Patterns of polymorphism resulting from long-range colonization in the Mediterranean conifer Aleppo...

Patterns of polymorphism resulting from long-rangecolonization in the Mediterranean conifer Aleppo pine

Delphine Grivet1*, Federico Sebastiani2,3*, Santiago C. Gonzalez-Martınez1 and Giovanni G. Vendramin3

1Department of Forest Systems and Resources, Forest Research Institute, CIFOR-INIA, Carretera de la Coruna km 7.5, ES–28040 Madrid, Spain;

2Department of Agricultural Biotechnology, Genexpress, University of Florence, Via della Lastruccia 14, I–50019 Sesto Fiorentino (FI), Italy;

3Plant Genetics Institute, Division of Florence, National Research Council, via Madonna del Piano 10, I–50019 Sesto Fiorentino (FI), Italy

Author for correspondence:Santiago C. Gonzalez-Martınez

Tel: +34 913471499

Email: [email protected]

Received: 16 April 2009Accepted: 12 July 2009

New Phytologist (2009) 184: 1016–1028doi: 10.1111/j.1469-8137.2009.03015.x

Key words: candidate genes, colonization,demography, drought response, genetic drift,local adaptation, Pinus halepensis, selection.

Summary

• The evolutionary outcomes of range expansion ⁄ contraction depend on the

biological system considered and the interactions among the evolutionary forces in

place. In this study, we examined the demographic history and the local polymor-

phism patterns of candidate genes linked to drought tolerance of a widespread

Mediterranean conifer (Pinus halepensis).

• To that end, we used cpSSRs and coalescence modelling of nuclear genes to

infer the demographic history of natural populations covering the species range.

Ten drought-response candidate genes were then examined for their patterns

of polymorphism and tested for selection considering plausible demographic

scenarios.

• Our results revealed a marked loss of genetic diversity from the relictual Greek

population towards the western range of the species, as well as molecular signa-

tures of intense bottlenecks. Moreover, we found an excess of derived polymor-

phisms in several genes sampled in the western part of the range – a potential

result of the action of natural selection on populations confronted with new

environments following long-range colonization.

• Wide-range expansions–contractions of forest trees are accompanied by strong

selective pressures, resulting in distinct evolutionary units. This knowledge is of

crucial importance for the conservation and management of forests in the face of

climate change.

Introduction

Predicting how demographic and selective processes interactduring colonization and affect the adaptive evolution of agiven species is a question of central importance in evolu-tionary and conservation genetics that remains a dauntingchallenge. For example, it is expected that populations thathave been through bottlenecks or founder effects duringcolonization will exhibit genetic erosion (Nei et al., 1975;Hewitt, 2000); also, declining genetic diversity with increas-ing distance from source population has been reported inseveral study cases (Grivet and Petit, 2003; Ramachandranet al., 2005; Eckert et al., 2008). However, that does notnecessarily mean that expanding populations are genetically

depleted or that refugial populations always harbour thehighest diversity, as has been shown in other studies (e.g.Petit et al., 1999, 2003; Comps et al., 2001; Walter & Ep-person, 2001). During range expansion, populations will beconfronted with selection and adaptation to different envi-ronments as well as to new conspecific and heterospecificinteractions (Hewitt, 2000). Whether the mechanisms thatgenerate different genetic patterns in expansion and refugialcore populations are able to influence the adaptive potentialof a given species will depend on the interplay among allthe evolutionary forces acting upon it. In the present study,we examined the genetic consequences of long-range coloni-zation of a widespread Mediterranean conifer.

Aleppo pine (Pinus halepensis) is a conifer that lives incontrasting environments, growing on all substrates andalmost all bioclimates of the Mediterranean region, thusreflecting high population adaptability. It is then remark-able that, in large parts of its range, various genetic studies

*These authors contributed equally to this work.

Original sequences have been deposited with the EMBL ⁄ GenBankData Libraries under accession numbers FJ588494–FJ588503.

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have shown low levels of genetic diversity both in neutralmolecular markers and in quantitative traits (Farjon, 1984;Schiller et al., 1986; Bucci et al., 1998; Climent et al.,2008). In addition, neutral genetic diversity in this speciesappears to be structured along a longitudinal gradient, witheastern Mediterranean populations harbouring greaterdiversity and more ancient lineages than western ones(Bucci et al., 1998; Morgante et al., 1998; Gomez et al.,2001). These genetic results, combined with commongarden experiments pointing to Greece as an outlier(Climent et al., 2008), data on flavonoid content (Barberoet al., 1998) and few available palynological and dendro-archaeological records (Schiller et al., 1986; Pons, 1992),suggest a scenario in which Aleppo pine populationsexpanded westwards all around the Mediterranean basinfrom some refugia located in the southern part of theBalkan Peninsula. This particular population dynamics ofAleppo pine (i.e. long-range colonization probably accom-panied by recurrent contractions–expansions caused, forexample, by forest fires), combined with its current wideecological range and scattered distribution across the Medi-terranean basin, makes this species especially suitable tostudy the impact of demographic and genetic processes, aswell as their interactions, on the adaptive potential of treepopulations.

To look at an extensive number of markers for outliers(i.e. the ‘population genomics’ approach; Luikart et al.,2003) is a recent approach generally used to understand theevolutionary forces at the origin of adaptation, and howthese forces shape the spatial–temporal pattern of variabilitywithin and between populations. Indeed, all loci of a gen-ome will be affected similarly by demographic processeswhile only a subset of them will be influenced by selection(but see Begun et al., 2007– the first population geneticsstudy based on whole-genome analysis) and depart from theaverage loci. Thus, looking at outlier loci among an exten-sive number of markers allows detection of loci subject toselection (see Namroud et al., 2008 for a recent example ina conifer). In nonmodel species with complex demographi-cal history (i.e. not fitting the simple models often assumedin outlier detection methods), such as Aleppo pine, forwhich only limited genetic resources are available, an alter-native approach to outlier loci detection would be the com-bination of inferences from putatively neutral markers, suchas chloroplast or nuclear microsatellites, which are theoreti-cally affected by demographic history but not by selection,and from candidate genes coding for known biologicalfunctions, potentially correlated with adaptive traits, such asphenology (Heuertz et al., 2006; Pyhajarvi et al., 2007),drought tolerance (Gonzalez-Martınez et al., 2006;Pyhajarvi et al., 2007; Eveno et al., 2008) or cold resistance(Wachowiak et al., 2009).

Tree species in the Mediterranean climate are facingspecific environmental constraints, among them drought.

Aleppo pine is well adapted to drought, although this factorcan still constitute an important threat to individual growthand survival at all life stages (Sathyan et al., 2005). In thecontext of climate change in the Mediterranean area, and itsconsequences on the adaptive potential of Mediterraneanvegetation, it is essential to understand the molecular basesunderlying adaptation of populations along colonizationgradients and how they are affected by population expan-sions and contractions. Moreover, from a practical point ofview, identifying populations harbouring the genotypes bestadapted for drought is essential not only for afforestation incurrent zones of extreme climatic conditions, such as inIsrael (Grunwald et al., 1986) and in Morocco (Boulliet al., 2001) in the case of P. halepensis, but also in the lightof future climatic changes, as most of the Mediterraneanarea is predicted to experience a significant increase in tem-peratures and aridity in the near future (Petit et al., 2005).The widespread distribution of Aleppo pine suggests that itspopulations may be differentially adapted to local climaticconditions and may show different capacities to survivewater-deficit stress (WDS). This view is reinforced byvarious studies showing differences in drought toleranceamong provenances using ecophysiological data and geneexpression (Atzmon et al., 2004 and references therein;Sathyan et al., 2005; Voltas et al., 2008). Consequently,genes associated with traits related to stress tolerance (e.g.cellular resistance to low water potentials or high secondarycompound production) have probably been main targetsfor natural selection in Aleppo pine and therefore constituterelevant candidates for testing the effects of long-rangecolonization and environment heterogeneity on adaptivepatterns in this species.

In this study, we used a set of neutral chloroplast micro-satellites (cpSSRs) to screen representative natural popula-tions covering Aleppo pine range and distributed along alongitudinal gradient. Different methods based on cpSSRs(F-statistics, SAMOVA and mismatch distributions) were usedto investigate the geographic structure of diversity and toinfer the demographic history of Aleppo pine populations.Populations were then examined for their local polymor-phism patterns at 10 candidate genes related to drought tol-erance to (1) describe geographical patterns of functionalnucleotide variation across Aleppo pine range and (2) inferthe past demographic history of this species using coales-cent-based modelling at the local scale. To do so, we simu-lated numerous demographic scenarios (in particular,expansion after bottleneck models, as suggested by cpSSRanalyses) to determine which one best fitted our empiricaldata and (3) examined whether different selective pressuresfor drought-response candidate genes exist along the longi-tudinal gradient sampled (covering putative refugialand expansion zones) by comparing the patterns of nucleo-tide variation observed at each locus with that expectedunder best-fitting demographic models. Overall, this study

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contributes to the identification of the evolutionary forcesshaping polymorphism patterns in the widespread Mediter-ranean conifer Aleppo pine.

Materials and Methods

Study species and sampling

Aleppo pine (P. halepensis Mill.) is a species with a scattereddistribution occupying extensive areas in the western Medi-terranean, while fewer and more disjoint populations arepresent in the eastern Mediterranean (Fig. 1). This speciesis well adapted to high-intensity fire regimes (Tapias et al.,2004), as well as dry conditions, and is therefore a species ofchoice for afforestation in Mediterranean areas. Pinus halep-ensis coexists with Pinus brutia in its eastern range (Fig. 1)and the two species can intercross but, as they occupy differ-ent geographical ranges and bioclimates, hybridization israre (Bucci et al., 1998).

In this study, samples were collected from six populationscovering the Aleppo pine natural range, from east to west:Shaharia (Israel), Elea (Greece), Imperia (northern Italy),Aures Beni Melloul (Algeria), Tarrasa (north-eastern Spain)and Zaouia Ifrane (Middle Atlas, Morocco) (Fig. 1). Seeds

were collected in each population from several trees, sepa-rated by at least 50 m to avoid collecting related individu-als, and used to screen chloroplast microsatellites andcandidate genes related to drought resistance. Samples usedfor the two sets of markers were not necessarily collectedfrom the same trees, although they were collected from thesame populations.

DNA extraction, amplification, genotyping

Genomic DNA from megagametophyte – the haploidmaternal tissue surrounding the embryo in conifer seeds –was extracted with the DNeasy Mini Kit (Qiagen). Twelveto twenty-five samples per population (n = 126, cf.Table 1) were used to genotype six chloroplast microsatel-lites markers: Pt15169, Pt26081, Pt36480, Pt63718,Pt71936 and Pt87268 (Vendramin et al., 1996). Polymer-ase chain reaction reactions were performed as follows: the25 ll mix for reaction contained 0.2 mM ⁄ dNTP, 2.5 mM

MgCl2, 0.2 mM of each primer (the forward primer beinglabelled with a fluorescent dye, FAM, HEX or TAMRA(Eurofins MWG Operon, Ebersberg, Germany)), 10· reac-tion buffer (GE Healthcare, Little Chalfont, UK), 25 ngDNA and one unit Taq polymerase (GE Healthcare). The

Fig. 1 Distribution of Pinus halepensis (greyareas and small dots) and Pinus brutia (greyhatched areas) along with the localities of thesix study populations (large circles: Shaharia(Israel), Elea (Greece), Imperia (northernItaly), Aures Beni Melloul (Algeria), Tarrasa(north-eastern Spain), Zaouia Ifrane (MiddleAtlas, Morocco)).

Table 1 Population characteristics and genetic diversity for the four polymorphic chloroplast microsatellites

Population Origin N nh nhp nhe�D2

shði; jÞ He

Elea Greece 17 11 10 7.05 2.85 0.91Shaharia Israel 23 7 3 2.42 0.27 0.61Imperia Italy 12 3 2 1.41 0.29 0.32Tarrasa Spain 25 5 3 1.40 1.54 0.30Aures Beni Melloul Algeria 24 1 0 1 0 0Zaouia Ifrane Morocco 25 1 0 1 0 0

North Africa 49 1 0 1 0 0

Statistics are computed per population and also for the North African populations together.nh, total number of haplotypes; nhp, total number of private haplotypes; nhe, effective number of haplotypes; �D2

shði; jÞ, average distancebetween pairs of individuals; He, unbiased haplotype diversity.

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PCR conditions were 5 min at 95�C, 25 cycles of 1 min at94�C, 1 min 55�C, 1 min 72�C, followed by 8 min at72�C. Genotyping was performed using a 96-capillaryMegaBACE 1000 (GE Healthcare) automatic sequencer.Allele sizes were assessed according to the ET400-R sizestandards (GE Healthcare) using MegaBACE fragment pro-filer version 1.2 (GE Healthcare).

At least eight samples per population (except for locusrd21A-like, see the Supporting Information, Table S1) wereused to sequence 10 candidate genes related to droughttolerance identified on the basis of functional studiesperformed in Pinus taeda and other conifers, or derivedfrom model species such as Arabidopsis thaliana: cpk3(calcium-dependent protein kinase), sod-chl (Cu : Znsuperoxide dismutase, nuclear gene for chloroplast prod-uct), dhn-1 (dehydrin), erd3 (early response to drought 3),Aqua-MIP (aquaporin, membrane intrinsic protein), pal-1(phenylalanine ammonia-lyase 1), lp3-1 (water-stress-induc-ible protein), rd21A-like (cysteine protease similar to rd21Ain Arabidopsis), sams-2 (S-adenosylmethionine synthetase 2)and ug-2_498 (unknown function) (Chang et al., 1996;Richard et al., 2000; see also Gonzalez-Martınez et al.,2006 and references therein). All PCR products werepurified and sequenced from both ends using the DYEnam-ic ET Dye Terminator Kit and an automatic capillarysequencer MegaBACE 1000 (GE HealthCare). Multiplesequence alignments were done using CLUSTALW (Thompsonet al., 1994) and adjusted manually using BIOEDIT (http://www.mbio.ncsu.edu/BioEdit/page2.html).

Statistical analyses

Patterns of neutral genetic diversity at cpSSRs Total num-ber of haplotypes and private haplotypes (nh and nhp,respectively, direct count), effective number of haplotypes(nhe, Kimura & Crow, 1964), average distance between allpairs of individuals according to a microsatellite stepwisemutation model ( �D2

shði; jÞ; Echt et al., 1998; Vendraminet al., 1998), and unbiased haplotype diversity (He, Nei,1978) were computed for each population using our ownspreadsheets. Global FST (Weir & Cockerham, 1984) andglobal RST (Rousset, 1996) among the six populations, andthe pairwise FST estimator were calculated with FSTAT v.2.9.3 (Goudet, 2001). In addition, to define the distribu-tion of genetic diversity among populations without explicita priori definition of population structure, we carried out aspatial analysis of molecular variance (SAMOVA; Dupanloupet al., 2002), which defines groups of populations that aregeographically homogeneous and maximally differentiatedfrom each other. The significance of the variance compo-nents of populations permutated among groups (RCT), ofgenotypes permutated among populations within groups(RSC) and of genotypes permutated among populations andamong groups (RST) was tested by 1000 permutations of

individuals for each of the hierarchical levels. We testedK = 2 to six groups of localities to explore the possiblegenetic structure. The number of groups was selectedaccording to the highest RCT value using the sum of squaredsize differences between haplotypes with 100 simulatedannealing processes (Dupanloup et al., 2002).

Demographic inferences: mismatch distributions atcpSSRs Mismatch distributions (distribution of theobserved number of differences between pairs of haplo-types) were computed using the ARLEQUIN version 3.1 soft-ware (Excoffier et al., 2005). Chloroplast microsatelliteswere coded in a binary fashion, following Navascues et al.(2006). These authors also showed, using extensive numeri-cal simulations, that mismatch distributions based on chlo-roplast microsatellites are sensitive to population growthand therefore suitable to estimate demographic parametersrelated to population expansion (see also Navascues et al.,2009). Then, Rogers & Harpending’s (1992) demographicmodel was fitted on the mismatch distributions followingSchneider & Excoffier (1999; but see also Excoffier et al.,2005). The goodness-of-fit of the demographic model wastested using 10 000 bootstraps. Mismatch distributions(and fitted demographic models) were examined for each ofthe six populations except Morocco and Algeria that lackedgenetic diversity and were fixed for the same haplotype.

Estimators of population-scaled gene diversity at candi-date genes Gene diversity is controlled by the parametertheta (h = 4 Ne l, where Ne is the effective population sizeand l the per generation mutation rate). Several sample-based estimators of theta (h) exist, all based on the site-frequency spectrum of the mutations (SFS), that is, thedistribution of the proportion of sites where the mutant isat frequency x. In our study, four of them were examined,as defined by Watterson (hw, Watterson, 1975), Tajima(hp, Tajima, 1989), Fay & Wu (hH, Fay & Wu, 2000) andZeng et al. (hL, Zeng et al., 2006). These various estimatorsof population-scaled nucleotide diversity were computed toestimate five summary statistics that were used to, first, inferthe demographic history of Aleppo populations (cf. sectionon Demographic inferences: coalescent simulations atcandidate genes), and, second, to perform some neutralitytests aiming at detecting whether some genes were underselection (cf. section on Neutrality tests at candidate genes).The following tests were chosen because, together, theycapture most information contained in the SFS and aretherefore relevant in inferring the evolutionary processesinvolved, either demographic or selective (Zeng et al.,2006): (1) Tajima’s D (Tajima, 1989) compares hp and hw

asking about the occurrence of rare and common variants;(2) Fu’s Fs (Fu, 1997) is based on the haplotype (gene)frequency distribution conditional on the value of hp;(3) Fay and Wu’s H (Fay & Wu, 2000) compares hp and hH




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and takes into consideration the abundance of derived high-frequency variants in comparison with intermediate-fre-quency ones; (4) normalized Fay and Wu’s H (Zeng et al.,2006) is a normalization by the variance of hH and hL; and(5) Zeng et al.’s E (Zeng et al., 2006) compares hw and hL

and addresses the relative abundance of very high-frequencyand very low-frequency variant classes. As Fay and Wu’s Hstatistics as well as Zeng et al.’s E require the use of out-groups, two genes out of our 10 (sams-2 and ug-2_498) weredisregarded, as outgroups were not available for them.Summary statistics were calculated independently for allpopulations except the two North African ones, which werecombined (as they were genetically homogeneous), usingscripts kindly provided by S. E. Ramos-Onsins.

Demographic inferences: coalescent simulations at candi-date genes To further infer the past demography of Aleppopine populations, we simulated numerous demographicscenarios – standard coalescent (i.e. no changes in popula-tion size), exponential growth and several bottleneck models– and examined which one best fitted our empirical data.Our approach was similar to that suggested by Richardset al.’s (2007) in that we used coalescence simulations andsummary statistics to test different demographic hypothesis.To do so, we compared the observed distributions of fivesummary statistics – Tajima’s D, Fu’s Fs, Fay and Wu’s H,normalized Fay and Wu’s H (Hnorm) and Zeng et al.’s E (asdefined in the section Estimators of population-scaled genediversity at candidate genes) – for the 10 candidate geneswith those obtained from the different demographicalmodels using the mlcoalsim multilocus coalescent simulator(Ramos-Onsins and Mitchell-Olds, 2007). Coalescentsimulations were run independently for each populationand for the two North African populations combined (asthey were genetically homogeneous): a standard neutralmodel (SNM) was run first, and when this model was notcompatible with our observed data, which was the case forall populations but the Greek one, different models inwhich population size changed across time were simulated.Because only models consisting of bottlenecks followed byexpansion produced simulated values compatible with theobserved ones, a more intensive search of the space para-meter was done for them. More specifically, times from theend of the bottleneck ranging from 0.0005 to 0.0040 (inunits of 4N0 generations from the present) and bottleneckseverities (i.e. the magnitude of the reduction in populationsize) from 0.0100 to 0.0008, keeping the duration of thebottleneck fixed at 0.0015 as in Heuertz et al. (2006) weretested. To simulate population growth after the bottleneck,a logistic curve fixing the shape parameter, ts, to half thegrowth duration to produce exponential-like growth curveswas used. Ancestral effective population size was assumed tobe similar to the current one (N0). More specific detailsabout simulated bottleneck models can be found in Fig. S1.

Coalescent simulations were performed with recombinationusing the average value of 0.00365 per site for the 10 genesobtained from composite-likelihood methods (LDhat;McVean et al., 2002). Watterson’s estimate of population-scaled nucleotide diversity (hw) was used to control forsequence polymorphism in the simulations. When morethan one model was compatible with the observed data,likelihood values were computed across all genes and allstatistics to obtain an overall likelihood indicating themost compatible model (for details see Ramos-Onsins &Mitchell-Olds, 2007).

Neutrality tests at candidate genes To test whether somecandidate genes were the target of positive selection inP. halepensis populations, five neutrality tests (as defined inthe section Estimators of population-scaled gene diversity atcandidate genes) were computed: Tajima’s D, Fu’s Fs, Fayand Wu’s H, normalized Fay and Wu’s H (Hnorm) andZeng et al.’s E. Although under neutrality the differentpopulation-scaled diversity estimators (h) have the sameexpectation, each of them is sensitive to changes in differentparts of the site-frequency spectrum (SFS), thus providinginformation on the evolutionary forces that have acted onthe genes under study (Zeng et al., 2006). Neutrality testswere performed separately for each population (except forthe two genetically homogeneous North African popula-tions), as the existence of population structure or differencesin demographic history would bias the outcome of thesetests. For each test under the best-fitting demographicscenario in each Aleppo pine population (the standardneutral model with recombination for Greece and variousbottlenecks followed by expansion models for Israel, Spain,Italy and Morocco–Algeria, see the Results section), 10 000simulated values were first generated using mlcoalsim(Ramos-Onsins & Mitchell-Olds, 2007). The significanceof the tests was then obtained by comparing observed valueswith the expected distribution obtained from the coalescentsimulations.

Results

Patterns and distribution of neutral genetic diversity(cpSSRs)

Out of six chloroplast microsatellite loci, two were mono-morphic (Pt3665 and Pt6375); the four polymorphic frag-ments resolved 21 haplotypes across the six populationssampled. The Greek population displayed the highest valuesfor all diversity parameters assessed (nh, nhp, nhe,

�D2shði; jÞ;

and He) and a progressive loss of genetic variation forpopulations located away from Greece was observed: to theeast, Israel showed intermediate levels of diversity, to thewest, Italian and Spanish populations showed reduced levelsof diversity, while North African populations (Morocco

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and Algeria) were fixed for one common haplotype(Table 1).

When looking at the spatial population structure of thechloroplast markers, a high level of differentiation amongpopulations (FST = 0.394 and RST = 0.255) was observed.Pairwise genetic differentiation estimates indicate that thepopulation in Greece is significantly different from all otherpopulations (P = 0.001), and the Israel population is signif-icantly different from those of Algeria and Morocco(P = 0.010) (Table 2). Morocco and Algeria harbour thesame haplotype and are geographically close, thus forming ahomogeneous group. The Spain and Italy populations arenot differentiated with cpSSRs, but they were still treatedseparately because they present quite distinct levels ofnuclear genetic diversity (cf. section on Nucleotide diversityand natural selection at candidate genes). Israel’s populationwas studied on its own because it is geographically separatefrom the western populations and harbours higher levels ofwithin-population genetic diversity (in contrast to the wes-tern populations), suggesting a distinct evolutionary historyfor this population.

The SAMOVA delimited two groups (K = 2): the Greekpopulation and the pool of all the other populations (Israel,Italy, Spain and Algeria-Morocco) for the highest RCT valueof 0.550. In each test of hierarchical structure the values ofthe among-group component (RCT = 0.540 for K = 2) andthat of the among-population and among-group compo-nent (RST = 0.550) are similar and much higher than thatof the among-population within-groups component(RSC = 0.002). These results highlight that populationsfrom Greece are genetically alike and, at the same time,genetically distant from Greece.

The results from neutral cpSSRs point out to differentgroups of genetically distinct populations with a contrastingevolutionary history: the outlier population (Greece), whichis genetically distinct from all other populations and dis-plays a high genetic diversity, the eastern population (Israel)with an intermediate level of diversity, the western popula-tions (Italy and Spain) with reduced levels of diversity, andthe North African group (Algeria and Morocco), which isgenetically depleted.

Demographic inferences

The wavy pattern of the cpSSR-based mismatch distribu-tion for the Greek population indicated a stable demogra-phy (Fig. 2). Moreover, Rogers & Harpending’s model ofpopulation expansion was rejected for this region (P(simu-lated ssd ‡ Observed ssd): 0.033). By contrast, Israel, Spainand Italy showed a unimodal mismatch distribution(mismatch observed mean of 0.917, 0.967 and 0.667, withP-values for the Rogers and Harpending’s model of 0.475,0.757 and 0.749, respectively) that fitted into a pattern ofpopulations undergoing recent range expansion (Fig. 2).Mismatch distributions for Morocco–Algeria could notbe computed as these populations are fixed for a singlehaplotype.

Gene-based summary statistics (Tajima’s D, Fu’s Fs, Fayand Wu’s H, Fay and Wu’s Hnorm and Zeng et al.’s E) alsorevealed a marked contrast among populations (Table 3).Greece, in particular, displayed extreme values (more posi-tive for H and Hnorm and less positive for Fs and E), whichsuggests a distinct evolutionary history for this population.Based on these results, and those from the cpSSR analyses,we examined various demographic models for Greece,Israel, Italy, Spain and Morocco–Algeria separately,although those of Spain and Morocco–Algeria are notdiscussed in detail because of the low power, owing to thelack of variation (only four and five polymorphic genesrespectively, with an average of 1.6 segregating sites pergene for both) to fit the models for these populations(Fig. S2).

For the Greek population, the observed pattern of poly-morphism was compatible with the standard coalescencemodel for all five summary statistics (probability thatsimulated values be smaller than or equal to the observedvalues: D = 0.736, Fs = 0.694, H = 0.224, Hnorm = 0.138and E = 0.902), suggesting an ancient population with a

Table 2 Pairwise FST in Pinus halepensis populations based on sixchloroplast microsatellites

Greece Israel Italy Spain Algeria Morocco

Greece –Israel 0.38*** –Italy 0.43*** 0.04 –Spain 0.51*** 0.05 )0.01 –Algeria 0.57*** 0.14** 0.06 0.06 –Morocco 0.58*** 0.15** 0.07 0.06 0.00 –

Probability obtained after 15 000 permutations and followingstandard Bonferroni corrections: **P = 0.01; ***P = 0.001.

Fig. 2 Chloroplast microsatellite mismatch distributions for Greece(closed symbols), Israel (open symbols), Spain (light tinted symbols)and Italy (dark tinted symbols).




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stable demography, in agreement with our interpretation ofmismatch distributions based on cpSSRs for this population.For the rest of the range of Aleppo pine, the standard coales-cence model and the different population growth modelscould be rejected because of the observed negative values ofH and Hnorm summary statistics (except for Spain, for whichthe rejection was based on the observed negative value of D),which contrast with the close-to-zero or positive valuesalways obtained in the simulations (data not shown).

Among the 20 different bottleneck models tested, thefive summary statistics for Israel were compatible with 12scenarios. Likelihood values indicate an ancient (t = 0.0040)bottleneck of weak intensity (severity of 0.0100) as the mostprobable scenario. By contrast, the five summary statisticsfor Italy were compatible with only four scenarios of a

relatively recent bottleneck (t = 0.0018 and 0.0020) ofstrong intensity (severity of 0.0010 and 0.0008). Likelihoodvalues point to the most recent scenario as the most proba-ble one (t = 0.0018 and severity of 0.0010) (Fig. 3).

Nucleotide diversity and natural selection at candidategenes

Out of the 10 candidate genes, one was monomorphicacross all populations (sams-2) while another was mono-morphic in all populations except Greece (ug-2_498).Thus, nucleotide diversity parameters could not be com-puted for these two genes (Table S1). Similar generalpatterns of polymorphism were found for nuclear genes andchloroplast microsatellites: the number of segregating sites

Table 3 Neutrality tests (against best-fitting demographic scenarios) for the 10 drought-response nuclear candidate genes

Population Gene

Neutrality testa

D Fs H Hnorm E

Greece cpk3 0.820 0.818 na na nasod-chl na na na na nadhn-1 0.330 0.356 1.867 1.035 )0.778erd3 )0.432 )0.363 1.333 0.819 )1.092Aqua-MIP )0.839 )0.329 )4.500*** )4.045*** 3.445pal-1 0.384 )0.197 )0.357 )0.321 0.534lp3-1 0.986 0.849 0.250 0.540 0.026rd21A-like 0.709 2.608 )1.200 )0.534 1.124sams-2 na na na na naug-2_498 )0.689 0.736 na na naAverage 0.159 0.560 )0.435 )0.418 0.543

Israel cpk3 na na na na nasod-chl 1.167 0.866 na na nadhn-1 )1.471* 0.553* )2.571 )1.267 0.243erd3 )1.069 0.009* 0.833 0.567 )1.258**Aqua-MIP 1.948 3.276 0.083 0.092 1.054pal-1 na na na na nalp3-1 )1.310 0.762 )3.000** )4.242** 3.138rd21A-like 1.799 5.621 )1.500 )0.667 1.826sams-2 na na na na naug-2_498 na na na na naAverage 0.177 1.848 )1.231 )1.103 1.000

Italy cpk3 )1.362 0.671 )1.361* )1.950 0.994sod-chl 0.334 0.536 na na nadhn-1 )1.595 2.407 )2.357* )1.811 0.739erd3 0.446 )1.298*** 1.667 1.011 )0.683Aqua-MIP )0.654 )0.133 0.361 0.281 )0.687pal-1 1.371 3.003 )0.750 )0.829 1.559lp3-1 )1.310 0.762 )3.000** )4.242** 3.138rd21A-like 1.573 2.429 )0.900* )0.950 1.659sams-2 na na na na naug-2_498 na na na na naAverage )0.150 1.047 )0.906 )1.213 0.960

Summary statistics are given by gene and for the averaged loci, and were computed for Greece, Israel and Italy for which reliable demographicinferences have been obtained.D, Tajima’s D (1989); Fs, Fu’s Fs (Fu, 1997); H, Fay and Wu’s H (2000); Hnorm, normalized Fay and Wu’s H (Zeng et al., 2006); E, Zeng et al.’sE (2006); na, not available or undefined (not enough segregating sites or no outgroup sequence available).aP-values for neutrality tests were obtained by comparison of observed values with those obtained from coalescent simulations under best-fitting demographic scenarios in each range (see text for details): *, 0.05 £ P £ 0.10; **, 0.01 £ P £ 0.05; ***, P £ 0.01.

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0.0040na

0.0030

0.0020

0.0018

0.0005

0.0008 0.0010 0.0025 0.0100Severity

Tim

ing

IsraelTajima's D

0.0040na

0.0030

0.0020

0.0018

0.0005

0.0008 0.0010 0.0025 0.0100Severity

Tim

ing

Fu's S

0.0040na

0.0030

0.0020

0.0018

0.0005

Fay and Wu's H

0.0008 0.0010 0.0025 0.0100Severity

Tim

ing

0.0040na

0.0030

0.0020

0.0018

0.0005

Fay and Wu's H norm

0.0008 0.0010 0.0025 0.0100Severity

Tim

ing

0.0040na

0.0030

0.0020

0.0018

0.0005

Zeng et al.'s E

0.0008 0.0010 0.0025 0.0100Severity

Tim

ing

Overall likelihood values

0.0040na –221.82 –171.15

–172.44–279.74

–273.10 –249.05 –181.01 –174.15

–179.38–248.80–267.97

–241.60

0.0030

0.0020

0.0018

0.0005

0.0008 0.0010 0.0025 0.0100Severity

Tim

ing

na

Tim

ing

ItalyTajima's D

0.0008 0.0010 0.0025 0.0100Severity

0.0040

0.0030

0.0020

0.0018

0.0005

na

Fu's S

0.0008 0.0010 0.0025 0.0100Severity

Tim

ing

0.0040

0.0030

0.0020

0.0018

0.0005

na

Fay and Wu's H

0.0008 0.0010 0.0025 0.0100Severity

Tim

ing

0.0040

0.0030

0.0020

0.0018

0.0005

na

Fay and Wu's H norm

0.0008 0.0010 0.0025 0.0100Severity

Tim

ing

0.0040

0.0030

0.0020

0.0018

0.0005

na

Zeng et al.'s E

0.0008 0.0010 0.0025 0.0100Severity

Tim

ing

0.0040

0.0030

0.0020

0.0018

0.0005

overall likelihood values

na

–418.79 –378.27

–409.51 –371.20

0.0008 0.0010 0.0025 0.0100Severity

Tim

ing

0.0040

0.0030

0.0020

0.0018

0.0005

(a)

(b)

Fig. 3 Probability of rejection of different population bottleneck models for Israel and Italy based on Tajima’s D, Fu’s Fs, Fay and Wu’s H,normalized Fay and Wu’s H (Hnorm), and Zeng et al.’s E summary statistics. Incompatible models at P = 0.05 are indicated in by dark tint,incompatible models at P = 0.1 are indicated in light tint, and compatible models across all five summary statistics are indicated by a black dot.Highest values of overall likelihood (bottom right diagram) indicate the best-fitting model (see text for details) and are indicated with a doubleframe. On the y-axis is the time of the end of a bottleneck in units of 4N0 generations. On the x-axis is the severity of the bottleneck (i.e. themagnitude of the reduction in population size) in units of N0.




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(S) and different nucleotide diversity estimators (hp, hw, hH

and hL) were highest in the Greek population, while thosefor Israel and Italy were intermediate and those for NorthAfrican and Iberian populations were the lowest (Fig. 4a).

For the Greek population, summary statistics (H andHnorm) revealed only one gene (Aqua-MIP, Table 3) thatdeparted from expectations under the standard coalescencemodel (which, in this case, is the best-fitting demographicscenario). By contrast, summary statistics for the otherpopulations detected many more genes that departed fromexpectations under the best-fitting bottleneck models, asillustrated by Israel and Italy (Table 3), suggesting theaction of natural selection.

When looking at the polymorphism pattern of the geneswith putative imprints of natural selection, a common trendwas observed: for most genes, various haplotypes that werepresent in Greece were only present in the other popula-tions in the form of a reduced subset, one of these haplo-types (or a closely derived one) often being fixed in thepopulations furthest away from Greece. Interestingly, thehaplotype that was generally fixed in these populations wasnot, as could have been expected, an ancestral one butpresented a substantial number of derived mutations (seeexample of cpk3 in Fig. 4b): out of six genes for which wecould determine ancestral and derived states, four were fixed

for a derived haplotype in Spain (cpk3, pal-1, rd21A-likeand dhn-1), Morocco (cpk3, pal-1, rd21A-like and lp3-1)and Algeria (cpk3, pal-1, rd21A-like and dhn-1), and twowere fixed in Israel (cpk3 and pal-1). When they were notfixed, the derived haplotypes in these populations (includ-ing those for the two other genes, Aqua-MIP and lp3-1)were the most frequent in all but two cases. This resultsuggests the action of natural selection in the Aleppo pinepopulations with distinct outcomes in Greece as opposed toall other populations.

Discussion

The main finding of our study is that Aleppo pine showscontrasting patterns of polymorphism at neutral andfunctional loci in eastern vs western populations, the likelyconsequence of genetic drift and adaptation to new environ-ments in its western range. Our results highlight the impor-tance of colonization processes in evolution, not only inthat they shape the spatial genetic pattern of current popula-tions but also in that migration interacts with demographicprocesses and selection, all eventually affecting the potentialfor population adaptation. Moreover, the spatial character-ization of patterns of polymorphism in Aleppo pine popula-tions identified zones with distinctive evolutionary histories,

P.taeda

(a)

(b)

Fig. 4 (a) Genetic diversity of Pinus halepen-

sis for nuclear genes plotted according toeach population: segregating sites (S) areplotted on the left y-axis, while Tajima’s (hp),Watterson’s (hw), Fay and Wu’s (hH) andZeng et al.’s (hL) population-scaled nucleo-tide diversity estimators are plotted on theright y-axis. (b) Neighbour joining tree ofcpk3 ancestral and derived haplotypes withPinus taeda used as outgroup. The pie chartscorrespond to the haplotype distribution foreach sampled population.

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information that is critical for the management of thisconifer across its wide range.

The clear genetic differentiation of the Greek population,and the loss of genetic diversity away from this population,combined with the demographic scenarios inferred fromcpSSRs and nuclear gene sequences, point towards olderand more stable populations in the east side of the Mediter-ranean Basin. In the western and probably more recentlycolonized area, reliable demographic inferences wereobtained only for Italy, which presents the population withthe highest level of variation. The pattern of polymorphismin Italy is in agreement with a recent (t = 0.0018) andstrong (f = 0.0010) bottleneck (Fig. 3). If we consider aneffective population size for pine of c. 100 000 (Willyardet al., 2007) and a generation time of 25 yr (as in Brownet al., 2004 and in Willyard et al., 2007), the time estimatefor the bottleneck compatible with our empirical data forItaly translates into 18 000 yr before present (BP), corre-sponding roughly to the time of the last glacial maximum(LGM) in Europe.

Although time estimates should be taken with caution, asthey are derived from demographic models fitted using arelatively low number of genes and are based on approxi-mate biological information of the species, the inferreddemographic scenario for Aleppo pine – ancient and stablerelictual Greek population and expanding western Mediter-ranean Basin populations around the LGM – is consistentwith previous genetic analyses made at the species distribu-tion level (Bucci et al., 1998; Morgante et al., 1998; Gomezet al., 2001), as well as with some flavonoid data (Barberoet al., 1998) and palaeoecological and fossil records thatsuggest a relatively recent expansion of the species west-wards within the last 10 000 yr (Nahal, 1962; Schilleret al., 1986; Pons, 1992). Thus, the indicative timescaleinferred for Aleppo pine bottlenecks for western popula-tions could correspond to a period situated just after theLGM (25 000–18 000 yr BP). This time-frame is muchmore recent than that estimated for cold-tolerant conifersso far: multilocus patterns of 22 gene loci in Picea abies werefound to be compatible with a severe bottleneck occurringsome several hundreds of thousands of years ago (Heuertzet al., 2006), while those of 16 loci in Pinus sylvestrismatched well an ancient bottleneck taking place c. 2 millionyr ago (Pyhajarvi et al., 2007). Our capacity to detect bot-tlenecks in the Mediterranean Aleppo pine could be con-nected to a high intensity of founder events combined witha recent and ⁄ or rapid colonization process from a limitednumber of refugia, together leading to a reduced geneticvariability in the expanding western populations. Bycontrast, bottlenecks for cold-tolerant conifers would havebeen of weaker intensity, and recolonization might havetaken place from lineages coming from multiple refugia (assuggested, for example, by mtDNA studies in Pinus sylves-tris, Soranzo et al., 2000), which would make the detection

of the signatures of demographic events on the polymor-phism patterns found in present-day populations moredifficult (Pyhajarvi et al., 2007).

Identifying the evolutionary forces that may have leftimprints on a species’ patterns of polymorphism and howthey influence adaptive evolution is complex because theyinteract with each other in multiple ways. Gene flow(or migration) can have two opposite effects on selection(Lenormand, 2002; Stockwell et al., 2003; Garant et al.,2007): in some cases it counteracts inbreeding depressionby increasing genetic variation within populations thusincreasing evolutionary potential (Hedrick, 1995; Ebertet al., 2002; Alleaume-Benharira et al., 2006), whereas inother cases it tends to oppose the effect of selection by intro-ducing (as well as maintaining by recurrent immigration)foreign genes to locally well-adapted populations, thereforelimiting local adaptation (Stearns and Sage, 1980; Riechert,1993; Tufto, 2001) and constraining adaptive divergence(Hendry & Taylor, 2004; Nosil & Crespi, 2004). Demo-graphic processes such as population bottlenecks and foun-der effects will lead to a reduced overall genetic diversitywithin the populations, which will limit the effectiveness ofselection and may cause deviations of mean phenotype fromthe local optimum leading to maladapted genotypes (Neiet al., 1975; Novella et al., 1995). In the case of Aleppopine, different scenarios can be considered to explain thedata following the bottleneck events:(1) Most of the genotypes would have contributed to theexpanding populations, and natural selection would havefavoured a wide range of well adapted genotypes, depend-ing on environmental conditions, from the original coloni-zation source. However, the overall reduction of geneticdiversity in expanding populations showed by both neutraland functional markers is incompatible with such ascenario.(2) A reduced number of genotypes selected at randomand with no particular selective advantage would have beenspread across the species range, and would have survivedunder various conditions, given the plasticity of P. halepen-sis. This scenario, however, does not explain why mostgenes display derived mutations in expanding areas.(3) Selection through interspecific and intraspecific com-petition and ⁄ or contrasted environmental conditions wouldhave favoured some genotypes now associated with areduced overall genetic variability in some populations ofthe expanding areas.

This last scenario would explain both the reduced level ofneutral and functional diversity (drift depends only onpopulation demography and affects all genes equally) as wellas the distinct mutations selected across genes in expansionversus core populations. Frequent recurrent forest fires,which play a major role in regulating population dynamicsin this species (Barbero et al., 1998; Tapias et al., 2004),could have contributed to maintaining this pattern of




New Phytologist (2009) 184: 1016–1028


selection and bottlenecks. The evolutionary outcomes ofcolonization depend greatly on the biological system con-sidered and on the interactions among the evolutionaryforces in place, leading from local extinction to reducedresponse to selection (Pujol & Pannell, 2008) to adaptation(Rosenblum et al., 2007). Here we propose that the currentobserved genetic pattern in Aleppo pine resulted from bothgenetic drift and selection.

Although Aleppo pine is a particularly plastic species, asshown for example by the different levels of resistance topests, frost and low water potentials or the reproductivefeatures displayed by phenotypic changes across differentenvironmental conditions (Chambel et al., 2007), theinfluence of selective forces on its adaptive potential shouldnot be underestimated. For example, common-garden testshave shown that total precipitation and dry season durationare important selective factors for water-use efficiency inAleppo pine provenances spanning the entire species range.These results highlight the selective role of climate variablesin determining intraspecific fitness in this species (Voltaset al., 2008; see also Climent et al., 2008 for reproductivefeatures in P. halepensis). In tree species, the role of naturalselection in promoting local adaptation has been largelydemonstrated in common garden experiments (Eveno et al.,2008).

The present study, in addition to providing insights intothe past demography of Aleppo pine, represents a first stepin the characterization of populations for genes potentiallylinked to drought tolerance across an entire species’ range.Some of the candidate genes that depart from expectationsunder the best-fitting demographic models have well-estab-lished functions. Aquaporins (Aqua-MIP) are membranepore proteins (i.e. water channels) that play a critical role incontrolling the water content of cells. Dehydrins (dhn-1),for their part, have a major role in cell protection againstdesiccation (via stabilization of endomembrane structures,metal-binding activity and protection from oxidative dam-age). Remarkably, some of the genes found under selectionin this study (erd3, dhn-1, lp3-1) are overexpressed underdrought-stress conditions and have been reported to beunder selection in other pine species, such as Pinus taeda(erd3; Gonzalez-Martınez et al., 2006) and Pinus pinaster(erd3, dhn-1, lp3-1; Eveno et al., 2008). They thus consti-tute relevant candidates to develop future drought-toleranceand adaptation studies in Aleppo pine, and probably otherconifers.

From a practical point of view, our work points to dif-ferent evolutionary and demographically independentunits. One of them, represented by the Greek population,encompasses most of the neutral and functional variationfound in this study and constitutes, therefore, an importanttarget for conservation purposes. Moreover, the Aleppopine forests of Greece and the Middle East grow in morearid conditions than those of the western Mediterranean

(Barbero et al., 1998; Arianoutsou & Ne’eman, 2000) andGreek provenances stand out from the others for variousadaptive features (e.g. reproductive allocation, ontogeny;cf. Climent et al., 2008 and references therein), pointingto these populations as a potential source material thatshould be selected not only for its adaptation to currentenvironmental conditions, but also because it will be ableto cope with future, more arid and hot conditions. Ulti-mately, causal inferences on the genetic basis of adaptation,that is, the links between genotypes and phenotypes, willhave to be assessed by association studies or complementa-tion and transgenic analyses (Storz, 2005; Wright & Gaut,2005).

Acknowledgements

We thank Sebastian E. Ramos-Onsins for kindly providingthe scripts used to compute various diversity and summarystatistics, Ricardo Alıa and Jose Climent for useful insightson Aleppo pine life-history and quantitative genetics, aswell as three anonymous reviewers and the Editor forconstructive comments on a first version of this manuscript.Thanks are extended to Patricia C. Grant for Englishlanguage review. The EUFORGEN program (BioversityInternational) kindly provided the distribution map used inFig. 1. This research was funded by the European Commis-sion, EVOLTREE Network of Excellence (http://www.evoltree.eu), the Collaborative Project on ‘Conserva-tion of Forest Genetic Resources’ between the SpanishMinistry of Environment and INIA AEG06-054 andProject CGL2008-05289-C02-02 ⁄ BOS (VaMPiro) fromthe Spanish Ministry of Science and Innovation.

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Supporting Information

Additional supporting information may be found in theonline version of this article.

Fig. S1 Details of the simulated bottleneck models.

Fig. S2 Probability of rejection of different populationbottleneck models for Spain and Morocco–Algeria.

Table S1 Nucleotide diversity for the 10 drought-responsenuclear candidate genes. Summary statistics are given bygene and for the averaged loci, and were computed for eachpopulation separately and for all populations together

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Patterns of polymorphism resulting from long-range colonization in the Mediterranean conifer Aleppo...

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