni et al. 2005).

We amplified a 416-bp fragment of the control

region for 188 individuals by polymerase chain

reaction (PCR) using the primers described in

Summerer et al. (2001) and Kocher et al. (1989).

Reactions of 24 ml total volume contain 2.5 ml of

10� buffer (Ecogen); 1.5 mM MgCl2, (Ecogen);

200 mM dNTP mix; 0.5 U Taq DNA polymerase

(Ecogen); 0.25 mM of each primer; 5�50 ng of DNA.

PCR cycles were performed on a Biometra T3

Thermocycler under the following conditions: 3

min at 948C, followed by 30 cycles of denaturing

at 948C for 30 s, annealing at 558C for 40 s and

extension at 728C for 40 s; finishing with an exten-

sion step at 728C for 5 min.

PCR products were electrophoresed and purified

using the ExoSAP-IT (USBEurope GmbH). Puri-

fied DNA was sequenced with an ABI sequenc-

ing kit (Big Dye Terminator Cycle Sequencing

Figure 1. Location of the sampling sites and simplified schematic representation of surface circulation (grey discontinuous lines) in the

North Atlantic and Mediterranean Sea (Gysels et al. 2004; Aboim et al. 2005; Domingues et al. 2007). AZ, Azores; CN, Canary Islands;

GL, Galicia; F, Faro; SW, Murcia; ML, Mallorca; BY, Banyuls; TN, Tunisia; CT, Castellamare; NAC, North Atlantic Current; SEC,

Southwest European Current; AC, Azores Current; AOF, Almerıa�Oran Front.

252 M. Gonzalez-Wanguemert et al.

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v. 2.0-ABI PRISM, Applied Biosystems) and then

analysed with an ABI 3700 automated sequencer.

Chromatograms obtained from the automated se-

quencer were read using BIOEDIT Sequence Align-

ment editor (Hall 1999).

Population genetic analysis

Genetic diversity parameters, FST values and results

of AMOVA analysis and Mantel’s test (cyt-b) were

taken from a previous paper (Gonzalez-Wanguemert

et al. 2010) to compare with the control region data.

To evaluate the genetic diversity of populations,

haplotype and nucleotide diversities were calculated

from control region data using ARLEQUIN version

2.000 (Schneider et al. 2000). The same program and

region was used to calculate the pairwise FST values

among populations and among groups (coastal/island

and Mediterranean/Atlantic groups). Their signifi-

cance was tested by performing 10,000 permutations.

Correlations between geographic distances and

FST values were tested using Mantel’s (1967) test.

Probabilities were calculated from the distribution

of 1000 randomized matrices computed by permu-

tation. Mantel’s test was performed using the MANTEL

procedure from the ‘Genetix’ package (Belkhir et al.

1996�2004).

We also performed an analysis of molecular var-

iance (AMOVA) to examine hierarchical population

structure, pooling the localities into two groups,

‘island’ (the Azores and Canary Islands, Mallorca

and Castellamare) and ‘coastal’ (Banyuls, Tunisia,

Galicia, Faro and Murcia). Also, another two groups,

Mediterranean (Murcia, Banyuls, Tunisia, Castella-

mare and Mallorca) and Atlantic (the Azores and

Canary Islands, Galicia and Faro), were tested as an

independent nesting design. Significance was esti-

mated with 10,000 permutations of the data matrix

(Guo 1992).

An exact test of population differentiation based

on haplotype frequencies (Raymond & Rousset

1995) was performed to test the null hypothesis

that observed haplotype distribution is random with

respect to sampling location. The significance of

individual tests was estimated by comparison with

simulated distributions constructed from 10,000

random permutations of the original data matrix.

Demographic history

To study the historical demography of the popula-

tions, we analysed the mismatch distributions of

pairwise differences between all individuals grouped

by region (Mediterranean and Atlantic) and insular-

ity (island and coastal) using control region and cyt-b

(ARLEQUIN, v. 2.000). These analyses discriminate

whether a population has remained demographically

stable over time or has undergone a rapid population

expansion (Rogers & Harpending 1992).

ARLEQUIN was used to test for departures from

mutation-drift equilibrium with Tajima’s D test (Tajima

1989). We also assessed the history of effective

population size by means of other statistics such as

Fu’s FS (Fu 1997), using DNASP version 4.10.9

(Rozas et al. 2003). Fu (1997) has noticed that the

FS statistic was very sensitive to population demo-

graphic expansion, which generally lead to large

negative Fs values. The significance of the FS statistic

was tested by generating random samples under the

hypothesis of selective neutrality and population

equilibrium, using a coalescent simulation algorithm

adapted from Hudson (1990).

The time of possible population expansions (t)

was calculated through the relationship t�2ut (Rogers

& Harpending 1992), where t is the mode of the

mismatch distribution, u is the mutation rate of the

sequence considering that u�2mk (m is the mutation

rate per nucleotide and k is the number of nucleo-

tides). A mutation rate of 11% per nucleotide per

million years (Myr) was used for control region

(Bargelloni et al. 2003).

Results

Genetic diversity

A total of 188 individuals were sequenced for the

mtDNA control region (410 bp) and the overall

diversity was high with 131 different haplotypes

whose sequences were registered in Genbank

[EF428560, GQ995933�GQ996062]. We found a

total of 125 polymorphic sites and 140 mutations.

Only nine haplotypes were shared among different

individuals; the other 122 haplotypes were singletons.

Two of the shared haplotypes were represented in

more than one geographical area (Mediterranean Sea

and Atlantic Ocean), while the other seven were only

shared between individuals restricted to the same

geographical area. The number of haplotypes per

population ranged from 12 to 19 (Table I). The most

common haplotype (CR-15) was found in

five localities (Mallorca, Tunisia, Murcia, Faro and

Canary Islands) and 93.1% of haplotypes were

population-specific. The haplotype network with

many reticulate relationships was not conclusive for

control region. Haplotype diversity was high, but

nucleotide diversity within each population was

moderate to low (Table I).

Genetic differentiation among populations

AMOVAs (Table II) using control region data

and considering two different nesting designs

Phylogeography of white seabream 253

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(‘coastal�island’ and ‘Mediterranean�Atlantic’) indi-

cated that the highest proportion of the variance was

attributed to variation within populations (99.05%

and 98.89%, respectively) and a significant but small

variance component was attributable to variation

among populations within groups (1.10% and

0.90%) (Table II).

Significant FST values were found in pairwise

comparisons between some Mediterranean locali-

ties (Tunisia, Galicia and Murcia) and the Azores

(Table III). Significant genetic differentiation was

also detected between Mediterranean localities such

as Banyuls and Tunisia/Murcia. Similarly, exact tests

of population differentiation showed significant dif-

ferences (PB0.05) for the same pairs of populations

(data not shown).

FST values (control region data and previous

published cyt-b data) (Table III) showed that the

Azores sample was the most genetically differentiated

population, so we tested to confirm whether genetic

differentiation detected among Mediterranean/

Atlantic groups and ‘island/coastal’ groups was real

or whether it resulted from differentiation of the

Azores population.

A significant differentiation is shown between the

Mediterranean and Atlantic basins considering each

geographical region as a single panmictic metapo-

pulation (control region: FST�0.004, P�0.020;

previous cyt-b data: FST�0.036, P�0.008). This

genetic differentiation was still significant (cyt-b)

when we removed the Azores population from the

Atlantic group (cyt-b: FST�0.040, P�0.013; con-

trol region: FST�0.002, P�0.110). No differentia-

tion was detected between ‘coastal’ and ‘island’

groups using control region data (FST�0.001,

P�0.212). However, the same analysis detecteda

significant differentiation using cyt-b data (FST�0.016, P�0.05), but the FST value decreased

when Azores was not considered (FST��0.004,

P�0.517).

Mantel tests did not show evidence of a pattern

of genetic differentiation in relation to geographic

Table I. Diversity measures for the populations of Diplodus sargus. Abbreviations indicate number of individuals (N), number of haplotypes

(Nh), nucleotide diversity (p) and haplotype diversity (h). Cyt-b data published Gonzalez-Wanguemert et al. (2010).

Control region Cyt-b

Populations N Nh p h Nh p h

Azores 20 15 0.0230 0.9766 3 0.0011 0.6377

Canary 20 12 0.0247 0.9872 2 0.0003 0.2000

Galicia 23 16 0.0290 0.9933 6 0.0011 0.5507

Faro 20 12 0.0234 1.0000 4 0.0009 0.5524

Murcia 20 18 0.0214 0.9656 7 0.0022 0.8381

Banyuls 21 12 0.0244 0.9429 6 0.0022 0.8889

Mallorca 22 16 0.0218 0.9926 7 0.0019 0.7647

Tunisia 22 19 0.0271 1.0000 7 0.0014 0.6883

Castellamare 20 12 0.0176 1.0000 5 0.0015 0.7091

Total 188 131 0.0244 0.9874 23 0.0163 0.7200

Table II. Hierarchical analysis of molecular variance (AMOVA) results for Diplodus sargus. We have considered four groups: island group

(Azores and Canary Islands, Mallorca and Castellamare localities), coastal group (Banyuls, Tunisia, Galicia, Faro and Murcia localities),

Mediterranean group (Murcia, Banyuls, Tunisia, Castellamare and Mallorca localities) and Atlantic group (the Azores and Canary Islands,

Galicia and Faro). Significant values ( PB0.05) are in bold. Cyt-b data published in Gonzalez-Wanguemert et al. (2010).

Molecular marker Source of variation Total variance (%) Fixation indices

Control region Among groups (Coast�Island) �0.16 FCT��0.0016

Among populations within groups 1.10 FSC�0.0110

Within populations 99.05 FST�0.0095

Control region Among groups (Med�Atl) 0.21 FCT�0.0021

Among populations within groups 0.90 FSC�0.0090

Within populations 98.89 FST�0.0111

Cytb Among groups (Coast�Island) 0.68 FCT�0.0068

Among populations within groups 3.97 FSC�0.0399

Within populations 95.35 FST�0.0465

Cytb Among groups (Med�Atl) 2.97 FCT�0.0297

Among populations within groups 2.61 FSC�0.0269

Within populations 94.42 FST�0.0558

254 M. Gonzalez-Wanguemert et al.

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distance (control region: Z�1062.36; r�0.046;

P�0.417; cyt-b: Z�7310.23, r�0.392, P�0.087).

The same test considering only Iberian coastal

samples (Galicia, Faro, Murcia and Banyuls), which

are more likely to be connected by gene flow given

the habitat continuity, did not detect a pattern either

(control region: Z�125.84, r��0.629, P� 0.75;

cyt-b: Z�464.20, r�0.705, P�0.140).

Demographic history

We performed one set of analyses considering each

geographical region (Mediterranean and Atlantic)

using control region data (Figure 2). Mismatch

distributions were not different from the sudden

expansion model for the Mediterranean and Atlantic

groups (Figure 2a; Table IV). Tajima’s D was sig-

nificant for the Mediterranean group and Fu’s FS

were significant for both groups (Table IV). The

same analyses were performed using ‘coastal’ and

‘islands’ groups and only the FS values were sig-

nificant (Table IV; Figure 2b). Considering these

data, we only detected a significant Mediterranean

expansion which took place approximately 121,383

years ago.

Mismatch distribution analyses were also per-

formed using cyt-b data. The least-squares procedure

to fit model mismatch distribution and observed

distribution did not converge after 1800 steps.

Tajima’s D was significant for the Mediterranean

group and FS values were significant for Mediterra-

nean and Atlantic groups (Table IV). We carried out

another set of analyses considering ‘island’ and

‘coastal’ groups and Tajima’s D and Fu’s FS were

significant for both groups (Table IV; Figure 2d).

Discussion

Nucleotide and haplotype diversities can provide

some information on the history of white seabream

populations. High haplotype diversity (control

region) and low to moderate nucleotide diversity

(control region and cyt-b) were found in all popula-

tions analysed. This pattern of genetic diversity can

be attributed to a recent population expansion after

a low effective population size caused by founder

events or bottlenecks (Grant & Bowen 1998; Aboim

et al. 2005). This explanation is also consistent with

the star-shaped haplotype network detected for cyt-b

from populations of Diplodus sargus in previous work

(Gonzalez-Wanguemert et al. 2010) whose shape

is obtained after the expansion event. Our data

suggest that expansion may have happened in the

Mediterranean region 121,383 years ago. Similar

expansion events for D. sargus were detected using

control region data by Bargelloni et al. (2005). These

authors considered the loss of an ancestral Mediter-

ranean clade during environmentally less suitable

periods and a subsequent re-colonization of the

vacant niche by the Atlantic clade. This hypothesis

would also be supported by our data.

A greater number of unique haplotypes was found

in the populations from the Mediterranean Sea

(control region: 75 haplotypes; cytb: 13 haplotypes)

than in the Atlantic populations (control region:

54 haplotypes; cytb: five haplotypes). This would

point to historical isolation followed by secondary

contact with D. sargus invading the Mediterranean

Sea from the Atlantic Ocean but considering the

survival of Mediterranean haplotypes throughout.

Other factors could also explain these results, such

as a more stable effective population size over time in

the Mediterranean Sea or an older origin of D. sargus

in the Mediterranean Sea. However, this last hy-

pothesis does not seem likely considering Summerer

et al.’s (2001) D. sargus phylogeny.

Low differentiation among Mediterranean and

Atlantic groups was shown by the AMOVA (control

region and cyt-b) and FST values (control region).

Similar FST values using control region data were

Table III. Population pairwise FST values from cyt-b data (below the diagonal) and control region (above diagonal). Cyt-b data published

Gonzalez-Wanguemert et al. (2010).

BY ML TN F CN AZ GL SW CT

BY � 0.166 0.0278* 0.0070 0.0150 0.0123 0.0310* 0.0411* 0.0291

ML �0.0334 � �0.0048 �0.0111 �0.0036 0.0093 0.0070 �0.0035 �0.0011

TN �0.0054 �0.0170 � �0.0039 0.0026 0.0116* 0.0034 0.0053 �0.0039

F 0.0498 0.0045 �0.0356 � �0.0064 �0.0056 0.0035 0.0020 0.0000

CN 0.2334* 0.1275** 0.0863 0.0409 � �0.0020 0.0096 0.0094 0.0064

AZ 0.1011* 0.0857* 0.0886* 0.1044* 0.1865* � 0.0149* 0.0290* 0.0121

GL 0.0558 0.0054 �0.0219 �0.0450 0.0302 0.1087* � 0.0207* 0.0035

SW �0.0213 0.0226 0.0027 0.0403 0.2240* 0.1208* 0.0644 � 0.0156

CT �0.0387 �0.0294 �0.0563 �0.0488 0.1002 0.0779 �0.0343 �0.0230 �

*PB0.05. BY, Banyuls; ML, Mallorca; TN, Tunisia; F, Faro; CN, Canary Islands; AZ, Azores Islands; GL, Galicia; SW, Murcia; CT,

Castellamare.

Phylogeography of white seabream 255

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shown by Bargelloni et al. (2005), who did not reject

the null hypothesis of panmixia. The simplest

explanation for slight genetic structure is that gene

flow permits the homogenization of gene frequencies

across geographic populations. However, the lack of

population structure may not be solely the result of

exchange of sufficient individuals to prevent popula-

tion divergence, as the slight genetic differences found

among some populations of D. sargus are also con-

sistent with a recent divergence. So, the genetic

similarity of the populations from two regions could

be explained by the insufficient time to accumulate

genetic differences. The Strait of Gibraltar has been

proposed to be the divide between two marine bio-

geographical regions (Quignard 1978; Turon et al.

2003; Baus et al. 2005; Lemaire et al. 2005).

However, a clear phylogenetic break has never been

observed and some species from diverse taxa show

no differentiation at all between Atlantic and

Mediterranean populations (Bargelloni et al. 2003,

2005; Costagliola et al. 2004; Duran et al. 2004;

Stamatis et al. 2004; Zardoya et al. 2004; Domingues

Figure 2. Mismatch distributions (frequency distributions of pairwise differences among haplotypes) of Diplodus sargus for each of the

Mediterranean, Atlantic, coastal and island populations. (Control region, A�B and cyt-b, C�D; black bars, observed data; continuous line

with black dots, simulated data.)

256 M. Gonzalez-Wanguemert et al.

Downloaded By: [B-on Consortium - 2007] At: 15:41 18 March 2011

et al. 2006, 2007; Patarnello et al. 2007; Comesana

et al. 2008; Gonzalez-Wanguemert et al. 2010). We

found considerable levels of gene flow between the

Atlantic and the Mediterranean populations for D.

sargus (data not shown), with all the comparisons

showing Nm values greater than 1. Diplodus sargus

adults spawn in the open sea from March to June

with the larvae spending 3�4 weeks in the open sea

before reaching a favourable environment for recruit-

ment (Gonzalez-Wanguemert et al. 2004). So, the

most likely time for migration is during the extended

pelagic phase, when larvae are at the mercy of

prevailing currents. In fact, any gene flow at present

is likely to be unidirectional from the Atlantic into the

Mediterranean, because of the physical oceanography

at the Atlanto-Mediterranean boundary (Duran et al.

2004). Previous results from white seabream micro-

satellites corroborated the absence of significant

genetic differentiation between Atlantic and Medi-

terranean populations, although these markers de-

tected the highest FST values among the Azores and

other populations (Gonzalez-Wanguemert et al. 2010).

It is important to highlight the significant genetic

differentiation found between the Azores (control

region and cyt-b) and all other populations, but not

associated with the transition between Atlantic

Ocean and Mediterranean Sea. Pinera et al. (2007)

working on Pagellus bogaraveo confirmed this result

and suggested that the differences found between

Atlantic and Mediterranean regions in previous

works are the consequence of the high geographic

distances between the Azores Islands and the con-

tinental coast. It must also be noted that phyloge-

netic analysis (data not shown) and the haplotype

network for cyt-b data (Gonzalez-Wanguemert et al.

2010) revealed a small clade of haplotypes that only

occurred in the Azores population. This is suggestive

of some degree of isolation. The Azores islands

showed the lowest number of migrants compared

with the all other populations (Nem�0.97�1.87).

It is possible that the large geographical distance

between the European continental slope and the

Azores islands is a barrier to gene flow within this

region. The current system (Figure 1) can also

influence the isolation of the Azores. The scarce

dispersal routes from the Azores may include larval

dispersal via the Canary Current such as has been

described for Helicolenus dactylopterus (Aboim et al.

2005). However, our data do not support this

hypothesis because we detected high and significant

FST values (cyt-b and microsatellites) between the

Azores and Canarian localities. We must also con-

sider historical aspects. The results agree with the

predicted phylogeographic effects caused by the

decline of the Azorean sea surface temperatures to

values similar to those prevailing nowadays in

western Iberia (Domingues et al. 2006, 2007). The

Azores population survived glaciations and the large

Table IV. Parameters of the sudden expansion model and the goodness-of-fit test to the model with respective significances. S, number of

sites with substitutions; u0 pre-expansion population size; u1, post-expansion population size; t, time in number of generations; SSD, sum of

squared deviations. Tajima’s D and Fu’s FS values and their statistical significance are shown.

Groups Mediterranean Atlantic Island Coastal

Control region Parameters

S 97 97 83 105

u0 3.320 0.012 1.952 1.986

u1 54.346 92.734 42.474 61.582

t 7.688 12.298 10.009 10.007

Goodness-of-fit-test

SSD 0.001 0.0005 0.002 0.001

P 0.759 0.949 0.800 0.649

Tajima’s D-test �1.618 �1.504 �1.452 �1.538

P 0.042 0.058 0.067 0.052

Fu’s FS �24.528 �24.445 �24.570 �24.382

P 0.000 0.000 0.000 0.000

Cyt-b Parameters

S 19 8 12 15

u0 0.000 0.000

u1 2657.500 1123.594

t 1.004 1.072

Goodness-of-fit-test

SSD No fit No fit 0.0135 0.007

P 0.000 0.05

Tajima’s D-test �2.074 �1.470 �1.800 �1.929

P 0.008 0.063 0.024 0.015

Fu’s FS �15.192 �6.744 �8.103 �11.796

P 0.000 0.000 0.000 0.000

Phylogeography of white seabream 257

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geographic distance to continental slope impeded

gene flow between populations; these facts would

explain the several private haplotypes detected in the

Azores population and the high FST values. How-

ever, the Canary Islands were severely affected by

the decrease of sea surface temperature (Barton

et al. 1998). In fact, this locality only showed two

cyt-b haplotypes and the lowest values of haplotype

diversity (hcyt-b�0.200) and observed heterozygosity

(Ho�0.467) (Gonzalez-Wanguemert et al. 2010).

Gene flow between coastal and island samples did

not seem to comply with an isolation by distance

model (IBD). Iberian coastal samples (Galicia, Faro,

Murcia and Banyuls) with geographic continuity did

not follow this model either. However, other authors

working with Mediterranean and Atlantic D. sargus

populations (Domingues et al. 2007) detected an

IBD from control region data. They considered that

this pattern stems from two white seabream features:

its benthopelagic nature and its survival during the

drops in sea surface temperatures during last glacia-

tions, allowing the possibility of movements of adults

from the Azores to the Iberian coast. We do not

agree with these considerations. Although the

benthopelagic nature of D. sargus is a fact, it is

unlikely that the mobility of the adults allows rapid

mixing between populations from the European

mainland coast and the Azores islands because of

the 1800 km distance and waters deeper than

4000 m. The presence of seamounts in this area

does not justify their use as stepping-stones such as

Domingues et al. (2007) argued. The depth of

waters between these seamounts and the lack of

prey (echinoderms) for D. sargus (Rosecchi 1985,

1987; Figueiredo et al. 2005) would prevent the

movements of D. sargus adults from the Azores

islands to the Iberian coast and vice versa. The

white seabream is a diurnal omnivore, feeding on

algae, sea urchins, worms, gastropods and amphi-

pods. Algae were the most commonly consumed

items and Echinodermata were the second com-

posed mostly of the sea urchins Arbacia lixula,

Sphaerechinus granularis and Paracentrotus lividus

(Figueiredo et al. 2005) which do not inhabit greater

depths. Also, some ontogenetic differences can be

observed, such as an increase in consumption of sea

urchins with increasing size of fish, so it is very

difficult for adult white seabream to find prey

between the seamounts.

In general, isolation promotes differentiation by

reducing gene flow between insular and continental

populations (Sa-Pinto et al. 2008). However, we

have only detected clear differentiation between the

Azores islands and the continental populations. The

rest of island populations (Mallorca, Castellamare

and Canary Islands) did not show the same pattern.

In fact, AMOVA using control region and cyt-b data

did not detect a significant genetic differentiation

between the ‘island’ and ‘coast’ groups and the

FST value between these two groups without the

Azores population was low and non-significant.

Mediterranean island localities (Mallorca and Cas-

tellamare) have shown poor genetic differentiation

with the other populations, which indicates a high

gene flow. These islands are located near continental

coasts and show high connectivity favoured by the

oceanographic current system.

Conclusion

Low differentiation among coastal/island and Medi-

terranean/Atlantic groups was shown, although weak

evidence of a phylogeographic break was detected

using the cyt-b marker. However, we found a clear

island/distance effect with regard to the Azores.

Significant genetic differentiation between the Azores

islands compared to all populations has been de-

tected. The large geographical distance between the

European continental slope and the Azores Islands is

a barrier to gene flow within this region and historical

events such as glaciations have also had an influence

on this genetic differentiation.

Acknowledgements

We thank Dr Sofıa Gamito, Dr Philippe Lenfant,

Dr Ben Stobart, Dr Lilia Barhi and Tomas Vega for

providing samples from Faro, Banyuls, Mallorca,

Tunisia and Castellamare, respectively. We are grate-

ful to Dr Fernando Canovas for useful comments.

This work received partial financial support from the

SENECA Program Murcia University (PB/56/FS/

02-03000/PI/05) and the AECI Program (Agencia

Espanola de Cooperacion Internacional. Ministerio

de Asuntos Exteriores, A/4396/05- A/6704/06). M.G.W.

was supported by a M.E.C. (Ministerio de Educa-

cion y Ciencia) postdoctoral grant.

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