Adaptation of Arabidopsis halleri (Brassicaceae) to sites recently polluted by high amount of zinc and cadmium:

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1 Adaptation of Arabidopsis halleri (Brassicaceae) to sites recently polluted by high amount of zinc and cadmium: Pierre Saumitou-Laprade Laboratoire de Génétique et Évolution des Populations Végétales UMR CNRS 8016, FR CNRS 1818, Université de Lille 1- France

2 Pollution of soils by heavy metals High selective pressure on plants Strong decrease of Biodiversity Health problems Erosion of soils, water contamination... non cultivable area Sociological and economical problems

3 Definitions Tolerance: «Ability of a plant to survive on soils that are toxic or inimical to most other plants because the soils contain elevated levels of one or more metals.» Hyperaccumulation: Accumulation in aerial parts of concentration of metal 10 times higher than in normal plants (Brooks, 1998). Eg: Zn hyperaccumulator µg/g dw (1983) phytoremediation?

4 Ecological genetics Candidate genes -omics History of populations Biogeography Genetics and Association studies Signature of selection SNPs, sequences Molecular Evolution Purrugganan & Gibson 2003 Molecular Ecology May 2003 Special issue «Genes in Ecology»

5 Arabidopsis halleri subsp. halleri (Brassicaceae) Zn and Cd tolerant ; Zn and Cd hyperaccumulator Pseudo-metallophyte Neighbor-joining distance tree based on Adh sequences. (Koch et al, 2000) Allogamous Self incompatible stoloniferous

6 Northern Carpathians ( m) Slovakia Poland Southern Carpathians Fagaras, Romania ( m)

7 Alps Trentin, Italy ( m) [Zn]: 4-5 % [Cd]: 0,017-0,023 % [Pb]: 0,16 % Silesia, Poland ( m) Recent industrial sites

8 Geographic variation for Zn tolerance in Arabidopsis halleri subsp. Halleri Does Zn tolerance polymorphism exists in A. halleri? How is this polymorphism distributed? Population screening in hydroponics from species-scale sampling

9 Sampling of the A. halleri subsp. halleri in Europe 67 populations M population NM population

10 Survival curves in 28 populations Sequential test established by (Schat et Ten Bookum 1992) Survival 1,0 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0, NT vs NM [Zinc] (µm) 11 M pop ssp halleri 17 NM pop ssp halleri Control A. lyrata petrea A. thaliana Test logrank: NT vs M P<0.001 P<0.001

11 Population survival (S) curves obtained from fitting to a sigmoïdal dose-response model. S =100/(1+10^((log(EC50)-log(X))*B)). S = Survival % EC50 = Effective Concentration, 50% B = Slope

12 Box plots of EC50 and B NM: non metallicolous populations; NMp: non metallicolous populations in polluted area; M: metallicolous populations ovirensis: A. h. ovirensis populations ; *: outlier a; b; c : significant difference at the 5% level. EC50 : M > NM Higher tolerance in M populations B : M > NM Less variability for tolerance in M population

13 Conclusions Zn tolerance is a constitutive trait in A. halleri subsp. halleri: the origin should be investigated at inter-specific level Occurrence of enhanced tolerance in metallicolous populations with a quantitative variation from the NM populations to the most tolerant M ones Prop. survie [Zinc] (µm) M pop PL6 NM pop A5 Control A. lyrata (Pauwels et al JEB 19, )

14 Evolution of metallicolous populations in Arabidopsis halleri subsp. halleri Ecological genetics Candidate genes -omics History of populations Biogeography Genetics and Association studies Signature of selection SNPs, sequences Molecular Evolution

15 Can we explain the distribution of the observed polymorphism? What are the origin and history of the NM / M populations? F, E F,E C,A F,E F, E C,B a single origin several origins

16 2/ Can we explain the distribution of the observed polymorphism? What are the origin and history of the NM / M populations? Broad-scale population genetic structure analysis Phylogeographic approach using cpdna Why chloroplast? non-recombinant maternally inherited A marker of choice for phylogeographic studies

17 Phylogeography (Avise et al., 1987) Fst (Wright, 1951): estimate of among population differentiation based on allelic frequencies Φst (Excoffier et al, 1992) and Nst (Pons et Petit, 1996) take into account the molecular information (i.e. the number of differences between haplotypes) C,A F,E C,B Φst and Nst >> Fst derived ancestral Phylogeographic structure??? Φst and Nst = Fst No phylogeographic structure modified from Schaal, 1998

18 Genetic analysis of the A. halleri chloroplast DNA M population NM population 67 populations 1255 accessions 3 Fragments : matk, psbc-trns, trnc-trnd PCR-RFLP, SNPs, fragment length polymorphism

19 Phylogenetic relationships among haplotypes : mutation : missing haplotype relative frequencies NM M computed using Arlequin (Schneider et al., 2000) M + NM

20 Geographic distribution of haplotypes M population NM population Gst Gst = 0.73*** = Nst = 0.77 N ST >G ST, P = 0.11 mean Gst = ±0.002 for 124 Angiosperm species (Petit et al., 2005)

21 N S Φ ct = 0.41***

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23 A NM = 2.064, A M = 2.002, P =0.42 H SNM = 0.237, H SM = 0.349, P = 0.31

24 Neigbour Joining population tree using Cavalli Sforza distances. A NM = 2.064, A M = 2.002, P =0.42 H SNM = 0.237, H SM = 0.349, P = 0.31 Strong differentiation among the metallicolous regions Φ ct = 0.58 p < 10-3 No differentiation among population from different edaphic type in a same region Φ ct = 0.04 p = 0.11 Pauwels et al Mol Ecol 14,

25 Conclusions Strong differentiation between northern and southern parts of the A. halleri subsp. halleri distribution area role of the Alps as a geographic barrier to seed and pollen flows? Geographic differentiation rather than edaphic differentiation Genetic differentiation among metallicolous sites The foundation of M populations occurred several times The increased tolerance observed in M populations has a polyphyletic origin Are genes involved in enhanced tolerance in Silesia and Harz different? M populations represent a a genetic resource that should be preserved

26 Participants to this project: University of Lille 1; GEPV Pierre Saumitou-Laprade Fabienne Van Rossum Hélène Frérot Nancy Roosens Maxime Pauwels Glenda Willems Alicja Kostecka Claire Lise Meyer Cécile Godé Xavier Vekemans Vincent Castric Collaborations: National Isabelle Bonnin (Moulon) Michel Lebrun (Montpellier) Stéphane Mari (Montpellier) Eric Lacombe (Montpellier) International Partners of the Metalhome RTN project Nathalie Verbruggen (Brussels) Mikael Courbot Ute Krämer (Golm) Dörthe Dräger (Golm) Henk Schat (Amsterdam) Pierre Meerts (Brussels) Thomas Mitchell-Olds (Jena) Outi Savolainen (Oulu; Finlande) Krystyna Grodzinska (Krakow Poland)

27 Thank you for your attention