Population genetics, gene flow, and biogeographical boundaries of Carcinus aestuarii (Crustacea:...

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Population genetics, gene flow, and biogeographicalboundaries of Carcinus aestuarii (Crustacea:Brachyura: Carcinidae) along the EuropeanMediterranean coast

LAPO RAGIONIERI1* and CHRISTOPH D. SCHUBART2

1RNA Biology Laboratory, Department of Biology & CESAM, University of Aveiro, 3810-193 Aveiro,Portugal2Fakultät für Biologie I, Universität Regensburg, D-93040 Regensburg, Germany

Received 3 December 2012; revised 6 February 2013; accepted for publication 7 February 2013

Carcinus aestuarii Nardo, 1847 is a widespread coastal crab species throughout the Mediterranean Sea with apelagic larval phase. This species tolerates a wide range of environmental conditions and typically inhabitsfragmented habitats, such as embayments, lagoons and estuaries. It is therefore a good candidate species forstudying and testing different phylogeographical hypotheses in the Mediterranean Sea. By contrast to its Atlanticsister species, Carcinus maenas, studies on the population genetic structure of C. aestuarii in its native range arestill scarce. In the present study, specimens from along the European Mediterranean Sea were collected andDNA-sequenced and analyses were applied to discriminate between present day and historical factors influencingthe population genetic structure of this species. The results obtained demonstrate the existence of two geneticallydistinct geographical groups, corresponding to the eastern and western Mediterranean, with further subdivisionwithin the East Mediterranean Basin. A strong asymmetric gene flow was recorded toward the Eastern Basin,which may play a crucial role in shaping the present day biogeographical patterns of this species and potentiallyother sympatric ones with pelagic larvae. © 2013 The Linnean Society of London, Biological Journal of theLinnean Society, 2013, 109, 771–790.

ADDITIONAL KEYWORDS: biodiversity – cox1 – demographic history – larval dispersal – MediterraneanSea – phylogeography.

INTRODUCTION

The Mediterranean Sea only comprises 0.82% of theWorld oceanic area and 0.3% of its whole volume.However, it hosts between 4–18% of the known marinespecies worldwide, depending on the phylum (Bianchi& Morri, 2000). Such overproportional species densityhas led many researchers to consider the Mediterra-nean Sea as a hotspot of species diversity, not forget-ting the role of an observation bias because of intenseresearch over various centuries (Lejeusne et al., 2009).The high species diversity is probably a result of three

characteristics of the Mediterranean Sea: its geologicalhistory, the variety of climatic conditions, and thevariety of hydrological conditions (Patarnello,Volckaert & Castilho, 2007). These factors simultane-ously may have contributed to produce a strong habitatdiversification within the Mediterranean Sea, whichproduced a consequent high rate of endemism.

The complex geological history of the Mediterra-nean Sea and its semi-enclosed geography resulted inperiods of complete isolation from the Atlantic Ocean,such as during the Messinian Salinity Crisis(5.3 Mya), which may have lasted no longer than0.01 Myr (Krijgsman et al., 1999). During this period,the Mediterranean Sea was desiccated, or almost so.Subsequently, the re-opening of the Strait of Gibraltarallowed re-colonization of the Mediterranean Sea

*Corresponding author. E-mail: lapo.ragionieri@ua.pt;lapo.ragionieri@gmail.com

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Biological Journal of the Linnean Society, 2013, 109, 771–790. With 3 figures

with Atlantic species, thus becoming an Atlantic prov-ince (Briggs, 1974). Also during the Pliocene andQuaternary, the Mediterranean Sea was affected bymany sea level regressions, repeatedly interruptingthe biotic exchange through the Strait of Gibraltarand resulting in a high rate of Mediterranean ende-mism (Vermeij, 1978; Patarnello et al., 2007). Atpresent, the thermohaline circulation system andother physical factors (e.g. winter surface isotherms)play a crucial role in shaping the present day speciesdistribution within the Mediterranean Sea (Bianchi &Morri, 2000; Bianchi, 2007). The thermohaline circu-lation system of the Mediterranean Sea is character-ized by an influx of cold Atlantic surface watersthrough the Strait of Gibraltar sinking into the threecoldest basins of the Mediterranean Sea (Gulf ofLyon, northern Adriatic Sea, and the northern AegeanSea). The circulation system within the Mediterra-nean Sea is characterized by the presence of manysteady surface flows associated with eddies, gyres,and fronts, which in turn show an inter-seasonal andinter-annual variability (Lascaratos & Nittis, 1998;Lascaratos et al., 1999; Korres, Pinardi & Lascaratos,2000; Millot & Taupier-Letage, 2005; Hamad, Millot& Taupier-Letage, 2006; Skliris, Sofianos &Lascaratos, 2007).

The phylogeographical break of the Atlanto-Mediterranean transition was responsible for the evo-lution of many new species, with genetically wellseparated sister taxa in the Atlantic and Mediterra-nean (Schubart, Cuesta & Rodríguez, 2001; Wilke,2003; Reuschel, Cuesta & Schubart, 2010). Demeusy(1958) suggested that the isolation between Atlanticand Mediterranean populations of the crab genusCarcinus during the Messinian Salinity Crisis couldhave provided the geographical framework for allo-patric speciation. This hypothesis was later tested byGeller et al. (1997) and Roman & Palumbi (2004), whoinvestigated the phylogenetic and phylogeographicalrelationships between the two Mediterranean sisterspecies of the genus Carcinus: Carcinus maenas(Linnaeus, 1758) and Carcinus aestuarii Nardo, 1847,which, for a long time (also after Nardo), were con-sidered synonyms because of difficulties in separatingthem morphologically. The native range of thecommon ancestor lies most likely in the easternAtlantic-Mediterranean area according to Geller et al.(1997), Roman & Palumbi (2004), and Darling et al.(2008). Today, autochthonous populations ofC. maenas are found along the north eastern Atlanticcoastline and in the Sea of Alboran, whereas C. aes-tuarii, is present throughout the Mediterranean Sea.These two sister species have probably separatedbetween 5 and 8 Myr and this separation is currentlymaintained near the Almería-Oran Front (Gelleret al., 1997; Roman & Palumbi, 2004; Marino, Pujolar

& Zane, 2011). Both species share similar life-historycharacteristics, such as wide environmental toler-ance, high fecundity, and long larval pelagic phases ofapproximately 6 weeks (Darling et al., 2008; Marinoet al., 2011). However, they differ in their habitatpreferences: C. maenas is a generalist, typically foundalong rocky shores among pebbles, in rock pools, onharbour walls, and in estuaries (Domingues et al.,2010), whereas C. aestuarii is more specialized andrestricted to estuaries, lagoons, and shallow embay-ments of the Mediterranean Sea (I. A. M. Marino,J. M. Pujolar & L. Zane, 2010, pers. observ.), resultingin an overall patchy distribution. The absence of acontinuous habitat, interrupted by long stretches ofrocky or sandy beaches, may promote divergenceamong populations, as well as the presence of geneti-cally isolated populations (DiBacco, Levin & Sala,2006; Ragionieri et al., 2010; Silva, Mesquita &Paula, 2010).

Although many studies have investigated the popu-lation genetic structure and geographical variabilityof C. maenas in its native range, as well as in manyof the allochthonous populations (Roman & Palumbi,2004; Roman, 2006; Darling et al., 2008), there is ascarcity of such studies for C. aestuarii in its nativerange. Only recently, Marino et al. (2011) reported thepresence of three main phylogroups within the Medi-terranean Sea: Adriatic-Ionian, Tyrrhenian, andwestern Mediterranean.

In general, assessing and confirming the presenceof biogeographical breaks in the marine environmentis a difficult task because of the lack of obviousbarriers to mobility of pelagic life stages. For manyyears, it was assumed that species with a larvaldispersal phase would spread over wide distances,resulting in a high connectivity among even distantly-related populations. However, gene flow in marinespecies may be constrained by dispersal barriers suchas sharp salinity and temperature gradients, cur-rents, and retention mechanisms leading to popula-tion structure, even in highly dispersive species(DiBacco et al., 2006; Zulliger et al., 2009). Geneticmethods are a useful novel tool for recognizing bio-geographical boundaries and to determine the extantand historical factors affecting the species distribu-tion worldwide.

Some of the best studied cases of genetic separationwithin the Mediterranean Sea highlight the biogeo-graphical importance of the Strait of Sicily(Patarnello et al., 2007). To assess the presence ofphylogeographical breaks within the MediterraneanSea in a marine coastal species with a patchy distri-bution, we investigated the population genetic struc-ture and gene flow of the Mediterranean endemic crabspecies C. aestuarii. The results are used to test, andpossibly generalize, phylogeographical hypotheses on

772 L. RAGIONIERI and C. D. SCHUBART

patterns of gene flow in the Mediterranean Sea.Based on the geological history, the main circulationfeatures, and the peculiar geography of the Mediter-ranean Sea, we intend to test four specific hypothesesof population genetic differentiation: (1) genetic isola-tion between western and eastern Mediterraneanbasins; (2) further eastern Mediterranean partition-ing between Adriatic and Ionian seas; (3) asymmetricgene flow between and within the two basins; and (4)the presence of transitional populations across knownphylogeographical breaks.

MATERIAL AND METHODSSAMPLE COLLECTIONS AND DNA EXTRACTION

A total of 199 specimens of C. aestuarii (classified inthe superfamily Portunoidea and the family Carcini-dae sensu Schubart & Reuschel, 2009) were collectedfrom 12 localities throughout the European Mediter-ranean Sea from eastern Spain to western Greece andused for genetic comparisons (Fig. 1, Table 1): twopopulations from the north-western Mediterranean,Ebro Delta, Spain, and Camargue, France; three fromthe Italian northern Tyrrhenian Sea, Livorno, ElbaIsland, and northern Sardinia; three from the Italiansouthern Tyrrhenian Sea, Lago Lungo, Fusaro, andsouth-eastern Sicily; two from the northern AdriaticSea, Venice, Italy, and Pomer, Croatia; one from theGreek Ionian Sea, Amvrakikos Lagoon; and one fromPeloponnesus, Greece.

A pereiopod from each specimen was removed andimmediately placed in absolute ethanol. Most animalswere subsequently released; a few specimens werekept as voucher specimens and have been depositedin the Senckenberg Museum in Frankfurt a.M.(Germany, collection numbers SMF 43890 to SMF43901). The genomic DNA was extracted using thePuregene Kit (Gentra Systems) and resuspended in20 mL of TE buffer and stored at -20 °C.

DNA AMPLIFICATION

A fragment of 658 bp of the cytochrome oxidasesubunit 1 gene (cox1, excluding primers) was ampli-fied by polymerase chain reaction (PCR) using theprimers: COL6b 5′-ACA AAT CAT AAA GAT ATYGG-3′ and COH6 5′-TAA ACT TCA GGG TGA CCAAAA AAT CA-3′ (Schubart & Huber, 2006). The PCRamplification was performed with 40 cycles of 45 s at94 °C for denaturation, 1 min at 48 °C for annealing,1 min at 72 °C for extension, preceded by 5 min at94 °C for initial denaturation and finished by 10 minat 72 °C for final extension. Subsequently, PCR prod-ucts were visualized on agarose gels, purified byprecipitation with Sure Clean (Bioline) and thenresuspended in water. The sequence reactions wereperformed with the Big Dye terminator mix (Big DyeTerminator, Version 1.1 Cycle Sequencing kit; AppliedBiosystems), followed by electrophoreses in an ABIPrism automated sequencer (ABI Prism 310 Genetic

Figure 1. Map of collection sites, the main circulation system of the Mediterranean Sea with black arrows, interannualcurrents with grey arrows, and main phylogeographic breaks as grey dotted lines (for locality abbreviations, see Table 1).

BIGEOGRAPHY OF C. AESTUARII IN THE MEDITERRANEAN SEA 773

Analyser; Applied Biosystems). Alternatively, theywere outsourced for sequencing to LGC Genomics(Berlin). The sequences were visually corrected withFLINCH TV, version 1.4.0 (Geospiza), aligned withBIOEDIT (Hall, 1999), and trimmed to a 546-bp frag-ment for subsequent analyses. To avoid pseudogenes,we tested different decapod-specific primer combina-tions without obtaining different products and con-trolled the resulting sequences for pseudogenes; therewere no double peaks among our sequences.Sequences of all haplotypes were deposited atGenBank under accession numbers (HF952775–HF952835).

GENETIC DIVERSITY INDICES AND POPULATION

GENETIC STRUCTURE

The nucleotide composition was estimated usingMEGA, version 5 (Tamura et al., 2011). The geneticheterogeneity within each population was estimatedas haplotype diversity (HD) (Nei & Tajima, 1981) andnucleotide diversity (p) (Nei, 1987) as implementedin ARLEQUIN, version 3.11 (Excoffier, Laval &Schneider, 2005). Differentiation among populationswas estimated by analysis of molecular variance(AMOVA) (Excoffier, Smouse & Quattro, 1992) usingARLEQUIN. We calculated the FST indices (Excoffieret al., 1992) using both haplotypic frequencies andgenetic distances based on the model of Tajima & Nei(1984). Significance of the fixation indices, under thenull hypothesis of no differentiation among popula-tions, was tested using a nonparametric permutation

approach (10 000 permutations of haplotypes amongpopulations). In this case, the P-value of the test iscalculated according to the proportion of permuta-tions with FST values larger or equal to the observedone (Excoffier et al., 1992). Additional hierarchicalAMOVA analyses were run to test our phylogeo-graphical hypotheses, with the groups of populations:western Mediterranean versus eastern Mediterra-nean populations; and western Mediterranean versusIonian Sea versus North Adriatic Sea.

Spatial analysis of molecular variance (SAMOVA)was used to define groups of populations that aregeographically homogenous and maximally differen-tiated from each other as implement in SAMOVA,version 1.1 (Dupanloup, Schneider & Excoffier,2002). The aim of this approach is to define groupsof populations (K) that maximize the proportion oftotal genetic variance as a result of differencesbetween these defined groups of populations, bymeans of an annealing procedure. Moreover, to inferwhether the genetic divergences (pairwise FST

values calculated from genetic distance values) arecorrelated with geographical distances, a MantelTest was employed as implemented in ARLEQUIN(Mantel, 1967). Significance was assessed by meansof 10 000 permutations of rows and columns of onematrix.

DEMOGRAPHIC HISTORY

The historic demographic history of the twelvesampled populations of C. aestuarii within the Medi-

Table 1. Locality names of the twelve sampled populations of Carcinus aestuarii within the northern Mediterranean Seawith GPS coordinates

Locality Abbreviation Geographic coordinates N sample Na HD p (%)

Camargue: Port St. Louis,France

CAM 43°20′56″N 4°49′32″E 20 9 0.65 ± 0.12 0.16 ± 0.13

Ebro Delta, Spain EBR 40°37′43″N 0°44′27″E 28 12 0.83 ± 0.05 0.38 ± 0.24Livorno, Italy LIV 43°34′56″N 10°17′58″E 16 6 0.74 ± 0.10 0.26 ± 0.19Elba: Laguna Mola, Italy ELB 42°45′30.5″N 10°23′11″E 16 9 0.91 ± 0.05 0.31 ± 0.21Sardinia: Palau, Italy SAR 41°10′24″N 9°24′26″E 18 7 0.78 ± 0.08 0.23 ± 0.17Lago Lungo, Italy LLG 41°16′20″N 13°24′16″E 15 8 0.87 ± 0.07 0.32 ± 0.22Gulf of Naples: Fusaro,

ItalyFUS 40°49′21.5″N 14°3′02.5″E 15 5 0.75 ± 0.08 0.33 ± 0.23

Sicily: Marina di Modica,Italy

SIC 36°42′34″N 14°46′54″E 10 6 0.78 ± 0.14 0.22 ± 0.17

Venice, Italy VEN 45°27′38″N 12°16′33″E 15 12 0.97 ± 0.03 0.62 ± 0.38Pomer, Croatia POM 44°49′08″N 13°53′52″E 20 14 0.93 ± 0.04 0.41 ± 0.26Amvrakikos Kolpos, Greece AMV 39°1′10″N 20°45′19″E 16 9 0.77 ± 0.11 0.26 ± 0.19Peloponnesus: Pylos, Greece PEL 36°57′08″N 21°39′41″E 6 4 0.60 ± 0.21 0.12 ± 0.12Total 195 63 0.84 ± 0.02 0.33 ± 0.21

Sample size for each population (N sample), number of haplotypes for each population (Na), haplotype diversity (HD),nucleotide diversity in percent (p).

terranean Sea was reconstructed using the mismatchdistribution (i.e. the distribution of the observednumber of differences between pairs of individuals),as implemented in ARLEQUIN. Populations at theequilibrium are expected to show a multimodaldistribution, whereas populations having recentlypassed through a demographic or spatial expansionare predicted to have a unimodal distribution (Rogers& Harpending, 1992). Because the unimodal distribu-tion of the mismatch distribution may be a result ofboth recent population expansions or recent bottle-neck events (Ramírez-Soriano et al., 2008), three neu-trality tests were applied: Tajima’s D-test, R2 test andFu’s FS test. The first two tests use information onthe mutation frequencies, whereas the latter FS testuses information on the distribution of haplotypes(Ramos-Onsins & Rozas, 2002). Significance levels ofthe D-test and FS test were estimated by generatingrandom samples under the null hypotheses of selec-tive neutrality and population equilibrium using coa-lescent simulations (Hudson, 1990) as implementedin ARLEQUIN. The R2 test significance level wasestimated using DNASP, version 5.10, based on1000 simulated re-sampling replicates. Finally, theraggedness index (rg), which uses information frommismatch distribution, was employed to assess theout of equilibrium state in single populations(Harpending et al., 1993; Harpending, 1994). The rgsignificance was tested by a parametric bootstrapapproach (10 000 replicates) under the null hypoth-esis of population expansion as implemented inARLEQUIN.

The expansion parameter tau (t), used for testingdemographic or spatial expansion hypotheses, wasestimated for the overall population of C. aestuariifrom the Mediterranean Sea. The time (t) at whichthe demographic or spatial expansion began wascalculated applying the formula of Li (1977): t = t/2m, where m is the mutation rate per site per year.To obtain a more comprehensive view of the demo-graphic history of C. aestuarii in the MediterraneanSea, we carried out age estimates by applying threedifferent mutation rates. The first and second aremutation rates commonly used during the last twodecades: 1.4% per Myr for cox1 in the snappingshrimp Alpheus (see Knowlton & Weigt, 1998) and2.33% per Myr for tropical coastal crabs Sesarma(see Schubart, Diesel & Hedges, 1998). The thirdrate is a mutation rate that was specificallyestimated for our study species by using deep cali-bration points derived from biogeographical eventssuch as the Messinian Crisis: 3.5% per Myr forC. aestuarii (Marino et al., 2011). Approximateconfidence intervals for the demographic para-meters were obtained by 1000 parametric bootstrapreplicates.

COALESCENT-BASED ANALYSIS

To discriminate between population genetic structureand demographic history of C. aestuarii, an analysismethod based on coalescent theory was employed(i.e. nested clade phylogeographical analysis; NCPA)(Templeton, Routman & Phillips, 1995; Templeton,1998, 2001). The NCPA was based on a minimumspanning network built with TCS, version 1.21(Clement, Posada & Crandall, 2000) with a 95% par-simony level of plausible sets for linkages of haplo-types. The NCPA nesting design was constructed byhand on the resulting statistical parsimony network,solving ambiguities (such as loops) by following therules derived from coalescent theory (Templeton,Crandall & Sing, 1992; Crandall & Templeton, 1993;Pfenninger & Posada, 2002). GEODIS, version 2.6(Posada, Crandall & Templeton, 2000) was used forcalculating the categorical test of significant associa-tions between nested clades and geography, using10 000 random permutations. For a single-locusnested clade analysis, the significance of the test iscorrected with the Dunn–Sidak correction (Posadaet al., 2000) incorporated in GEODIS, version 2.6.Finally, the historical and current processes respon-sible for the observed pattern of genetic variationwere inferred using the latest version of the inferencekey given in Templeton (2004) (updated online on 15December 2008).

GENE FLOW ANALYSIS

Gene flow among the twelve sampled populations ofC. aestuarii was investigated using MIGRATE,version 3.0.3 (Beerli & Felsenstein, 2001). We esti-mated the mutation scaled effective population sizetheta parameter (q = xNef*mu, where x is a multiplierdepending on the ploid phase x = 2 for mitochondrialdata, Nef is the effective female population size andmu is the mutation rate per site per generation m) andmutation-scaled migration rate (M = m/m, where m isthe immigration rate and m is the mutation rate). ABayesian search strategy (Beerli, 2006) was selectedwith three replicates of four chains, each running forone million generations. The effective number ofimmigrants per generation was calculated multiply-ing q by M (as the equation: Nemji = qi ¥ Mji).

RESULTSGENETIC DIVERSITY INDICES AND POPULATION

GENETIC STRUCTURE

The nucleotide composition of the analyzed fragmentshowed an A-T bias (C = 18.45%; T = 36.85%;A = 26.68%; G = 18.02%) as previously reported forarthropod mitochondrial DNA (Simon et al., 1994).

Four individuals from the Peloponnesus belong to anobviously unrelated haplogroup, which is discussedbelow. Of the 546 bp from the remaining 195 indi-viduals, 56 sites were polymorphic and 16 wereparsimony-informative, resulting in 61 haplotypes(see Appendix, Table A1). All but two mutations aresilent. The substitution at position 34 is between aglycine and a serine and at position 99 between aphenylalanine and a leucine.

The haplotype diversity (HD) and nucleotide diver-sity (p) values for the whole dataset, as well as for thesingle populations, are characterized by high haplo-typic diversity values and low nucleotide diversity(Table 1). Haplotype 5 (ht5) is the most common hap-lotype of the European Mediterranean populations ofC. aestuarii. Haplotype 25 is common in easternMediterranean populations, whereas, in the west, itwas only reported from the islands of Elba and Sicily.Except for haplotype 22, which is present in Elba andPomer, haplotypes 1–33 (excluding ht5) are presentexclusively in the western Mediterranean popula-tions, whereas haplotypes 34–61 belong to easternpopulations (Table 2). Except for the omnipresenthaplotype 5, the presence of two main clusters in thehaplotype distribution of C. aestuarii strongly sup-ports a genetic partitioning between the western andeastern basins of the Mediterranean Sea.

The AMOVA tests, both based on haplotypic fre-quencies and genetic divergence data, reveal theexistence of population genetic structure along theEuropean Mediterranean coast (FST = 0.03, P = 0.003from haplotypic frequencies; FST = 0.045, P < 0.001from sequence divergence data). The pairwise FST

values, based on nucleotide diversity values, reveal aclear genetic separation between west and east Medi-terranean populations, especially in the populationsof Venice, Pomer, and Amvrakikos (Table 3, below thediagonal). This genetic separation is weaker in thepairwise FST values based on haplotypic frequencies(Table 3, above the diagonal). Based on the presentresults and various phylogeographical hypotheses,two additional AMOVA were performed with groupedpopulations. With the first hypothesis (i.e. the pres-ence of two groups), we tested the genetic separationbetween west (CAM + EBR + LIV + ELB + SAR+ LLG + FUS + SIC) and east (VEN + POM +AMV + PEL; for abbreviations, see Table 1) Mediter-ranean populations. With the second hypothesis (i.e.the presence of three groups), we tested furthergenetic subdivision within the eastern MediterraneanSea, by splitting it into the north Adriatic popula-tions (VEN + POM) and Ionian Sea populations(AMV + PEL). This three-group hypothesis hadhigher values of FCT, both based on haplotypic fre-quencies and sequence divergence data (Table 4). Bycontrast, the FSC value decreased in the three-group

hypothesis, indicating low genetic differentiationamong populations within groups.

A SAMOVA was used to infer the relationshipbetween genetic distance and geographical locationof the twelve studied populations of C. aestuarii.The number of groups (K) was variably set between2 and 5. The highest and most significant FCT valuewas recorded when the number of user-definedgroups was set to K = 3 (Table 5). In that case, thefirst group comprises exclusively the population ofVenice, in agreement with results of the pairwiseFST, whereas, in the second group, there are twopopulations from the western Mediterranean (Elbaand Fusaro) and, in the third group, there were allthe other populations, which appear to be geneti-cally homogenous (Table 5).

Despite the presence of two or more genetic breaksin the overall population of C. aestuarii within theMediterranean Sea, such separation may not beattributed to geographical distances, in so far as theMantel test did not reveal any significant relationshipbetween FST and geographical distances for theoverall population (based on GPS coordinates:Z = 5.94, P = 0.7; based on the coastline distances:Z = 7.3, P = 0.08), as well as within western Mediter-ranean populations (Z = 2.28, P = 0.26) and easternMediterranean populations (Z = -0.22, P = 0.69).

DEMOGRAPHIC HISTORY

The demographic history of C. aestuarii was recon-structed by means of mismatch distribution and threeneutrality tests. The mismatch distribution showed aunimodal distribution of pairwise differences, typicalfor a species or population that recently underwentdemographic or spatial expansion (Fig. 2) (Rogers &Harpending, 1992; Ray, Currat & Excoffier, 2003;Excoffier, 2004). According to the mismatch distribu-tion and three neutrality tests, the overall populationof C. aestuarii of the Mediterranean Sea is out ofequilibrium (Table 6). Moreover, the FS test and theR2 test resulted in significant values in the majorityof the studied populations. Considering that these twotests are more sensitive to reveal recent populationexpansion, the pooled European population of C. aes-tuarii, as well as the majority of the studied singlepopulations, recently underwent a spatial or demo-graphic expansion. In addition, the raggedness index(rg) was never significant, which does not allow therejection of the hypothesis of populations being out ofequilibrium. Under the assumption of a demographicexpansion, the value of t for the pooled EuropeanMediterranean population of C. aestuarii is 1.83, cor-responding to a population expansion that beganbetween 653 000–400 000 years ago [according tothe mutation rates of Knowlton & Weigt (1998) or

Schubart et al. (1998)]. Under the spatial expansionhypothesis, the value of t was similar 1.82, corre-sponding to an expansion time of approximately650 000–400 000 years ago. Using the species-specificmutation rate recently suggested by Marino et al.(2011) for the genus Carcinus assuming a separationof the two species during the Messinian Crisis, weobtain quite different results, with a populationexpansion that began approximately 237 000 yearsago.

COALESCENT-BASED ANALYSIS

The minimum-spanning network analysis recordedtwo nonconnected haplogroups: one including thepooled populations of C. aeastuarii from the EuropeanMediterranean Sea (Fig. 3) and a second one compris-ing four specimens from Peloponnesus, which aredifferentiated from the overall population by up to 14mutation steps (not shown). Haplotype 5, which ispresent in all the populations of C. aestuarii, hadthe highest root probability (P = 0.15) and is thusassumed to be the ancestral haplotype of the studiedpopulations (Castelloe & Templeton, 1994). TheNCPA recorded significant association between hap-lotype clades and geographical distribution in threeclades (Table 7). Within Clade 3-1, restricted geneflow with isolation-by-distance was recorded. In Clade2-3, which comprises haplotypes from the westernMediterranean Sea (haplotype 2 and 26) and fromboth Mediterranean basins (haplotypes 22 and 46), acontiguous range expansion (CRE) was recorded.

GENE FLOW ANALYSIS

Gene flow analyses were run following hierarchicalcriteria based on population genetic structure resultsand our hypotheses. In the first run, all the popula-tions were split into two groups: western populations(CAM, EBR, LIV, ELB, SAR, LLG, FUS and SIC) andeastern populations (VEN, POM, AMV and PEL; forabbreviations, see Table 1). MIGRATE recorded astrong asymmetric gene flow from western Mediter-ranean populations towards eastern Mediterraneanpopulations (Table 8). In the second analysis, threegroups were identified according to our hypothesis:western group (CAM, EBR, LIV, ELB, SAR, LLG,FUS, and SIC), northern Adriatic group (VEN andPOM) and Ionian group (AMV and PEL). Even here,strong asymmetric gene flow was recorded betweenthe western group towards the north Adriatic and theIonian groups, whereas no strong asymmetric geneflow was recorded between north Adriatic and Ionianpopulation groups (Table 8). Finally, we ran two dif-ferent analyses: one within the western group andanother one within the eastern group. In the firstT

analysis, stronger asymmetric gene flow was recordedin most of the comparisons between the two popula-tions from the north-western Mediterranean (CAMand EBR) towards all other populations. Pairwiseasymmetric gene flow was observed between Sardiniaand Lago Lungo, as well as between Sardinia and

Sicily (Table 8). In the analysis of the eastern group,the strongest asymmetric gene flow was recordedbetween Venice, which is also the population withthe largest sample size, towards Peloponnesus,between Pomer and Peloponnesus, and between Amv-rakikos and Peloponnesus. However, all comparisons

Table 4. Analysis of molecular variance testing for partitioning the genetic variation under two biogeographic hypoth-eses, using both haplotypes and nucleotide diversities

Source of variation d.f. SS Variation %Fixationindices P

Haplotype frequencyWestern Mediterranean

versus EasternMediterranean

Among groups 1 1.227 1.95 FCT = 0.019 0.002Among populations

within groups10 5.533 2.17 FSC = 0.022 0.001

Within populations 183 74.292 95.88 FST = 0.041 0.002Western Mediterranean

versus Ionian versusNorth Adriatic

Among groups 2 2.301 11 : 31 FCT = 0.035 0.007Among populations

Within populations 183 74.292 95.22 FST = 0.048 0.003

Nucleotide diversityWestern Mediterranean

versus EasternMediterranean

Among groups 1 4.605 4.46 FCT = 0.044 0.001Among populations

within groups10 12.292 2.40 FSC = 0,025 0.013

Within populations 183 159.183 93.14 FST = 0.068 < 0.001Western Mediterranean

versus IonianMediterranean versusNorth Adriatic

Among groups 2 6.457 0 : 14 FCT = 0.050 0.003Among populations

Within populations 183 159.138 93.07 FST = 0.069 < 0.001

Degrees of freedom (d.f.), sum of squares (SS), percentage of total variation (Variation %), F-statistics (FST, varianceamong populations; FSC, variance among populations within groups; FCT, variance among groups defined a priori), andP-values. Significant P-values are shown in bold. Group: Western Mediterranean Basin (CAM, EBR, LIV ELB, SAR, LLG,FUS, SIC); Eastern Mediterranean Basin (VEN, POM, AMV, PEL); North Adriatic (VEN, POM).

Mismatch Distribution

0 1 2 3 4 5 6 7 8 9

Number of differences

Figure 2. Mismatch distribution for pooled populations (195 individuals not including four deviant sequences fromPeloponnesus) of Carcinus aestuarii of the European Mediterranean Sea.

including Peloponnesus have to be taken with cautionas a result of the smaller number of samples from thispopulation (N = 7).

DISCUSSION

According to the results of the present study, thepopulations of C. aestuarii from the European Medi-terranean Sea can be subdivided into two main

groups, corresponding to the western and easternMediterranean basins, which are separated by theStrait of Sicily. In the Western Basin, all of thesampled populations from the Tyrrhenian and Bal-earic seas, as well as the population of Sicily, weregenetically homogenous, with the only exceptionbeing the populations of Fusaro and Elba, whichappear to be genetically differentiated from the popu-lations of the Balearic Sea, and group togetheraccording to SAMOVA. In the Eastern Basin, the onlysignificant values of population genetic structurewere recorded in the comparison between the popu-lation of Venice and Amvrakikos, suggesting possiblefurther subdivision. This hypothesis was confirmed bya hierarchical AMOVA that recorded the highestvalues of population genetic structure among groups,when populations were grouped according to threegroups: western group, north Adriatic group, andIonian group. These results might be explained inpart by present day physical characteristics of theMediterranean Sea, such as the presence of a unidi-rectional eastward current from Atlantic Oceanflowing along the North African coast and turning offthe Tunisian coast at the entrance of the Strait ofSicily. This steady flow may trigger an isolation effectbetween north-western populations from those of theEastern Basin (Zitari-Chatti et al., 2009). Moreover,even different physical features of the two basins andepeiric seas may maintain the genetic isolation of theeastern populations; for example, the semi-enclosedAdriatic with its own characteristics of salinity, tem-perature, and depth (Maggio et al., 2009). Concerning

Table 5. Results of spatial analysis of molecular variance showing the F-values for the sampling areas

Numberof groups Sampling areas FSC FST FCT

2 VEN 0.031* 0.117* 0.088CAM + EBR + LIV + ELB + SAR + LLG + FUS + SIC + POM + AMV + PEL

3 VEN 0.009* 0.090* 0.081*ELB + FUSCAM + EBR + LIV + SAR + LLG + SIC + POM + AMV + PEL

4 VEN 0.001* 0.077* 0.078*ELB + FUSPOMCAM + EBR + LIV + SAR + LLG + SIC + AMV + PEL

5 VEN 0.014* 0.064* 0.077*ELB + FUSPOMSIC + AMV + PELCAM + EBR + LIV + SAR + LLG

The number of hierarchical groups for the sampling areas in each group is shown. Partitioning of variance among groups(FCT) is highest when there are three hierarchical groups (shown in bold). *P < 0.05. FSC, variance among populationswithin groups; FST, variance among populations.

Table 6. Neutrality tests (Tajima’s D-test, R2 test, andFu’s FS test) for each population and for the whole popu-lation, as well as raggedness index (rg) for the mismatchdistribution

Localities D FS R2 rg

Camargue -2.22 -7.32 0.07 0.10Ebro -1.83 -5.61 0.06 0.03Livorno -1.16 -1.64 0.10 0.04Elba -1.05 -4.91 0.09 0.15Sardinia -1.30 -2.96 0.08 0.10Lago Lungo -1.09 -3.63 0.10 0.08Fusaro -0.57 -0.05 0.14 0.08Sicily -1.80 -3.29 0.11 0.12Venice -1.06 -6.90 0.09 0.05Pomer -1.74 -10.88 0.06 0.06Amvrakikos -1.94 -5.71 0.07 0.06Pelopponesus -1.13 -0.86 0.29 0.24Total -2.44 -27.64 0.02 0.04

Significant P-values are shown in bold.

334143

5730 4548

1-1 1-2 1-3 1-4 1-5

1-131-141-15

1-191-20

2-52-6

PELAMVPOMVENSICFUSLLGSARELBLIVEBRCAM

Western Mediterranean Eastern Mediterranean

334143

5730 4548

1-1 1-2 1-3 1-4 1-5

1-131-141-15

1-191-20

2-52-6

PELAMVPOMVENSICFUSLLGSARELBLIVEBRCAM

Western Mediterranean Eastern Mediterranean

Figure 3. Statistical parsimony cladogram with complete nesting design of 195 individuals of Carcinus aestuarii from theEuropean Mediterranean Sea. The square-shaped haplotype is the proposed ancestral haplotype. The size of haplotypesis proportional to the frequencies (for locality abbreviations, see Table 1).

Table 7. Results of nested clade phylogeographic analysis with a chi-squared test of geographical association of clades

Cladesnested with:

Permutationalc2 statistic P Chain of inference Inference

2-3 17.0591 0.0142 1-2-11-12-NO CRE3-1 56.8352 0.0078 1-2-11-17-4-NO Restricted gene flow with IBD3-2 22.5926 0.0109 1-2-11-17-NO Inconclusive

The results of nonsignificant tests have been omitted. P, probabilities of obtaining a chi-squared value larger than orequal to the observed statistic by randomly permuting the original contingency table 10 000 times. Inferences wereobtained in accordance with the most recent key by Templeton (2004). CRE, continuous range expansion; IBD,isolation by distance.

(106 )

–0.0

.60–

.61–

–0.0

9–67

0–13

01–0

.6–6

.00–

–0.0

.00–

0–9.

0–5.

–0.0

0–9.

0–5.

04–0

.05–

.00–

–0.0

2–13

0–4.

04–0

4–9.

0–5.

02–0

0–16

0–4.

–0.0

.17–

.00–

01–0

1–12

0–4.

.03–

.00–

0–17

0–4.

0–9.

0–5.

0–17

0–3.

0–7.

0–10

0–6.

0–9.

.00–

0–22

0–7.

0–6.

0–7.

0–10

0–9.

.00–

0–18

0–5.

0–11

0–4.

.00–

0–19

0–4.

.00–

0–17

0–7.

0–17

0–8.

(106 )

–0.0

.19–

.02–

–0.0

8–12

2–11

–0.0

0–63

0–5.

–0.0

.06–

.08–

0–66

0–5.

0–58

0–7.

–97.

the southern Ionian Sea, the presence of a quasi-circular anticyclonic front south-west of the Pelopon-nesus Peninsula may further promote geneticisolation from the Adriatic Sea (Borrero-Pérez et al.,2011). Overall, our results are in agreement with thegeographical structuring reported by Marino et al.(2011), although Marino et al. (2011) considerablywiden their dataset by many populations fromthe western Mediterranean Basin. In addition, ourresults are in agreement with other published exam-ples in which genetic differentiation was recordedbetween western and eastern Mediterranean popula-tions across the Strait of Sicily, with further subdivi-sions between the northern and southern AdriaticSea, as well as of the Ionian Sea (Maes & Volckaert,2002; Peijnenburg et al., 2004; Zardoya et al., 2004;Magoulas et al., 2006; Maggio et al., 2009; Zitari-Chatti et al., 2009; Serra et al., 2010; Borrero-Pérezet al., 2011).

One common haplotype (ht5) was present in all thesampled populations of the European MediterraneanSea, having much higher frequencies in populationsfrom the western and Greek populations than in thetwo populations from the northern Adriatic Sea. Oth-erwise, both of the major basins appear to have specifichaplotypes, such as haplotype 2 in western populationsand haplotype 25 in eastern populations, with theexceptions of Elba and Sicily, suggesting the presenceof a possible transition area close to the southern coastof Sicily in the Ionian Sea. Overall, all sampled popu-lations of C. aestuarii in the Mediterranean Seaappear to be connected to a certain degree and can beconsidered as a metapopulation, with haplotype 5having the highest root probability and probably rep-resenting an ancestral haplotype. The four aberranthaplotypes from Peloponnesus deserve further studyand may correspond to a haplogroup that is morecommon in the eastern or southern Mediterranean.

The analysis of gene flow, with populations groupedaccording to the western and eastern basins, recordedstrong asymmetric gene flow with a comparablehigher number of migrants from the Western towardsthe Eastern Mediterranean Basin. Similar resultswere also recorded when the same analysis was runamong the three population groups (western groups,northern Adriatic group, and Ionian group), recordinga weak asymmetric gene flow from northern Adriaticpopulations towards the Greek populations. In both ofthese analyses, the Western Basin had comparablelarger values of effective population size. Such resultsindicate that the Western Basin played a fundamen-tal role as source of genetic variability for the EasternBasin, at least during the recent past. A possibleexplanation of the reported asymmetric gene flow inthe European Mediterranean populations of C. aestu-arii comes from the main circulation systems of the

Mediterranean Sea. The steady flow along the NorthAfrican coast, which splits in front of the Siciliancoast in two streams (one directed towards the Tyr-rhenian coast and another one towards Tunisia), maypromote the genetic isolation of the two basins. At thesame time, the presence of an interannual streamalong the southern coast of Sicily toward the centralIonian Sea, may favour a reduced connectivity fromthe Western towards the Eastern Basin (Millot &Taupier-Letage, 2005; Hamad et al., 2006).

All these results taken together support a complexscenario, in which there is a spread from western toeastern populations, with the latter possibly beingadapted to different local environmental characteris-tics (such as winter isotherms and different salinityregimes). This is also supported by the presence ofnumerous gyres and anticyclones in both basins thatare able to produce mechanisms of larval retention innearshore areas.

The population of C. aestuarii from the Lagoon ofVenice is a good example of local adaptation. Usingmicrosatellite analyes, Marino et al. (2010) found thatthe genetic variability within this lagoon, which issubdivided into three sub-basins with stable ecologi-cal differences in time and space, is higher thanamong neighbouring lagoons. Moreover, a geneticpatchiness was recorded within Venice Lagoon atthree different sites, which was probably caused byselection on the larval population, larval immigrationfrom different populations, and stochastic processesrelated to both reproduction and recruitment events.As far as the larval pool in front of the lagoon appearsto be homogeneous, Marino et al. (2010) argued thatselective forces acting during the recruitment shouldhave produced the genetic differentiation among localsamples. In our dataset, the population of the VeniceLagoon turned out to be strongly differentiated fromall others, according to SAMOVA. Indeed, within theEastern Basin, significant differences were onlyrecorded for comparisons with the population of Amv-rakikos, whereas weak asymmetric gene flow wasrecorded from northern Adriatic Pomer towardsVenice. The latter result is in agreement with the ideathat the larval population in front of the lagoon ishomogenous, with few individuals arriving from theeastern coast of the Adriatic Sea, probably driven byboth surface currents and winds (Cavaleri & Sclavo,2006).

Concerning the demographic history of C. aestuariiin the Mediterranean Sea, the overall population isout of equilibrium, as visible from the unimodal shapeof the mismatch distribution and from the neutralitytests (with the overall population, as well as withmost of the single populations), and may be underspatial expansion. In addition, we confirmed that allof the populations, especially the Adriatic populations

of Venice and Pomer, were under expansion, with theonly exceptions being the populations of Livorno andFusaro. An additional confirmation of recent popula-tion expansion comes from the haplotypic and nucleo-tidic diversity for all of the single populations and forthe overall population (Grant & Bowen, 1998).

It is more difficult to understand when such apopulation expansion may have begun. According tothe mutation rate used in many previous studies(Geller et al., 1997; Roman & Palumbi, 2004; Darlinget al., 2008), the spatial expansion of C. aestuarii mayhave begun within a time range from approximately650 000 to 400 000 years ago. A lower value ofapproximately 237 000 years ago is calculated withthe mutation rate of Marino et al. (2011). According tothese estimations, the Mediterranean population ofC. aestuarii appears to have undergone populationexpansion only during the mid Pleistocene. At thebeginning of the Pleistocene (approximately 2 Mya),glacial cycles started lowering sea levels and tem-perature, affecting the Mediterranean fauna. Duringthis period, the populations of C. aestuarii of theMediterranean Sea may have experienced numerousfluctuations in population size, with isolated popula-tions confined to local refugia. Moreover, duringglacial cycles, a large part of the Adriatic Sea wasrepeatedly desiccated, suggesting that C. aestuariipopulations of the Eastern Basin colonized the Adri-atic Sea relative recently, possibly originating fromrefugia present in the Eastern Mediterranean Basin,such as the Sea of Marmara (Peijnenburg et al.,2004). This aspect is clearly reflected in our neutralitytests, which support a population expansion ratherthan a bottleneck effect of the two populations of thenorthern Adriatic Sea.

The NCPA only recorded a significant associationbetween haplotypes and geographical position inClade 3-1, which comprises most of the haplotypesfrom the western Mediterranean populations and afew haplotypes from eastern populations, and inClade 2-3. In the former clade, restricted gene flowwith isolation-by-distance was recorded, emphasizingthe reduced effective larval exchange between thetwo main basins, or different survival rates in thetwo different basins. In the latter clade, a contiguousrange expansion was recorded, probably as a resultof the presence of three out of four haplotypes thatwere only present in the populations of Elba andPomer. The fourth haplotype present in this clade ishaplotype 2, which is exclusively present in popula-tions from the Western Basin. The fact that thepopulations of Elba and Pomer share common hap-lotypes may be a consequence of anthropogenic influ-ence (similar to ships), which are among the mainvectors for marine species with larval phase(Occhipinti-Ambrogi, 2007).

ACKNOWLEDGEMENTS

We are indebted to Sara Fratini, Carsten Müller,Yvan Perez, Pere Abelló, Florian Gmeiner, SilkeReuschel, Ruth Jesse, Sebastian Klaus, and HenrikM. Schubart, as well as several students of the Uni-versity of Regensburg for their help in collecting andsequencing specimens of C. aestuarii. We thank threeanonymous reviewers for their helpful comments.Travel for this study was partly financed throughDAAD exchange programmes to Italy (VIGONI D/04/47157 thanks to Marco Vannini) and Spain (AccionesIntegradas Hispano-Alemanas D/03/40344 thanks toJosé A. Cuesta). The authors declare that there are noconflicts of interest.

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