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Two markers and one history: phylogeography of the edible common
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sea urchin Paracentrotus lividus in the Lusitanian region
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I. Calderón*, G. Giribet+, X. Turon#
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*Department of Animal Biology, Faculty of Biology, University of Barcelona, 645 Diagonal
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Ave, 08028 Barcelona, Spain. +Department of Organismic and Evolutionary Biology & Museum
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of Comparative Zoology, Harvard University, 26 Oxford Street, Cambridge, MA 02138, USA.
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#
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Center for Advanced Studies of Blanes (CSIC), C. d’Accés a la Cala S. Francesc 14, 17300
Blanes (Girona), Spain.
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Corresponding author:
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I. Calderón
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Department of Animal Biology, Faculty of Biology, University of Barcelona, 645 Diagonal
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Ave, 08028 Barcelona, Spain.
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Telephone: +34934021441
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Fax: +34934035740
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E-mail: calderon@ub.edu
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Abstract
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Benthic marine invertebrates with long-lived larvae are believed to have dispersal capabilities
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that contribute to maintaining genetic uniformity among populations over large geographical
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scales. However, both hydrological and biological factors may limit the actual dispersal of such
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larvae. We studied the population genetic structure of the edible common sea urchin
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Paracentrotus lividus (Lamarck, 1816) to explore its dispersal patterns in the Atlanto-
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Mediterranean region and, more specifically, to ascertain the role of the Strait of Gibraltar in
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shaping the genetic structure of this species. For this purpose, we analysed 158 individuals for
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the mitochondrial 16S rRNA gene and 151 of these for the nuclear single-copy intron ANT
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(Adenine Nucleotide Transporter) from 16 localities from the Atlantic and Mediterranean
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basins, spanning over 4000 km. Mitochondrial 16S rRNA shows higher genetic diversity in the
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Mediterranean than in the Atlantic and reveals a sharp break between the populations of both
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basins, probably as a consequence of the barrier imposed by the Almería-Orán hydrological
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front, situated east of the Strait of Gibraltar. Both markers suggest that a recent population
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expansion has taken place in both basins, most probably following the Messinian salinity crisis.
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Introduction
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The last decades have witnessed an ever-increasing interest in discerning the role of historical
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and current processes in shaping observed genetic structure at the intraspecific level (reviewed
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in Avise 2000). In marine benthic invertebrates, gene flow between populations is mainly driven
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by dispersal of larvae that, in some cases, can remain in the water column for weeks or even
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months. Nevertheless, recent studies in several benthic invertebrates have shown little
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correlation between larval lifespan and dispersal ability (e.g., Benzie 2000; Hellberg et al. 2002;
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Palumbi 2004). Indeed, barriers to gene flow are not always conspicuous, especially in marine
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habitats where geographical barriers, currents, temporal and spatial spawning patterns (Hart and
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Scheibling 1998), physical and behavioural properties of larvae (Thomas 1994), juvenile
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mortality (Gosselin and Qian 1996, 1997; Hunt and Scheibling 1997) and many other abiotic or
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biotic factors can ultimately determine population structure over space and time (Moberg and
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Burton 2000; Sponaugle et al. 2002).
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Current and historical barriers to gene flow may leave a strong footprint on population
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structure. A good model to study the role of such barriers on marine organisms is the Strait of
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Gibraltar, which constitutes the limit between two marine biogeographical regions, the north-
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eastern Atlantic Ocean and the Mediterranean Sea. Historically, the exchange of waters between
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both basins was interrupted during the Messinian salinity crisis (Maldonado 1985; Pérès 1989),
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which constituted one of the most dramatic events during the Cenozoic era (Duggen et al. 2003).
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The opening of a new connection through the Strait of Gibraltar re-established water exchanges
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between basins, leading to the recolonisation of the Mediterranean by organisms from the
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Atlantic. Besides, fluctuations in sea level during the Quaternary also produced sporadic
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separations between both basins (Nilsson 1982; Waelbroeck et al. 2002). Population
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differentiation across the Atlantic-Mediterranean divide has been described in a number of
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marine species (e.g., Borsa et al. 1997; Bargelloni et al. 2003; Baus et al. 2005). The
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concordance in intraspecific patterns in species with diverse life history traits points to the
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importance of the historical processes that occurred in this area. Nowadays, the so-called
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Almería-Orán hydrological front, situated east of the Strait (Tintore et al. 1998) still hinders
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migration between basins for numerous marine species (Patarnello et al. 2007 and references
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therein).
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Sea urchins (Echinodermata, Echinoidea) are a diverse group of marine deuterostomes
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(Smith et al. 2006) that play an important role in structuring benthic communities (e.g., Palacín
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et al. 1998; Sala et al. 1998; Sivertsen 2006). The common edible sea urchin Paracentrotus
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lividus (Lamarck, 1816) is a commercially exploited species found in the north-eastern Atlantic
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and throughout the Mediterranean (Boudouresque and Verlaque 2001). Larval lifespan has been
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estimated between 20 and 40 days (Fenaux et al. 1985; Pedrotti 1993) and thus, individuals of
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this species are potentially able to disperse over long distances. Nonetheless, high spatial,
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bathymetric and temporal variability in settlement suggests that biotic or abiotic factors may
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affect dispersal (Hereu et al. 2004; Tomas et al. 2004).
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Notwithstanding the vast amount of literature on the ecology and biology of
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Paracentrotus lividus (e.g., Savy 1987; Turon et al. 1995; López et al. 1998; Tomas et al. 2006),
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little is still known about its genetic structure. Iuri et al. (2007), in a study based on two
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mitochondrial and one nuclear markers, stated that P. lividus presents no genetic differentiation
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within the Gulf of Naples. At a larger scale, Duran et al. (2004) observed panmixia within
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Atlantic and Mediterranean basins using cytochrome c oxidase subunit I (hereafter COI) DNA
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sequences, but detected also a slight but significant pattern of genetic differentiation between the
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two basins.
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The aim of our study was to obtain a more detailed picture of the population genetic
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structure of Paracentrotus lividus throughout the Atlanto-Mediterranean region and, especially,
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to ascertain the role of the Strait of Gibraltar in shaping the genetic structure of this species. In
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order to achieve such a goal, we sampled the common sea urchin in16 locations from the
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Atlantic and Mediterranean basins and analysed two molecular markers of different
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characteristics: a mitochondrial ribosomal gene and a nuclear intron.
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Material and methods
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Sixteen locations were sampled for Paracentrotus lividus by scuba along the Atlanto-
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Mediterranean arch (Figure 1). Distances between sampling sites ranged from 20 to around 4400
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km. The gonads were dissected from live specimens, fixed in 96% ethanol and stored at –80ºC
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until processing.
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Genomic DNA was extracted using the REALPURE extraction kit (Durviz, Spain) and
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two molecular markers were analysed. A fragment of the mitochondrial 16S rRNA gene was
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amplified for 158 individuals with universal primers 16Sa and 16Sb (Kessing et al. 1989). The
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single-copy intron of the nuclear Adenine Nucleotide Transporter (ANT) gene was amplified for
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151 individuals by EPIC-PCR, using degenerate universal primers designed by Jarman et al.
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(2002). In both cases, amplifications were performed in a final volume of 25 μL using 2.5 mM
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of MgCl2, 1 mM of dNTPs, 0.5 μM of each primer and 1 U of Taq polymerase. For the
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mitochondrial 16S rRNA, 1 μL of DMSO was added per sample. PCR amplicons were vacuum-
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cleaned and labelled using BigDye® Terminator v.3.1 (Applied Biosystems, Branchburg, New
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Jersey, USA). Sequences were obtained on an ABI 3730 and 3100 Genetic Analyzer (Applied
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Biosystems) for 16S and ANT, respectively.
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In order to reconstruct the allelic phase from the ANT genotypic data we used the
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program PHASE v2.1 (Stephens et al. 2001; Stephens and Scheet 2005). To confirm the
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existence of only two alleles per individual and to check the results provided by PHASE, PCR
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products of six individuals from different populations were cloned with the pGEM-Easy Vector
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cloning kit (Promega, Wisconsin, USA) following manufacturer’s instructions. Four to six
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colonies per individual were sequenced.
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Population genetics analyses
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Haplotype and nucleotide diversity values were calculated with DnaSP v.4.10.3 (Rozas et al.
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2003). Genetix v 4.05.2 (Belkhir et al. 2004) was used to calculate haplotype frequencies and
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inbreeding coefficients from the data obtained for ANT with PHASE. Pairwise genetic distances
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(FST) for both markers were calculated with Arlequin ver. 3.1 (Excoffier et al. 2005) and their
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significance was assessed by performing 10,000 permutations. A Multidimensional Scaling
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(MDS) was performed with Systat 11 (SPSS) to graphically visualise these results.
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SAMOVA 1.0 (Dupanloup et al. 2002) was used to define groups of populations that are
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geographically homogeneous and with the highest differentiation among each other. Analyses
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were performed for K=2 groups with 10,000 simulated annealing procedures. AMOVA, as
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implemented in Arlequin, was performed to further examine hierarchical population structure.
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Finally, the correlation of genetic and geographical distances was tested with the Mantel test
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procedure available in Arlequin.
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Haplotype network
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For 16S rRNA, the complete data set was used to build a median-joining network using
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Network v4.2.0.1 (Bandelt et al. 1999). For ANT, however, due to the high number of
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haplotypes found, we limited the network to populations surrounding the Strait of Gibraltar. The
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loops observed in the networks were solved using criteria derived from coalescent theory
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(Templeton et al. 1987; Templeton and Sing 1993).
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Demographic analyses
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Neutrality tests and mismatch distribution analyses can provide hints to infer population
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demographic events. Tajima’s D (Tajima 1989a), Fu’s FS (Fu 1997) and R2 (Ramos-Onsins and
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Rozas 2002) were calculated with DnaSP. Mismatch distributions (Rogers and Harpending
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1992; Harpending 1994), as well as goodness-of-fit tests for demographic and spatial
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expansions, were calculated with Arlequin.
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Results
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Diversity and population structure
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For the mitochondrial 16S rRNA, a fragment of 582 bp was sequenced from 158 individuals. A
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multi-T region was observed in the amplified fragment, containing from 6 to 9 Ts. Except for
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these, all other changes observed were substitutions. Thirty-one polymorphic sites and 38
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haplotypes were observed. Of these, 16 haplotypes were present in Atlantic samples (9 of these
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were exclusive to this basin) whereas Mediterranean samples comprised 29 (22 haplotypes
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exclusive to this basin; Table 1 in Appendix I). Haplotype diversity is thus higher in the
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Mediterranean than in the Atlantic basin, and so is nucleotide diversity (Table 1).
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For the ANT intron, 323 bp were sequenced for 151 out of the 158 individuals sequenced
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for 16S rRNA. Fifty-seven variable sites were observed. Cloning of six randomly chosen
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individuals proved that ANT was a single-copy marker and confirmed in every case the
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haplotype assignment provided by PHASE. The allelic reconstruction estimated 142 haplotypes
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(Tables 2 and 3 in Appendix I), 86 in the Atlantic (60 haplotypes exclusive to this basin) and 82
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in the Mediterranean (56 of which were exclusive to this basin). Haplotype and nucleotide
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diversity did not differ between basins (Table 1). Twenty-nine out of 151 individuals appeared
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to be homozygotes, the number of homozygote individuals being evenly distributed in both
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basins. Eight populations showed a significant departure from Hardy-Weinberg equilibrium
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(Table 1).
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FST for 16S rRNA showed higher levels of population differentiation between basins
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(mean FST = 0.109) than within the Atlantic and Mediterranean basins (means of 0.021 and –
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0.004 respectively; Table 4 in Appendix I). As a consequence, a sharp separation between
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Atlantic and Mediterranean populations was observed in the MDS representation (Figure 2a).
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On the contrary, differences between basins as revealed by FST measures were not as clear for
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the ANT data, mostly due to the high variability observed in the sampled populations (Table 4 in
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Appendix I). Likewise, MDS did not show a clear separation between basins (Figure 2b).
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For 16S rRNA, SAMOVA for K=2 clustered Atlantic populations, plus Ceuta and
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Tarifa, in one group, and the remaining Mediterranean populations in another group. Ceuta and
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Tarifa, although located in the Mediterranean Sea, are placed west of the Almería-Orán
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hydrological front. Results from AMOVA showed a significant variance component associated
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with the differentiation between basins (11.20%, considering Ceuta and Tarifa as Atlantic
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populations). The variance between populations within basins was low and not significant
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(0.08%), but increased to 6.39% and became significant when no groups were specified (Table
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2a). The Mantel test showed a significant correlation coefficient between genetic and
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geographical distances (r=0.395, p=0.009) for the whole sample. However, neither the
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correlation coefficients estimated within basins (r=0.05 and r=0.225 for the Atlantic and the
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Mediterranean, respectively) nor the coefficient for between-basins pairs alone (r=0.184) were
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significant, indicating that the overall significant results may, in fact, be an artefact. Indeed, this
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outcome may stem from the fact that the global analysis lumped together comparisons within
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basins, which corresponded to populations poorly differentiated and geographically close, with
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between-basin comparisons, which relied on populations that tended to be more divergent and
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widely separated. This suggests that there is no isolation by distance between our basins, but
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only a sharp genetic break at the Almería-Orán front.
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As for ANT, SAMOVA showed a clear differentiation between Nao and all other populations
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when K=2 groups. When K>2, populations were separated one by one from the main group and
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the sharp distinction between the Atlantic and Mediterranean basins observed for 16S rRNA was
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not observed. For the sake of comparison, we computed AMOVA (and subsequent analyses)
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with the same Atlantic and Mediterranean groups as for 16S rRNA. A much higher variability
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within populations was observed, accounting for more than 97% of the overall variation (Table
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2b). Variation among populations within basins was in general small but significant (between 2
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and 3%) whereas variation between groups (basins) was not significant. As expected, the Mantel
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test provided a smaller, non-significant correlation coefficient (r=0.137, p>0.05).
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Haplotype network
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The network obtained for the 16S rRNA data suggests that haplotype 1 is the ancestral
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haplotype due to its high frequency, its wide geographical distribution and its central position in
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the network. All haplotypes are separated by a few mutational steps (Figure 3). For ANT, the
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haplotype network was only built for the 4 populations around the Strait of Gibraltar (Cádiz,
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Ceuta, Tarifa and Gata), comprising 78 individuals representing 51 haplotypes. The single
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network obtained presented a high amount of loops that were almost always unambiguously
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resolved (Figure S1).
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Demographic analyses
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Neutrality tests for the 16S rRNA detected a population expansion for the whole sample set,
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with significant values for all three tests. However, only the Fs and R2 statistics detected
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significant expansions within each basin (Table 3). In the case of ANT, neutrality tests provided
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the same results observed for 16S rRNA, although R2 did not detect a significant expansion for
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the Atlantic basin (Table 3).
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The mismatch distributions for both 16S rRNA and ANT presented a unimodal
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distribution, characteristic of a sudden expansion model (Rogers and Harpending 1992) for each
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basin separately as well as for the whole area (Figure 4 and Table 4). The test for spatial
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expansion for the 16S rRNA in Mediterranean populations failed to converge with the algorithm
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implemented in Arlequin ver. 3.1.
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According to Rogers and Harpending (1992), the wave’s crest is determined at τ=2ut,
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where τ is the mode of mismatch distribution and t represents the approximate time of
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expansion. In this equation, u is the mutation rate of the entire region under study (mutation rate
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per nucleotide times the number of nucleotides of the fragment analysed). A mutation rate of
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0.5% per nucleotide per million years (Myr) was used for the 16S rRNA, following that in other
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sea urchins (Chenuil and Féral 2003). Assuming independent demographic events within each
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basin, the expansion in the Atlantic would have taken place around 270,000 years ago, whereas
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in the Mediterranean it would have occurred 360,000 years ago. According to previous studies
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(e.g., Lozano et al. 1995; Turon et al. 1995) Paracentrotus lividus has a generation time of 3
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years. Therefore, expansions in each basin would have occurred 90,000 and 120,000 generations
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ago, respectively. On the contrary, if we assume that a single expansion occurred in the whole
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area, this would have taken place 351,000 years or 117,600 generations ago.
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Likewise, the intron data detected population expansions both for the Atlantic and
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Mediterranean basins, as well as for the whole distribution. Lack of data for the mutation rate of
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nuclear introns prevented us from estimating expansions times.
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Discussion
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The study of population genetics of Paracentrotus lividus in the Atlanto-Mediterranean arch
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using a mitochondrial ribosomal gene and a nuclear intron reveals two salient points: a lack of
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differentiation within each basin, suggesting long range dispersal, and the role of the Strait of
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Gibraltar, and more specifically the Almería-Orán hydrological front, in restricting gene flow
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between Atlantic and Mediterranean populations.
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The use of multiple molecular markers with different properties can yield considerably
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more sensitive results than a single marker (e.g., Chow and Takeyama 2000; Buonaccorsi et al.
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2001). MtDNA has been classically used in population genetics and phylogeographic studies
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(reviewed in Avise 2000, but see Ballard and Whitlock 2004; Hurst and Jiggins 2005). The use
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of introns for such studies is much more recent (e.g., Villablanca et al. 1998; Daguin et al. 2001;
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Berrebi et al. 2005). These non-coding regions are expected to evolve at a higher rate than
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coding regions or than ribosomal genes constituting good markers for inferring recent processes
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at the intraspecific level (but see Kreitman 1983; Villablanca et al. 1998; Zhang and Hewitt
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2003). One of the difficulties encountered when working with nuclear DNA is the existence of
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allele polymorphisms. In the case of Paracentrotus lividus, the presence of homozygotes in our
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sample (19.2% of our individuals) allowed the most likely reconstruction of the allelic phase for
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all our individuals following a Bayesian approach (Clark 1990; Excoffier and Slatkin 1995;
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Stephens et al. 2001). Although some populations showed departures from Hardy-Weinberg
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equilibrium, the biological significance of this is questionable. Most populations showing excess
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of homozygosis had only one homozygote individual, which was enough to be significant in the
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HWE test, due to the high expected heterozygosity in the whole sample (Table 1; Table 2 and 3
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in Appendix I). The level of variability of ANT was probably excessive for detecting genetic
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structure at the studied geographical scale. Therefore, most of the variability is found within
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populations, rendering relationships between populations difficult to infer.
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MtDNA is a single molecule that is maternally inherited without generally overcoming
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recombination (but see Rokas et al. 2003; Tsaousis et al. 2005). Linkage of COI and 16S rRNA
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implies a shared evolutionary history and, thus, any difference in the patterns inferred using these
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two genes may be due to differences in mutation rates (0.5% for 16S rRNA [Chenuil and Féral
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2003], and 1.6-3.5% for COI [Lessios et al. 1999; McCartney et al. 2000] in sea urchins),
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ultimately determined by their functional constraints. Iuri et al. (2007) found COI to be more
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efficient than both mitochondrial 16S rRNA and nuclear ITS-2 to detect population structure at a
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small geographic scale in the Gulf of Naples (maximum sampled distance <80 km). On the
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contrary, our results for 16S rRNA provided a stronger signal of population differentiation
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between Atlantic and Mediterranean basins than that observed for COI by Duran et al. (2004) at a
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similar geographic scale. In our study, differentiation between basins explained 11.2% of the
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total variance while it only accounted for 1.5% in Duran et al. (2004). The 16S rRNA data reveal
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significant differentiation between groups (basins) but differentiation is negligible among
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populations between groups (Table 2). The reverse pattern occurred for ANT data. This
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apparently counter-intuitive result can be explained by the high mutation rate observed for ANT,
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that may lead to saturation and thus lack of signal when comparing the more divergent
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populations at both sides of the Gibraltar divide, while some signal is still appreciable between
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populations that have diverged less (i.e., within basins).
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Following the Messinian salinity crisis, populations of marine species in the
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Mediterranean became for the most part extinct, since this basin was reduced to an assemblage
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of hypersaline lakes (Hsü 1972; Briggs 1974; Blondel and Aronson 1999; Duggen et al. 2003).
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After the opening of the Strait (5.33 Mya) the Mediterranean was refilled with Atlantic waters,
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with the concomitant entry of biota. For many species, therefore, current Mediterranean
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populations are at most around 5 Myr old. It is unlikely that the Mediterranean hosted the
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common sea urchin Paracentrotus lividus or its ancestors during the Messinian episode due to
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the strict saline conditions following the closure of the Strait of Gibraltar, especially considering
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the poor osmoregulatory abilities of echinoderms. Taking into account that the only other
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species of the same genus, P. gaimardi (de Blainville, 1825), inhabits both coasts of the
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Southern Atlantic Ocean (Mortensen 1943), the most likely hypothesis is that P. lividus
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originated from an Atlantic stock that colonised the Mediterranean after the salinity crisis.
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Population growth generates an excess of (recent) mutations and therefore an excess of
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singletons (Avise et al. 1984; Watterson 1984; Tajima 1989a,b; Slatkin and Hudson 1991;
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Ramos-Onsins and Rozas 2002). The pattern observed in our data of few frequent haplotypes
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and many low-frequency haplotypes with few differences, similar to that observed by Duran et
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al. (2004) and also found in other marine invertebrates (e.g., Edmands et al. 1996; Zane et al.
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2000; Lejeusne and Chevaldonné 2006), is compatible with this prediction. Neutrality tests and
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mismatch distributions are also sensitive to such excess of low frequency haplotypes. Ramos-
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Onsins and Rozas (2002) suggested that Fs and R2 are more powerful tests than Tajima’s D in
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detecting population changes, a prediction confirmed by our results (Table 3). These significant
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values can be due to changes in demographic parameters but also a consequence of selection
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acting upon the studied genes. In our case, the agreement between data from unlinked markers,
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including an intron, points to demographic changes and not selection as the most likely
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explanation for the pattern found.
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Mismatch distribution analyses for both markers also suggest that demographic
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expansions took place in Paracentrotus lividus (Figure 4 and Table 4), after becoming
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established in the Mediterranean, corroborating the data from Duran et al. (2004). Interestingly,
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when the mismatch distribution is tested for populations one by one using 16S rRNA, only
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Roscoff and Cádiz seem to have experienced an expansion in the Atlantic basin whereas all
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populations in the Mediterranean show a unimodal distribution indicative of population growth
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(data not shown). This indicates that the expansion may have been more important in the
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Mediterranean than in the Atlantic basin. Our data suggest that expansions occurred some
307
300,000 years ago, corresponding to the Mindel-Riss interglaciary period (Kukla 2005), and
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possibly earlier in the Mediterranean than in the Atlantic. These expansions may have been
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determinant of the present-day genetic structure of the common sea urchin along its
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distributional range. In particular, the lower haplotypic and nucleotidic diversity of 16S rRNA in
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the Atlantic basin could be the result of the arrival of Mediterranean lineages into the Atlantic
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during this expansion episode, which may have erased the genetic signal of older Atlantic
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populations.
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In conclusion, the origin of the populations of Paracentrotus lividus in the area studied
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probably dates back to the end of the Messinian period and, afterwards, the interplay of glacial
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and interglacial periods during the Quaternary and associated demographic changes would have
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shaped the present-day distribution of the species. Restricted gene flow between the Atlantic and
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the Mediterranean basins and a high connectivity within basins are nowadays the prevailing
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processes acting upon populations of Paracentrotus lividus.
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Acknowledgements
513
We thank S. Duran, S. López-Legentil, J. Sánchez-Fontenla, M. Rius, C. Palacín, A. Blanquer,
514
A. Corbacho, E. Cebrián and E. Macpherson for providing samples for this study. Two
515
anonymous reviewers and Associate Editor Thorsten Reusch provided comments that helped to
516
improve this manuscript. This study was funded by projects CTM2004-05265 and CTM2007-
517
66635 of the Spanish Government and by internal funds from the Museum of Comparative
518
Zoology, Harvard University. We declare that all the experiments done comply with current
519
Spanish laws.
520
16
521
Figure captions
522
Figure 1. Sampling scheme for Paracentrotus lividus. Codes for populations: 1) Ros: Roscoff;
523
2) San: Santander; 3) Fer: Ferrol; 4) Lis: Lisbon; 5) Cdz: Cádiz; 6) Tfe: Tenerife; 7) Ceu: Ceuta;
524
8) Tar: Tarifa; 9) Gata: Gata; 10) Nao: Nao; 11) Tos: Tossa de Mar; 13) Cad: Cadaqués; 14)
525
Cab: Cabrera; 15) Cor: Corsica; 16); Gre: Greece.
526
Figure 2. Multidimensional scaling (MDS) for 16S rRNA (a) and ANT (b). Open circles
527
represent Atlantic populations (including Ceuta and Tarifa) whereas filled circles represent
528
Mediterranean populations.
529
Figure 3. Minimum Spanning Network for 16S rRNA. Circles represent the 38 haplotypes
530
observed in our sample. Areas of the circles are proportional to the number of sampled
531
individuals. Partitions inside the circles represent the proportion of each population within each
532
haplotype. Red dots represent missing, probably unsampled haplotypes or extinct sequences.
533
Figure 4. Mismatch distribution for 16S rRNA and ANT for each basin. The dashed line
534
represents the expected distribution under a sudden expansion model; the solid line represents
535
the observed distribution. Ceuta and Melilla are considered as belonging to the Atlantic
536
group of populations.
537
17
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
567
Ros
45ºN
Fer
San
Cor
Cad
Tos
Col
Lis
Nao
Tar
Cdz
Gata
Ceu
Cab
Gre
Tfe
0º
15ºE
18
Pairwise nucleotide differences
(a)
(b)
568
569
19
570
571
572
573
574
575
576
577
578
579
580
Roscoff
Santander
Ferrol
Lisbon
Cádiz
Tenerife
Ceuta
Tarifa
Gata
Nao
Columbretes
Tossa
Cadaqués
Cabrera
Corsica
Greece
20
581
16S (Atlantic)
1200
Observed
Expected
1000
Frequency
800
600
400
200
0
0
1
2
3
4
5
6
7
Pairwise differences
582
16S (Mediterranean)
1000
Observed
Expected
800
Frequency
600
400
200
0
0
2
4
6
8
10
Pairwise differences
583
ANT (Atlantic)
1800
Observed
Expected
1600
1400
Frequency
1200
1000
800
600
400
200
0
0
584
2
4
6
8
10
12
14
Pairwise differences
21
585
ANT (Mediterranean)
2000
Observed
Expected
Frequency
1500
1000
500
0
0
586
587
2
4
6
8
10
12
14
16
Pairwise differences
22
588
589
590
591
592
593
594
Table 1. Diversity measures for the populations of Paracentrotus lividus studied. Number of individuals per
population (N). Number of haplotypes per population (Nh), out of which the number of private alleles is shown in
brackets. Two haplotypes in the Atlantic and 5 in the Mediterranean are exclusive from each basin, but shared by
more than one population. Haplotypic (H) and nucleotidic (π) diversity, with standard deviations presented in
brackets. Inbreeding coefficient for ANT. Asterisks represent significant coefficients at p<0.05. H exp represents the
expected heterozygosity and Hobs represents the observed heterozygosity. Ceuta and Tarifa were considered in the
Atlantic group of populations following results from SAMOVA (see text).
Population
16S
N
H
0.563
(±0.062)
0.839
Roscoff
9
(±0.110)
0.378
Santander
10
(±0.181)
0.733
Ferrol
10
(±0.101)
0.564
Lisbon
11
(±0.134)
0.818
Cádiz
11
(±0.119)
0.250
Tenerife
8
(±0.18)
0.533
Ceuta
10
(±0.180)
0.200
Tarifa
10
(±0.154)
0.839
79
MED
(±0.020)
0.667
Gata
10
(±0.163)
0.952
Nao
7
(±0.96)
0.894
Columbretes 12
(±0.63)
0.885
Tossa
13
(±0.064)
0.929
Cadaqués
8
(±0.084)
0.944
Cabrera
9
(±0.070)
0.929
Corsica
8
(±0.84)
0.970
Greece
12
(±0.044)
158 0.785
Total
(±0.032)
ATL
79
Π
0.00189
(±0.00027)
0.00239
(±0.00057)
0.00072
(±0.00037)
0.00259
(±0.00057)
0.00184
(±0.00049)
0.00262
(±0.00067)
0.00043
(±0.00031)
0.00179
(±0.00075)
0.0076
(±0.00058)
0.00407
(±0.00028)
0.00261
(±0.00102)
0.00375
(±0.00080)
0.00390
(±0.00052)
0.00406
(±0.00064)
0.00413
(±0.00070)
0.00466
(±0.00076)
0.00429
(±0.00068)
0.00374
(±0.00066)
0.00333
(±0.00025)
ANT
Nh
(private)
N
H
Π
16 (9)
75
4 (1)
9
4
9
7 (2)
10
4
10
7 (3)
11
3
8
6
10
3 (1)
8
29 (22)
76
7 (4)
10
7
6
7 (1)
11
7 (4)
13
6 (2)
8
7 (2)
9
6
8
10 (4)
11
38
151
0.975
(±0.006)
0.961
(±0.034)
0.928
(±0.040)
0.968
(±0.028)
1
(±0.016)
0.961
(±0.028)
0.933
(±0.48)
0.995
(±0.018)
0.992
(±0.025)
0.959
(±0.011)
0.953
(±0.028)
0.939
(±0.058)
0.931
(±0.046)
0.975
(±0.021)
0.958
(±0.036)
0.958
(±0.033)
0.942
(±0.048)
0.961
(±0.024)
0.9672
(±0.0060)
0.01397
(±0.00058)
0.01360
(±0.00191)
0.01267
(±0.00185)
0.01367
(±0.00162)
0.01623
(±0.00149)
0.01330
(±0.00118)
0.01026
(±0.00112)
0.01486
(±0.00131)
0.01442
(±0.00193)
0.01230
(±0.00064)
0.01014
(±0.00141)
0.00577
(±0.00077)
0.01072
(±0.00180)
0.01259
(±0.00131)
0.01164
(±0.00148)
0.01409
(±0.00189)
0.01099
(±0.00110)
0.01608
(±0.00168)
0.01316
(±0.00044)
Nh
Inbreeding
H exp.
(private) Coefficient
87 (60)
0.0621*
0.913
H obs.
0.8108
14 (5)
0.200*
0.9074
0.778
11 (6)
0.045
0.8765
0.889
16 (5)
0.182*
0.920
0.800
20 (12)
-0.006
0.945
1.000
16 (8)
0.349*
0.917
0.636
11 (4)
0.611*
0.867
0.375
19 (10)
-0.006
0.945
1.000
15 (4)
-0.0091
0.930
1.000
82 (56)
0.098*
0,901
0.803
13 (3)
0.383*
0.905
0.600
9 (2)
-0.071
0.861
1.000
16 (8)
0.024
0.888
0.909
21 (12)
0.143*
0.944
0.846
12 (3)
0.229*
0.898
0.750
15 (8)
0.069
0.915
0.900
12 (9)
0.075
0.883
0.875
15 (10)
0.444*
0.917
0.546
142
0.080*
0.907
0.806
595
596
23
597
598
599
Table 2. Analyses of Molecular Variance for 16S rRNA (a) and ANT (b). Ceuta and Tarifa were considered as
600
(a) 16S
Source of variation
belonging to the Atlantic group of populations. Asterisks represent significant tests at p<0.05 after 10,000
permutations. Va, Vb and Vc are the associate covariance components. FSC, FST and FCT are the F-statistics.
Df
Sum of
squares
Variance
components
% of
variation
Fixation
indices
AMOVA between basins
Among groups
1
10.646
0.12237 Va
11.20*
FCT: 0.00086
Among populations within groups
14
13.689
0.00084 Vb
0.08
FSC: 0.11275
Within populations
142
137.678
0.96956 Vc
88.72*
FST: 0.11198
Total
157
162.013
1.09277
Among populations without groups
15
24.335
0.06622 Va
6.39*
FST: 0.06393
Within populations
142
137.678
0.96956 Vb
93.61
Total
157
162.013
1.03578
AMOVA without groups
601
602
(b) ANT
Source of variation
Df
Sum of
squares
Variance
components
% of
variation
Fixation
indices
Among groups
1
3.561
0.00207 Va
0.10
FCT: 0.02848
Among populations within groups
14
44.924
0.06050 Vb
2.84*
FSC: 0.02942
Within populations
288
594.455
2.06408 Vc
97.06*
FST: 0.00098
Total
303
642.941
2.12665
Among populations without groups
15
47.260
0.05706 Va
2.68*
FST: 0.02684
Within populations
288
595.782
2.06869 Vb
97.32
Total
303
643.043
2.12575
AMOVA between basins
AMOVA without groups
603
24
604
605
Table 3. Tests of neutrality for 16S rRNA and ANT. Ceuta and Tarifa were considered as belonging to the Atlantic
group of populations. Asterisks represent significant results: * p<0.05; ** p<0.01; *** p<0.002.
16S
Atlantic
Mediterranean Total
Tajima’s D
-1.73683
-1.65461
-1.90111*
Fu’s Fs
-7.010***
-18.888***
-30.171***
R2
0.042*
0.043*
0.029*
ANT
Atlantic
Mediterranean Total
Tajima’s D
-1.47953
-1.89368
-1.85294*
Fu’s Fs
-123.389***
-116.093***
-246.361***
R2
0.0501
0.0395*
0.0369*
606
607
25
608
609
610
611
Table 4. Parameters of population expansion for both markers, for each basin separately and for the whole sample.
Ceuta and Tarifa were considered as belonging to the Atlantic group of populations. Parameters are estimated with a
confidence interval of 0.01 for 10,000 bootstrap replicates. SDD (sum of square deviations) with its respective p
values in brackets.
16S
Parameters
ANT
Atlantic
Mediterranean
Total
Atlantic
Mediterranean
Total
Τ
1.581
2.097
2.043
4.544
4.176
4.510
Θo
0.113
0.217
0.159
0.643
0.580
0.560
Θ1
2031.363
4330.372
3687.971
1379.746
1149.083
1210.135
0.003005
(0.14)
0.000574
(0.12)
0.00057
(0.080)
0.002188
(0.29)
0.0016
(0.34)
0.001098
(0.61)
0.0013
(054)
0.001279
(0.42)
0.0017
(0.39)
Goodness-of-fit test (SDD)
Demographic
expansion
Spatial
expansion
0.00108
(0.54)
0.000982
(0.60)
-------
612
26
613
APPENDIX
614
Table 1. Absolute haplotype frequencies for 16S. Population codes correspond to codes in Figure 1.
615
Hapl Ros San Fer Lis Cdz Tfe Ceu Tar ATL Gata Nao Col Tos Cad Cab Cor Gre MED
1
4
4
3
2
2
1
1
3
2
4
1
3
4
5
5
3
8
36
1
6
4
3
1
1
2
2
1
1
2
1
1
2
2
1
12
8
1
1
9
1
1
1
1
4
1
2
1
12
1
1
12
1
1
13
1
1
14
1
1
1
1
2
1
5
1
1
1
1
1
1
1
1
1
1
3
11
1
1
3
1
1
4
2
2
14
1
1
17
1
1
1
2
4
2
19
1
1
2
2
20
1
18
2
10
1
16
1
4
7
15
1
1
5
10
4
2
2
10
3
1
1
6
21
1
1
22
1
1
3
1
23
2
2
24
1
1
25
1
1
26
1
1
27
1
1
28
1
1
29
1
1
30
1
1
31
1
1
32
1
1
33
1
1
34
1
1
35
1
1
36
1
1
27
37
1
1
38
1
1
Total
9
10
11 10 11
8
10
10
79
10
7
12 13
8
9
8
12
79
616
617
28
618
619
Table 2. Haplotypic phase for each individual form results obtained with PHASE. Homozygotic individuals are
marked with an asterisk. Population codes correspond to codes in Figure 1.
ATLANTIC
Individual
Haplotye
Individual
Haplotye
ROSCOFF
CÁDIZ
H1, H2
H8, H49
Ros1
Cdz1
MEDITERRANEAN
Individual
Haplotye
Individual
Haplotye
GATA
CADAQUÉS
H134, H135 Cad2
H7, H85
Gata2
H3, H4
Ros2
H5, H6
Ros3
H7, H7
Ros4*
H8, H9
Ros5
H10, H10
Ros6*
H11, H12
Ros8
H7, H13
Ros9
H10, H14
Ros10
SANTANDER
H1, H15
San2
H7, H10
San3
H16, H17
San4
H18, H18
San5*
H19, H20
San7
H21, 22
San8
H1, H7
San9
H7, H17
San10
H7, H17
San11
FERROL
H1, H23
Fer1
H7, H7
Fer2*
H24, H25
Fer3
H11, H26
Fer4
H1, H1
Fer5*
H27, H28
Fer9
H7, H17
Fer11
H29, H30
Fer13
H31, H32
Fer14
H33, H34
Fer17
LISBON
H7, H27
Lis2
H35, H36
Lis3
H10, H37
Lis6
H34, H38
Lis7
H39, H40
Lis8
H41, H42
Lis9
H8, H42
Lis10
H43, H44
Lis15
H45, H46
Lis16
H47, H48
Lis17
Gata3
Gata4
Gata10*
Gata11*
Gata13
Gata15
Gata16
Gata17*
Gata18
H8, H50
H26, H51
H7, H7
H52, H52
H53, H53
H8, H8
H54, H55
H56, H57
H10, H58
H29, H59
TENERIFE
H7, H60
Tfe1
H12, H61
Tfe3
H10, H62
Tfe4
H1, H1
Tfe6*
H63, H63
Tfe7*
H1, H1
Tfe10*
H64, H64
Tfe11*
H65, H65
Tfe13*
CEUTA
H66, H67
Ceu1
H28, H68
Ceu2
H69, H70
Ceu4
H17, H71
Ceu6
H72, H73
Ceu8
H7, H74
Ceu11
H75, H76
Ceu12
H12, H77
Ceu13
H49, H78
Ceu14
H7, H79
Ceu15
TARIFA
H1, H7
Tar1
H7, H80
Tar2
H28, H68
Tar3
H81, H82
Tar4
H83, H84
Tar5
H12, H49
Tar7
H10, H85
Tar9
H29, H86
Tar14
Cdz2
Cdz3
Cdz4*
Cdz5*
Cdz6*
Cdz7*
Cdz8
Cdz9
Cdz10
Cdz11
H68, H136
H137, H137
H68, H68
H1, H1
H7, H138
H7, H139
H7, H140
H141, H141
H2, H142
NAO
H7, H85
Nao5
H96, H97
Nao6
H31, H85
Nao11
H10, H37
Nao12
H17, H83
Nao14
H10, H85
Nao20
COLUMBRETES
H7, H8
Col1
H7, H91
Col3
H92, H93
Col4
H7, H87
Col8
H31, H88
Col9
H31, H85
Col10
H89, H90
Col12
H10, H94
Col15
H72, H95
Col16
H7, H7
Col17*
H7, H28
Col18
TOSSA
H85, H98
Tos1
H92, H92
Tos2*
H8, H12
Tos4
H7, H99
Tos5
H100, H101
Tos6
H1, H102
Tos7
H103, H104
Tos8
H105, H106
Tos9
H8, H84
Tos10
H22, H53
Tos11
H17, H107
Tos18
H7, H7
Tos33*
H108, H109
Tos34
H1, H110
Cad4
H17, H17
Cad10*
H8, H51
Cad15
H7, H7
Cad17*
H13, H51
Cad18
H111, H112
Cad19
H9, H61
Cad23
CABRERA
H7, H83
Cab19
H113, H114
Cab22
H7, H9
Cab23
H38, H115
Cab24
H116, H117
Cab25
H118, H119
Cab26
H7, H120
Cab27
H7, H8
Cab34
H72, H72
Cab40*
CORSICA
H7, H7
Cor5*
H7, H8
Cor6
H92, H131
Cor12
H31, H85
Cor13
H132, H133
Cor17
H8, H30
Cor18
H1, H2
Cor19
H7, H17
Cor20
GREECE
H7, H7
Gre1*
H121, H121
Gre2*
H122, H123
Gre5
H7,H60
Gre7
H124, H125
Gre8
H1, H1
Gre9*
H126, H127
Gre10
H128, H128
Gre12*
H129, H129
Gre14*
H34, H73
Gre16
H1, H130
Gre17
620
29
621
622
623
624
625
626
627
628
Table 3. Absolute frequencies of each ANT haplotype per basin.
Haplotype ATL
MED
Total
Haplotype ATL
MED Total Haplotype ATL MED To
11
8
19
1
1
2
1
1
H1
H37
H73
1
2
3
1
1
2
1
H2
H38
H74
1
1
1
1
1
H3
H39
H75
1
1
1
1
1
H4
H40
H76
1
1
1
1
1
H5
H41
H77
1
1
2
2
1
H6
H42
H78
18
27
45
1
1
1
H7
H43
H79
6
7
13
1
1
1
H8
H44
H80
1
2
3
1
1
1
H9
H45
H81
8
3
11
1
1
1
H10
H46
H82
2
2
1
1
1
2
H11
H47
H83
4
1
5
1
1
1
1
H12
H48
H84
1
1
2
3
3
1
7
H13
H49
H85
1
1
1
1
1
H14
H50
H86
1
1
1
2
3
1
H15
H51
H87
1
1
2
2
1
H16
H52
H88
5
5
10
2
1
3
1
H17
H53
H89
2
2
1
1
1
H18
H54
H90
1
1
1
1
1
H19
H55
H91
1
1
1
1
4
H20
H56
H92
1
1
1
1
1
H21
H57
H93
1
1
2
1
1
1
H22
H58
H94
1
1
1
1
1
H23
H59
H95
1
1
1
1
2
1
H24
H60
H96
1
1
1
1
2
1
H25
H61
H97
2
2
1
1
1
H26
H62
H98
2
2
2
2
1
H27
H63
H99
3
1
4
2
2
1
H28
H64
H100
3
3
2
2
1
H29
H65
H101
1
1
2
1
1
1
H30
H66
H102
1
4
5
1
1
1
H31
H67
H103
1
1
2
3
5
1
H32
H68
H104
1
1
1
1
1
H33
H69
H105
2
1
3
1
1
1
H34
H70
H106
1
1
1
1
1
H35
H71
H107
1
1
1
3
4
1
H36
H72
H108
Table 4. FST values for 16S (lower diagonal) and for ANT (upper diagonal) based on pairwise nucleotide
differences. Population codes correspond to codes in Figure 1. Values in bold represent significant comparisons
for p<0.05. The empty diagonal represents FST=0. Note that, for 16S (lower diagonal) more than 50% of
comparisons among basins are significant whereas only when comparing Ferrol to Santander and Tarifa in the
Atlantic FST values were significant within basins. As for ANT, Nao shows a striking difference with all other
populations, all comparisons being significant.
-0.00235 -0.01301 -0.00096
0.04941
-0.02116 0.03136
0.00810 0.16986
0.01153
-0.07614 0.06913 -0.02859
-0.02640 -0.01190 0.07542 -0.00100
0.06756
0.03762
0.06147
0.08728
-0.00596
0.00192
-0.02138
0.00801
0.07553
0.01111 -0.02075
0.01399 -0.01079
0.01868 -0.02204
0.04769 0.01527
0.08201 0.05606
0.04582
0.01231
0.02500
0.07672
0.04519
-0.01875 0.
-0.00307 -0.0
0.01228 -0.0
0.04081 0.0
0.04909 0.0
0.10380
0.14511
0.17063
0.15730
0.17424
30
0.02391 -0.07781
-0.01734 -0.00168
0.05868 0.06504
0.05262 0.02597
0.07612 0.13354
0.12152 0.10075
0.08484 0.11534
0.17250 0.21468
0.16156 0.16475
0.15419 0.15304
0.02811 0.03854
Ros
San
0.15995
0.07407
0.22087
0.15500
0.19000
0.19393
0.17737
0.25810
0.22565
0.21641
0.12745
Fer
0.04469 -0.06833
0.04238 0.00362 0.03855 0.24229 0.03381 0.0
-0.02882 -0.03894 -0.04876
-0.02963 0.08477 0.14719 0.02407 0.0
0.07231 -0.00458 -0.02132 0.00383
0.05282
0.13716 0.00021 -0.0
0.06420 0.01650 -0.01112 -0.00473 0.01961
0.17311 0.03043 0.0
0.10487 0.03466 0.07082 0.02015 0.09386 -0.04184
0.06629 0.1
0.09510
0.07812
0.08016
0.10733
-0.03670
-0.05160
0.13220
-0.0
0.10061 0.07158 0.06797 0.04523 0.06030 -0.02893 -0.03872 0.00282
0.20163 0.17081 0.19481 0.12778 0.22381 0.01189 -0.02028 -0.00445 -0.0
0.17570 0.13886 0.14214 0.13680 0.18024 0.01538 -0.02448 -0.04575 0.0
0.16740 0.12880 0.13393 0.09005 0.17764 -0.04002 -0.04965 -0.07413 -0.0
0.04849 0.02787 0.00836 -0.01103 0.03115 -0.05117 0.00128 0.02273 -0.0
Lis
Cdz
Tfe
Ceu
Tar
Gata
Nao
Col
T
629
630
631
31