Molecular Ecology (2007) 16, 5259–5266 doi: 10.1111/j.1365-294X.2007.03587.x

© 2007 The AuthorsJournal compilation © 2007 Blackwell Publishing Ltd

Blackwell Publishing LtdThe distribution of Quercus suber chloroplast haplotypes matches the palaeogeographical history of the western Mediterranean

D. MAGRI ,* S . F INESCHI ,† R . BELLAROSA,‡ A. BUONAMICI ,§ F. SEBASTIANI ,§ B . SCHIRONE,‡ M. C . S IMEONE‡ and G. G. VENDRAMIN¶*Dipartimento di Biologia Vegetale, Università di Roma ‘La Sapienza’, Piazzale Aldo Moro 5, 00185 Roma, Italy, †Consiglio Nazionale delle Ricerche, Istituto per la Protezione delle Piante, via Madonna del Piano 10, 50019 Sesto Fiorentino (Firenze), Italy, ‡Dipartimento di Tecnologie, Ingegneria e Scienze dell’Ambiente e delle Foreste, Università della Tuscia, Via S. Camillo de Lellis, 01100 Viterbo, Italy, §Dipartimento di Biotecnologie Agrarie, Università degli Studi di Firenze, Via della Lastruccia 14, 50019, Sesto Fiorentino (Firenze), Italy, ¶Consiglio Nazionale delle Ricerche, Istituto di Genetica Vegetale, Via Madonna del Piano 10, 50019 Sesto Fiorentino (Firenze), Italy

Abstract

Combining molecular analyses with geological and palaeontological data may reveal timingand modes for the divergence of lineages within species. The Mediterranean Basin isparticularly appropriate for this kind of multidisciplinary studies, because of its complexgeological history and biological diversity. Here, we investigated chloroplast DNA of Quercussuber populations in order to detect possible relationships between their geographicaldistribution and the palaeogeographical history of the western Mediterranean domain. Weanalysed 110 cork oak populations, covering the whole distribution range of the species, by14 chloroplast microsatellite markers, among which eight displayed variation amongpopulations. We identified five haplotypes whose distribution is clearly geographicallystructured. Results demonstrated that cork oak populations have undergone a genetic driftgeographically consistent with the Oligocene and Miocene break-up events of the European–Iberian continental margin and suggested that they have persisted in a number of separatemicroplates, currently found in Tunisia, Sardinia, Corsica, and Provence, without detectablechloroplast DNA modifications for a time span of over 15 million years. A similar distributionpattern of mitochondrial DNA of Pinus pinaster supports the hypothesis of such long-termpersistence, in spite of Quaternary climate oscillations and of isolation due to insularity,and suggests that part of the modern geographical structure of Mediterranean populationsmay be traced back to the Tertiary history of taxa.

Keywords: chloroplast DNA, genetic drift, geographical structure, Neogene, Quercus suber, westernMediterranean

Received 23 March 2007; revision received 21 August 2007; accepted 28 August 2007

Introduction

The Mediterranean Basin is a hotspot for biogeographicaland evolutionary studies, showing an exceptional level ofbiodiversity (Greuter 1995; Quézel 1995; Blondel & Aronson1999; Comes 2004; Thompson 2005). A complex geologicalhistory, characterized by orogenic processes and widespread

extensional tectonics, a considerable physiographic andclimatic heterogeneity and a long-term impact of humanactivities have caused repeated isolations of individualplant populations and admixtures of populations of differentorigins. Another prominent feature of the Mediterraneanflora is its high rate of regional endemism, due partly to arecent diversification (neo-endemics), and partly to theconservation of taxa with relictual distribution, resultingfrom a long-term regional permanence without evolutionarychanges (palaeo-endemics) (Comes 2004; Thompson 2005).

Correspondence: Giovanni G. Vendramin, Fax: +39-055-5225729;E-mail: giovanni.vendramin@igv.cnr.it

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In addition to a very high level of species diversity, theMediterranean region shows a considerable diversityamong and within plant populations, documented by alarge number of studies on different genetic markers(Fady-Welterlen 2005). The main factor for the differenti-ation of European gene resources is generally found inQuaternary climate oscillations, causing repeated retreatsand re-advances of plant populations into and from refugeareas (Comes & Kadereit 1998; Taberlet et al. 1998; Hewitt1999, 2000). Recently, combined phylogeographical andpalaeobotanical studies have shown that within-speciesdiversity for the European beech was shaped over timesmuch longer than the last interglacial–glacial cycle (Magriet al. 2006). However, a possible age for the genetic diver-gence of tree populations remains a challenge and urgesnew investigations. The Mediterranean flora is especiallyappropriate for this purpose, as many species with a Tertiaryorigin have persisted through the Quaternary up to present,and so may bear witness of long-term changes of geneticcharacters.

With this purpose in mind, we have focused our attentionon cork oak (Quercus suber L), an emblematic Mediterraneanevergreen sclerophyllous tree. Cork oak is a slow growing,extremely long-lived evergreen tree. It is a monoeciouswind-pollinated species with a protandrous system to ensurecross-pollination. Reproductive system (mostly sexual)and dissemination (gravity and zoochory) of cork oak arecoherent with those of most oak species.

It may reach about 20 m in height, with massivebranches forming a round crown. Its thick and soft barkis the source of cork, which is stripped every 10–12 yearsfrom the outer layer of the bark along the lower portion ofthe trunk (Gellini & Grossoni 1997). The modern distrib-ution of Q. suber, rather discontinuous, ranges from theAtlantic coasts of North Africa and Iberian Peninsula to the

southeastern regions of Italy, and includes the main westMediterranean islands as well as the coastal belts ofMaghreb (Algeria and Tunisia), Provence (France) andCatalonia (Spain) (Fig. 1).

Quercus suber is widely cultivated within its naturalrange for the production of cork. According to Carrión et al.(2000a), without human activities Q. suber would neverdevelop pure stands in the Iberian Peninsula, and wouldform mixed forests with xerophyllous and deciduous oakstogether with Pinus pinaster instead.

In recent years, cork oak has been the subject of intensivegenetic studies by means of different markers, and a strongdifferentiation among populations has been detectedaround the Mediterranean Basin by investigating bothchloroplast and mitochondrial DNA (which are maternallyinherited in oaks, as demonstrated by Dumolin et al. 1995),as well as allozyme variations; moreover, evidence ofcytoplasmic introgression of Q. suber by Quercus ilex geneshas been reported (Toumi & Lumaret 1998, 2001; Belahbibet al. 2001; Lumaret et al. 2002, 2005; López de Heredia et al.2005). Although both Q. suber and Q. ilex are evergreenoaks, they belong to different subgenera (Q. suber, subgenusCerris; Q. ilex, subgenus Sclerophyllodrys) according to classicaloak taxonomy (Schwarz 1993). However, a more recentclassification includes both species within the same Eurasiansection Cerris (Manos et al. 1999). In their survey on theMediterranean evergreen oak species, Jiménez et al. (2004)and López de Heredia et al. (2005) detected three lineagesof chloroplast DNA haplotypes, one of them (suber lineage)being specific of cork oak populations from peninsularItaly, Sardinia, Sicily, Corsica, northern Africa and theisland of Minorca, whereas cork oak populations fromthe Spanish mainland and from the island of Majorcawere characterized by one maternal lineage also shared byQ. ilex and Quercus coccifera.

Fig. 1 Distribution of cpDNA haplotypeswithin Quercus suber populations andphylogenetic reconstruction of the rela-tionships among haplotypes usingnetwork 4.2.0.1. (Fluxus Technology2007). The black circle in the networkindicates a hypothesized mutation, whichis required to connect existing haplotypeswithin the network with maximumparsimony. The grey area correspondsto the modern distribution of Q. suber(EUFORGEN database www.bioversityinternational.org/networks/euforgen).

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Several hypotheses have been advanced concerning theevolutionary history of cork oak as well as the geographicallocation of its centre of origin, which has been suggested tobe in the eastern countries of the western MediterraneanBasin (Bellarosa et al. 2005; Lumaret et al. 2005). However,the details of its differentiation processes are still largelyunknown.

We have analysed cork oak populations throughout thespecies distribution range using chloroplast DNA (cpDNA)markers (microsatellites) and have combined the geneticresults with palaeobotanical data and geodynamic modelsto suggest new insights on the modes and timing of thegenetic divergence of cork oak populations.

Materials and methods

Genomic DNA was extracted from fresh leaves of 110populations from Italy (including Sicily, Sardinia and theTuscan Islands), France and Corsica, Spain and Balearics,Portugal, Morocco, Algeria and Tunisia. At least threeindividuals were analysed for each population. Invitek(Invisorb Spin Plant Mini Kit) protocol was utilized for DNAextraction. List of populations, number of individuals perpopulation, and geographical coordinates are available asSupplementary material (S1). Fourteen chloroplast microsa-tellite regions were amplified using primers: ccmp 2,ccmp 4, ccmp 6, ccmp 7, ccmp 10 (Weising & Gardner 1999);and cmcs 1, cmcs 2, cmcs 3, cmcs 6, cmcs 7, cmcs 8, cmcs 9,cmcs 13, cmcs 14 (Sebastiani et al. 2004). Polymerase chainreaction (PCR) amplification and sizing of the fragmentsfollowed procedures described by Fineschi et al. (2005).Amplification conditions were: 4 min at 95 °C, 25 cycleseach consisting of 95 °C for 30 s, 50 °C for 30 s, 72 °C for30 s; then 72 °C for 8 min. PCR products were loaded ona capillary automatic sequencer MegaBACE 1000 (GEHealthcare). MegaBACE ET400 (GE Healthcare) was usedas size standard. Fragment lengths were determined usingMegaBACE fragment profiler software version 1.2(GE Healthcare).

In order to check for introgression between Quercus suberand Quercus ilex, microsatellite fragments polymorphic inQ. suber were also analysed in Q. ilex, choosing individualspreviously characterized as having different haplotypes(Fineschi et al. 2005). Amplified fragments of the polymor-phic microsatellites were cloned into plasmid vectors usingthe Invitrogen TOPO cloning kit and then sequenced fromboth ends using an automatic sequencer MegaBACE 1000(GE Healthcare). Each fragment was sequenced twice. Thesoftware permut (Pons & Petit 1996) (www.pierroton.inra.fr/genetics/labo/Software) was used to measure differentia-tion for unordered alleles (GST) and for ordered alleles(NST). One thousand random permutations of haplotypeidentities were made, keeping the haplotype frequenciesand the matrix of pairwise haplotype differences as in the

original study (Burban et al. 1999). If NST, which takes thegenetic differences between the haplotypes into account, ishigher than GST, this indicates the presence of a phylogeo-graphical structure (Pons & Petit 1996), that is closely relatedhaplotypes are more often found in the same geographicalarea than would be expected by chance.

Phylogenetic relationships among haplotypes wereinferred with network 4.2.0.1 (Fluxus Technology Ltd. atwww.fluxus-engineering.com) using the median-joiningmethod (Bandelt et al. 1999).

Results

Polymorphisms at eight (ccmp4, ccmp6, cmcs1, cmcs6,cmcs7, cmcs8, cmcs9, cmcs14) of 14 microsatellite lociidentified only five different haplotypes (S2) with a stronggeographical structure (Fig. 1), as demonstrated by thehigh value of genetic differentiation between populationsfor unordered alleles: GST = 0.965 (SE 0.016) as well as forordered alleles NST = 0.962 (SE 0.018). Most populations arefixed for one haplotype. Only five populations (one perregion in Spain, Morocco, and Corsica, and two in Italy)contain two different haplotypes. Sequence data confirmedthat the detected variation is due to differences in thenumber of repeats within the microsatellite stretches. Inaddition, an insertion/deletion of 25 bp was detected in theflanking regions of the microsatellite CmCs9 (see GenBankAccession nos EU088193 and EU088194).

In the Italian peninsula and Sicily, two closely relatedhaplotypes (H1 and H2) of cork oak are present. HaplotypeH3 is found along the Mediterranean coast of Provence andLiguria, in the islands of Corsica, Sardinia, and northernTuscany, as well as in the northern part of Tunisia andAlgeria. Haplotype H4 is distributed in the westernmostpart of the range (southwest France, Portugal, southwestSpain and northern Morocco). Haplotype H5 shows a dis-continuous distribution in Catalonia, the Balearic Islandsand the Rif mountain range in Morocco.

The haplotype network (Fig. 1) indicates that the twoItalian haplotypes, H1 and H2, differ from each other byonly one mutation, and are very divergent from haplotypesH4 (Portugal–western Spain–southwest France–northernMorocco) and H3 (north Africa–Sardinia–Corsica–Provence).H5 corresponds to a cpDNA lineage shared with Quercusilex, interpreted as the result of multiple and mainly uni-directional cytoplasmic introgression of Quercus suber by Q. ilex(Belahbib et al. 2001; López de Heredia et al. 2005; Lumaretet al. 2005) resulting in populations morphologicallyidentified as cork oak, which maintain the chloroplastgenome of the seed parent species Q. ilex. Belahbib et al.(2001), López de Heredia et al. (2005) and Lumaret et al.(2005) suggest that hybridization between holm oak andcork oak must be an ancient event (repeated hybridizationevents and backcrosses with the pollen parent). Similarly,

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haplotypes H1 and H2 may be the result of unidirectionalcytoplasmic introgression by Quercus cerris, an easternEuropean deciduous oak that is widespread in the Italianpeninsula. In fact, our unpublished data (not shown)indicate a sharing of haplotypes H1 and H2 between Q. cerrisand Q. suber in Italian populations.

Discussion

The modern history of Quercus suber is closely related tohuman activity for cork production. For this reason,humans have been considered responsible for a reductionin genetic variation in some stands of cork oak, as well asfor hybridization with congeners (Blondel & Aronson 1999;Thompson 2005). However, the geographical distributionof the cork oak haplotypes does not appear to be related tocultivation. In fact, fossil pollen and wood records suggestthat cork oak was distributed in approximately the sameareas as today even before the Neolithic (Fig. 2). In theIberian Peninsula and North Africa, there is palynologicalevidence for a long-term persistence of cork oak datingback to the last glacial, indicating that at least some of theareas where cork oak is presently found had glacial climateconditions suitable for its survival. The pollen records ofcork-oak of postglacial age do not exclude the presence ofQ. suber in much earlier times at a level not detected bypollen analysis. In Italy, the distributions of Q. suber andQuercus cerris overlap. As the pollen of these two oak speciescannot be distinguished, it is not possible to use fossilpollen to reconstruct the history of cork oak from the Italianpeninsula.

Other cultivated tree species in the Mediterraneandisplay low geographical structure in genetic variation,arguing for a multidirectional diffusion of the cultivatedtaxa because of human activity. For example, in Castaneasativa (Fineschi et al. 2000), the low geographical structure

of the chloroplast diversity may be explained with a stronghuman impact, documented by a wealth of fossil dataduring the last few thousands of years (Conedera et al. 2004).The phylogeographical pattern of Olea europaea (Besnardet al. 2002; Besnard & Bervillé 2002; Besnard et al. 2007),although slightly different from that of chestnut, confirmsthe important role played by humans in transferring pro-pagation material throughout the Mediterranean, mainlyfollowing an east–west direction. Contrary to this pattern,distinct cork oak haplotypes are found even in neighbourgeographical areas such as Corsica and Italy, Tunisia andSicily, and western and eastern Morocco, indicating thathuman activity did not blur the original genetic structure.

Another possible hypothesis to explain the moderndistribution of the cpDNA haplotypes of cork oak is its post-glacial population expansion from potential glacial refugesin Italy, North Africa and Iberia (Lumaret et al. 2005). Corkoak populations are expected to migrate from one regionto the other along continental areas. Excluding exceptionaland unproven cases of long-distance dissemination, themodern distribution of haplotype H3 cannot be ascribedto a postglacial colonization route starting from Tunisia,crossing the Mediterranean Sea and reaching southernFrance through Sardinia and Corsica. In fact, the geologicalrecord suggests that there have been no land connectionsbetween northern Africa and Europe, or between Sardiniaand Provence during the Quaternary, in spite of substantialsea-level oscillations.

Only during a short time interval of the Messinian salinitycrisis, between 5.59 and 5.50 million years ago (Krijgsmanet al. 1999), might there have been such connections, but theMediterranean Basin may not have been completely dryeven during this time (Cipollari et al. 1999; Manzi et al. 2005)and the exposed land may not have been suitable for plantlife (Quézel 1995; Thompson 2005). More importantly, ourdata are not consistent with a Messinian migration of

Fig. 2 Location and calendar ages offossil woods (�) and pollen records ( )of cork oak. References are listed in Fig.S1, Supplementary material.

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cork oak. In fact, Tunisia and Sicily, which could have beenconnected to each other, show different haplotypes (H3and H1, respectively), while Tunisia and Sardinia, whichwould have remained separated even in the event of asea-level drop of 1000 m, share the same haplotype (H3).

Mediterranean biogeographers agree that the presentdistributional patterns of many taxa in the region are wellexplained by the late Cenozoic palaeogeographical evolutionof the Mediterranean (Greuter 1995; Quézel 1995; Blondel& Aronson 1999; Comes 2004; Thompson 2005). In the last40 years, a number of geodynamic reconstructions of theMediterranean basin have been advanced (Rosenbaumet al. 2002). Although some aspects of these models are stillcontroversial, there is a general agreement that duringthe Oligocene, the European–Iberian continental marginassembled continental terranes that are now found inCalabria, Sicily, Corsica, Sardinia, Kabylies (Algeria),Balearic Islands and Rif range in Morocco (Fig. 3). Startingin the late Oligocene (30–25 million years) and during theMiocene, these microplates underwent separation fromthe mainland and drifted towards south or southeastreaching their current positions (Gueguen et al. 1998; Rocaet al. 1999; Gelabert et al. 2002; Rosenbaum et al. 2002; Cavazza& Wezel 2003; Carminati & Doglioni 2005).

Cork oak is presently found on all these plates togetherwith many other Mediterranean palaeo-endemics. Thus, itseems reasonable to infer an early Cenozoic origin for thisspecies in the Iberian continent and subsequent displace-ment on the drifted microterranes. This is consistent withtwo fossil records of cork oak of Miocene age in Portugal(de Carvalho 1958; Mai 1995) and two Pliocene finds inTunisia and Galicia, respectively (Losa Quintana 1978;Quézel 1995).

If we assign the colours of the modern cork oak haplo-types (Fig. 1) to the corresponding microplates rifted offthe European continent during the Miocene (Fig. 3), wefind a remarkable conformity between the position of thedrifting blocks and the distribution of haplotypes. Corkoak populations, isolated from the Iberian populationand island-rafted across the Mediterranean to their presentposition, appear to have undergone a genetic drift consistentwith the break-up of the main continent.

The distribution of haplotypes suggests a westernMediterranean origin for cork oak, according to the followingpalaeogeographical history. Provence, Corsica, Sardiniaand Kabylies, which were connected to each other untilthe early Miocene (Gueguen et al. 1998; Roca et al. 1999;Rosenbaum et al. 2002), share haplotype H3 that differsfrom the Iberian haplotype (H4) by two mutations. There-fore haplotype H3 presumably differentiated when Provence,Corsica, Sardinia and Tunisia were still in connection witheach other, but already separated from Iberia (H4). TheCalabrian haplotype H1, very different from both H4 andH3, indicates an early isolation of the Calabrian microplate

from Iberia and the Corsica–Sardinia–Kabylies block. Thisscenario is supported by the observation that Corsica andSardinia have closer floristic affinities to the Balearics thanto Calabria (Gamisans & Jeanmonod 1995). Alternatively,the differences between H3 and H1 might be due to a possiblecytoplasmic introgression with Q. cerris in the Italianpeninsula and Sicily, which may have occurred rather late,after the detachment of Calabria from the Corsican–Sardinianblock. This second hypothesis is supported by: (i) a late(10–8 million years ago) detachment of Calabria and Sicily(Peloritani mountains) from the Corsica–Sardinia block(Rosenbaum et al. 2002), and (ii) geological studies indicatingthat Sicily moves together with the African Plate in itscollision against Calabria (Goes et al. 2004), and that Sicilyand Tunisia may have shared a geological connection until

Fig. 3 Reconstructions of the Western Mediterranean palaeo-geography (modified from (Rosenbaum et al. 2002) and possiblelocation of Quercus suber haplotypes (colours as in Fig. 1). Continentalmicroterranes rifted off the European–Iberian continental margin:Rif (R), Betic range (B), Balearics (Ba), Kabylies (Ka), Corsica (Co),Sardinia (Sa), Calabria (Ca).

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at least the Late Pliocene–Pleistocene across the Maghrebides(Rosenbaum et al. 2002).

The Balearic Islands and Rif (sharing haplotype H5)underwent a break-up leading to the displacement of theRif chain from the Iberian mainland to Morocco, whichmust have occurred after the introgression of Quercus ilexby Q. suber. Our hypothesis still holds if we assume that onlyH3 and H4 are the ancestral Q. suber haplotypes, with H1,H2 and H5 originating through either ancient or recentintrogression with Q. cerris (H1 and H2) and Q. ilex (H5).

The geodynamic models for the western Mediterraneando not provide detailed ages for break-up events, whichare believed to have occurred between 25 million years and15 million years (Gueguen et al. 1998; Roca et al. 1999; Gelab-ert et al. 2002; Rosenbaum et al. 2002; Cavazza & Wezel 2003;Carminati & Doglioni 2005). Even with this large ageuncertainty, it is possible to highlight some evolutionarypatterns. In particular, the broken distribution of haplotypeH3 suggests that cpDNA differentiation occurred at atime of intense tectonic activity along the Iberian margin,which produced the detachment of the Corsica–Sardinia–Kabylies block from the mainland during the late Oligocene,and was followed by the fragmentation of Corsica, Sardiniaand Kabylies in separate microplates after 20 million years(Gueguen et al. 1998; Roca et al. 1999; Gelabert et al. 2002;Rosenbaum et al. 2002; Cavazza & Wezel 2003; Carminati& Doglioni 2005). After the fragmentation of the Corsica–Sardinia–Kabylies block, for a time span of at least 15 millionyears, cork oak populations appear to have persisted withineach microplate without detectable cpDNA modifications.

Long-term permanence in situ and prolonged evolutionarystandstill of Mediterranean palaeo-endemic species, implyinglong periods without speciation, are well known to biogeo-graphers and evolutionists (Greuter 1995; Quézel 1995;Blondel & Aronson 1999; Comes 2004). Our data suggestthe possibility of a long-lasting stability not only at the

species level, but also at the level of cpDNA microsatellites,in spite of isolation due to insularity conditions and of popu-lation responses to Quaternary climate oscillations. Low ratesof evolution of tree species were highlighted and discussedby Petit & Hampe (2006). Indeed, perennials evolve moreslowly than other plants at the DNA sequence level, forchloroplast, mitochondrial, and nuclear genes, particularlyat silent sites, as reported by Petit & Hampe (2006).

This issue raises the question whether other geneticmarkers, evolving at faster rates than cpDNA, would providea different microevolutionary scenario and whether otherMediterranean palaeo-endemics have similar evolutionaryhistories. Only few published phylogeographical studiesrefer to such taxa. Among them, Pinus pinaster Aiton hasa modern distribution similar to Q. suber, as it is limitedto the western Mediterranean basin, where it occurs asscattered nuclei in Portugal, Spain, southern France, Italy,Morocco, Tunisia, Algeria, and in several islands, such asSardinia, Corsica, Pantelleria and Minorca (Fig. 4). Pinussect. Pinaster is considered to be a palaeomediterraneanelement, being found with many other evergreen taxa inearly Cenozoic deposits (Mai 1995). Although it is oftendescribed as an invasive species, P. pinaster is certainlynative in the Iberian Peninsula, where both fossil wood(Figueiral 1995) and pollen (Carrión et al. 2000b) indicatethat it survived during the last glacial, sharing its refugiawith Q. suber.

Molecular analysis of mitochondrial (maternallyinherited) markers of P. pinaster provides a clear picture ofthree nonoverlapping mitochondrial haplotypes (Burban& Petit 2003). In particular, one mitotype is found in Morocco,a second mitotype is present in the Iberian peninsula, anda third one is found in all populations from southeasternFrance, Corsica, Italy, Pantelleria Island, Tunisia and Algeria(Fig. 4). The similarity of the distribution of the thirdmitotype of P. pinaster with the distribution of haplotype

Fig. 4 Distribution of mitotypes withinPinus pinaster populations (modifiedfrom Burban & Petit 2003). The greyarea represents the modern distributionof Pinus pinaster (EUFORGEN databasewww.bioversityinternational.org/networks/euforgen).

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H3 of Q. suber strongly supports our hypothesis that: (i) agenetic differentiation occurred when Corsica, Sardiniaand the Kabylie blocks were already separated from theIberian mainland, but still in connection among themand with southern France; (ii) survival of trees in the riftedmicroterranes persisted until present without significantgenetic modifications, even at the population level; and(iii) Neogene tectonic events may leave their footprint onmodern population genetic diversity/differentiation. It isremarkable that such scenario is supported by the distri-bution of diversity detected using two different molecularmarkers (cpDNA for Q. suber and mitochondrial DNA forP. pinaster).

This evidence puts forward the question of how much ofthe modern genetic diversification of Mediterranean popu-lations is due to isolation in glacial refugia, as generallyassumed, and how much is to be traced back to the Tertiaryhistory of taxa, as suggested by the distributions of Q. suberand P. pinaster. There is therefore a need for new analyseson different taxa and molecular markers. This might leadto a reassessment of the age of processes shaping the phylo-geographical structure of tree species.

Acknowledgements

We thank K.E. Holsinger (Storrs, USA), Ch. Lexer (Kew, UK), A. Lowe(Adelaide, Australia), and R.J. Petit (Pierroton, France) for criticalcomments on the manuscript and the following colleaguesfor discussions and/or collection of cork oak material: L. Baldoni,S. Cozzolino, P. Degli Antoni, G. Della Rocca, A. De Natale, J.Flexas, N. Hasnaoui, F. Loreto, B. Moya Sanchez, A. Musacchio, P.Nascetti, I. Parra, A. Porceddu, J. Peñuelas, J.A. Rossello, I. Scotti.

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Donatella Magri is a palaeoecologist interested in past Mediter-ranean vegetation. Rosanna Bellarosa and Bartolomeo Schironeare molecular botanists interested in oak phylogenesis. This workrepresents part of the PhD thesis of Marco C. Simeone. A. Buonamiciis a PhD student working on the phylogeography of Angiosperms.F. Sebastiani is experienced in the development and applicationof molecular markers for the analysis of diversity in trees. G. G.Vendramin’s and Silvia Fineschi’s studies focus on conservationand population genetics, with particular emphasis on range-widephylogeography and fine-scale population gene dynamics.

Supplementary material

The following supplementary material is available for this article:

Table S1 Location and frequency of SSR haplotypes withinQuercus suber populations

Table S2 Description of SSR haplotypes identified in Quercussuber (in bold polymorphic fragments)

Fig S1 Location and references of palaeobotanical sites reportedin Fig. 2

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