OAKFLOW -  WORKPLAN

            The workplan is organised sequentially in three main steps. Step one aims at detecting and quantifying gene flow. Gene flow includes not only natural dispersal of pollen and seed, but also artificial transfer of material. Of particular interest is also natural hybridisation which is a special case of gene flow between two species. The detection of gene flow is based on parentage analysis between offspring and adult trees using highly polymorphic genetic markers. The second step consists in evaluating genetic and ecological consequences of gene transfer. Systematic comparisons of material stemming from hybrid or allochthonous origin will be made with parental or autochthonous material, both in situ and under controlled environmental conditions. Step two seeks also to identify the main genomic regions that are affected by hybridisation, which will bring hybridisation into an evolutionary significance. Finally step three will specifically address management consequences on gene flow and hybridisation.

Workpackage 1: Quantification of pollen dispersal and hybridisation

            The detection of pollen flow is achieved through paternity analysis. Parentage analysis consists of comparing genetic fingerprints of offspring with their potential parents by using exclusion procedures. The methodology requires exhaustive genotyping of the potential parents in a given area. The methods therefore relies on two major tools:

-the study site where exhaustive genotyping of trees is done. These stands are called ISP (Intensive Study Plots), and are about 5 to 10 ha in size, thus corresponding to a management compartment. All trees in an ISP are mapped and fingerprinted. Most of the ISPs have already been selected and the adult trees fingerprinted in the previous FAIR project. The ISPs vary in species composition and comprise usually at least two interfertile oak species (Q. robur, Q. petraea, Q. pubescens, Q. pyrenaica), allowing sources of gene flow variation to be identified.

-the genetic markers to obtain fingerprints. In a previous FAIR project (EU number) we have developed a set of six microsatellite loci that are now routinely applied in oak genetics These loci are highly variable (from 15 to 31 alleles/locus) and enable exclusion probabilities as high as 0.9999 to be obtained.

The 12 partners involved in this workpackage will use this methodology for depicting hybridisation (task 2.2) and pollen dispersal (task 2.3) in their own ISP.

Workpackage 2: Detection and quantification of artificial and natural seed transfer

In a similar way to the assessment of pollen movement, detection and quantification of seed movement is based on genetic markers. However the method varies according to the type of dispersal: natural vs artificial.

Natural dispersal by gravity, rodents or jays will be retraced by parental analysis based on microsatellites. In this case both parents, and not only the male parent need to be identified. However parental assignment cannot separate the female and male contributions. Additional molecular markers in the chloroplast genome, which is maternally inherited in oaks, will be used to help identify maternal contribution. The eleven partners involved in this workpackage will use this methodology for depicting natural dispersal in their own ISP.

Artificial seed transfer is detected by studying the spatial distribution of cpDNA polymorphism within given stands. Due to their mode of evolution, cpDNA polymorphism tends to become fixed in populations after only a few generations. Hence a stand of autochthonous origin should show no variation for cpDNA. The occurrence of within stand cpDNA diversity is therefore an indication of introduction of foreign material. In the previous FAIR project a systematic map of cpDNA haplotypes was constructed on a European scale based on a systematic sampling of countries on a 50 km grid system. Several thousand populations were analysed which permitted the construction of a fine scale geographic map of the distribution of cpDNA haplotypes across Europe. This map will be used as reference to retrace past seed introductions. Scoring of cpDNA haplotypes has been standardised in the frame of a previous EU supported projects, and all partners involved in this task use this scoring procedure.

Workpackage 3: Genetic consequences of gene flow and hybridisation

Hybridisation between species, and  between autochtonous and allochthonous stands are likely to produce new genomic recombinations and assortments, that will act as a further source of "beneficial" and "detrimental" diversity. The balance between "beneficial" and "detrimental" consequences of gene flow and hybridisation is largely unknown. Before separating these effects, we will attempt to identify regions of the genomes that are likely to be modified by hybridisation, and to assess the association between these regions and quantitative traits of phenotypic and adaptive significance. These regions will be localized on intra and interspecific genetic maps that have been constructed, either by QTL or loacalizing candidate genes involved in species differentiation.

Workpackage 4: Ecological consequences of gene flow and hybridisation

            Hybridisation and gene flow create novel genotypes and gene associations that will differ from their parents in their abilities to exploit environmental resources. The comparative fitness of hybrids and parents is a central question to evolutionary biology. Reviews of other plant examples indicate that hybrid fitness is environment-dependent. Hybrids are often more fit than their parents in certain habitats and less fit in others and was witnessed in the previous investigations in oaks. In the frame of the previous EU supported project, it was found that the occurrence of hybrids between Q. petraea and Q.robur was more important on the northern limits of the natural distributions than in the central and southern regions. We will compared various adaptive traits in hybrids and parental species. These comparisons are made either in situ in natural stands or ex situ (greenhouse) by using inters  and intraspecific full sib crosses.

Workpackage 5: Impact of gene flow and hybridisation on the management of oak stands

            Seed and pollen flow are basic genetic processes that shape the genetic structure of forest stands. They not only modify the spatial distribution of diversity, but also determine the genetic relatedness between trees and the population size of stands. Despite the strong impact of the dispersal mechanisms on stand genetic makeup, there has been scarcely any incorporation of these considerations into management strategies, mostly because the tools to monitor pollen and seed flow were not available. In the context of this project, pollen and seed movement will be traced in a number of different ISPs, characterised by different silvicultural regimes and different stand compositions. The results obtained will allow the interaction between forest management strategies and gene flow on the distribution of genetic diversity in oak stands to be identified. workpackage 5 will not require additional molecular analysis or assessment. It is more oriented towards data analysis and computation in co-operation with the different end-users associated to the project as subcontractors (National forest services, Conservation agencies).