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

Botanical chemotaxonomyIntro-duction

Conclusions

II. Developmentof experimental

palaeochemotaxonomy

Introduction

IV. Perspectives& future works

I. Objectives& procedure ofexperimentalpalaeochemo

taxonomy

III. Applicationto Coniferales

Terpenoids have a chemotaxonomic value and are

thus specific of certain taxa

Chemical composition :- lignin- carbohydrates- lipids, e.g. terpenoids

conifers

Abietic acid

angio-sperms

lupeol

From bioterpenoids to geoterpenoidsIntro-duction

BIOSPHERE

GEOSPHERE

Sedimentary basin

transport

sediment

bioterpenoids

Geoterpenoids can keeptheir initial

chemotaxonomic value

conifers

angio-sperms

Geoterpenoids ormolecular biomarkers

Diagenetic

transformations

angio-sperms

conifers

Conclusions

II. Developmentof experimental

palaeochemotaxonomy

Introduction

IV. Perspectives& future works

I. Objectives& procedure ofexperimentalpalaeochemo

taxonomy

III. Applicationto Coniferales

Distribution of plantbiomarkers

Palaeofloristic compositionon emerged lands

Intro-duction Palaeofloristic and palaeoclimatic reconstructions

The distribution of plant biomarkers reflect the palaeofloristic composition during the deposition

cypres

fern

Interpretation in terms of palaeofloristic composition

pine pineangio-

sperms

sequoia

Conclusions

II. Developmentof experimental

palaeochemotaxonomy

Introduction

IV. Perspectives& future works

I. Objectives& procedure ofexperimentalpalaeochemo

taxonomy

III. Applicationto Coniferales

Intro-duction

desertic climate

temperate climate

tropical climate

polar climate flora ↔ climate

Relations between floras and climates

Conclusions

II. Developmentof experimental

palaeochemotaxonomy

Introduction

IV. Perspectives& future works

I. Objectives& procedure ofexperimentalpalaeochemo

taxonomy

III. Applicationto Coniferales

Chemostratigraphy of vascular plant biomarkersIntro-duction

stratigraphic record

palaeobiodiversitypalaeoflora

palaeoclimateT°, humidity

desertic climate

temperate climate

molecular facies

Geolo

gic

al ti

mes

tropical climate

Plant biomarkers : Are they really interesting compared to other proxies ?

Do they really bear pertinent information ?Conclusions

II. Developmentof experimental

palaeochemotaxonomy

Introduction

IV. Perspectives& future works

I. Objectives& procedure ofexperimentalpalaeochemo

taxonomy

III. Applicationto Coniferales

Advantages of palaeochemotaxonomy

but fossils are scarse

Improved approaches for palaeofloristic

and palaeoclimatic reconstructions

PALAEOBOTANY(fossil plants)

PALYNOLOGY(spore & pollen)

but spores & pollen are not easily reliable to plant taxa

BOTANICAL PALEOCHEMOTAXONOMY (plant biomarkers)

-widespread in the sedimentary record

- related to plant taxa if they have a palaeochemotaxonic value

however plant biomarkers are :

BUT, our current knowledge in paleochemotaxonomyis weak and very lacunar

Intro-duction

Conclusions

II. Developmentof experimental

palaeochemotaxonomy

Introduction

IV. Perspectives& future works

I. Objectives& procedure ofexperimentalpalaeochemo

taxonomy

III. Applicationto Coniferales

Aims of experimental palaeochemotaxonomy

plantbiomarkers

palaeofloristic and palaeoclimatic proxies

➜ new technique of artificial maturation of living plants (confined pyrolysis).

➜ experimental "reproduction" of the plant diagenesis & fossilisation (at the molecular scale).

➜ Aim :

Moleculartaxonomy

Botanicaltaxonomy

I.01

Conclusions

II. Developmentof experimental

palaeochemotaxonomy

Introduction

IV. Perspectives& future works

I. Objectives& procedure ofexperimentalpalaeochemo

taxonomy

III. Applicationto Coniferales

I.02 Experimental and analytical procedures

Fresh plant Sealed gold tubes Confinedpyrolysis

Solubilisation ofterpenoids (CH2Cl2)

Molecular analysis(GC-MS)

Aliphatic

Aromatic

Polar

FractionationConclusions

II. Developmentof experimental

palaeochemotaxonomy

Introduction

IV. Perspectives& future works

I. Objectives& procedure ofexperimentalpalaeochemo

taxonomy

III. Applicationto Coniferales

I.03 Development of experimental palaeochemotaxonomy

Diagenesis of bioterpenoids : ➜ progressive removal of oxygenated functions

➜ reduction of double bonds ➜ saturation vs. aromatisation of 6C cycles (depending on redox conditions

during diagenesis)

Experimental diagenesis : ➜ in accordance with these transformations ➜ generate the broadest possible distribution of biomarkers

(functionalised & hydrocarbon ; aromatic & saturate)

Development of the pyrolysis procedure :

➜ well known diagenetic pathway of a bioterpenoid

➜ abietanoic acids like abietic acid

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.Conclusions

II. Developmentof experimental

palaeochemotaxonomy

Introduction

IV. Perspectives& future works

I. Objectives& procedure ofexperimentalpalaeochemo

taxonomy

III. Applicationto Coniferales

Choice of the plant and its initial compositionII.01

Fresh Abies pinsapoMethylated total fraction

Retention time

Diagenetic pathway of abietanoic acids

Fresh Abies pinsapo contains large amounts

of abietanoic acids

Abies pinsapo (Spanish fir)

Conclusions

II. Developmentof experimental

palaeochemotaxonomy

Introduction

IV. Perspectives& future works

I. Objectives& procedure ofexperimentalpalaeochemo

taxonomy

III. Applicationto Coniferales

II.02

150°C

200°C

250°C

280°C

300°C

Other pyrolysis parameters :duration : 24 h ;pressure : 700 bars.

Diagenetic pathway of abietic acid

280°C Presence ofaromatic diterpanes

Pyrolysed Abies pinsapoTotal fractionm/z 219, 223, 237, 239, 241

Determination of the ideal pyrolysis temperature

Conclusions

II. Developmentof experimental

palaeochemotaxonomy

Introduction

IV. Perspectives& future works

I. Objectives& procedure ofexperimentalpalaeochemo

taxonomy

III. Applicationto Coniferales

II.03

phytene

280°C

280°C presence of diterpanesclassically detected

in the geosphereLiAlH4

Unsaturated abietanes

not satisfying

Pyrolysis with LiAlH4

Pyrolysed Abies pinsapoAliphatic fraction TIC

280°CSaturatedabietanes

labdanes

Diterpane diagenesis

Pyrolysed Abies pinsapoAliphatic fraction TIC

Generation of saturated diterpanes

Conclusions

II. Developmentof experimental

palaeochemotaxonomy

Introduction

IV. Perspectives& future works

I. Objectives& procedure ofexperimentalpalaeochemo

taxonomy

III. Applicationto Coniferales

II.04Pyrolysed Abies pinsapoAliphatic fraction TIC

Pyrolysed Abies pinsapoAromatic fraction TIC

pyrolysed Abies pinsapoPolar fraction TIC

with LiAlH4

280°C

WithoutLiAlH4

280°C

withoutLiAlH4

280°C

Palaeochemotaxonomy of a virtual fossil Abies pinsapo

Typical molecular signature of fossil

Pinaceae

Conclusions

II. Developmentof experimental

palaeochemotaxonomy

Introduction

IV. Perspectives& future works

I. Objectives& procedure ofexperimentalpalaeochemo

taxonomy

III. Applicationto Coniferales

II.05

Aliphatic fractionTime : 24 h, pressure : 700 bar, temperature : 280°C, WITH LiAlH4

Aromatic fractionTime : 24 h, pressure : 700 bar, temperature : 280°C, WITHOUT LiAlH4

Polar fractionTime : 24 h, pressure : 700 bar, temperature : 280°C, WITHOUT LiAlH4

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

Summary of the experimental procedure

Conclusions

II. Developmentof experimental

palaeochemotaxonomy

Introduction

IV. Perspectives& future works

I. Objectives& procedure ofexperimentalpalaeochemo

taxonomy

III. Applicationto Coniferales

Conifers currently studied

Coniferal order is composed of 7 familiesAraucariaceae

3 Agathis, 8 Araucaria & 1 Wollemia

Cupressaceae1 Calocedrus, 4 Chamaecyparis, 2 Cupressus, 5 Juniperus,

1 Microbiota, 3 Thuja & 1 Thujopsis

Pinaceae 4 Abies, 3 Cedrus, 4 Larix, 5 Picea, 4 Pinus, 1 Pseudotsuga &

1 Tsuga

Podocarpaceae4 Podocarpus

Sciadopityaceae1 Sciadopitys

Taxaceae2 Taxus, 2 Cephalotaxus, 1 Torreya

Taxodiaceae 1 Cryptomeria, 2 Cunninghamia, 1 Sequoiadendron, 1 Meta-

sequoia, 1 Sequoia & 2 Taxodium

69 species studied forexperimental palaeochemotaxonomy

III.01

Conclusions

II. Developmentof experimental

palaeochemotaxonomy

Introduction

IV. Perspectives& future works

I. Objectives& procedure ofexperimentalpalaeochemo

taxonomy

III. Applicationto Coniferales

Example of Araucariaceae (sesquiterpenoids)

Araucariaangustifolia

Araucariaaraucana

Araucarialaubenfelsii

n-C14 n-C15

Aliphatic fraction Aromatic fraction

farnesane

bisabolanes cadalanescadalenepentaMedi

hydroindenes

chamazulene ?

curcumenes

III.02

Conclusions

II. Developmentof experimental

palaeochemotaxonomy

Introduction

IV. Perspectives& future works

I. Objectives& procedure ofexperimentalpalaeochemo

taxonomy

III. Applicationto Coniferales

III.03

Araucariaangustifolia

Araucariaaraucana

Araucarialaubenfelsii

labdanes

(iso)pimaranes

MonoaromaticLabdane ?

Monoaromatictetracyclic diterpane

Aromaticabietanes

Aliphatic fraction Aromatic fraction

Example of Araucariaceae (diterpenoids)

beyerane

phyllocladaneskauranes

phyllocladanes

Conclusions

II. Developmentof experimental

palaeochemotaxonomy

Introduction

IV. Perspectives& future works

I. Objectives& procedure ofexperimentalpalaeochemo

taxonomy

III. Applicationto Coniferales

Results on the whole Coniferale orderIII.04

AraucariaceaeHigh abundance of tetracyclic diterpanes

Low abundance of tricyclic diterpanes & polar terpenoids

CupressaceaeHigh diversity between the different genera

Cuparene, cedrane and totaranes seem specific

Systematic occurrence of ferruginol and occasional occurrence of tetracylic diterpanes

Conclusions

II. Developmentof experimental

palaeochemotaxonomy

Introduction

IV. Perspectives& future works

I. Objectives& procedure ofexperimentalpalaeochemo

taxonomy

III. Applicationto Coniferales

PinaceaeSystematic presence of dehydroabietic acid and dehydroabietol

Some fonctionnalised compouds seem to be specific for some genera

TaxodiaceaeHigh diversity between the different genera

Presence of ferruginol & sugiol

Occasional occurrence of tetracyclic diterpanes

Results on the whole Coniferale orderIII.05

Conclusions

II. Developmentof experimental

palaeochemotaxonomy

Introduction

IV. Perspectives& future works

I. Objectives& procedure ofexperimentalpalaeochemo

taxonomy

III. Applicationto Coniferales

Difficulties of experimental palaeochemotaxonomyIV.01

Huge mass of data acquired on 69 species of conifers

Fresh plants

pyrolysed plants (with & without LiAlH4)

Presented data "Iceberg's point"

many information remainsto be discovered

a lots of compounds have never been

reported and remain to be identifed

Many peaks "orphan" spectra➜

Their future identification will supply much more

palaeochemotaxonic dataConclusions

II. Developmentof experimental

palaeochemotaxonomy

Introduction

IV. Perspectives& future works

I. Objectives& procedure ofexperimentalpalaeochemo

taxonomy

III. Applicationto Coniferales

Future identification of unknown biomarkersIV.02

Araucariaangustifolia

Aromaticfraction

Monoaromatictetracyclic diterpane

?M+ : 240 ➜ C18H24

Loss of 5 C atoms on

cycles D & E

Ellis et al. (1996)pyrolysis of pure compounds

pyrolysis of commercial essential oils

pyrolysis of commercial resinsConclusions

II. Developmentof experimental

palaeochemotaxonomy

Introduction

IV. Perspectives& future works

I. Objectives& procedure ofexperimentalpalaeochemo

taxonomy

III. Applicationto Coniferales

Perspectives of experimental palaeochemotaxonomyIV.03

Enlarge on other botanical groups :

- Angiosperms ;

- Bryophytes, pteridophytes (as ferns)and cycadophytes.

In targeting the organisms which have a palaeoenvironmental interest

Enlarge on other living organisms :

- Bacteria (anoxygenic, psychrophile bacteria, cyanobacteria, etc…)

- Planktonic organisms ;

- Animals, etc…

Conclusions

II. Developmentof experimental

palaeochemotaxonomy

Introduction

IV. Perspectives& future works

I. Objectives& procedure ofexperimentalpalaeochemo

taxonomy

III. Applicationto Coniferales

ConclusionsConclusion

Experimental Palaeochemotaxonomy ➜ pertinent and innovative approach

investigate the molecular composition of fossil plants from their present

representatives

BOTANICAL PALAEOCHEMOTAXONOMY(plant biomarkers)

PALEOBOTANY(fossil plants)

PALYNOLOGY(spore & pollen)

Molecularsystematic

Botanicalsystematic

Conclusions

II. Developmentof experimental

palaeochemotaxonomy

Introduction

IV. Perspectives& future works

I. Objectives& procedure ofexperimentalpalaeochemo

taxonomy

III. Applicationto Coniferales

Y. HautevelleR. Michels, B. Farre, F. Lannuzel, F. Malartre

Determination of the molecular signature of fossil conifers by experimental

palaeochemotaxonomy.

Contribution to palaeofloristic and palaeoclimatic reconstructions.