1. Artificial maturation of conifers by confined pyrolysis Contribution to palaeofloral and palaeoclimatic reconstructions HAUTEVELLE Yann, MICHELS Raymond, LANNUZEL Frédéric *, FARRE Bastien, MALARTRE Fabrice, TROUILLER Alain May 21-26, 2006 17th International Symposium on Analytical & Applied Pyrolysis, Budapest, Hungary

2. Plant chemotaxonomy Many bioterpenoids have a chemotaxonomic value and are specific of certain plant taxa Molecular composition : - lignin - carbohydrates - lipids like terpenoids conifers abietic acid Angio- sperms lupeol

3. Transport and diagenesis of terpenoids BIOSPHERE GEOSPHERE Sedimentary basins transport sediment bioterpenoids Geoterpenoids or molecular biomarkers Diagenetic transformations Geoterpenoids can keep the initial chemotaxonomic value conifers Angio- sperms conifers Angio- sperms

4. Plant biomarkers distribution as palaeofloristic proxy Terrestrial flora at the time of the deposition angiosperms pines cypress sequoia Land plant biomarkers distribution in sediment ferns paleoflora reconstruction The distribution of plant biomarkers in sediments & rocks is a proxy of the terrestrial palaeoflora at the time of the deposition

5. Relations between floras and climates Desertic climate Temperate climate Tropical climate Polar climate flora ↔ climate

6. Chemostratigraphy of vascular plant biomarkers Stratigraphic record palaeobiodiversity paleoflora  palaeoclimate T°, humidity  desertic climate  Geological times  tropical climate   temperate climate  Molecular facies

7. Advantages & disadvantages of palaeochemotaxonomy BOTANICAL PALEOCHEMOTAXONOMY (plant biomarkers assemblages) BUT macrofossil plants are rare classically used for the reconstruction of palaeofloras and palaeoclimates PALEOBOTANY (fossil plants assemblages) PALYNOLOGY (spore & pollen assemblages) BUT spores and pollen are not easily related to plant taxa - widespread in the stratigraphic record - related to plant taxa when they have a palaeochemotaxonomic value WHILE plant biomarkers are : HOWEVER, presently molecular databases contain many gaps

8. Aims of experimental palaeochemotaxonomy ➜ innovative experimental approach based on artificial maturation of extant plants by confined pyrolysis ➜ simulation of the diagenesis ➜ Aims 1 : determine the pyrolysis parameters for which the broadest distribution of plant biomarkers is obtained In order to fill these gaps : ➜ Aims 2 : verify the consistency with the plant biomarkers of the geological record ➜ Aims 3 : apply the procedure to a large set of extant plants in order to construct a palaeochemotaxonomic database

9. Aims 1 and 2 ➜ Pyrolysis of a Pinaceae (Abies pinsapo) Fresh Abies pinsapo Methylated total fraction Retention time Diagenetic evolution of abietanoic acids Fresh Abies pinsapo contain large amounts of abietanoic acids

10. Experimental procedure Fresh plantSealed gold tubesConfined pyrolysis Extraction of terpenoids CH 2 Cl 2 Analysis by GC-MS Aliphatics Aromatics Polar Fractionation

11. Calibration of the pyrolysis temperature 150 °C 200 °C 250 °C 280 °C 300 °C Other pyrolysis parameters : duration : 24 h ; pressure : 700 bar. Diagenetic pathway of abietic acid 280°C Presence of aromatic diterpanes Pyrolysed Abies pinsapo Total fraction m/z 219, 223, 237, 239, 241

12. Generation of saturated diterpanes phytene 280 °C presence of diterpanes classically detected in the geosphere LiAlH 4 Unsaturated abietanes not satisfying Pyrolysis with LiAlH 4 Pyrolysed Abies pinsapo Aliphatic fraction TIC 280 °C Saturated abietanes labdanes Diterpane diagenesis Pyrolysed Abies pinsapo Aliphatic fraction TIC

13. Palaeochemotaxonomy of a virtual fossil Abies pinsapo Pyrolysed Abies pinsapo Aliphatic fraction TIC Pyrolysed Abies pinsapo Aromatic fraction TIC pyrolysed Abies pinsapo Polar fraction TIC with LiAlH 4 280°C Without LiAlH 4 280°C without LiAlH 4 280°C

14. Summary of the experimental procedure Aliphatic fraction Time : 24 h, pressure : 700 bar, temperature : 280°C, WITH LiAlH 4 Aromatic fraction Time : 24 h, pressure : 700 bar, temperature : 280°C, WITHOUT LiAlH 4 Polar fraction Time : 24 h, pressure : 700 bar, temperature : 280°C, WITHOUT LiAlH4 Determination/prediction of the fossil molecular signature of the pyrolysed plant The reproduction of this procedure on a great number of plant taxa will considerably increase our knowledge in palaeochemotaxonomy and contribute to the reconstruction of ancient flora

15. Experimental palaeochemotaxonomy of conifers ➜ Aim 3 : investigate the palaeochemotaxonomy of the conifers using our experimental procedure The conifers order is composed of 7 families : Araucariaceae 2 Araucaria Cupressaceae 1 Chamaecyparis & 2 Juniperus Pinaceae 1 Abies (fir), 1 Cedrus (cedar) & 1 Picea (spruce) Podocarpaceae 2 Podocarpus Sciadopityaceae 1 Sciadopitys Taxaceae 1 Taxus (yew) Taxodiaceae 1 Sequoiadendron, 1 Metasequoia, 1 Cryptomeria & 1 Taxodium 17 conifer species were studied for experimental palaeochemotaxonomy

16. Experimental palaeochemotaxonomy of conifers Each conifer family is characterized by its own molecular signature Araucariaceae Cupressaceae Chamaecyparis Juniperus Araucaria Tetracyclic diterpanes Abietic acid derived abietanes Keto- phenolic abietanes cedrane ++++ + absent … (ent-beyerane or kauranes) ++++ (ent-beyerane) + ++++ ++ Relative abundance in chromatograms High: ++++Low: + ++ ++++ absent

17. 1) Our confined pyrolysis procedure of extant land plants allows to experimentally generate biomarkers consistent with those encountered in the fossil record Confined pyrolysis of extant land plants allows to significantly improve the paleochemotaxonomy of fossil plants Conclusions Thank you for your attention 2) The reaction products are consistent with the diagenetic pathways of fossilization for biomarkers 3) Many biomarkers distributions obtained are of palaeochemotaxonomic value

19. Conclusions Thank you for your attention Many compounds in the pyrolysates remain unidentified and may have a useful palaeochemotaxonomic value

20. Experimental palaeochemotaxonomy of conifers - Pinaceae + Absence of tetracyclic diterpanes + High abundance of abietanes deriving from abietic acid and not from cétono-phenolic abietanes - Podocarpaceae + Absence of tetracyclic diterpanes + High abundance of abietanes deriving from cétono-phenolic abietanes and not from abietic acid - Sciadopityaceae + Abundance of labdanes and tetracyclic diterpanes + Low abundance of abietanes deriving from abietic acid and not from cétono-phenolic abietanes The pyrolysis of these conifers show : 2) Each conifer family is characterized by its own molecular signature

21. The pyrolysis of these conifers show : These biomarkers originate from all conifers families and are very frequent in sediments & sedimentary rocks 1) Many biomarkers are present in all pyrolysates : - Many sesquiterpenoids cadinanes dihydro-ar- curcumene many sesquiterpenoids e.g. eudesmanes, ionene - C29 steranes and steroids Experimental palaeochemotaxonomy of conifers