the atlantic–mediterranean transition: discordant genetic patterns in two seabream species,...
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Molecular Phylogenetics and Evolution 36 (2005) 523–535
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The Atlantic–Mediterranean transition: Discordant genetic patterns in two seabream species, Diplodus puntazzo (Cetti) and Diplodus
sargus (L.)
L. Bargelloni a, J.A. Alarcon b, M.C. Alvarez b, E. Penzo a, A. Magoulas c, J. Palma d, T. Patarnello a,e,¤
a Dipartimento di Biologia, Università di Padova, Via G. Colombo, 3, I-35121 Padova, Italyb Departamento de Genética, Facultad de Ciencias, Universidad de Málaga, 29071, Málaga, Spain
c Institute of Marine Biology of Crete, P.O. Box 2214, 71003 Iraklio, Greeced CCmar, Universidade do Algarve, UCTRA, Campus de Gambelas, 8000-810 Faro, Portugal
e Facoltà di Medicina Veterinaria-Agripolis, Università di Padova, Via Romea 16 I-35020 Legnaro, Italy
Received 29 June 2004; revised 5 April 2005Available online 4 June 2005
Abstract
Sparids are a group of demersal perciform Wsh of high commercial value, which have experienced an extensive radiation, particu-larly in the Mediterranean, where they occupy a variety of diVerent niches. The present study focuses on two species: Diplodus sargusand D. puntazzo, presenting a wide distribution from the Mediterranean to the eastern Atlantic coasts. They display similar ecologicalbehaviour and are evolutionary closely related. Both are highly appreciated in Wsheries and D. puntazzo is currently under domestica-tion process. However, little is know on their population structure and it is an open question whether any genetic diVerentiation existsat the geographic level. To address this issue we examined sequence variation of a portion of the mitochondrial DNA (mtDNA) con-trol region in population samples of each of the two species collected over a wide geographic range. In addition to the mtDNA, analy-sis of nuclear loci (allozymes) was included in the study to compare patterns revealed by nuclear and mitochondrial markers. Thestudied samples covered an area from the eastern Mediterranean to the Portuguese coasts immediately outside the Gibraltar Strait.The two species revealed a level of sequence polymorphism remarkably diVerent for the control region with the D. puntazzo and D.sargus showing 111 and 28 haplotypes, respectively. Such a diVerence was not detected with allozyme markers. The two species alsoshowed large diVerences in their population structure. While D. puntazzo presented a marked genetic divergence between the Atlanticand Mediterranean samples, D. sargus showed little intraspeciWc diVerentiation. These results were supported using both mtDNA andallozyme markers, and were interpreted as the consequence of diVerences in the history of the two species such as Xuctuations in theeVective population size due to bottlenecks and expansions, possibly combined with present-day diVerences in levels of gene Xow. 2005 Elsevier Inc. All rights reserved.
Keywords: Sparid Wsh; Phylogeography; Atlantic–Mediterranean transition; mtDNA; Allozymes
* Corresponding author. Fax: +39 0498276209.E-mail addresses: [email protected] (M.C. Alvarez), magoulas@
ns0.imbc.gr (A. Magoulas), [email protected] (J. Palma),[email protected] (T. Patarnello).
1055-7903/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2005.04.017
1. Introduction
The perciform family Sparidae comprises more than100 species worldwide, with a peak of diversity in theNortheast Atlantic and the Mediterranean, where 24species, belonging to 11 genera, have been described(Bauchot and Hureau, 1986). Sparids, or seabreams, aredemersal Wshes living in coastal waters and occupying a
524 L. Bargelloni et al. / Molecular Phylogenetics and Evolution 36 (2005) 523–535
variety of trophic niches; they are generally gregarious,though adults might be solitary in some species. In theMediterranean area, sparids are of great interest forWsheries and aquaculture. Most sparid species are, infact, excellent foodWshes, with high commercial value.In the recent years, seabreams have also gained consid-erable importance for aquaculture. For instance, thegilthead seabream, Sparus aurata, has become one ofthe most important cultured species in the Mediterra-nean region. Several other sparids are cultivated in Wshfarms, or potential candidates for aquaculture. Despitethe growing interest in this group of Wshes, however,many aspects of their biology remain unknown. This isparticularly worrying if we consider that a correctmanagement of biological resources should begrounded on the most complete information about thenatural genetic diversity of the species involved. WhileinterspeciWc relationships within the family Sparidaehave been investigated also at the molecular level(Hanel and Sturmbauer, 2000; Orrell and Carpenter,2004), little is known about the partition of DNA poly-morphism among geographic populations within thesame species.
The present work is aimed at investigating the popu-lation structure of two sparid species of the genus Diplo-dus with relevance both in Wshery and aquaculture,
namely D. puntazzo (the sharpsnout seabream) and D.sargus (the white seabream). Population samples werecollected at four geographic locations, three into theMediterranean Sea (Greece, Italy, Spain) and one intoAtlantic waters immediately outside the Strait of Gibral-tar strait (Faro, Portugal) roughly covering three geo-graphic areas : the Northeast Atlantic just outside thestrait of Gibraltar, the Western and the Eastern Mediter-ranean Sea (Fig. 1). These samples span only partiallythe natural distribution range of the species. D. puntazzois present in the Mediterranean Sea, the Black Sea, andEastern Atlantic (oV the African costs). D. sargus wasdescribed as a species complex which includes D. s. sar-gus in the Mediterranean and Black Sea, D. s. cadenati inthe Eastern Atlantic (from Bay of Biscay to Senegalincluding Canary and Azores Islands), D. s. capensis(from Angola to Mozambique and Southern Madagas-car) and D. s. lineatus endemic to the Cape Verde (De laPaz et al., 1973). However, to avoid confusion, we willuse in the present work the species name D. sargus ratherthan the sub-species one since, on the base of the geo-graphic origin of the investigated populations, we shouldname D. s. sargus the Mediterranean samples whereasthe Atlantic one (Faro) should be considered as part ofthe sub-spececies D. sargus cadenati (Summer et al.,2001).
Fig. 1. Sampling locations for D. sargus and D. puntazzo. Samples were collected directly or through local Wshermen in Faro (Portugal, FAR),Alicante (Spain, ALI), Otranto (Italy, OTR), Mesolongi (Greece, MSL, D. puntazzo), Iraklion (Greece, IRK, D. sargus). Sample sizes are indicatedwithin squares for D. sargus, within circles for D. puntazzo. The circulation in the Alboran Sea and location of the Orian-Almeria Front (OAF) arealso shown.
Almeria
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Despite the limited sampling scheme, that covers onlythe northernmost distribution of the species, it was par-ticularly important to have obtained samples from bothsides of the Gibraltar Straits. In fact, this divide has beenhypothesised to represent the boundary between the twobiogeographic provinces, the north-east Atlantic and theMediterranean (Quignard, 1978), while studies on intra-speciWc genetic variation in a variety of organisms haveshowed, for several of the examined species, a reductionof gene Xow in relation to the transition between theMediterranean and the Atlantic (reviewed in Borsa et al.,1997; but see also Bargelloni et al., 2003; Naciri et al.,1999; Pannacciulli et al., 1997; Perez-Losada et al., 1999;Zane et al., 2000).
We report here data on allozyme and mtDNA vari-ation in D. puntazzo and D. sargus: both species arevery common throughout the Mediterranean Sea, andare quite similar in terms of habitat preference and lifehistory. We show that, despite the aforementionedsimilarities, when genetic variation is examined at theintraspeciWc level, both nuclear and mitochondrialmarkers suggest a marked diVerence between the twoseabream species. D. puntazzo show a strong geneticdivergence between Atlantic and Mediterranean sam-ples, while for D. sargus there is no evidence for diVer-entiation when individuals from these two geographicregions are compared. Such discordant results are dis-cussed in light of the historical and present-day fea-tures that characterise the Atlantic–Mediterraneanbiogeographic region(s), we also considered the WnediVerences in ecology between the two species, thatmight have an impact on microevolutionaryprocesses.
2. Materials and methods
2.1. Collection of samples
Adult specimens of both species were collected fromthe wild at Wve diVerent locations (see Fig. 1). Individualswere shipped in dry ice to the lab, where they were keptat ¡40 °C until analysis. Eye, liver, and muscle tissuewere removed for protein and DNA analyses.
Due to practical and technical reasons, data ongenetic variation from the two species diVer in terms ofsample size, number of scored allozyme loci, and lengthof sequenced mtDNA region. To allow for a bettercomparison between the two species, for D. puntazzo, areduced data set was obtained as follows: sample sizewas made equal to that of D. sargus by randomlyselecting the appropriate number of individuals, allo-zyme loci were reduced to the same ones scored forD. sargus, and sequence length was shortened to matchlength and position of the fragment analysed by SSCPin D. sargus.
2.2. Allozymes scoring
Frozen samples were subjected to horizontal starch gelelectrophoresis. Electrophoresis protocols, staining proce-dures, genetic interpretation of zymogram patterns, andlocus designation, were done according to Reina et al.(1994). Scored loci for D. sargus were: adenilate kinase(AK*), alcohol dehydrogenase (ADH*), endopeptidase(ENDOP), esterase (EST), glucosephosphate isomerase(two loci: GPI-1*, GPI-2*), glycerol-3-phosphate dehy-drogenase (G3PDH*), lactate dehydrogenase (three loci:LDH-1*, LDH-2*, LDH-3*), malate dehydrogenase(three loci: MDH-1*, MDH-2*, MDH-3*), malic enzyme(two loci: MEP-1*, MEP-2*), phosphoglucomutase(PGM*), 6-phosphogluconate dehydrogenase (PGDH*),superoxide dismutase (two loci: SOD-1*, SOD-2*). ForD. puntazzo, in addition to the above mentioned loci, twoother allozyme systems were scored: adenosine deaminase(ADA*) and iditol dehydrogenase (IDDH*). Raw allo-zyme data are reported in Appendix A.
2.3. MtDNA scoring
Total genomic DNA was extracted for each individ-ual from few milligrams of muscle tissue using a proto-col based on Chelex resin (Walsh et al., 1991) with somemodiWcations (Ostellari et al., 1996).
Two microliters of Chelex extracted DNA, were usedin 20 �l of PCR mix containing 0.5 U of Taq polymerase(Promega), 1£ reaction buVer, 2.5 mM MgCl2, 50 �M ofeach dNTPs and 250 nM of each primer. PCR primerswere designed based on previously obtained sequenceinformation (i.e., 400–500 bp of 5� end of the mitochon-drial D-loop region, for eight sparid species), asdescribed in Ostellari et al. (1996). Primers were species-speciWc (accessible through Accession No. AF373417–373527 and AF373528–373555) and allowed amplifyinga fragment of approximately 220 bp. Individual PCRproducts were then subjected to single strand conforma-tion polymorphism (SSCP) analysis as previouslydescribed (Ostellari et al., 1996). After the Wrst electro-phoretic run, PCR products showing similar mobilitypatterns were run side by side and compared to alreadyidentiWed mobility classes. On average, each sample wasrun two-three times under identical conditions. A fewindividuals from each mobility group (1–10, dependingon SSCP group frequency) were randomly chosen, andtheir nucleotide sequence was determined as follows: alarge fragment (400 bp) of the control region was PCRampliWed for each selected individual, using primers spe-ciWcally designed for sparid species (Ostellari et al.,1996). An aliquot (5 �l) of each PCR was then run on a1.8% agarose gel to verify the quality and quantity of thePCR product obtained. A variable amount (5–20 ng) ofthe ampliWed DNA was directly sequenced, without fur-ther puriWcation, using a cycle-sequencing commercial
526 L. Bargelloni et al. / Molecular Phylogenetics and Evolution 36 (2005) 523–535
kit (Amersham Premixed Cycle-sequencing kit). A Xuo-rescent (Texas Red 5� labelling) internal oligo was usedas sequencing primer. Sequencing products were ana-lysed on an automatic DNA sequencer (Amersham Vis-tra 725). The use of an internal primer and the fact thatthe sequenced region was larger (about 300 bp) than theSSCP fragment, allowed to determine without ambigui-ties the complete sequence of the region analysed bymeans of SSCP. Computer-produced DNA data werealigned using ClustalX (Thompson et al., 1997) withdefault settings, and individual sequences were re-exam-ined manually by visual inspection of raw Xuorogram-data, with special attention being paid to those sitesinterested by nucleotide substitutions. MtDNA data forD. puntazzo and D. sargus are presented in Appendix Band C, respectively.
2.4. Data analysis
Diversity indexes for allozymes and mtDNA werecalculated using the program Arlequin (ver. 2.000,Schneider et al., 2000), and Genepop (ver. 3.1d) (Ray-mond and Rousset, 1995a). The latter software packagewas used to perform an exact test of Hardy–Weinbergproportions for multiple alleles (Guo and Thompson,1992). A similar approach was used to assess indepen-dence of allozyme loci. DiVerentiation between popula-tion samples was tested by means of an exact test ofpopulation diVerentiation based on allelic frequencies(Raymond and Rousset, 1995b).
Similarly, Fst values (single locus and multilocus) wereestimated both for population pairs and for all popula-tions, using a “weighted” analysis of variance (Weir andCockerham, 1984). In all tests, signiWcantly deviationsfrom the null hypothesis of genetic homogeneity wereassessed by means of a non-parametric approach with100,000 permutations (reshuZing individuals among pop-ulations). For mtDNA, Fst values were estimated eithertaking into account haplotype frequencies only or consid-
ering also genetic distances between haplotypes. Molecu-lar distances were estimated as uncorrected pairwisenucleotide diVerences. Rate heterogeneity among sites wasalso evaluated, as described in Bargelloni et al. (2000).
Phylogenetic relationships among haplotypes werereconstructed using diVerent approaches. A reducedmedian network (MN, Bandelt et al., 1995) of mtDNAhaplotypes was constructed using the program Network(ver. 2.0b http://www.Xuxus-engineering.com). Insertion–deletion events were recoded as transversions.
MtDNA sequence data were also tested for depar-tures from mutation-drift equilibrium, using Tajima’s(1989) D statistics as implemented in Arlequin 2000.
A mismatch analysis (Rogers and Harpending, 1992)was also applied. Nucleotide diVerences are counted ineach possible pairwise comparison between individualsequences. Frequencies for each mismatch class (0, 1, 2,3,ƒ diVerences) are then plotted on a graph. Demo-graphic expansions are expected to generate “waves” inthe mismatch distribution, while a stable population sizeshould show a less bell-shaped distribution (Rogers andHarpending, 1992). Mismatch distribution for each pop-ulation was calculated using Taijma and Nei model toestimate diVerences between haplotypes, parameters ofthe expansion �0, �1, � were estimated using a non linearleast square method (Schneider and ExcoYer, 1999) asimplemented in the program Arlequin 2000.
3. Results
3.1. Genetic diversity
Estimates of genetic variability for both species arereported in Table 1. As for D. puntazzo, results ongenetic diversity and population diVerentiation weresimilar when analysing the complete data set and thereduced one (see Section 2), therefore only the former setof data will be presented hereafter.
Table 1Genetic diversity at allozyme and mtDNA loci
He and Ho are respectively expected and observed heterozygosity (associated standard errors are shown in brackets). Average number of allele perlocus over all allozyme loci is also reported. For mtDNA haplotype diversity (h) and nucleotide diversity (�) are presented. Probability values associ-ated to Tajima’s D statistics are *P < 0.05 and **P < 0.01.
Sample Allozymes mtDNA
He Ho n alleles/locus h � Tajima’s D
D. puntazzoFAR 0.0287 (0.0682) 0.0286 (0.0659) 1.2857 0.978 0.05 0.79ALI 0.0501 (0.1275) 0.0451 (0.1260) 1.4762 0.994 0.040 ¡0.14OTR 0.0482 (0.1228) 0.0564 (0.1498) 1.2857 0.995 0.037 ¡0.32MES 0.0449 (0.1288) 0.0436 (0.1288) 1.2381 0.986 0.035 ¡0.27
D. sargusFAR 0.0775 (0.1362) 0.0662 (0.1114) 1.3684 0.810 0.011 ¡1.54*
ALI 0.0874 (0.1616) 0.0875 (0.1626) 1.4211 0.619 0.006 ¡1.29OTR 0.0888 (0.1492) 0.0694 (0.1156) 1.4737 0.574 0.007 ¡2.08**
IRK 0.0788 (0.1282) 0.0638 (0.1047) 1.4211 0.797 0.009 ¡1.31
L. Bargelloni et al. / Molecular Phylogenetics and Evolution 36 (2005) 523–535 527
The same allozyme loci (GPI-1*, GPI-2*, MDH-2*,MDH-3*, MEP-1*, MEP-2*, PGM*, PGDH*) arefound to be variable in both species, whereas, when a95% frequency criterion is assumed, less polymorphicloci are found in D. puntazzo (two loci: GPI-1* andMDH-3*) as compared to D. sargus, which shows Wvepolymorphic loci (GPI-2*, MDH-3*, MEP-2*, PGM*,PGDH*). On the other hand, mean number of alleles perlocus is similar in both species (1.6). Mean expected het-erozygosity (He) calculated for each population rangesfrom 0.078 to 0.089 for D. sargus; in D. puntazzo He isslightly lower (0.029–0.050). When observed genotypefrequencies are compared to expectations under HWequilibrium, only one signiWcant exception is observedfor a single locus in a single population [D. puntazzo(ALI), MDH-2*], and samples are considered to be atHW equilibrium for the analysed loci. In any case, subse-quent analyses on genetic diVerentiation show no diVer-ences when performed either including or excludinglocus MDH-2. Likewise, no signiWcant exception, aftersequential Bonferroni correction, is observed when inde-pendence of loci is tested.
While genetic diversity at allozyme loci is comparable,analysis of mtDNA variation reveals a clear diVerencebetween the two species. The SSCP approach applied to
D. sargus yielded 28 SSCP mobility classes. Sequenceanalysis on a number of individuals larger than that ofmobility groups (a total of 50 randomly selected individ-uals were sequenced, nine of which were taken out of 44from the most frequent class) conWrmed the sensitivity ofthe method, in agreement with a previous work wherethe method was more thoroughly validated on anothersparid species (Ostellari et al., 1996).
When the SSCP method was used to evaluate varia-tion at the mtDNA control region in D. puntazzo, almosteach individual appeared to belong to a diVerent mobilityclass. For this reason it was decided to directly sequencethe ampliWed fragment for all D. puntazzo individuals.This procedure revealed 111 diVerent haplotypes.
The two species share similar features in terms ofsequence evolution (compositional and transition–trans-version bias, rate heterogeneity across sites; data notshown). However, D. puntazzo shows a much highermtDNA polymorphism than D. sargus (Table 1). In thelatter species both haplotype (h) and nucleotide diversity(�) are similar to those observed in other marine teleosts,whereas in D. puntazzo h is always close to one (0.98–0.99), and � (0.035–0.05) is in the high range of valuesreported for teleost species (Grant and Bowen, 1998;Viñas et al., 2004).
Fig. 2. Median network based on mtDNA sequences of D. sargus. Circles represent haplotypes, with size proportional to relative frequencies. Foreach haplotype present in more than one population sample, sectors of diVerent colours (black, Portugal; dark grey Spain; light grey Italy; and white,Greece) refer to absolute number of haplotype counts in each population. Haplotype deWnitions are as in Appendix C.
528 L. Bargelloni et al. / Molecular Phylogenetics and Evolution 36 (2005) 523–535
3.2. Historical and demographic factors
Diplodus sargus values of D statistic in the Tajima’stest are substantially negative for all populations, withsigniWcant deviations from neutrality for OTR and FARsamples (Table 1). These values may also be underesti-mated if we consider that strong mutation rate heteroge-neity as the one observed in this study (data not shown)tends to shift D toward more positive values (Aris-Bro-sou and ExcoYer, 1996). Negative D values are oftenassociated with past changes in population size, namelybottlenecks followed by population expansion. D. sarguspopulations therefore might have experienced largedemographic Xuctuations in the past. On the opposite,no signiWcant D value is obtained for D. puntazzo.
To investigate the presence of past demographicevents we carried out the analysis of mismatch distribu-
tion in the two species. Sudden demographic expansionsare expected to produce unimodal (“waves”) distribu-tions of pairwise nucleotide diVerences (mismatch)between alleles (Rogers and Harpending, 1992), whilesuch distribution shape is unlikely under population sta-tionarity. In the case of the two species under study, inconsideration of the deviations observed in the neutral-ity tests, the method of Schneider and ExcoYer (1999)was applied to test whether the data Wtted a suddenexpansion model. For both species, the sum of squaredeviations (SSD, Schneider and ExcoYer, 1999) for10,000 simulated data sets on the basis of estimatedparameters is larger than SSD for observed data (Figs. 4,5), thereby the model is not rejected. As observed by theauthors (Schneider and ExcoYer, 1999), however, thismethod is quite conservative, rarely rejecting the expan-sion model. Associated P values, in any case, are much
Fig. 3. Median network based on mtDNA sequences of D. puntazzo. Branches represent single substitutional events, if not otherwise indicated (blackbars refer to multiple events). Circles represent haplotypes, with size proportional to relative frequencies. For each haplotype present in more thanone population sample, sectors of diVerent colours (black, Portugal; dark grey, Spain; light grey, Italy; and white, Greece) refer to absolute number ofhaplotype counts in each population. Haplotype deWnitions are as in Appendix B. Only haplotypes present more than once are indicated. Haplo-groups were squared in dashed lines and numbered with roman numbers (see text).
L. Bargelloni et al. / Molecular Phylogenetics and Evolution 36 (2005) 523–535 529
lower for D. puntazzo (P D 0.1–0.2, Fig. 4) compared toD. sargus (P D 0.6–0.8, Fig. 5), suggesting that modelWtting is rather poor for the former species. Similar evi-dence is obtained from estimation of raggedness index(Harpending, 1994) for each sample (data not shown), aswell as from visual inspection of mismatch distributionsfor D. puntazzo, which always show a multimodal proWle(Fig. 4).
Estimates of expansion parameter � for D. puntazzosamples are larger �D 4.2–19.4, Fig. 4 than those calcu-lated for D. sargus �D 1.4–3.2, Fig. 5. The parameter � isequal to 2t*� where t is the time of the expansion and �is the mutation rate. If we assume the same mutationrate for the two species, then the timing of the possibleexpansion is much more recent for D. sargus than forD. puntazzo. Regarding to the magnitude of expansion,simulation studies (Schneider and ExcoYer, 1999) haveshowed that while the time of expansion is adequatelyrecovered with valid conWdence intervals, estimate of ini-tial population size (�0) is relatively accurate, but with anoverly conservative conWdence interval due to a toolarge upper limit of the interval, and Wnal population size(�1) is generally biased upward, with an overly largeupper bound. Although less reliable than estimates of �,values of �0 indicate that even assuming sudden popula-tion expansion, the starting population size was large forD. puntazzo (at least for some populations), whereasD. sargus appears to have undergone a drastic bottle-
neck before the expansion, as estimates of the �0 resultextremely low (�0 D 0–0.5) in all samples.
3.3. MtDNA networks
Evolutionary relationships among haplotypesequences are represented in the form of reduced mediannetworks (MNs), which take into account haplotype fre-quencies, and allow for parallel substitutions. For D. sar-gus, the network shows a clear star-like shape (Fig. 2). Asingle haplotype (A) at high frequency in all populationsamples is present, from which the majority of remainingsequences stems out, being removed by one or few muta-tional steps. Evidence of moderate homoplasy is alsoobserved in the form of few network reticulations.
In D. puntazzo, network reconstruction reveals amore complex pattern, where four groups of haplotypes(haplogroups) are observed, separated by 8–14 substitu-tion steps (Fig. 3), therefore suggesting a “deep” evolu-tionary history. The number of haplotypes andindividuals, however, is not equal in each haplogroup,with a vast majority of sequences clustering into groupII. With regard to the geographical origin of individualhaplotypes, three haplogroups (I, II, III in Fig. 3) con-tain sequences from all four geographic samples, whilethe fourth one (IV in Fig. 3) is composed by haplotypesfound only in the Portuguese sample, except for a singleindividual collected in Alicante (Spain). The entire
Fig. 4. Mismatch distribution for D. sargus mtDNA data. On the x-axis are classes of pairwise diVerences (0, 1, 2,ƒ), on the y-axis the absolute num-ber of comparisons yielding a certain number of diVerences. Estimated values for the expansion model are reported for each population with theexception of the Iraklion population for which the program was unable to provide a mismatch distribution analysis.
530 L. Bargelloni et al. / Molecular Phylogenetics and Evolution 36 (2005) 523–535
network is also characterised by several reticulatedinstead of linear relationships connecting haplotypes.This evidence seems to suggest a considerable degree ofhomoplasy.
3.4. Population structure
Results from both allozyme and mtDNA data indi-cate that partitioning of genetic diversity among geo-graphic samples is profoundly diVerent betweenD. puntazzo and D. sargus. The latter species shows noevidence of genetic structure, whereas a remarkablegenetic divergence is present between Atlantic and Med-iterranean samples of D. puntazzo.
An exact test of population diVerentiation across allo-zyme loci in D. puntazzo reveals that gene frequenciesare signiWcantly (P < 0.0001) diVerent among populationsamples. At the level of single locus, two loci (GPI-1*and MDH-3*) show signiWcant heterogeneity in bothallele and genotype distribution (P < 0.0001 after correc-tion for multiple tests). These two loci are also the onlypolymorphic ones if a 95% criterion is assumed. Pairwisetests of population diVerentiation (data not shown)reveal that in all comparisons involving the Atlanticsample (FAR) the distribution of allele frequencies sig-niWcantly (P < 0.0001) deviates from that expected underthe hypothesis of genetic homogeneity. A similar albeitless signiWcant diVerentiation is observed between sam-ples from East and West Mediterranean, as OTR andMSL samples are signiWcantly diVerent from ALI sam-ple (P < 0.01). Estimates of Fst values suggest a similarpattern of genetic divergence among populations in D.puntazzo. Global Fst, calculated across loci and popula-
tions shows a highly signiWcant value (Table 2), and inpairwise comparisons the highest Fst values are foundwhen the Atlantic sample is compared to Mediterraneanones, especially to the easternmost ones (OTR andMSL). As for the exact test, signiWcant Fst values are alsoobserved comparing the ALI sample with OTR andMSL.
For D. sargus, analysis of allozyme variation revealsno evidence for diVerentiation among or between popu-lation samples (data not shown). Global Fst across lociand populations is one order of magnitude lower thanthe one observed in D. puntazzo (Table 2). Similarly, allpairwise Fst values are much lower and never signiWcant.
MtDNA data delineate a comparable pattern. In thecase of D. puntazzo a strong diVerentiation is observedamong population samples (Fst D 0.08 P < 0.0001). Esti-mates of Fst take into account also molecular distancesbetween haplotypes, because in case of large values of �(eVective size £ mutation rate), it is known that statisti-cal tests of population diVerentiation based on haplo-type frequencies are less powerful than sequence-basedstatistics (Hudson et al., 1992). The highly signiWcantglobal Fst value appears to be inXuenced by the stronggenetic divergence of the Atlantic sample compared tothe Mediterranean ones. When populations are exam-ined in pairwise comparisons, the FAR sample results tobe signiWcantly diVerent from all three Mediterraneanones (Table 2), with increasing Fst values along a West-East gradient suggesting a possible mechanism ofisolation by distance as revealed by the statistically sig-niWcant correlation (P < 0.005) between pairwise Fst andgeographic distances (Mantel test with 1000 permuta-tions). SigniWcant genetic diVerentiation between
Fig. 5. Mismatch distribution for D. puntazzo mtDNA data. On the x-axis are classes of pairwise diVerences (0, 1, 2,ƒ), on the y-axis the absolutenumber of comparisons yielding a certain number of diVerences. Estimated values for the expansion model are reported for each population.
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Mediterranean samples is observed only when compar-ing the two most distant locations: ALI and MSL.
Likewise, the distribution of haplotypes is signiW-cantly heterogeneous (P < 0.00001) when sampling loca-tions and the four haplogroups (I, II, III, IV in Fig. 3) areused as categorical variables in an exact test. This resultindicates that individuals collected at diVerent locationsare not randomly assorted among the four major clades.
In D. sargus the obtained results are rather diVerent.The null hypothesis of panmixia is never rejected whenestimating Fst values for population pairs (Table 2) oracross all samples (Fst D 0.007, P D 0.2), while the exacttest of overall population diVerentiation reveals just amarginally signiWcant heterogeneity (P < 0.05) amongsamples that could be partly ascribed to divergent haplo-type distribution in the IRK sample (data not shown).
4. Discussion
As mentioned above, evidence of genetic discontinuitybetween Atlantic and Mediterranean populations hasbeen reported for several marine species. The transitionbetween the two basins, however, seems to be perceivedby diVerent organisms in a diVerent way. Strong to mod-erate genetic divergence is observed in some specieswhereas others show no diVerentiation at all. The degreeof genetic diVerentiation cannot be easily related to thespecies biology, as diVerent patterns are found acrossorganisms with similar ecological features, and vice versa.For instance, in the case of the blueWn tuna and theswordWsh, which are both large pelagic Wsh, only in thelatter species Atlantic samples are genetically distinctfrom Mediterranean ones (Chow and Takeyama, 2000;Ely et al., 2002). Divergent patterns are reported alsoamong bivalves or benthic Wsh species (Borsa et al., 1997).
Likewise, in the present study D. puntazzo and D. sar-gus display a remarkable discordance. DiVerences are
observed in the relative levels of genetic diversity, at leastfor mtDNA, and particularly in the degree of populationdiVerentiation. These results provide us with relevantinformation on population structure separately for thetwo species, and at the same time, allow us a compara-tive approach to understand the more general impor-tance of the Atlantic–Mediterranean transition onbiogeography of marine organisms.
Starting from the perspective of a single species, forD. puntazzo geographic distance between sampling loca-tions appears to be correlated with degree of geneticdiVerentiation within the Mediterranean as well as whencomparing Mediterranean samples with the Atlanticone. Despite sampling sites are rather sparse, this mightbe considered as preliminary evidence that reduction ofgene Xow is associated with increasing geographic dis-tances as suggested by the signiWcant correlationbetween pairwise Fst and geographic distance betweensampling sites. Potential for dispersal is supposed to berelatively limited in D. puntazzo. After a pelagic larvalphase, which is relatively short (1 month), juveniles settleinto very shallow benthic habitats, which are abandonedseveral months later to join the adult population indeeper waters (Macpherson, 1998). Adults are consid-ered to be sedentary (Bauchot and Hureau, 1986). There-fore, a model of isolation by distance appears inagreement with the species biology. However, the abruptchange in genetic composition between Atlantic andMediterranean samples, cannot be explained solely asthe eVect of geographic distance. A marked reduction ofgenetic exchanges appears to be associated with the tran-sition between the Atlantic and the Mediterranean.Moreover, mtDNA analysis shows the presence of fourhaplotype groups, phylogenetically distinct and stronglyassociated with sampling locations (especially clade IV,which contains Atlantic haplotypes but one that is fromSpain). Although such large phylogenetic distances aregenerally associated with phylogeographic discontinuities,
Table 2Fixation indexes (Fst) across samples and between samples overall allozyme loci and mtDNA
For pairwise Fst estimated values are in brackets. Abbreviations of sampling locations are as in Fig. 1. Probability values after sequential Bonferronicorrection: *P < 0.05; **P < 0.01; ***P < 0.001;****P < 0.0001.
Species Allozymes mtDNA
Global Fst Pairwise Fst Global Fst Pairwise Fst
D. puntazzo 0.160*** FAR-ALI (0.130)** 0.085*** FAR-ALI (0.120)***
FAR-OTR (0.300)**** FAR-OTR (0.162)***
FAR-MSL (0.320)**** FAR-MSL (0.202)****
ALI-OTR (0.06)* ALI-OTR (¡0.004)ALI-MSL (0.07)* ALI-MSL (0.040)*
OTR-MSL (0.0) OTR-MSL (0.003)
D. sargus 0.009 FAR-ALI (0.07) 0.007 FAR-ALI (0.0)FAR-OTR (0.02) FAR-OTR (0.016)FAR-IRK (0.0) FAR-IRK (¡0.004)ALI-OTR (0.0) ALI-OTR (¡0.012)ALI-IRK (0.01) ALI-IRK (0.034)OTR-IRK (0.0) OTR-IRK (0.048)*
532 L. Bargelloni et al. / Molecular Phylogenetics and Evolution 36 (2005) 523–535
reciprocal monophyly of Atlantic and Mediterraneanhaplotypes is not present in the mtDNA network ofD. puntazzo.
To explain this observation two hypotheses can beput forward: either gene Xow between the Atlantic andMediterranean regions has been constantly very low orabsent, but time since separation of populations hasbeen insuYcient to reach reciprocal monophyly, orrecent episodes of genetic exchanges between the tworegions have mixed previously separated “regional”clades. It has been shown (Neigel and Avise, 1986) thatgene trees are likely to be concordant with populationsubdivisions after 2 £ Ne generations where Ne is theeVective population size (female eVective size in the caseof mtDNA). Assume for the sake of discussion that thetwo regions have been isolated since the separation ofthe branch leading to clade 4 from the lineage ancestralto the rest of haplotypes. Uncorrected mean sequencedivergence between these two lineages is 0.068, whichtranslates, under a conventional evolutionary rate forWsh control region (McMillan and Palumbi, 1997) of0.1–0.12 uncorrected sequence divergence per millionyear (myr), in a divergence time of 0.56–0.68 myr, or340,000–187,000 generations, (assuming a 2–3 years gen-eration time for D. puntazzo). This means that Ne ofD. puntazzo should have been larger than 2 £ (340,000–187,000) to prevent complete sorting of haplotypes intoreciprocal monophyletic clades. Based on FAO catchdata, just in the year 1997, 200 metric tons of D. pun-tazzo were landed from Wshing vessels only in the NorthAdriatic, meaning 400,000–600,000 individuals for anaverage size of 300–500 g. Although admittedly crude,this evidence suggests a census size for D. puntazzo atleast in the order of 106–107. A female eVective size ofseveral hundreds thousands individuals therefore is notincompatible with present-day census size of D. pun-tazzo, and the hypothesis of incomplete monophyly asthe consequence of large Ne could not be deWnitivelyrejected. It should be noticed, however, that in a largenumber of taxa, especially marine ones, Ne is severalorders of magnitude lower than census size (Frankham,1995). Moreover, some individuals of the FAR samplehave haplotypes closely related to sequences foundwithin the Mediterranean region, and included in all theremaining three clades (I, II, and III). For these reasons,the hypothesis that reciprocal monophyly might havebeen reached once and then lost because of episodes ofgenetic exchange between the two regions seems morelikely. The latter scenario seems also more congruentwith the hypothesis that levels of exchanges between theAtlantic and the Mediterranean are variable at diVerenttime scales, with short-term, annual changes as well ashistorical Xuctuations associated with glacial and inter-glacial periods. In the latter case, geological data (Nils-son, 1982) suggest that historical events of habitatfragmentation have occurred, followed by episodes of
increased circulation between the Atlantic and the Medi-terranean. For instance, during the Mindel Glaciation(400,000 years ago) the sea level dropped 115–120 mbelow the present level, reducing both width and depthof the Gibraltar Passage. Similar drops are suggested forthe Riss Glaciation (130,000–170,000 years ago), andother glacial maxima, until the most recent one, occurred22,000 years ago. During interglacial periods, on theother hand, sea level drops were followed by suddenrises, with movement of large water masses.
On a much shorter time scale, oceanographic surveyshave demonstrated that water Xow at Gibraltar is asym-metrical, with inXow consisting of surface waters fromthe Atlantic, while outXow composed by deeper Mediter-ranean waters. The Xow is also signiWcantly smaller thanpreviously estimated, and variable during the year (Mil-lot, 1999). Under this regard, it should be observed thatin D. puntazzo several “Atlantic” individuals are includedin clades I, II, and III, while only a single individual fromthe ALI sample falls into clade IV. However, additionaldata are needed to make clear whether this result is dueto asymmetrical gene Xow favouring immigration fromthe Atlantic into the Mediterranean (therefore in coun-tercurrent with respect to surface water Xow) or it is justthe consequence of unequal sample and clade size.
More importantly, the inXowing Atlantic waterdescribes in the Alboran Sea a quasi permanent anticy-clonic gyre in the West, a more variable one in the East.The particular water circulation in the Alboran Sea gen-erates an oceanographic front located from Oran(Morocco) to Almeira (Spain), called the Oran-AlmeriaFront (OAF) (Fig. 1). To date, it is unclear whether inthe past, especially during the mentioned climaticchanges, the hydrological regime was similar to the pres-ent one. Simulation studies suggest that the volume Xowrate into the strait inXuences the growth rate of thegyre(s), but not its general structure (Gleizon et al.,1996). Thereby changes in the section of the GibraltarStrait associated with climate modiWcations might haveled to changes in levels of water Xow, but not to reversalof hydrological conditions. The relevance of the OAF, inthe present as well as in the past, is suggested also bygenetic evidence. Population genetics studies on sea bass(Naciri et al., 1999) and the mussel Mytilus galloprovin-cialis (Quesada et al., 1995 and reference therein) wheresamples from the Alboran Sea are included, demonstratethat a clear shift in gene frequencies (at microsatelliteloci in the sea bass, or mtDNA and allozymes in themussel) is observed to be associated to the OAF. Alsothe genetic pattern found for D. puntazzo is congruenttemporally (large divergence among haplotypes, incom-plete monophyly) and spatially (sharp change in geneticcomposition between Atlantic and Mediterranean sam-ples) with the hypothesis that the mentioned historicaland contemporary factors might have reduced, althoughnot constantly, gene Xow between the two marine basins.
L. Bargelloni et al. / Molecular Phylogenetics and Evolution 36 (2005) 523–535 533
Rather diVerent are the results for D. sargus. Onlyweak genetic diVerentiation is found, never associatedwith the transition between Atlantic and Mediterra-nean.This observation partly contradict the proposedseparation in distinct sub-species of the Mediterraneanand Atlantic D. sargus populations, classiWed as D. s.sargus and D. s. cadenati, respectively (De la Paz et al.,1973; Bauchot and Hureau, 1986). The present results infact do not support this view as no appreciable geneticdiVerences were found between Atlantic and Mediterra-nean samples. Similar Wndings were also reported inanother molecular investigation which grouped togetherin a phylogenetic tree D. sargus samples collected in theMediterranean Sea (Calvi, Corsica, France) and oV theAtlantic African coast (Agadir, Morocco) (Summeret al., 2001).
Previous allozyme studies showed diVerences betweenpopulation collected within the Mediterranean basin(Lenfant and Planes, 1996; González-Wangüemert et al.,2004). In the present study no indication for D. sarguspopulation structure at allozyme loci was evidenced nei-ther between Mediterranean and Atlantic samples norwithin Mediterranean populations. It is however worthto note that the average observed heterozygosity at thescored allozyme loci is much lower in the samples ana-lysed in this work as compared to allozyme data whichrecently appeared in the literature on this species (Gon-zález-Wangüemert et al., 2004). It diYcult to explainsuch a large diVerence, polymorphism reduction wasreported in this D. sargus as the consequence of selectiveprocesses during recruitment (Planes and Romans,2004). However, drop in the observed heterozygositycould be alternatively due low eVective population sizeassociated to genetic drift.
The simplest explanation for (near) absence of geneticstructure in D. sargus is that gene Xow is suYciently highto homogenise gene frequencies across geographic popu-lations. Alternatively, even if migration between contem-porary populations is eVectively low or null, the eVect ofpast migrations might still be evident because migration-drift equilibrium has not been reached yet. Life-historyof D. sargus is very similar to that described for D. pun-tazzo, with a relatively short (1 month) plancktonic lar-val stage, settlement of juveniles in shallow benthichabitats, and a sedentary adult stage. Capacity for dis-persal is thereby supposed to be comparable between thetwo species (Vigliola et al., 1998). Moreover, experimen-tal conditions (sampling scheme, used markers) arealmost identical for the two species. For these reasons,absence of genetic structure in D. sargus is even morestriking, if compared to the genetic pattern observed forD. puntazzo.
How can be explained this sharp diVerence betweenthe two species? Incongruent phylogeographic patternsmight be caused by ecological, genetic or historical fac-tors (Zink, 1996). As already mentioned, the two Diplo-
dus species under study have similar dispersal capacity,distributional range and ecological features. DiVerencesin ecology therefore appear unlikely to be responsiblefor divergent genetic patterns. Some relevance for diVer-ential dispersal might have the observation that D. sar-gus is more abundant than D. puntazzo both as adults(Macpherson, 1998), and juveniles (Vigliola et al., 1998).If larger census size translates into greater Ne for D. sar-gus, then higher levels of gene Xow are expected for thisspecies even for dispersal capacity equivalent to D. pun-tazzo. While this hypothesis might explain the generallylower level of genetic population diVerentiationobserved for D. sargus, it seems insuYcient to accountfor the peculiar diVerence between the two speciesregarding the Atlantic–Mediterranean transition. SomediVerentiation is indeed detected for D. sargus, but onlywithin the Mediterranean region (signiWcant only in thecomparison between Italian and Greek samples for themtDNA). Moreover, levels of genetic diversity appear toindicate a comparable (allozymes) or much higher(mtDNA) long-term Ne for D. puntazzo compared toD. sargus, thereby not supporting the above hypothesis(D. sargus Ne > D. puntazzo Ne). EVective populationsize, however, could have experienced important histori-cal variations. For instance, population size might benormally large (in the case of D. sargus larger than D.puntazzo), but bottlenecks may occasionally occur,strongly reducing long-term Ne. Results of Tajima’s neu-trality test and mismatch analysis on D. sargus mtDNAdata indeed suggests that this species underwent a bot-tleneck and a subsequent expansion, as all samples showhighly negative D values, two of which signiWcantly. Ifstrong population bottlenecks have characterised theevolutionary history of D. sargus, what is the possibleinXuence on genetic divergence among populations? TheeVect of variation in population size on estimates of pop-ulation diVerentiation has been explored, suggesting thatthe classical results for constant population size holdstrue also for varying population size, with the obvioussubstitution of long-term eVective size as the harmonicmean of single-generation population sizes (Hudson,1998). Therefore, lack of genetic diVerentiation in D. sar-gus cannot be simply ascribed to small long-term eVec-tive size due to past population bottlenecks. However,reductions in population size and subsequent expansionsmight have been historically associated with occasional,but signiWcant migration events. For instance, if D. sar-gus had in the past a more limited distribution, notincluding the Mediterranean region, which might havebeen colonised only recently, then lack of a phylogeo-graphic pattern could be due to insuYcient time to accu-mulate genetic diVerences between the two regions. Thisseems in keeping with the suggestion that the diversiWca-tion within D. sargus clade has its origin in the central-eastern Atlantic, (probably near Cape Verde Islands)(Summer et al., 2001) which imply that Mediterranean
534 L. Bargelloni et al. / Molecular Phylogenetics and Evolution 36 (2005) 523–535
colonization was a secondary event. Alternatively, eventsof extinction and recolonization could have produced asimilar eVect. Both scenarios are in agreement with evi-dence of past demographic reduction and sudden expan-sion of population size of D. sargus, and with the“troubled” paleoclimatic history of the Mediterranean.
In conclusion, available evidence seems to suggestthat discordant genetic patterns might be associated toaccidental factors rather than to ecological diVerencesacross species. While this suggestion is in agreement withresults from other species, studies based on faster evolv-ing loci (e.g. microsatellites) will help to better evaluatethe importance of recent and/or present-day phenom-ena, thereby deWnitively supporting or rejecting thehypothesis that the observed discordant patterns are thelegacy of historical events.
Acknowledgment
This work was part of a project funded by the Euro-pean Union, contract No. AIR3-CT94-1926.
Appendix A. Supplementary data
Supplementary data associated with this article canbe found, in the online version, at doi:10.1016/j.ympev.2005.04.017
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