Determination of the molecular signature of fossil conifers by experimental palaeochemotaxonomy.

1. Determination of the molecular signature of fossil conifers by experimental palaeochemotaxonomy. Contribution to palaeofloristic and palaeoclimatic reconstructions. Y. Hautevelle*, R. Michels, B. Farre, F. Lannuzel, F. Malartre * UMR G2R 7566, Université Henri Poincaré, Vandoeuvre-lès-Nancy, France Current address : UMR 7509, Laboratoire de Chimie Bioorganique, Strasbourg, France

2. Intro- duction Botanical chemotaxonomy • Chemical composition : • lignin • carbohydrates • lipids, e.g. terpenoids Abietic acid conifers lupeol angio- sperms Terpenoids have a chemotaxonomic value and are thus specific of certain taxa Introduction I.Objectives & procedure of experimental palaeochemo taxonomy II. Development of experimental palaeochemo taxonomy III. Application to Coniferales IV.Perspectives & future works Conclusions

3. Intro- duction From bioterpenoids to geoterpenoids transport Diagenetic transformations conifers angio- sperms Geoterpenoids can keep their initial chemotaxonomic value Geoterpenoids or molecular biomarkers BIOSPHERE Introduction I.Objectives & procedure of experimental palaeochemo taxonomy conifers II. Development of experimental palaeochemo taxonomy Sedimentary basin angio- sperms sediment bioterpenoids GEOSPHERE III. Application to Coniferales IV.Perspectives & future works Conclusions

4. Intro- duction Palaeofloristic and palaeoclimatic reconstructions Distribution of plant biomarkers Palaeofloristic composition on emerged lands Interpretation in terms of palaeofloristic composition angio- sperms pine pine cypres fern sequoia The distribution of plant biomarkers reflect the palaeofloristic composition during the deposition Introduction I.Objectives & procedure of experimental palaeochemo taxonomy II. Development of experimental palaeochemo taxonomy III. Application to Coniferales IV.Perspectives & future works Conclusions

5. Relations between floras and climates Intro- duction flora ↔ climate Introduction I.Objectives & procedure of experimental palaeochemo taxonomy temperate climate II. Development of experimental palaeochemo taxonomy desertic climate III. Application to Coniferales tropical climate IV.Perspectives & future works polar climate Conclusions

6. Intro- duction Chemostratigraphy of vascular plant biomarkers    temperate climate    Geological times tropical climate   desertic climate palaeobiodiversity palaeoclimate T°, humidity palaeoflora Plant biomarkers : Are they really interesting compared to other proxies ? Do they really bear pertinent information ? Introduction I.Objectives & procedure of experimental palaeochemo taxonomy II. Development of experimental palaeochemo taxonomy  III. Application to Coniferales stratigraphic record molecular facies IV.Perspectives & future works Conclusions

7. Intro- duction Advantages of palaeochemotaxonomy Improved approaches for palaeofloristic and palaeoclimatic reconstructions but fossils are scarse but spores & pollen are not easily reliable to plant taxa BOTANICAL PALEOCHEMOTAXONOMY(plant biomarkers) however plant biomarkers are : • widespread in the sedimentary record • - related to plant taxa if they have a palaeochemotaxonic value BUT, our current knowledge in paleochemotaxonomy is weak and very lacunar Introduction PALAEOBOTANY (fossil plants) I.Objectives & procedure of experimental palaeochemo taxonomy PALYNOLOGY (spore & pollen) II. Development of experimental palaeochemo taxonomy III. Application to Coniferales IV.Perspectives & future works Conclusions

8. Aims of experimental palaeochemotaxonomy I.01 Introduction I.Objectives & procedure of experimental palaeochemo taxonomy plant biomarkers palaeofloristic and palaeoclimatic proxies II. Development of experimental palaeochemo taxonomy ➜ new technique of artificial maturation of living plants(confined pyrolysis). ➜ experimental "reproduction" of the plant diagenesis & fossilisation(at the molecular scale). III. Application to Coniferales ➜ Aim : IV.Perspectives & future works Molecular taxonomy Botanical taxonomy Conclusions

9. Experimental and analytical procedures Fresh plant Sealed gold tubes Confined pyrolysis Aliphatic Aromatic Polar Molecular analysis (GC-MS) Solubilisation of terpenoids (CH2Cl2) Fractionation I.02 Introduction I.Objectives & procedure of experimental palaeochemo taxonomy II. Development of experimental palaeochemo taxonomy III. Application to Coniferales IV.Perspectives & future works Conclusions

10. Development of experimental palaeochemotaxonomy Diagenetic pathway of abietanoic acids Laflamme and Hites, 1978 ; Wakeham et al., 1980; Simoneit, 1986; Otto and Simoneit, 2001, 2002; Marchand-Genest and Carpy, 2003, etc. I.03 Introduction Diagenesis of bioterpenoids : ➜ progressive removal of oxygenated functions ➜reduction of double bonds ➜ saturation vs. aromatisation of 6C cycles (depending on redox conditions during diagenesis) I.Objectives & procedure of experimental palaeochemo taxonomy Experimental diagenesis : ➜ in accordance with these transformations ➜ generate the broadest possible distribution of biomarkers (functionalised & hydrocarbon ; aromatic & saturate) II. Development of experimental palaeochemo taxonomy Development of the pyrolysis procedure : ➜ well known diagenetic pathway of a bioterpenoid ➜ abietanoic acids like abietic acid III. Application to Coniferales IV.Perspectives & future works Conclusions

11. Choice of the plant and its initial composition Abies pinsapo(Spanish fir) Fresh Abiespinsapo Methylated total fraction Diagenetic pathway of abietanoic acids Retention time II.01 Introduction I.Objectives & procedure of experimental palaeochemo taxonomy II. Development of experimental palaeochemo taxonomy Fresh Abies pinsapo contains large amounts of abietanoic acids III. Application to Coniferales IV.Perspectives & future works Conclusions

12. Determination of the ideal pyrolysis temperature 280°C Presence of aromatic diterpanes Pyrolysed Abiespinsapo Total fraction m/z 219, 223, 237, 239, 241 II.02 Introduction 150°C I.Objectives & procedure of experimental palaeochemo taxonomy 200°C II. Development of experimental palaeochemo taxonomy Diagenetic pathway of abietic acid Other pyrolysis parameters : duration: 24 h ; pressure :700 bars. 250°C III. Application to Coniferales 280°C IV.Perspectives & future works 300°C Conclusions

13. Generation of saturated diterpanes Unsaturated abietanes not satisfying Pyrolysed Abiespinsapo Aliphatic fraction TIC Pyrolysis with LiAlH4 280°C Saturated abietanes 280°C presence of diterpanes classically detected in the geosphere labdanes LiAlH4 II.03 phytene Pyrolysed Abiespinsapo Aliphatic fraction TIC Introduction 280°C I.Objectives & procedure of experimental palaeochemo taxonomy II. Development of experimental palaeochemo taxonomy Diterpane diagenesis III. Application to Coniferales IV.Perspectives & future works Conclusions

14. Palaeochemotaxonomy of a virtual fossil Abies pinsapo 280°C with LiAlH4 Typical molecular signature of fossil Pinaceae 280°C Without LiAlH4 II.04 Pyrolysed Abiespinsapo Aliphatic fraction TIC Introduction I.Objectives & procedure of experimental palaeochemo taxonomy Pyrolysed Abiespinsapo Aromatic fraction TIC 280°C II. Development of experimental palaeochemo taxonomy without LiAlH4 III. Application to Coniferales pyrolysedAbiespinsapo Polar fraction TIC IV.Perspectives & future works Conclusions

15. Summary of the experimental procedure Determination/prediction of the molecular signature of the fossil counterpart of the pyrolysed plant The reproduction of this procedure on a great number of plant taxa should considerably increase our knowledge in palaeochemotaxonomy II.05 Aliphatic fraction Introduction Time :24 h, pressure :700 bar,temperature :280°C,WITH LiAlH4 I.Objectives & procedure of experimental palaeochemo taxonomy Aromatic fraction Time :24 h, pressure :700 bar, temperature:280°C,WITHOUT LiAlH4 Polar fraction II. Development of experimental palaeochemo taxonomy Time :24 h, pressure :700 bar, temperature:280°C,WITHOUT LiAlH4 III. Application to Coniferales IV.Perspectives & future works Conclusions

16. Conifers currently studied 69 species studied for experimental palaeochemotaxonomy III.01 Coniferal order is composed of 7 families Araucariaceae 3 Agathis, 8Araucaria&1Wollemia Cupressaceae 1Calocedrus,4Chamaecyparis,2Cupressus,5Juniperus, 1Microbiota,3Thuja&1Thujopsis Pinaceae 4Abies,3Cedrus,4Larix,5Picea,4Pinus,1Pseudotsuga&1Tsuga Podocarpaceae 4Podocarpus Sciadopityaceae 1Sciadopitys Taxaceae 2Taxus, 2Cephalotaxus, 1Torreya Taxodiaceae 1Cryptomeria, 2Cunninghamia, 1Sequoiadendron,1Meta-sequoia,1Sequoia&2Taxodium Introduction I.Objectives & procedure of experimental palaeochemo taxonomy II. Development of experimental palaeochemo taxonomy III. Application to Coniferales IV.Perspectives & future works Conclusions

17. Example of Araucariaceae (sesquiterpenoids) farnesane chama zulene ? cadalene pentaMedi hydroindenes bisabolanes cadalanes curcumenes Araucaria angustifolia III.02 Aliphatic fraction Aromatic fraction Introduction I.Objectives & procedure of experimental palaeochemo taxonomy n-C14 n-C15 II. Development of experimental palaeochemo taxonomy Araucaria araucana III. Application to Coniferales IV.Perspectives & future works Araucaria laubenfelsii Conclusions

18. Example of Araucariaceae (diterpenoids) Monoaromatic tetracyclic diterpane Aromatic abietanes Monoaromatic Labdane ? phyllocladanes labdanes beyerane (iso)pimaranes kauranes phyllocladanes III.03 Aliphatic fraction Aromatic fraction Introduction I.Objectives & procedure of experimental palaeochemo taxonomy Araucaria angustifolia II. Development of experimental palaeochemo taxonomy Araucaria araucana III. Application to Coniferales IV.Perspectives & future works Araucaria laubenfelsii Conclusions

19. Results on the whole Coniferale order Araucariaceae High abundance of tetracyclicditerpanes Low abundance of tricyclicditerpanes &polar terpenoids Cupressaceae High diversity between the different genera Cuparene, cedrane and totaranesseem specific Systematic occurrence of ferruginol and occasional occurrenceof tetracylicditerpanes III.04 Introduction I.Objectives & procedure of experimental palaeochemo taxonomy II. Development of experimental palaeochemo taxonomy III. Application to Coniferales IV.Perspectives & future works Conclusions

20. Results on the whole Coniferale order Pinaceae Systematic presence of dehydroabieticacid and dehydroabietol Some fonctionnalisedcompouds seem to be specific for some genera Taxodiaceae High diversity between the different genera Presence of ferruginol & sugiol Occasional occurrence of tetracyclicditerpanes III.05 Introduction I.Objectives & procedure of experimental palaeochemo taxonomy II. Development of experimental palaeochemo taxonomy III. Application to Coniferales IV.Perspectives & future works Conclusions

21. Difficulties of experimental palaeochemotaxonomy Presented data "Iceberg's point" many information remains to be discovered Many peaks ➜ "orphan" spectra a lots of compounds have never been reported and remain to be identifed Their future identification will supply much more palaeochemotaxonic data IV.01 Introduction Huge mass of data acquired on 69 species of conifers Fresh plants I.Objectives & procedure of experimental palaeochemo taxonomy pyrolysed plants (with & without LiAlH4) II. Development of experimental palaeochemo taxonomy III. Application to Coniferales IV.Perspectives & future works Conclusions

22. Future identification of unknown biomarkers M+ : 240 ➜C18H24 ? Loss of 5 C atoms on cycles D & E Monoaromatic tetracyclic diterpane pyrolysis of pure compounds Ellis et al. (1996) pyrolysis of commercial essential oils pyrolysis of commercial resins IV.02 Introduction I.Objectives & procedure of experimental palaeochemo taxonomy Araucaria angustifolia Aromatic fraction II. Development of experimental palaeochemo taxonomy III. Application to Coniferales IV.Perspectives & future works Conclusions

23. Perspectives of experimental palaeochemotaxonomy Enlarge on other living organisms : - Bacteria (anoxygenic, psychrophile bacteria, cyanobacteria, etc…) - Planktonic organisms ; - Animals, etc… In targeting the organisms which have a palaeoenvironmental interest IV.03 Introduction Enlarge on other botanical groups : - Angiosperms ; I.Objectives & procedure of experimental palaeochemo taxonomy - Bryophytes, pteridophytes (as ferns) and cycadophytes. II. Development of experimental palaeochemo taxonomy III. Application to Coniferales IV.Perspectives & future works Conclusions

24. investigate the molecular composition of fossil plants from their present representatives PALEOBOTANY (fossil plants) PALYNOLOGY (spore & pollen) Conclusions Conclusion Experimental Palaeochemotaxonomy ➜ pertinent and innovativeapproach Introduction I.Objectives & procedure of experimental palaeochemo taxonomy Molecular systematic Botanical systematic II. Development of experimental palaeochemo taxonomy BOTANICAL PALAEOCHEMOTAXONOMY (plant biomarkers) III. Application to Coniferales IV.Perspectives & future works Conclusions

25. Determination of the molecular signature of fossil conifers by experimental palaeochemotaxonomy. Contribution to palaeofloristic and palaeoclimatic reconstructions. Y. Hautevelle R. Michels, B. Farre, F. Lannuzel, F. Malartre Thank you for your attention !