WP1– Micro-evolutionary potential of tree populations in response to Global Change (GC).  
Population response to new selection exerted by GC depends on the amount of genetic diversity at adaptive traits residing within populations (i.e. the standing genetic variation). Genomic technologies now  provide  the  opportunity  to  discover  the  genes  and  the  allelic  variants  in  these  genes  that underlie complex phenotypic traits on which selection acts. In this WP, we will first assess the levels of  diversity  at  gene  level,  and  characterize  selection  forces  causing  allele  frequency  changes between  generations  within  environment  and  across  environment  at  genes  underlying  adaptive traits. Then in a second step, we will assess levels of phenotypic diversity for putatively adaptive traits  and  characterize  selection  forces  on  these  traits,  based  on  traits  measured  in  native populations, reciprocal transplants or common garden experiments. 

Objective 1: Gene diversity and inference of selection from allele frequencies
The original contribution of TipTree is that we will study both divergent selection along ecological gradients and contemporary selection occurring between adult and offspring generations within  a given population. To this end, we will sample large numbers of adults and seedlings (at least 1000 individuals and 6 populations in total per species) in contrasted environment conditions. We will partly rely on existing local gradient experiments (LGE) already set up in previous project for TipTree  study  species;  each  LGE  typically  consists  of  two  populations  (~30  adult/population) experiencing contrasted environmental challenges (stressed versus unstressed for low-temperature, drought/flooding stress and fire response), but still within the reach of gene flow (typically large in trees, up to several kilometres, (Petit and Hampe 2006)). In TipTree, we will increase the number of sampled  adult  trees,  sample  established  seedlings,  and  install  some  new  LGE  experiment  at margins of species range.Large arrays of polymorphisms (1000-2000 per species) will be generated by combining  (1) SNP-genotyping  for  candidate  genes  identified  in  previous  studies  (e.g.  Linktree)  and  (2)  new  SNP identified by Next-Generation Sequencing (NGS) of short DNA fragments containing the loci, based on gene capture or on the Restriction Associated DNA (RAD) technique (Miller et al. 2007). RAD and  related  approaches  (GBS,  (Elshire  et  al.  2011);  MSG,  (Andolfatto  et  al.  2011))  are  novel methods based on NGS to investigate directly the inter-individual variability of potentially functional regions of whole genomes, without the need for prior sequence information. To detect contemporary selection acting through adult trees, we will develop a new approach based on existing parentage-based methods that take as input the genotypes and spatial positions of seedlings and adults to jointly estimate the seed and pollen dispersal kernels and the variation of adult tree fecundities (Oddou-Muratorio and Klein 2008). We will modify the method that presently considers gene flow and the spatial position and fecundity of adults (neutral model), to additionally consider that the parental genotypes can determine reproductive success. Hence, using as input large arrays of SNP data at candidate loci, we will be able to detect the outlier SNP loci for which the frequency of an allele in the offspring cohort differs significantly from the neutral expectation. This approach was already applied to study ongoing balancing selection at self-incompatibility locus (see (Stoeckel et al. 2012). The advantage of this new contemporary estimates of selection over historical estimates of selection (based e.g. on Fst genome scan (Foll and Gaggiotti 2008), is that they  make  no  hypotheses  on  the  evolutionary  forces  (gene  flow,  genetic  drift,  selection)  at  work within the population, and are not sensitive to demographic history. Additionally, we will investigate the historical response to divergent selection among populations along LGE, by studying the correlation of allelic frequencies with the environment (SNP-environment association).  Depending  on  the  species/site,  the  relevant  gradient  will  consist  in  hydrological, pedological and/or temperature variation, characterised on the sites (characterisation of soil water content and composition, records from climate station). To study the SNP-environment association, we  will  use  methods  that  take  simultaneously  into  account  genome-wide  estimates  of demographical processes and population structure, thereby allowing identifying individual loci under selection  (Foll  and Gaggiotti  2008). The  originality  of TipTree  is  that  we  will  compare  patterns of SNP-environment association across cohorts; though seedlings may not have been through all the selection steps the adults went through, such spatiotemporal comparison has already been shown to highlight the selective forces at work within populations (Jump et al. 2006). Moreover, we will also compare  patterns  of  SNP-environment  association  across  different  environmental  conditions  (ie different LEG), which should highlight the effect of environment on adaptive divergence. 

Objective  2:  Phenotypic  diversity,  genotype-phenotype  association  and  inference  of selection on adaptive traits

We  will  first  use  classical  quantitative  genetic  methods  to  estimate  levels  of  additive  genetic variation  (and  related  quantitative  genetic  parameters)  for  targeted  traits  putatively  involved  or proven to be involved in the response to environmental stresses (listed in Table 1). Targeted traits will  be  selected  based  on  published  studies,  on  our  own  results  (including  Linktree  and  related projects) and on exploratory simulations performed in WP2. Moreover, for all TipTree species, some controlled experiments based on LGEs were already set up (in LinkTree and other projects) in order to obtain a more direct view of genetic variation of adaptive traits. Typically, half-sib progenies (a minimum of 12 mother-plants per species and two populations of contrasted environment, but often much larger experiments) were grown in common environments either in a common garden or in reciprocal  transplantation  sites  corresponding  to  the  populations  of  origin  (experimental  units consisting  of  10-15  half-sibs  from  each  mother).  These  experiments  will  be  completed  and  fully analysed in TipTree (including measurement of additional traits). Additionally,  we  will test whether the loci, identified in the previous task, to be under selection in natural  populations  contribute  to  the  genetic  control  of  the  traits.  To  that  aim,  we  will  obtain genotypic  data  at  those  loci  and  at  a  set  of  control  loci  for  plants  with  contrasted  phenotype  or fitness (min 500 seedlings/species). As the reciprocal transplant and common garden populations are recurrently measured for growth, physiological and survival traits, SNP markers will be checked for  association  with  those  traits  to  validate  the  link  between  selection  at  the  molecular  level  and phenotypes. The simultaneous detection of selection at a locus and of its involvement in the control of an adaptive trait is compelling evidence that the locus acts on adaptive processes through the expression of the trait. Finally, the form and the intensity of the selection acting on targeted traits will also be estimated based on traits measurement in adult trees in a subset of native populations. The studied traits and protocols of measurement will be defined in close coordination with managers of functional trait TRY database (Kattge et al. 2011). Then, we will test whether some value of the trait confer a fitness advantage  and  estimate  ongoing  selection  gradient  on  adaptive  traits,  using  parentage-based methods and adult phenotypic values (Oddou-Muratorio et al. 2005).