Common microgeographical selection patterns revealed in four European conifers.

Microgeographical adaptation occurs when the effects of directional selection persist despite gene flow. Traits and genetic loci under selection can then show adaptive divergence, against the backdrop of little differentiation at other traits or loci. How common such events are and how strong the selection is that underlies them remain open questions. Here, we discovered and analysed microgeographical patterns of genomic divergence in four European and Mediterranean conifers with widely differing life-history traits and ecological requirements (Abies alba MIll., Cedrus atlantica [Endl.] Manetti, Pinus halepensis Mill. and Pinus pinaster Aiton) by screening pairs from geographically close forest stands sampled along steep ecological gradients. We inferred patterns of genomic divergence by applying a combination of divergence outlier detection methods, demographic modelling, Approximate Bayesian Computation inferences and genomic annotation to genomic data. Surprisingly for such small geographical scales, we showed that selection is strong in all species but generally affects different loci in each. A clear signature of selection was systematically detected on a fraction of the genome, of the order of 0.1%-1% of the loci depending on the species. The novel modelling method we designed for estimating selection coefficients showed that the microgeographical selection coefficient scaled by population size (Ns) was 2-30. Our results convincingly suggest that selection maintains within-population diversity at microgeographical scales in spatially heterogeneous environments. Such genetic diversity is likely to be a major reservoir of adaptive potential, helping populations to adapt under fluctuating environmental conditions.

Polygenic adaptation and negative selection across traits, years and environments in a long-lived plant species (Pinus pinaster Ait., Pinaceae).

A decade of genetic association studies in multiple organisms suggests that most complex traits are polygenic; that is, they have a genetic architecture determined by numerous loci, each with small effect-size. Thus, determining the degree of polygenicity and its variation across traits, environments and time is crucial to understand the genetic basis of phenotypic variation. We applied multilocus approaches to estimate the degree of polygenicity of fitness-related traits in a long-lived plant (Pinus pinaster Ait., maritime pine) and to analyse this variation across environments and years. We evaluated five categories of fitness-related traits (survival, height, phenology, functional, and biotic-stress response) in a clonal common-garden network planted in contrasted environments (over 20,500 trees). Most of the analysed traits showed evidence of local adaptation based on Qst -Fst comparisons. We further observed a remarkably stable degree of polygenicity, averaging 6% (range of 0%-27%), across traits, environments and years. We detected evidence of negative selection, which could explain, at least partially, the high degree of polygenicity. Because polygenic adaptation can occur rapidly, our results suggest that current predictions on the capacity of natural forest tree populations to adapt to new environments should be revised, especially in the current context of climate change.

A draft genome of sweet cherry (Prunus avium L.) reveals genome-wide and local effects of domestication.

Sweet cherry (Prunus avium L.) trees are both economically important fruit crops but also important components of natural forest ecosystems in Europe, Asia and Africa. Wild and domesticated trees currently coexist in the same geographic areas with important questions arising on their historical relationships. Little is known about the effects of the domestication process on the evolution of the sweet cherry genome. We assembled and annotated the genome of the cultivated variety "Big Star*" and assessed the genetic diversity among 97 sweet cherry accessions representing three different stages in the domestication and breeding process (wild trees, landraces and modern varieties). The genetic diversity analysis revealed significant genome-wide losses of variation among the three stages and supports a clear distinction between wild and domesticated trees, with only limited gene flow being detected between wild trees and domesticated landraces. We identified 11 domestication sweeps and five breeding sweeps covering, respectively, 11.0 and 2.4 Mb of the P. avium genome. A considerable fraction of the domestication sweeps overlaps with those detected in the related species, Prunus persica (peach), indicating that artificial selection during domestication may have acted independently on the same regions and genes in the two species. We detected 104 candidate genes in sweep regions involved in different processes, such as the determination of fruit texture, the regulation of flowering and fruit ripening and the resistance to pathogens. The signatures of selection identified will enable future evolutionary studies and provide a valuable resource for genetic improvement and conservation programs in sweet cherry.

Evolutionary history of the mediterranean Pinus halepensis-brutia species complex using gene-resequencing and transcriptomic approaches.

KEY MESSAGE: Complementary gene-resequencing and transcriptomic approaches reveal contrasted evolutionary histories in a species complex. Pinus halepensis and Pinus brutia are closely related species that can intercross, but occupy different geographical ranges and bioclimates. To study the evolution of this species complex and to provide genomic resources for further research, we produce and analyze two new complementary sets of genetic resources: (i) a set of 172 re-sequenced genomic target loci analyzed in 45 individuals, and (ii) a set of 11 transcriptome assemblies. These two datasets provide insights congruent with previous studies: P. brutia displays high level of genetic diversity and no genetic sub-structure, while P. halepensis shows three main genetic clusters, the western Mediterranean and North African clusters displaying much lower genetic diversity than the eastern Mediterranean cluster, the latter cluster having similar genetic diversity to P. brutia. In addition, these datasets provide new insights on the timing of the species-complex history: the two species would have split at the end of the tertiary, and the changing climatic conditions of the Mediterranean region at the end of the Tertiary-beginning of the Quaternary, together with the distinct species tolerance to harsh climatic conditions would have resulted in different geographic distributions, demographic histories and genetic patterns of the two pines. The multiple glacial-interglacial cycles during the Quaternary would have led to the expansion of P. brutia in the Middle East, while P. halepensis would have been through bottlenecks. The last glaciations, from 0.6 Mya on, would have affected further the Western genetic pool of P. halepensis.

Genomic and phenotypic divergence unveil microgeographic adaptation in the Amazonian hyperdominant tree Eperua falcata Aubl. (Fabaceae).

Plant populations can undergo very localized adaptation, allowing widely distributed populations to adapt to divergent habitats in spite of recurrent gene flow. Neotropical trees-whose large and undisturbed populations often span a variety of environmental conditions and local habitats-are particularly good models to study this process. Here, we explore patterns of adaptive divergence from large (i.e., regional) to small (i.e., microgeographic) spatial scales in the hyperdominant Amazonian tree Eperua falcata Aubl. (Fabaceae) under a replicated design involving two microhabitats (~300 m apart) in two study sites (~300 km apart). A three-year reciprocal transplant illustrates that, beyond strong maternal effects and phenotypic plasticity, genetically driven divergence in seedling growth and leaf traits was detected both between seedlings originating from different regions, and between seedlings from different microhabitats. In parallel, a complementary genome scan for selection was carried out through whole-genome sequencing of tree population pools. A set of 290 divergence outlier SNPs was detected at the regional scale (between study sites), while 185 SNPs located in the vicinity of 106 protein-coding genes were detected as replicated outliers between microhabitats within regions. Outlier-surrounding genomic regions are involved in a variety of physiological processes, including plant responses to stress (e.g., oxidative stress, hypoxia and metal toxicity) and biotic interactions. Together with evidence of microgeographic divergence in functional traits, the discovery of genomic candidates for microgeographic adaptive divergence represents a promising advance in our understanding of local adaptation, which probably operates across multiple spatial scales and underpins divergence and diversification in Neotropical trees.

Fine-scale spatial genetic structure across the species range reflects recent colonization of high elevation habitats in silver fir (Abies alba Mill.).

Variation in genetic diversity across species ranges has long been recognized as highly informative for assessing populations' resilience and adaptive potential. The spatial distribution of genetic diversity within populations, referred to as fine-scale spatial genetic structure (FSGS), also carries information about recent demographic changes, yet it has rarely been connected to range scale processes. We studied eight silver fir (Abies alba Mill.) population pairs (sites), growing at high and low elevations, representative of the main genetic lineages of the species. A total of 1,368 adult trees and 540 seedlings were genotyped using 137 and 116 single nucleotide polymorphisms (SNPs), respectively. Sites revealed a clear east-west isolation-by-distance pattern consistent with the post-glacial colonization history of the species. Genetic differentiation among sites (FCT = 0.148) was an order of magnitude greater than between elevations within sites (FSC = 0.031), nevertheless high elevation populations consistently exhibited a stronger FSGS. Structural equation modelling revealed that elevation and, to a lesser extent, post-glacial colonization history, but not climatic and habitat variables, were the best predictors of FSGS across populations. These results suggest that high elevation habitats have been colonized more recently across the species range. Additionally, paternity analysis revealed a high reproductive skew among adults and a stronger FSGS in seedlings than in adults, suggesting that FSGS may conserve the signature of demographic changes for several generations. Our results emphasize that spatial patterns of genetic diversity within populations provide information about demographic history complementary to non-spatial statistics, and could be used for genetic diversity monitoring, especially in forest trees.

Genetic signatures of divergent selection in European beech (Fagus sylvatica L.) are associated with the variation in temperature and precipitation across its distribution range.

High genetic variation and extensive gene flow may help forest trees with adapting to ongoing climate change, yet the genetic bases underlying their adaptive potential remain largely unknown. We investigated range-wide patterns of potentially adaptive genetic variation in 64 populations of European beech (Fagus sylvatica L.) using 270 SNPs from 139 candidate genes involved either in phenology or in stress responses. We inferred neutral genetic structure and processes (drift and gene flow) and performed differentiation outlier analyses and gene-environment association (GEA) analyses to detect signatures of divergent selection. Beech range-wide genetic structure was consistent with the species' previously identified postglacial expansion scenario and recolonization routes. Populations showed high diversity and low differentiation along the major expansion routes. A total of 52 loci were found to be putatively under selection and 15 of them turned up in multiple GEA analyses. Temperature and precipitation related variables were equally represented in significant genotype-climate associations. Signatures of divergent selection were detected in the same proportion for stress response and phenology-related genes. The range-wide adaptive genetic structure of beech appears highly integrated, suggesting a balanced contribution of phenology and stress-related genes to local adaptation, and of temperature and precipitation regimes to genetic clines. Our results imply a best-case scenario for the maintenance of high genetic diversity during range shifts in beech (and putatively other forest trees) with a combination of gene flow maintaining within-population neutral diversity and selection maintaining between-population adaptive differentiation.

Evolutionary rate and genetic load in an emblematic Mediterranean tree following an ancient and prolonged population collapse.

Severe bottlenecks significantly diminish the amount of genetic diversity and the speed at which it accumulates (i.e., evolutionary rate). They further compromise the efficiency of natural selection to eliminate deleterious variants, which may reach fixation in the surviving populations. Consequently, expanding and adapting to new environments may pose a significant challenge when strong bottlenecks result in genetic pauperization. Herein, we surveyed the patterns of nucleotide diversity, molecular adaptation and genetic load across 177 gene-loci in a circum-Mediterranean conifer (Pinus pinea L.) that represents one of the most extreme cases of genetic pauperization in widespread outbreeding taxa. We found very little genetic variation in both hypervariable nuclear microsatellites (SSRs) and gene-loci, which translated into genetic diversity estimates one order of magnitude lower than those previously reported for pines. Such values were consistent with a strong population decline that began some ~1 Ma. Comparisons with the related and parapatric maritime pine (Pinus pinaster Ait.) revealed reduced rates of adaptive evolution (α and ωa ) and a significant accumulation of genetic load. It is unlikely that these are the result from differences in mutation rate or linkage disequilibrium between the two species; instead they are the presumable outcome of contrasting demographic histories affecting both the speed at which these taxa accumulate genetic diversity, and the global efficacy of selection. Future studies, and programs for conservation and management, should thus start testing for the effects of genetic load on fitness, and integrating such effects into predictive models.

De novo assembly of English yew (Taxus baccata) transcriptome and its applications for intra- and inter-specific analyses.

KEY MESSAGE: We provide novel genomic resources for Taxus baccata in the form of a reference transcriptome, SSR and SNP markers, and orthologous single-copy genes, useful for phylogenomic and population genomic applications. English yew (T. baccata) is the only European representative of the Taxaceae family, a conifer group originated in the Jurassic period. The wide extent of environmental heterogeneity within the species' range, together with its long presence in Europe, make English yew an ideal species to investigate adaptive evolution in conifers. To enlarge the genomic resources available for this species, we used Illumina short read sequencing followed by de novo assembly to build the transcriptome of English yew. In addition to a fully annotated transcriptome as well as large sets of new potential SSR and SNP markers for T. baccata, we provide a data set of orthologous single-copy genes across three Taxus species using Picea sitchensis as outgroup, and discuss ortholog uses and limitations for phylogenomic and population genomic applications.

Inferring selection in instances of long-range colonization: The Aleppo pine (Pinus halepensis) in the Mediterranean Basin.

Teasing apart the effects of natural selection and demography on current allele frequencies is challenging, due to both processes leaving a similar molecular footprint. In particular, when attempting to identify selection in species that have undergone a recent range expansion, the increase in genetic drift at the edges of range expansions ("allele surfing") can be a confounding factor. To address this potential issue, we first assess the long-range colonization history of the Aleppo pine across the Mediterranean Basin, using molecular markers. We then look for single nucleotide polymorphisms (SNPs) involved in local adaptation using: (a) environmental correlation methods (bayenv2), focusing on bioclimatic variables important for the species' adaptation (i.e., temperature, precipitation and water availability); and (b) FST -related methods (pcadapt). To assess the rate of false positives caused by the allele surfing effect, these results are compared with results from simulated SNP data that mimics the species' past range expansions and the effect of genetic drift, but with no selection. We find that the Aleppo pine shows a previously unsuspected complex genetic structure across its range, as well as evidence of selection acting on SNPs involved with the response to bioclimatic variables such as drought. This study uses an original approach to disentangle the confounding effects of drift and selection in range margin populations. It also contributes to the increased evidence that plant populations are able to adapt to new environments despite the expected accumulation of deleterious mutations that takes place during long-range colonizations.

Increased fire frequency promotes stronger spatial genetic structure and natural selection at regional and local scales in Pinus halepensis Mill.

Background and Aims: The recurrence of wildfires is predicted to increase due to global climate change, resulting in severe impacts on biodiversity and ecosystem functioning. Recurrent fires can drive plant adaptation and reduce genetic diversity; however, the underlying population genetic processes have not been studied in detail. In this study, the neutral and adaptive evolutionary effects of contrasting fire regimes were examined in the keystone tree species Pinus halepensis Mill. (Aleppo pine), a fire-adapted conifer. The genetic diversity, demographic history and spatial genetic structure were assessed at local (within-population) and regional scales for populations exposed to different crown fire frequencies. Methods: Eight natural P. halepensis stands were sampled in the east of the Iberian Peninsula, five of them in a region exposed to frequent crown fires (HiFi) and three of them in an adjacent region with a low frequency of crown fires (LoFi). Samples were genotyped at nine neutral simple sequence repeats (SSRs) and at 251 single nucleotide polymorphisms (SNPs) from coding regions, some of them potentially important for fire adaptation. Key Results: Fire regime had no effects on genetic diversity or demographic history. Three high-differentiation outlier SNPs were identified between HiFi and LoFi stands, suggesting fire-related selection at the regional scale. At the local scale, fine-scale spatial genetic structure (SGS) was overall weak as expected for a wind-pollinated and wind-dispersed tree species. HiFi stands displayed a stronger SGS than LoFi stands at SNPs, which probably reflected the simultaneous post-fire recruitment of co-dispersed related seeds. SNPs with exceptionally strong SGS, a proxy for microenvironmental selection, were only reliably identified under the HiFi regime. Conclusions: An increasing fire frequency as predicted due to global change can promote increased SGS with stronger family structures and alter natural selection in P. halepensis and in plants with similar life history traits.

Genome-environment association study suggests local adaptation to climate at the regional scale in Fagus sylvatica.

The evolutionary potential of long-lived species, such as forest trees, is fundamental for their local persistence under climate change (CC). Genome-environment association (GEA) analyses reveal if species in heterogeneous environments at the regional scale are under differential selection resulting in populations with potential preadaptation to CC within this area. In 79 natural Fagus sylvatica populations, neutral genetic patterns were characterized using 12 simple sequence repeat (SSR) markers, and genomic variation (144 single nucleotide polymorphisms (SNPs) out of 52 candidate genes) was related to 87 environmental predictors in the latent factor mixed model, logistic regressions and isolation by distance/environmental (IBD/IBE) tests. SSR diversity revealed relatedness at up to 150 m intertree distance but an absence of large-scale spatial genetic structure and IBE. In the GEA analyses, 16 SNPs in 10 genes responded to one or several environmental predictors and IBE, corrected for IBD, was confirmed. The GEA often reflected the proposed gene functions, including indications for adaptation to water availability and temperature. Genomic divergence and the lack of large-scale neutral genetic patterns suggest that gene flow allows the spread of advantageous alleles in adaptive genes. Thereby, adaptation processes are likely to take place in species occurring in heterogeneous environments, which might reduce their regional extinction risk under CC.

Causes and consequences of large clonal assemblies in a poplar hybrid zone.

Asexual reproduction is a common and fundamental mode of reproduction in plants. Although persistence in adverse conditions underlies most known cases of clonal dominance, proximal genetic drivers remain unclear, in particular for populations dominated by a few large clones. In this study, we studied a clonal population of the riparian tree Populus alba in the Douro river basin (northwestern Iberian Peninsula) where it hybridizes with Populus tremula, a species that grows in highly contrasted ecological conditions. We used 73 nuclear microsatellites to test whether genomic background (species ancestry) is a relevant cause of clonal success, and to assess the evolutionary consequences of clonal dominance by a few genets. Additional genotyping-by-sequencing data were produced to estimate the age of the largest clones. We found that a few ancient (over a few thousand years old) and widespread genets dominate the population, both in terms of clone size and number of sexual offspring produced. Interestingly, large clones possessed two genomic regions introgressed from P. tremula, which may have favoured their spread under stressful environmental conditions. At the population level, the spread of large genets was accompanied by an overall ancient (>0.1 Myr) but soft decline of effective population size. Despite this decrease, and the high clonality and dominance of sexual reproduction by large clones, the Douro hybrid zone still displays considerable genetic diversity and low inbreeding. This suggests that even in extreme cases as in the Douro, asexual and sexual dominance of a few large, geographically extended individuals does not threaten population survival.

Evidence of divergent selection for drought and cold tolerance at landscape and local scales in Abies alba Mill. in the French Mediterranean Alps.

Understanding local adaptation in forest trees is currently a key research and societal priority. Geographically and ecologically marginal populations provide ideal case studies, because environmental stress along with reduced gene flow can facilitate the establishment of locally adapted populations. We sampled European silver fir (Abies alba Mill.) trees in the French Mediterranean Alps, along the margin of its distribution range, from pairs of high- and low-elevation plots on four different mountains situated along a 170-km east-west transect. The analysis of 267 SNP loci from 175 candidate genes suggested a neutral pattern of east-west isolation by distance among mountain sites. F(ST) outlier tests revealed 16 SNPs that showed patterns of divergent selection. Plot climate was characterized using both in situ measurements and gridded data that revealed marked differences between and within mountains with different trends depending on the season. Association between allelic frequencies and bioclimatic variables revealed eight genes that contained candidate SNPs, of which two were also detected using F(ST) outlier methods. All SNPs were associated with winter drought, and one of them showed strong evidence of selection with respect to elevation. Q(ST)-F(ST) tests for fitness-related traits measured in a common garden suggested adaptive divergence for the date of bud flush and for growth rate. Overall, our results suggest a complex adaptive picture for A. alba in the southern French Alps where, during the east-to-west Holocene recolonization, locally advantageous genetic variants established at both the landscape and local scales.

Local effects drive heterozygosity-fitness correlations in an outcrossing long-lived tree.

Heterozygosity-fitness correlations (HFCs) have been used to understand the complex interactions between inbreeding, genetic diversity and evolution. Although frequently reported for decades, evidence for HFCs was often based on underpowered studies or inappropriate methods, and hence their underlying mechanisms are still under debate. Here, we used 6100 genome-wide single nucleotide polymorphisms (SNPs) to test for general and local effect HFCs in maritime pine (Pinus pinaster Ait.), an iconic Mediterranean forest tree. Survival was used as a fitness proxy, and HFCs were assessed at a four-site common garden under contrasting environmental conditions (total of 16 288 trees). We found no significant correlations between genome-wide heterozygosity and fitness at any location, despite variation in inbreeding explaining a substantial proportion of the total variance for survival. However, four SNPs (including two non-synonymous mutations) were involved in significant associations with survival, in particular in the common gardens with higher environmental stress, as shown by a novel heterozygosity-fitness association test at the species-wide level. Fitness effects of SNPs involved in significant HFCs were stable across maritime pine gene pools naturally growing in distinct environments. These results led us to dismiss the general effect hypothesis and suggested a significant role of heterozygosity in specific candidate genes for increasing fitness in maritime pine. Our study highlights the importance of considering the species evolutionary and demographic history and different spatial scales and testing environments when assessing and interpreting HFCs.

Adapting through glacial cycles: insights from a long-lived tree (Taxus baccata).

Despite the large body of research devoted to understanding the role of Quaternary glacial cycles in the genetic divergence of European trees, the differential contribution of geographic isolation and/or environmental adaptation in creating population genetic divergence remains unexplored. In this study, we used a long-lived tree (Taxus baccata) as a model species to investigate the impact of Quaternary climatic changes on genetic diversity via neutral (isolation-by-distance) and selective (isolation-by-adaptation) processes. We applied approximate Bayesian computation to genetic data to infer its demographic history, and combined this information with past and present climatic data to assess the role of environment and geography in the observed patterns of genetic structure. We found evidence that yew colonized Europe from the East, and that European samples diverged into two groups (Western, Eastern) at the beginning of the Quaternary glaciations, c. 2.2 Myr before present. Apart from the expected effects of geographical isolation during glacials, we discovered a significant role of environmental adaptation during interglacials at the origin of genetic divergence between both groups. This process may be common in other organisms, providing new research lines to explore the effect of Quaternary climatic factors on present-day patterns of genetic diversity.

Halfway encounters: meeting points of colonization routes among the southern beeches Nothofagus pumilio and N. antarctica.

The Patagonian region is characterized by a complex biogeographic history, with evidence of deep phylogeographic breaks shared among species. Of particular interest to conservation is the nature of colonization and settlement patterns after the last glacial period, including the detection of secondary contact between different lineages and/or hybridization among related species around phylogeographic breaks. Here we studied population demography and past hybridization of two widespread tree species endemic to South America, Nothofagus pumilio and N. antarctica. Using 8 nuclear microsatellites we genotyped 41 populations of both species. Genetic variation and structure across the geographic region were evaluated within and among species and the past demographic history of hybridization between the two species was inferred using Approximate Bayesian Computation (ABC). Northern and southern lineages were identified in each species, and Bayesian clustering revealed their convergence at mid latitudes (42°S). Spatial genetic structure (SGS) also indicated the existence of a genetic discontinuity at these latitudes, which is in agreement with previous data from maternal DNA markers. Several populations around 42-44°S presented high levels of genetic diversity with a decrease toward southern populations. Even though the species are clearly differentiated (G'ST=0.335), admixed gene pools were observed in both species. Two independent runs of ABC suggested that inter species admixture-like patterns occurred within the timescale of the Last Glacial Maximum (around 20,000 BP). We also provide evidences of recent and bi-directional hybridization/introgression between the two Nothofagus species and describe features of the populations demography in the past. The settlement of a secondary contact zone in Nothofagus species around 42-44°S coincides with the phylogeographic breaks and hotspots of genetic diversity found in other plant and animal species in Patagonia, highlighting its importance as reservoir of diversity. The characterization of the population history of native species can contribute substantially to long-term conservation and management policies.

In situ genetic association for serotiny, a fire-related trait, in Mediterranean maritime pine (Pinus pinaster).

Wildfire is a major ecological driver of plant evolution. Understanding the genetic basis of plant adaptation to wildfire is crucial, because impending climate change will involve fire regime changes worldwide. We studied the molecular genetic basis of serotiny, a fire-related trait, in Mediterranean maritime pine using association genetics. A single nucleotide polymorphism (SNP) set was used to identify genotype : phenotype associations in situ in an unstructured natural population of maritime pine (eastern Iberian Peninsula) under a mixed-effects model framework. RR-BLUP was used to build predictive models for serotiny in this region. Model prediction power outside the focal region was tested using independent range-wide serotiny data. Seventeen SNPs were potentially associated with serotiny, explaining approximately 29% of the trait phenotypic variation in the eastern Iberian Peninsula. Similar prediction power was found for nearby geographical regions from the same maternal lineage, but not for other genetic lineages. Association genetics for ecologically relevant traits evaluated in situ is an attractive approach for forest trees provided that traits are under strong genetic control and populations are unstructured, with large phenotypic variability. This will help to extend the research focus to ecological keystone non-model species in their natural environments, where polymorphisms acquired their adaptive value.

Detecting short spatial scale local adaptation and epistatic selection in climate-related candidate genes in European beech (Fagus sylvatica) populations.

Detecting signatures of selection in tree populations threatened by climate change is currently a major research priority. Here, we investigated the signature of local adaptation over a short spatial scale using 96 European beech (Fagus sylvatica L.) individuals originating from two pairs of populations on the northern and southern slopes of Mont Ventoux (south-eastern France). We performed both single and multilocus analysis of selection based on 53 climate-related candidate genes containing 546 SNPs. FST outlier methods at the SNP level revealed a weak signal of selection, with three marginally significant outliers in the northern populations. At the gene level, considering haplotypes as alleles, two additional marginally significant outliers were detected, one on each slope. To account for the uncertainty of haplotype inference, we averaged the Bayes factors over many possible phase reconstructions. Epistatic selection offers a realistic multilocus model of selection in natural populations. Here, we used a test suggested by Ohta based on the decomposition of the variance of linkage disequilibrium. Overall populations, 0.23% of the SNP pairs (haplotypes) showed evidence of epistatic selection, with nearly 80% of them being within genes. One of the between gene epistatic selection signals arose between an FST outlier and a nonsynonymous mutation in a drought response gene. Additionally, we identified haplotypes containing selectively advantageous allele combinations which were unique to high or low elevations and northern or southern populations. Several haplotypes contained nonsynonymous mutations situated in genes with known functional importance for adaptation to climatic factors.

Adaptive evolution of Mediterranean pines.

Mediterranean pines represent an extremely heterogeneous assembly. Although they have evolved under similar environmental conditions, they diversified long ago, ca. 10 Mya, and present distinct biogeographic and demographic histories. Therefore, it is of special interest to understand whether and to what extent they have developed specific strategies of adaptive evolution through time and space. To explore evolutionary patterns, the Mediterranean pines' phylogeny was first reconstructed analyzing a new set of 21 low-copy nuclear genes with multilocus Bayesian tree reconstruction methods. Secondly, a phylogenetic approach was used to search for footprints of natural selection and to examine the evolution of multiple phenotypic traits. We identified two genes (involved in pines' defense and stress responses) that have likely played a role in the adaptation of Mediterranean pines to their environment. Moreover, few life-history traits showed historical or evolutionary adaptive convergence in Mediterranean lineages, while patterns of character evolution revealed various evolutionary trade-offs linking growth-development, reproduction and fire-related traits. Assessing the evolutionary path of important life-history traits, as well as the genomic basis of adaptive variation is central to understanding the past evolutionary success of Mediterranean pines and their future response to environmental changes.