Patterns of polymorphism resulting from long-range colonization in the Mediterranean conifer Aleppo pine Delphine Grivet1*, Federico Sebastiani2,3*, Santiago C. Gonzalez-Mart��nez1 �� �� and Giovanni G. Vendramin3 1 Department of Forest Systems and Resources, Forest Research Institute, CIFOR-INIA, Carretera de la Coruna �� km 7.5, ES���28040 Madrid, Spain 2 Department of Agricultural Biotechnology, Genexpress, University of Florence, Via della Lastruccia 14, I���50019 Sesto Fiorentino (FI), Italy 3 Plant 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-Mart�� �� ��nez Tel: +34 913471499 Email: santiago@inia.es Received: 16 April 2009 Accepted: 12 July 2009 New Phytologist (2009) 184: 1016���1028 doi: 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 interact during colonization and affect the adaptive evolution of a given species is a question of central importance in evolu- tionary and conservation genetics that remains a daunting challenge. For example, it is expected that populations that have been through bottlenecks or founder effects during colonization will exhibit genetic erosion (Nei et al., 1975 Hewitt, 2000) also, declining genetic diversity with increas- ing distance from source population has been reported in several study cases (Grivet and Petit, 2003 Ramachandran et al., 2005 Eckert et al., 2008). However, that does not necessarily mean that expanding populations are genetically depleted or that refugial populations always harbour the highest 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 be confronted with selection and adaptation to different envi- ronments as well as to new conspecific and heterospecific interactions (Hewitt, 2000). Whether the mechanisms that generate different genetic patterns in expansion and refugial core populations are able to influence the adaptive potential of a given species will depend on the interplay among all the 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 in contrasting environments, growing on all substrates and almost all bioclimates of the Mediterranean region, thus reflecting 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 ��� GenBank Data Libraries under accession numbers FJ588494���FJ588503. New Phytologist Research 1016 New Phytologist (2009) 184: 1016���1028 www.newphytologist.org �� The Authors (2009) Journal compilation �� New Phytologist (2009)

have shown low levels of genetic diversity both in neutral molecular 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 species appears to be structured along a longitudinal gradient, with eastern Mediterranean populations harbouring greater diversity and more ancient lineages than western ones (Bucci et al., 1998 Morgante et al., 1998 Gomez �� et al., 2001). These genetic results, combined with common garden experiments pointing to Greece as an outlier (Climent et al., 2008), data on flavonoid content (Barbero �� et al., 1998) and few available palynological and dendro- archaeological records (Schiller et al., 1986 Pons, 1992), suggest a scenario in which Aleppo pine populations expanded westwards all around the Mediterranean basin from some refugia located in the southern part of the Balkan Peninsula. This particular population dynamics of Aleppo pine (i.e. long-range colonization probably accom- panied by recurrent contractions���expansions caused, for example, by forest fires), combined with its current wide ecological range and scattered distribution across the Medi- terranean basin, makes this species especially suitable to study the impact of demographic and genetic processes, as well as their interactions, on the adaptive potential of tree populations. 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 the evolutionary forces at the origin of adaptation, and how these forces shape the spatial���temporal pattern of variability within and between populations. Indeed, all loci of a gen- ome will be affected similarly by demographic processes while only a subset of them will be influenced by selection (but see Begun et al., 2007��� the first population genetics study based on whole-genome analysis) and depart from the average loci. Thus, looking at outlier loci among an exten- sive number of markers allows detection of loci subject to selection (see Namroud et al., 2008 for a recent example in a conifer). In nonmodel species with complex demographi- cal history (i.e. not fitting the simple models often assumed in outlier detection methods), such as Aleppo pine, for which 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, such as chloroplast or nuclear microsatellites, which are theoreti- cally affected by demographic history but not by selection, and from candidate genes coding for known biological functions, potentially correlated with adaptive traits, such as phenology (Heuertz et al., 2006 Pyhajarvi �� �� et al., 2007), drought tolerance (Gonzalez-Mart��nez �� �� 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 facing specific environmental constraints, among them drought. Aleppo pine is well adapted to drought, although this factor can still constitute an important threat to individual growth and survival at all life stages (Sathyan et al., 2005). In the context of climate change in the Mediterranean area, and its consequences on the adaptive potential of Mediterranean vegetation, it is essential to understand the molecular bases underlying adaptation of populations along colonization gradients and how they are affected by population expan- sions and contractions. Moreover, from a practical point of view, identifying populations harbouring the genotypes best adapted for drought is essential not only for afforestation in current zones of extreme climatic conditions, such as in Israel (Grunwald et al., 1986) and in Morocco (Boulli et al., 2001) in the case of P. halepensis, but also in the light of future climatic changes, as most of the Mediterranean area 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 its populations may be differentially adapted to local climatic conditions and may show different capacities to survive water-deficit stress (WDS). This view is reinforced by various studies showing differences in drought tolerance among provenances using ecophysiological data and gene expression (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 secondary compound production) have probably been main targets for natural selection in Aleppo pine and therefore constitute relevant candidates for testing the effects of long-range colonization and environment heterogeneity on adaptive patterns 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 a longitudinal gradient. Different methods based on cpSSRs (F-statistics, SAMOVA and mismatch distributions) were used to investigate the geographic structure of diversity and to infer 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 functional nucleotide variation across Aleppo pine range and (2) infer the 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 cpSSR analyses) to determine which one best fitted our empirical data and (3) examined whether different selective pressures for drought-response candidate genes exist along the longi- tudinal gradient sampled (covering putative refugial and expansion zones) by comparing the patterns of nucleo- tide variation observed at each locus with that expected under best-fitting demographic models. Overall, this study New Phytologist Research 1017 �� The Authors (2009) Journal compilation �� New Phytologist (2009) New Phytologist (2009) 184: 1016���1028 www.newphytologist.org