Terpenoids in extracts of Lower Cretaceous ambers from the Basque-Cantabrian Basin

15 Pages·2010·1.51 MB·English

OrganicGeochemistryxxx(2010)xxx–xxx
ContentslistsavailableatScienceDirect
Organic Geochemistry
journal homepage: www.elsevier.com/locate/orggeochem
Terpenoids in extracts of Lower Cretaceous ambers from the Basque-Cantabrian
Basin (El Soplao, Cantabria, Spain): Paleochemotaxonomic aspects
César Menor-Salvána,*, Maria Najarrob, Francisco Velascoc, Idoia Rosalesb, Fernando Tornosb,
Bernd R.T. Simoneitd,e
aCentrodeAstrobiología(CSIC-INTA),28850TorrejóndeArdoz,Spain
bInstitutoGeológicoyMinerodeEspaña(IGME),RiosRosas23,28003Madrid,Spain
cUniversidaddelPaísVasco,DepartamentoMineralogíayPetrología,Apdo.644,48080Bilbao,Spain
dCOGER,KingSaudUniversity,Riyadh11451,SaudiArabia
eDepartmentofChemistry,OregonStateUniversity,Corvallis,OR97331,USA
a r t i c l e i n f o a b s t r a c t
Articlehistory: ThecompositionofterpenoidsfromwellpreservedCretaceousfossilresinsandplanttissuesfromthe
Received22June2010 amberbearingdepositsofElSoplaoandReocíninCantabria(northernSpain)havebeenanalyzedusing
Accepted30June2010 gaschromatography–massspectrometryandtheresultsarediscussedusingtheterpenoidcomposition
Availableonlinexxxx
ofextantconifersasareference.Amberispresentatmanyhorizonswithintwounitsofcoastaltoshallow
marinesiliciclasticsofAlbianandCenomanianage.Thefossilresinsareassociatedwithblackamber(jet)
andabundant,wellpreservedplantcuticlecompressions,especiallythoseoftheextinctconifergenus
Frenelopsis(Cheirolepidiaceae).
Wereportthemolecularcharacterizationoftwotypesofamberwithdifferentbotanicalorigins.Oneof
themischaracterizedbythesignificantpresenceofphenolicterpenoids(ferruginol,totarolandhinokiol)
andpimaric/isopimaricacids,aswellastheirdiageneticproducts.Thepresenceofphenolicditerpenoids
togetherwiththelackofabieticanddehydroabieticacidsexcludesbothPinaceaeandAraucariaceaeas
sourcesfor thistype ofamber. The biological diterpenoid composition issimilar to that observed for
extantCupressaceae.Thesecondtypeofamberischaracterizedbytheabsenceofphenolicterpenoids
andotherspecificbiomarkers.Someterpenoidswithuncertainstructureweredetected,aswellasthe
azulenederivativeguaiazulene.OurresultssuggestthattheamberfromCantabriacouldbefossilized
resinfromFrenelopsisandotherundeterminedbotanicalsources.Thebiologicalterpenoidassemblage
confirmsachemosystematicrelationshipbetweenFrenelopsisandmodernCupressaceae.
(cid:2)2010ElsevierLtd.Allrightsreserved.
1.Introduction Analysis of the chemical composition of fossil resins is not
straightforward, because the original biochemical fingerprints of
Amberisfossilizedresinproducedfromtheexudatesofconifers theresinsareusuallymodifiedduringdiagenesis,withthebioterp-
andcertainangiospermsandisconsideredtobeoneofthefewfos- enoids (unmodified biosynthetic natural products) being trans-
sil deposits of exceptional preservation (Konservat Lagerstätten), formed into geoterpenoids (diagenetic products of degraded
because it permits the conservation of fossil organisms with all bioterpenoidsthatarefoundinamberandfossilplanttissues;Otto
their delicate anatomical details. Fossil resins not only preserve etal.,2007).Despitethesediageneticalterations,geoterpenoidsre-
the anatomy of fossil life forms that were trapped as biological tainthebasicskeletalstructuresoftheirbiologicalprecursorsand
inclusions, but also constitute a valuable source of information canbeusedasmolecularmarkers(biomarkers;Petersetal.,2005;
about their own botanical origin, ancient terrigenous ecosystems Marynowskietal.,2007).Coniferssynthesizemainlyditerpenoids,
and climatic change by means of their chemical composition whichare,alongwithsesquiterpenoids,thecompoundsthatpro-
(AndersonandCrelling,1995). videthebestresultsasdiagnosticbiomarkersofconifersandtheir
resins(OttoandWilde,2001).Amongthediterpenoidspreserved
in amber, labdane derivatives and non-phenolic abietane diage-
* Correspondingauthor.Tel.:[phone];fax:[phone]. netic derivatives have the most limited chemotaxonomic value,
E-mailaddresses:[email protected],[email protected](C.Menor-Salván),m.na
as they occur in all conifer families. On the other hand, phenolic
[email protected](M.Najarro),[email protected](F.Velasco),[email protected]
(I.Rosales),[email protected](F.Tornos). terpenoids, such as ferruginol and totarol, are produced only by
0146-6380/$-seefrontmatter(cid:2)2010ElsevierLtd.Allrightsreserved.
doi:10.1016/j.orggeochem.2010.06.013
Pleasecitethisarticleinpressas:Menor-Salván,C.,etal.TerpenoidsinextractsofLowerCretaceousambersfromtheBasque-CantabrianBasin(ElSoplao,
Cantabria,Spain):Paleochemotaxonomicaspects.Org.Geochem.(2010),doi:10.1016/j.orggeochem.2010.06.013
2 C.Menor-Salvánetal./OrganicGeochemistryxxx(2010)xxx–xxx
themembersofthefamiliesCupressaceaeandPodocarpaceae(Cox omybasedonmodernresincompositionsandrelatedterpenoids
etal.,2007).Therefore,thechemotaxonomicvalueofthesecom- foundinamberassociatedfossils.
poundsisveryhighandtheirpresenceinamberprovidesveryuse-
ful palaeobotanical information. Although the preservation
potentialofpolarbiomarkersisconsideredtobelow(Ottoetal., 2.Samplesandmethods
2007), the oldest polar diterpenoids have been identified in ex-
tracts of Middle Jurassic fossil conifer wood from Poland (Mary- 2.1.Geologicalbackground
nowski et al., 2007), and diterpenoid derivatives could also be
liberated from a Carboniferous amber by pyrolysis (Bray and TheanalyzedsamplesbelongtotheCretaceoussuccessionatthe
Anderson,2009). northwestern margin of the Basque-Cantabria Basin in northern
Recently,anewCretaceousamberdepositwithexquisite,well Spain.DuringtheCretaceous,theevolutionofthisbasinwascon-
preserved fossil organisms, mostly insects, has been discovered trolledbyextensional,andperhapsstrike-slip,deformationassoci-
innorthernSpain(RábagovillageinElSoplaoterritory,Cantabria; atedwiththeopeningoftheNorthAtlanticOceanandtheBayof
Menor-Salvánetal.,2009;Najarroetal.,2009).Basedonprelimin- Biscay(e.g.LePichonandSibuet,1971;Rat,1988;García-Mondéjar
ary infrared spectroscopy of the El Soplao amber (Najarro et al., etal.,1996;Sotoetal.,2007).RiftingduringtheLateJurassic–Early
2009) and previous gas chromatography–mass spectrometry Cretaceousledtotheformationofseveralnarrowsub-basinscon-
(GC–MS)studiesonamberfromaneighboringsiteinÁlava(Alonso trolledbyE–W,NW–SEandSW–NWtrendingfaults;thesebasins
etal.,2000;ChalerandGrimalt,2005),ithasbeensuggestedthat hostbothcontinentalandmarinesedimentsofvariablethickness
exudate from Agathis (a conifer of the family Araucariaceae) was (García-Mondéjaretal.,1996;Sotoetal.,2007).
the most likely source of this amber, as has also been proposed ThestudyarealiesintheCantabriaregionimmediatelytothe
for other Cretaceous ambers (e.g. Lambert et al., 1996; Alonso north of the Cabuérniga Ridge (Fig. S1; supplementary material),
etal.,2000;Poinaretal.,2004;ChalerandGrimalt,2005;Delclòs anE–WfaultzonethatrepresentsaLate-Variscanstructurereacti-
et al., 2007). This speculation was largely based on the presence vatedfirstasapaleo-highboundedbynormalfaultsduringtheEarly
of some geoterpenoids that may have been derived from agathic Cretaceous,andlaterasreversalfaultsduringthewidespreadCeno-
andpimaricacids.However,althoughthosecompoundsandtheir zoic(Pyrenean)compression.TheLower–MiddleCretaceous(Barre-
diageneticderivativesarecharacteristicofAraucariaceae,theyare mian–Early Cenomanian) deposits in the study area are weakly
notdiagnostic,becausetheycanalsobefoundinotherextantconi- deformedandaffectedonlybygentlefolding.Theyarecomposed
ferfamilies(Ottoetal.,2007).Moreover,Alonsoetal.(2000)have ofarelativelythin((cid:2)200–800m)syn-riftsequencethatliesuncon-
reported the presence of the phenolic abietane ferruginol in the formablyonCarboniferoustoLowerJurassicbasement(Fig.S1).
Álavaambersamples,indicatingthatmoreextensivestudyofthe Asimplifiedsynthesisofthestratigraphy intheElSoplaoand
chemotaxonomicinformationcontainedintheamberisnecessary Reocín areas is shown in Fig. 1, with formation names according
toestablishitsdefinitebotanicalorigin. to Hines (1985) and revised by Najarro et al. (2009). The amber
Inaddition,meso-andmacrofossilplantremainsofAraucaria- bearingdepositatElSoplaoisincludedwithintheLasPeñosasFor-
ceae are absent in these amber bearing deposits, although there mation(Fig.1),aLowerAlbianunit((cid:2)112–110Ma)ofcontinental
areplentyofcuticlesandremainsofothervascularplants,especially to transitional marine siliciclastics. Detailed descriptions of field
thegeneraFrenelopsissp.andMiroviasp.,oftheextinctconiferfam- sections,depositionalenvironmentsandfossilcontentofthisunit
iliesCheirolepidiaceaeandMiroviaceae,respectively(Gomezetal., aregiveninNajarroetal.(2009).Withintheoutcrop,theElSoplao
2002a;Najarroetal.,2009).Thisisalsothecaseinmanyotheramber amberdepositischaracterizedbyabout1.5–2mofdark,carbona-
depositsfromtheCretaceousofSpainandFrance(e.g.Delclòsetal., ceouslutites,siltstonesandsandstoneswithinterbedded,centime-
2007;Néraudeauetal.,2008).Thus,therecurrentassociationofam- ter to decimeter layers with remarkable accumulations of plant
berwithcuticlesofCheirolepidiaceaeandMiroviaceae,alongwith remains and amber pieces of different sizes and forms (Fig. 2A
thelackofAraucariaceaeremains(exceptforasmallamountofpol- andB).Mostamberpiecesshowablue-purplecolorundernormal
lengrainsinthesediments)(Barrónetal.,2001),challengesthepro- sunlightandbrightmilkybluefluorescenceunderultravioletlight.
posedoriginoftheamber.Sincechemicalevidencehasnotyetgiven Plantcuticlesareveryabundantinthelevelsassociatedwitham-
adefinitiveanswer,moreconvincingproofisnecessarytoaccept ber(Fig.2C).TheyaremainlyassignedtotheconifergeneraFrenel-
Araucariaceaeasthesourceoftheresin. opsisandMirovia,alongwithothermoreoccasionalleavesofthe
Inthisstudy,amberpiecesandassociatedfossilleavesfromthe ginkgoalean genera Nehvizdya and Pseudotorellia (Najarro et al.,
Cretaceousamberbearing depositofEl Soplao (Cantabria;Fig.1) 2009). Inmostoftheamberbeds,leavesofthegenusFrenelopsis
were systematically analyzed using complementary techniques of the extinct conifer family Cheirolepidiaceae are the dominant
suchasinfraredspectroscopy(FTIR)andGC–MS.Theoverallaim macro-botanical remains. Frenelopsis were xeromorphic plants
was to identify the terpenoids preserved and their diagenetic adaptedtocoastalhabitatsandprobablygrewmainlyinbrackish
transformationproductsinthefossilresinandtodeterminetheir coastalmarshesandmangroves,butwereadaptedtoawiderrange
possible botanical sources. Due to exceptional preservation, the ofhabitats(Gomezetal.,2002a,2003).
amberbearingdepositatElSoplaooffersauniqueopportunityto TheamberdepositatReocín(Fig.S1;supplementarymaterial)is
compare the molecular composition of the amber with that of slightly younger than the El Soplao amber deposit. It is included
plantremainsfromthefamilyCheirolepidiaceaeandMiroviaceae, withintheBielvaFormation(Fig.1),aLatestAlbian–EarlyCenoma-
whichappearinthesamedeposit.Amorphologicalsimilaritybe- nian((cid:2)102–99Ma)unitcomposedofabout250moftidaldomi-
tweenextinctCheirolepidiaceaeandextantCupressaceaehasbeen nated, estuarine siliciclastic deposits in the study area (Hines,
described,buttheirrelationshipremainsspeculativeduemainlyto 1985). Within this unit, the amber accumulations are associated
thelackofmolecularevidence(BroutinandPons,1975;Alvinand withcarbonaceousclaystonesandtidalchannelsandstonesdevel-
Hluštík,1979;Seoane,1998;Miller,1999;Farjon,2008).AsChei- oped in estuarine mouth subtidal areas (López-Horgue et al.,
rolepidiaceae is an extinct family, the connection between the 2001).Despitethedifferencesinage,theReocínambershowsthe
two families could aid in the chemotaxonomical study of amber samecompositionastheElSoplaoamber.Thermalmaturityindica-
and in the confirmation of the botanical origin. We present data tors(vitrinitereflectance)ofmaceralsintheElSoplaodepositreveal
of two separate types of amber found in the El Soplao deposit minorchangesintheorganicmatteroftheresinsduringtheirdiage-
anddiscusstheirbotanicaloriginusingcomparativechemotaxon- netic history and maximum thermal conditions during burial of
Pleasecitethisarticleinpressas:Menor-Salván,C.,etal.TerpenoidsinextractsofLowerCretaceousambersfromtheBasque-CantabrianBasin(ElSoplao,
Cantabria,Spain):Paleochemotaxonomicaspects.Org.Geochem.(2010),doi:10.1016/j.orggeochem.2010.06.013
C.Menor-Salvánetal./OrganicGeochemistryxxx(2010)xxx–xxx 3
SW NE
STRATIGRAPHYOFELSOPLAO STRATIGRAPHYOFREOCÍN
93ma LITHOLOGY SECTOR SECTOR LITHOLOGY
N AT ALTAMIRAFm. ALTAMIRAFm.
E L
99ma C EAR BIELBAFm. BIELBAFm.
N ATE BARCENACIONESFm. BARCENACIONESFm.
A L
112ma EOUSALBI EARLY LASPEÑOSASFm. LASPEÑOSASFm.
C
CRETAAPTIAN AAELRTLY PRAETROSOACCNÍINNEISOFTmEFmB.A.NFm. SPRAAENRTOROEDOSCECTZÍIEANNSBIOFAFmNmF.mF.m..
125ma E RÁBAGOFm. UMBRERAFm. UMBRERAFm.
BAR PASGROUP(WEALD) PASGROUP.(WEALD)
HAU
VAL
145ma BER HIATUS
HIATUS
AC
200ma JURSSI LIAS
AC KEUPER
RISI
250ma TS BUNTSANDSTEIN BUNTSANDSTEIN
N
RA
PEMI HIATUS HIATUS
300ma
360ma ACRBONIFEROUS PALEOZOICBASEMENT PALEOZOICBASEMENT
Marls Mudstonesandgypsum Conglomerates
Limestones Mudstonesandsandstones Sandstones
Dolostones Mudstones Silts
Amber-bearingdeposit Lignite
Fig.1. Chrono-andlithostratigraphyoftheElSoplaoandReocínareas(modifiedfromHines,1985).ChronostratigraphyafterGradsteinetal.(2004).
(cid:2)60–70(cid:3)C(Supplementarymaterial).Consequentlyandduetoits 2.3.2.Extractionandfractionation
highertransparencyandlackofinclusionsandinterferences,theEl For the analytical characterization, two representative single
Soplao amber was used preferentially for the chemosystematic pieces(AandB;Table1)ofamberofabout50g,withthehighest
study. transparency available and free of major inclusions, crusts and
debris,werecollectedfromtheElSoplaodeposit.Eachpiecewas
crushed and extracted for 4h with dichloromethane:methanol
2.2.Sampling
(2:1v:v) using a Büchi model B-811 automatic extractor. The
extractablematerialconstitutes16%ofthetotalamberweighton
Amberpieces,jet(blackamber),fossilwoodandsedimentsrich
average.Onealiquotofextractwasinjecteddirectlyintotheport
inplantcuticleswerecollectedfromtheElSoplaodepositduringa
ofthegaschromatograph.
recent excavation in October 2008. Two types of amber pieces
Thebulkextractwasthenprocessedinordertopurifythepheno-
werefoundatthedepositinthesamesedimentologicalandtapho-
licterpenoidfractionandtheacidicfractionandtoidentifyunam-
nomicalcontext:Atype,characterizedbyastrongblue-purplecol-
biguously the minor components with higher chemosystematic
orundernaturallight,purple-reddishunderartificiallightandless
value.Theaimwastoestablishacompletedescriptivecomposition
abundant B type, yellow-honey under artificial light and honey
oftheambersample.Theextractwasconcentratedtoavolumeof
withabluishtingeundernaturallight.Wecollectedthetwotypes
20mlandfractionatedbyflashchromatographyonsilicagel.The
of amber present and the black amber associated with amber of
elutionwasperformedusingn-hexane,dichloromethane,dichloro-
type A and fossil plant tissue. Plant cuticles were obtained from
methane:methanol (1:1v:v), and methanol as eluents and 25
claystonesbyrinsingtheplantrichsedimentinanultrasonicbath
fractions of 1.5ml were collected using an automatic fraction
ofdistilledwatertoremovealltheclayandsiltsediment.Theor-
collector. Each fraction was concentrated by evaporation of the
ganic residue (Fig. 2C) was air dried. Plant fragments and leaves
solventunderN andanalyzedbyGC–MS.Thefractionswithsimilar
fromdifferentfamiliesweredistinguishedandseparatedundera 2
compositions were combined. The polar fraction (eluted with
stereomicroscope.
methanol)andthefractionscontainingferruginolwererecombined,
further separated using a glass column (20cm) filled with
2.3.Analyticalmethods chromatographic grade silica gel, and eluted sequentially with
n-hexane:dichloromethane (1:1v:v), pure dichloromethane,
2.3.1.Infraredspectroscopy(FTIR) dichloromethane:methanol(1:1v:v)andmethanol.Fourfractions
IR spectra of pulverized solid amber were obtained using a of20mlwerecollected,designatedAtoD.Allfractionsweredried
NexusNicoletFTIRspectrometerinthe4000–400cm(cid:3)1range. andthealcoholsandacidsconvertedtotrimethylsilylderivatives
Pleasecitethisarticleinpressas:Menor-Salván,C.,etal.TerpenoidsinextractsofLowerCretaceousambersfromtheBasque-CantabrianBasin(ElSoplao,
Cantabria,Spain):Paleochemotaxonomicaspects.Org.Geochem.(2010),doi:10.1016/j.orggeochem.2010.06.013
4 C.Menor-Salvánetal./OrganicGeochemistryxxx(2010)xxx–xxx
Fig.2. Photographsofamberandpalaeobotanicalcomponentsusedinthisstudy.(A)InsituamberpieceoftypeA(6cm)fromtheElSoplaodeposit,showingblue-purple
colorundersunlight.(B)AmberpieceoftypeB(3cm)onfossilwoodundersunlight.(C)Insitusedimentsshowingtheirpalaeobotanicalcomponents:fossilleavesofMirovia
andFrenelopsisandoneleafofGinkgoaceae.(D)SelectedleavesofFrenelopsissp.wereusedforbiomarkeranalysis.(Forinterpretationofthereferencestocolorinthisfigure
legend,thereaderisreferredtothewebversionofthisarticle.)
byreactionwithN,O-bis-(trimethylsilyl)trifluoroacetamide(BSTFA) identifiedbycomparingtheirmassspectrawiththoseofauthentic
containing1%trimethylchlorosilane(TMCS)at65(cid:3)Cforaperiodof standardsandwithpublisheddata(seeSection3.2).
3h. Finally, the derivatized fractions werediluted withn-hexane
andinjectedintotheportofthegaschromatograph. 3.Resultsanddiscussion
TostudythemolecularcontentoffossilFrenelopsisandMirovia
leaves(Fig.2),5gofleaveswereextractedfor4hwithdichloro- 3.1.Infraredspectroscopy
methane:methanol(2:1v:v)usingaBüchimodelB-811automated
extractor. The bulk extract was filtered and analyzed directly by TheapplicationofIRtothestudyofamberiswelldocumented
GC–MS.Theextractwasthenfractionatedusingsilicagelchroma- andconstitutesabasictechniqueforthecharacterizationoffossil
tographyintwofractionsbyelutionwithn-hexane:dichlorometh- resins(Langenheim,1969;Grimaltetal.,1988;Alonsoetal.,2000).
ane (3:1v:v) and dichloromethane:methanol (4:1v:v). The polar Becauseoftheinclinationofallambersandresins(evennon-fossil
fraction was dried and derivatized using the method described resins)toshowsimilarbulkinfraredspectra(duetotheircommon
above.Thesamplesofjet(blackamber)wereextractedusingthe chemicalfunctionalgroups),IRspectroscopyhasstronglimitations
sameprotocol.Duetothelesseravailabilityofleavesandjetand for the determination of their botanical origin (Yamamoto et al.,
the lower percentage of extractable organic matter, we used the 2006).TheIRspectrumoftheCantabrianamberisconsistentwith
simplifiedfractionationdescribedaboveinordertocomparetheir those observed for other amber samples (Fig. S2; supplementary
biomarkercompositionwiththatofamber. material)andcouldindicatethatitiscomposedofamixtureofter-
penoidsandlabdatrienecopolymers.Thebandpatternissimilarto
2.3.3.GC–MS theIRspectrumexpectedforthelabdatrienescommunicacidand
TheanalyseswereperformedonanAgilent6850GCcoupledto biformene and their polymers, consistent with the stated macro-
an Agilent5975C quadrupolemass spectrometer. Separation was molecularstructureofamber(Villanueva-Garcíaetal.,2005)and
achieved on a HP-5MS column coated with (5%-phenyl)-meth- withtheterpenoidcompositionfound(seeSection3.2).Theweak
ylpolysiloxane (30m(cid:4)0.25mm, 0.25lm film thickness). The bandat882cm(cid:3)1(Fig.S2,supplementarymaterial)ischaracteris-
operatingconditionswereasfollows:8psicarrierpressure,initial tic of the exocyclicmethylene moiety supporting the labdatriene
temperature held at 40(cid:3)C for 1.5min, increased from 40(cid:3)C to input.Thetwotypesofambersamplesfoundinthedepositshow
150(cid:3)C at a rate of 15(cid:3)C/min, held for 2min, increased from similarIRspectra.
150(cid:3)C to 255(cid:3)C at a rate of 5(cid:3)C/min, held constant for 20min
and finally increased to 300(cid:3)C at a rate of 5(cid:3)C/min. The sample 3.2.TerpenoidcompositionofCantabrianamber
was injected in the splitless mode with the injector temperature
at290(cid:3)C.Themassspectrometerwasoperatedintheelectronim- Thetotalextractsoftheambercontainmethylatednaphthalenes
pact mode at 70eV ionization energy and scanned from 40 to (di-,tri-andtetramethylnaphthalenes)anddi-andtrimethyltetra-
700Da. The temperature of the ion source was 230(cid:3)C and the lins,sesquiterpenoidsandbi-andtricyclicditerpenoids(Table1).
quadrupoletemperaturewas150(cid:3)C.Datawereacquiredandpro- GCanalysisofthebulkextractshowsthreedifferentzonesinthe
cessed using Chemstation software. Individual compounds were gaschromatogram(Fig.3).Thedominantcompoundsintheearly
Pleasecitethisarticleinpressas:Menor-Salván,C.,etal.TerpenoidsinextractsofLowerCretaceousambersfromtheBasque-CantabrianBasin(ElSoplao,
Cantabria,Spain):Paleochemotaxonomicaspects.Org.Geochem.(2010),doi:10.1016/j.orggeochem.2010.06.013
C.Menor-Salvánetal./OrganicGeochemistryxxx(2010)xxx–xxx 5
Table1
Terpenoidsandtheirdiageneticderivativesidentifiedinbulkandchromatographicfractionsofextractsfromtheambers(typesAandB)oftheElSoplaodeposit.
Noa Compound Composition MW Relativeabundanceb
A B
AbietanesandPodocarpanes
1(XVI) 16,17-Bisnorsimonellite C17H20 224 38.2 –
2(XXII) 16,17,18-Trisnorabieta-8,11,13-triene C17H24 228 100 100
3 16,17,19-Trisnorabieta-8,11,13-triene C17H24 228 23.4 20.9
4(XVII) Retene C18H18 234 1.0 –
5 16,17-Bisnordehydroabietane C18H26 242 4.6 42.5
6 Simonellite C19H24 252 9.3 1.5
7(XXVII) 14-Methyl-16,17-bisnordehydroabietane C19H28 256 2.5 6.9
8 1-Methyl-10,18-bisnorabieta-8,11,13-triene C19H28 256 – 6.6
9(I) 18-Norabietatriene(Dehydroabietin) C19H28 256 45.8 14.0
10(I) 19-Norabietatriene C19H28 256 3.6 4.1
11(IV) 18-Norabieta-7,13-diene C19H30 258 9.7 –
12(V) Norabiet-13-ene C19H32 260 57.0 23.0
13(III) Fichtelite C19H34 262 5.8 d
14(X) 12-Hydroxysimonellite C19H24O 268 11.8 –
15(II) Dehydroabietane C20H30 270 40.3 –
16(XXV) 16,17-Bisnordehydroabieticacidc C18H24O2 272 d d
17(VIII) Ferruginol C20H30O 286 24.3 –
18(XIII) Callitrisicacidc C20H28O2 300 d –
19(XII) Hinokiolc C20H30O2 302 d –
PimaranesandIsopimaranes
20(XXI) Pimaricacidc C20H30O2 302 d d
21(XXII) Isopimaricacidc C20H30O2 302 d d
22(XXVI) Pimar-8-en-18-oicacidc C20H32O2 304 d d
23 Pimaradiene C20H32 272 – 4.5
Labdanes
24 14,15-Bisnorlabda-8,12-dien-18-oicacidc C18H28O2 276 d d
25(XXXIV) E-19-Noragathicacid C19H30O2 290 10.6 3.1
26(XXXIII) Z-19-Noragathicacid C19H30O2 290 3.7 1.5
27(XXIX) 13-Dihydro-19-noragathicacidc C19H32O2 290 d –
28(XXVIII) 13-Dihydroagatholicacid C20H34O3 322 11.8 –
Othercompounds
29 Ionene C13H18 174 37.2 48.4
30 Methylionene C14H20 188 13.0 34.2
31 Tetrahydroeudalene C14H20 188 16.0 8.3
32(XXXVII) Guaiazulene C15H18 198 7.6 1.5
33(XXXVIII) Cadalene C15H18 198 8.2 7.5
34 Drimane C15H28 208 3.6 14.8
35 Homodrimane C16H30 222 3.3 3.9
36(XXXI) 2,5,8-Trimethyl-1-butyltetralin C17H26 230 79.1 57.3
37(XXXII) 2,5,8-Trimethyl-1-isopentyltetralin C18H28 244 d 10.8
38(XX) Diaromatictotarane C19H24 252 1.0 –
39(XI) Totarolc C20H30O 286 d –
a RomannumeralsinparenthesesrefertostructuresshowninAppendixA.
b Abundancerelativetothemajorpeak(100%)inthebulkextracts(GC–MSTIC).Occurrenceistabulatedoncompoundsdetectedonlyafterfractionationandderivatization
(d:detected;–:notdetected).
c AlsoanalyzedastheTMSderivative.
elution range are a-ionene, methylionene, trimethylnaphthalene Theabietaneclassterpenoidswereidentifiedbycomparisonoftheir
isomers, tetrahydroeudalene, calamenene isomers, drimane and massspectrawiththoseofstandardsorpublishedintheliterature
homodrimane(identifiedafterDzouetal.,1999andSonibareand (Czechowski et al., 1996; Otto and Simoneit, 2002; Otto et al.,
Ekweozor,2004).Thea-ionene,methylioneneanddrimanesmay 2002; Hautevelle et al., 2006; Cox et al., 2007), and comprised
bederivedfromlabdanesintheresinthroughdegradationprocesses 18- and 19-norabieta-8,11,13-triene (I; chemical structures cited
(Yamamoto et al., 2006; Pereira et al., 2009). Overall, these are shown in Appendix A), dehydroabietane (II), fichtelite (III),
componentsarehighlydegradeddiageneticproductsthathaveno 18-norabieta-7,13-diene (IV) and norabiet-13-ene (V). The latter
chemotaxonomic value due to their unrecognizable parent struc- compoundwastentativelyidentifiedbymatchwithamassspec-
tures. The second section of the gas chromatogram is dominated trumintheliterature(Hautevelleetal.,2006),characterizedbya
by non-oxygenated bi- and tricyclic diterpenoids and the third molecularionatm/z260andlossofanisopropylgroup(m/z217).
sectioncontainspolarbi-andtricyclicditerpenoids.Wedidnotfind 18-Norabieta-7,13-diene(IV)wasidentifiedonlyinsampleAbya
aliphaticlipids,hopanoids,fungalterpenoidsorplanttriterpenoids match with the published mass spectrum (Otto and Simoneit,
intheambersamples,discardinganangiospermcontributionand 2002). This compound has been described as a decarboxylation
majorcontamination. product of abietic acid during diagenesis (Otto and Simoneit,
2002).Inthiscase,theprecursormoleculehasnotbeenfound.The
3.2.1.Abietanediterpenoids lackofaclearbiologicalprecursorfornorabieta-7,13-diene(IV)sug-
Thediterpenoidsidentifiedintheextractsbelongtotheabietane, gestsanalternativeorigin,possiblybydoublebondisomerizationof
pimarane/isopimaraneandlabdanestructuralclasses(Fig.3).These unsaturatedabietanes.Thiscompositionisconsistentwiththedom-
diterpenoidsaretypicalofconifers(OttoandWilde,2001;Yamam- inanceofdehydroabietaneandabietanegeoterpenoidsinthetypeA
otoetal.,2006),confirmingsuchanoriginfortheCantabrianambers. amber.Thenorabietatrienes(dehydroabietins)foundinbothamber
Pleasecitethisarticleinpressas:Menor-Salván,C.,etal.TerpenoidsinextractsofLowerCretaceousambersfromtheBasque-CantabrianBasin(ElSoplao,
Cantabria,Spain):Paleochemotaxonomicaspects.Org.Geochem.(2010),doi:10.1016/j.orggeochem.2010.06.013
6 C.Menor-Salvánetal./OrganicGeochemistryxxx(2010)xxx–xxx
A
16,17,18-Trisnorabieta- 18-Norabieta-7,13- Norabiet-13-ene Dehydroabietin
8,11,13-triene diene
OH
O
16,17-
16,17- Bisnordehydroabietane
Bisnorsimonellite
2,5,6-trimethyl-
1-butyl tetralin
19-Noragathic
acid
Tetrahydroeudalene
OH
Dehydroabietane
O
e
c Guaiazulene OH
n
a
d
n α-Ionene
u HO
b
a 13-Dihydroagatholic
e Methylionene Ferruginol acid
v
ati OH
el Homodrimane
R
Simonellite
12-
Drimane hydroxysimonellite
B
2,5,6-Trimethyl-1-
16,17,18-Trisnorabieta- isopentyl tetralin
Methylionene 8,11,13-triene
Norabiet-13-ene
Pimaradiene
α-Ionene Dehydroabietin
2,5,6-Trimethyl-
1-butyl tetralin
HO OH
e 16,17- O O
c Bisnordehydroabietane
n Guaiazulene
a
d
n
u
b Drimane
a 19-Norlabda-
e 8(20),12-dien-15-oic
ativ bis1n4o-rdMeehtyhdyrlo-1a6b,i1e7ta-ne acids
el
R
10 20 30
Retention time (min)
Fig.3. GC–MStotalioncurrent(TIC)tracesoftheunderivatizedtotalextractsof:(A)ElSoplaoblueamber,typeA,(B)ElSoplaoyellow-blueamber,typeB,withthemain
terpenoidcompoundsidentified.Peaksnotannotatedareunidentified,tentativelyidentifiedorknowncompoundswithlittlechemotaxonomicvalue.(Forinterpretationof
thereferencestocolorinthisfigurelegend,thereaderisreferredtothewebversionofthisarticle.)
Pleasecitethisarticleinpressas:Menor-Salván,C.,etal.TerpenoidsinextractsofLowerCretaceousambersfromtheBasque-CantabrianBasin(ElSoplao,
Cantabria,Spain):Paleochemotaxonomicaspects.Org.Geochem.(2010),doi:10.1016/j.orggeochem.2010.06.013
C.Menor-Salvánetal./OrganicGeochemistryxxx(2010)xxx–xxx 7
samplescouldbederivedfromallabietaneprecursorsbydiagenetic presenceoftotarol(seebelow)andthefossilrecordofthedeposit,
alteration(Simoneit,1986;Hautevelleetal.,2006). suggestthatAraucariaceaedidnotcontributetothemaintypeof
Dehydroabietane(II)isanaturalproductofmanyPinaceaeres- amber found at the studied deposit (type A). On the other hand,
ins(Ottoetal.,2007)aswellassomeCupressaceaeresins.Inthese wedidnotfindphenolicabietanesinthetypeBambersample.This
samples, dehydroabietane is a significant component in amber fact,takentogetherwiththepresenceofdehydroabietaneinsam-
typeA,whereasitisnotdetectableinambertypeB,suggestinga pleAanditsabsenceinsampleB,constitutesthemainchemotaxo-
different paleobotanical origin for both types of amber samples. nomicdifferencebetweenthetwotypesofsamples.Asignificant
Theabsenceofabietic(VI)ordehydroabieticacids(VII)eliminates relationship between amber type A and modern Cupressaceae is
aPinaceaecontributiontotheamber,becauseabieticacidisama- thepresenceofalowquantityofcallitrisicacid(XIII)whichisan
jor component of such resins and dehydroabietic acid, its major epimerofdehydroabieticacid(VII,Fig.5).Thedifferenceinreten-
diageneticderivative,ispresentinambersderivedfromPinaceae tiontimewithdehydroabieticacidandthehigherrelativeintensity
(Yamamotoetal.,2006). oftheionatm/z357(M-CH )versusthemolecularionincallitrisic
3
PhenolicditerpenoidsoccurinpolarfractionBofthetypeAam- acidaredistinctivefeaturesbetweenthetwoepimersusedherefor
ber(Fig.4),withadominanceofferruginol(VIII)anditsoxidation theidentificationoftheacid(VandenBergetal.,2000;Coxetal.,
products 6,7-dehydroferruginol (IX) and 12-hydroxysimonellite 2007). Callitrisic acid has a higher chemotaxonomical value than
(X)andtotarol(XI)(OttoandSimoneit,2001;Ottoetal.,2002).Fer- dehydroabietic acid due to its scarcity. In modern conifer resins,
ruginol and 12-hydroxysimonellite are also identifiable (underiv- thesynthesisofcallitrisicacidseemstoberestrictedtocertaingen-
atized)inthebulkextractaspartofthemaincomponentsofthe eraoftheCupressaceaefamilyanditwasalsofoundinCenomanian
amber(Fig.3).Thepresenceofthesephenolicditerpenoidsisofsig- amberfromtheRaritanFormation(NewJersey,USA),suggestinga
nificantchemosystematicvalue,asferruginolisanabundantnatu- relationshipwithCupressaceae(Anderson,2006).
ral product in extant conifers of the families Cupressaceae and Degradationofphenolicditerpenoidscouldleadtotheabietane
Podocarpaceae and can be used as a characteristic biomarker of geoterpenoidsfoundinthetypeAamber(Ottoetal.,1997;Ottoand
thesefamilies(OttoandSimoneit,2001;Marynowskietal.,2007). Simoneit,2001;Stefanovaetal.,2002).Hautevelleetal.(2006)and
A minor amount of hinokiol (3-hydroxyferruginol, XII; Fig. 5A) Yamamotoetal.(2006)discussedthediageneticpathwaysofabie-
wasalsofoundinthetypeAamber.Hinokiolhasbeendescribed tane class bioterpenoids, suggesting that 18-norferruginol (XIV)
fromCupressaceae(Ottoetal.,2002;Coxetal.,2007).Thereisno couldbetheprecursorofdehydroabietin,andferruginol(VIII)could
reported presence of phenolic diterpenoids in modernAraucaria- leadto12-hydroxysimonellite(X),simonellite(XV),16,17-bisnorsi-
ceae(Coxetal.,2007),butOttoandWilde(2001)citedtheoccur- monellite(XVI)andretene(XVII),allfoundinthetypeAamber.Un-
rence of ferruginol in Araucaria. To avoid this ambiguity and to der the anaerobic depositional conditions of the amber (Najarro
testthisfindingunderourexperimentalconditions,resinsofAga- et al., 2009), we could not disregard redox reactions that lead to
thissp.andAraucariasp.wereanalyzedandferruginolwasnotde- theactual composition found(Pereira etal., 2009). If, as in some
tected in any Araucariaceae resins. Hence, these results, coupled modernCupressaceaegenera(i.e.Cupressus;Fig.S3,supplementary
with the absence of kaurane or phyllocladane diterpenoids, the material),theoriginalproportionofferruginol(VIII)washigh,its
OH
OH
12-
Ferruginol
Hydroxysimonellite
OH
e
c
n
a
d OH
n
u
b
a 6,7-Dehydroferruginol
e
v
ati
el
R Totarol
Retention time
Fig.4. GC–MSTICtraceoffractionBresultingfromthecolumnseparationofthepolarfractionofamberA.Thisfractioncontainsthepartiallypurifiedphenolicterpenoids.
CompoundsareanalyzedastheTMSderivatives.
Pleasecitethisarticleinpressas:Menor-Salván,C.,etal.TerpenoidsinextractsofLowerCretaceousambersfromtheBasque-CantabrianBasin(ElSoplao,
Cantabria,Spain):Paleochemotaxonomicaspects.Org.Geochem.(2010),doi:10.1016/j.orggeochem.2010.06.013
8 C.Menor-Salvánetal./OrganicGeochemistryxxx(2010)xxx–xxx
A
B
Fig.5. GC–MSTICtracesofthepolarfractionsCof:(A)amberAand(B)amberB.Thisfractioncontainsmainlypimaricandlabdenoicacids.Thefigureshowstheidentified
compoundsinthefraction.Unlabelledpeaksareconsideredasunidentifiedduetothelackofstandardsandpublishedreferencesordatabaseswithdetailsoftheirmass
spectra.CompoundsareanalyzedastheTMSderivatives.
diagenesis could ultimately have generated dehydroabietane (II) nolic fraction of the bulk extract and analysis as trimethylsilyl
andsimonellite(XV),bothsignificantinthetypeAamber. derivatives(Fig.4).Duetothesimilaritiesbetweenthemassspectra
ofphenolicditerpenoids,theretentiontimeandmassspectrumof
3.2.2.Totarol totarol (XI) were determined using a standard (Sigma–Aldrich).
Totarol(XI),atricyclicditerpenoidphenol,isconsideredasacon- Thepresenceoftotarolsuggestsarelationshipbetweenthepalae-
firmatorychemotaxonomicmarkerforCupressaceaeandPodocarp- obotanicoriginoftheamberandextantCupressaceaeorPodocarp-
aceae,evenatlowconcentrations(LeMétayeretal.,2008;Stefanova aceae.Totestthispossibility,thephenolicditerpenoidsoftheamber
andSimoneit,2008).Totarolisdetectableinthebulkextractusing werecomparedwiththosefromamodernCupressaceae(Cupressus
thecharacteristicmassfragmentsofm/z271and286.Theidentifica- arizonica;Fig.S3,supplementarymaterial). Bothextractscontain
tionisunambiguousinambersampleAafterpurificationofthephe- ferruginol(VIII),totarol(XI)andhinokiol(XII)asthemainphenolic
Pleasecitethisarticleinpressas:Menor-Salván,C.,etal.TerpenoidsinextractsofLowerCretaceousambersfromtheBasque-CantabrianBasin(ElSoplao,
Cantabria,Spain):Paleochemotaxonomicaspects.Org.Geochem.(2010),doi:10.1016/j.orggeochem.2010.06.013
C.Menor-Salvánetal./OrganicGeochemistryxxx(2010)xxx–xxx 9
diterpenoids,butsempervirol(XVIII)hasnotbeenobserved.Adif- tanecomposition,totarolisnotdetectableinthetypeBamber,con-
ferencebetweentheassemblageofpolarterpenoidsfromC.arizo- firmingthatbothambertypesfoundintheCantabriadepositsdiffer
nicaandtheamberisthepresenceofsugiol(XIX)andthelackof intheirbiologicalorigins.
callitrisicacid(XIII)intheformer.Weidentifiedadiaromatictotara-
ne(XX)asapossiblediageneticproductoftotarol(XI),whichmaybe 3.2.3.Pimarane/isopimaranediterpenoids
derivedbyaparalleldiageneticpathwayassimonellite(XV)from PolarfractionCofambercontainslowamountsofpimaric(XXI)
ferruginol(VIII)(Ottoetal.,1997).Inaccordwiththephenolicabie- and isopimaric (XXII) acids (Fig. 6). Diagenesis of pimarane
OH OH OH
6,7-Dehydroferruginol 12-Hydroxysimonellite
Ferruginol
Dehydroabietane Dehydroabietin Tetrahydroretene Simonellite 16,17-Bisnorsimonellite
?
Pimaradiene Isopimaradiene 14-Methyl-16,17- 16,17-
bisnordehydroabietane Bisnordehydroabietane
OH
O
OH
O
OH OH
Pimaric acid O O
Isopimaric acid 16,17- 16,17,18-trisnorabieta-
Pimar-8-en-18-oic
Bisnordehydroabietic 8,11,13-triene
acid
acid
OH
OH HO
O
O O
OH
HO
O O
E-and Z-19-norlabda-8(20),12-dien-15-oic acids
HO
Agathic acid
13-Dihydroagatholic
acid
2,5,8-Trimethyl-1-alkyltetralins
Fig.6. ProposeddiageneticpathwaysforthediterpenoidprecursorsfromtheCantabrianambers(basedonOttoandSimoneit(2002),Stefanovaetal.(2002),Hautevelleetal.
(2006),andPereiraetal.(2009)).Dottedbox:biologicalprecursors;solidbox:majorterpenoidfoundinthesamples.
Pleasecitethisarticleinpressas:Menor-Salván,C.,etal.TerpenoidsinextractsofLowerCretaceousambersfromtheBasque-CantabrianBasin(ElSoplao,
Cantabria,Spain):Paleochemotaxonomicaspects.Org.Geochem.(2010),doi:10.1016/j.orggeochem.2010.06.013
10 C.Menor-Salvánetal./OrganicGeochemistryxxx(2010)xxx–xxx
A
16,17-
Bisnorsimonellite Tetrahydro- Dehydroabietane
retene
Dehydroabietin
16,17,18-Trisnorabieta-
8,11,13-triene S Simonellite
8
2,5,8-Trimethyl- 14-methyl-16,17-
1-butyl tetralin bisnordehydroabietane
P
OH
r
e is
nc ta OH
a n
d e
n Cadalene
bu Retene Ferruginol
a
e Naphthaleneand
ativ tetralinderivatives 1s2i-mHoynderollixtey-
el
R
B
16,17,18-Trisnorabieta- Dehydroabietane
8,11,13-triene
Simonellite
16,17-
Cadalene
Bisnorsimonellite
2-Methylretene
Guaiazulene
OH
e
c Naphthalene and Retene
n
a tetralin derivatives
d
n
u
b
a
e Ferruginol 12-
v Hydroxy-
ati simonellite
el
R
10 20 30
Retention time (min)
Fig.7. GC–MSTICtracesofthetotalextractsfrom:(A)Frenelopsisleavesand(B)jet(blackamber),showingtheidentifiedbiomarkers.
diterpenoidscouldbeoneofthepossibleoriginsof16,17,19-tris- noids (Fig. 6; Otto et al., 2002; Pereira et al., 2009). Due to the
norabieta-8,11,13-triene (XXIII), a major compound identified in widespread distribution of the pimaric/isopimaric acids, the
the amber samples. Anotherpossible originfor thiscompound is chemotaxonomical interpretation of their presence in the Canta-
bydiagenesisofdehydroabietaneandotherabietanerelatedterpe- brian ambers must be taken with caution and comparisons with
Pleasecitethisarticleinpressas:Menor-Salván,C.,etal.TerpenoidsinextractsofLowerCretaceousambersfromtheBasque-CantabrianBasin(ElSoplao,
Cantabria,Spain):Paleochemotaxonomicaspects.Org.Geochem.(2010),doi:10.1016/j.orggeochem.2010.06.013