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Terpenoid Compositions and Botanical Origins of Late Cretaceous and Miocene Amber from China

Terpenoid Compositions and Botanical Origins of Late Cretaceous and Miocene Amber from China The terpenoid compositions of the Late Cretaceous Xixia amber from Central China and the middle Miocene Zhangpu amber from Southeast China were analyzed by gas chromatography-mass spectrometry (GC-MS) to elucidate their botanical origins. The Xixia amber is characterized by sesquiterpenoids, abietane and phyllocladane type diterpenoids, but lacks phenolic abietanes and labdane derivatives. The molecular compositions indicate that the Xixia amber is most likely contributed by the conifer family Araucariaceae, which is today distributed primarily in the Southern Hemisphere, but widely occurred in the Northern Hemisphere during the Mesozoic according to paleobotanical evidence. The middle Miocene Zhangpu amber is characterized by amyrin and amyrone-based triterpenoids and cadalene-based sesquiterpe- noids. It is considered derived from the tropical angiosperm family Dipterocarpaceae based on these compounds and the co-occurring fossil winged fruits of the family in Zhangpu. This provides new evidence for the occurrence of a dipterocarp forest in the middle Miocene of Southeast China. It is the first detailed biomarker study for amber from East Asia. Citation: Shi G, Dutta S, Paul S, Wang B, Jacques FMB (2014) Terpenoid Compositions and Botanical Origins of Late Cretaceous and Miocene Amber from China. PLoS ONE 9(10): e111303. doi:10.1371/journal.pone.0111303 Editor: Qi Wang, Institute of Botany, China Received August 12, 2014; Accepted September 30, 2014; Published October 29, 2014 Copyright:  2014 Shi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper. Funding: This work was supported by the 973 Project of China (2012CB821900, http://program.most.gov.cn/), National Natural Science Foundation of China (41206173, http://www.nsfc.gov.cn/), and the Humboldt Research Fellowship for Postdoctoral Researchers to BW (http://www.humboldt-foundation.de/web/ humboldt-fellowship-postdoc.html). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: glshi@nigpas.ac.cn As a kind of traditional Chinese medicine that could calm nerve, Introduction amber has been known and collected for a long history in China. Amber is fossilized natural resin that was produced by secretary Although amber has been reported from several sites in China, cells of ancient plants. It is known as a source for jewelry and also most of these sites were known based on very little amber pieces as an effective preservation medium for fossil insects and soft- [17,18]. Only in three sites amber has been extensively collected bodied microorganisms [1–4]. Resins are polymerized from a by local people, including Fushun in Northeast China, Xixia in broad range of isoprenoid compounds including primarily Central China, and Zhangpu in Southeast China (Fig. 1). The terpenoids, carboxylic acids and associated alcohols [5,6]. The early Eocene Fushun amber is well-known for containing a diverse function of resin is not clearly understood, but is considered to assemblage of fossil insects and plant remains, and has been protect plants from invasion by fungi and insects after injury [7–9]. studied in entomological, gemological and geochemical aspects Terpenoids are amongst the most diverse plant natural products, [4,19]. The terpenoid compositions indicate that the Fushun with about 25,000 known compounds and are often diagnostic for amber was derived from the conifer family Cupressaceae [4]. In certain plant groups [10]. Albeit various chemical transformations contrast to the Fushun amber, little attention has been paid to the during burial, the terpenoids in fossil resins and fossil plant Late Cretaceous Xixia amber (Fig. 2A) and middle Miocene remains often retain their characteristic basic structural skeletons Zhangpu amber (Fig. 2B) probably because they are minute or and can thus be used as biomarkers for botanical origins of the fragile and thus not suitable for making jewelry. In this paper we fossils [5,11]. Based on compositions of terpenoids, for example, investigated the terpenoid compositions of the Xixia and Zhangpu the Early Cretaceous Burmese amber was considered derived from amber and explored its botanical affinities based on biomarkers. the conifer family Pinaceae [12]; the early Eocene Cambay amber of India from the tropical angiosperm family Dipterocarpaceae Materials and Methods [13–15], and the Eocene Baltic amber of Europe from the conifer family Sciadopityaceae [6]. Other plant families from which Ethics Statement amber was probably produced include Araucariaceae, Cupressa- The collection of amber under study was permitted by the local ceae sensu lato (including Cupressaceae sensu stricto and governments of Xixia County, Henan Province and Zhangpu Taxodiaceae), Leguminosae, Burseraceae, Hamamelidaceae, County, Fujian Province. The field trip was done in non-National Combretaceae and the extinct conifer family Cheirolepidiaceae Nature Reserves and non-private areas. We did not violate the [2,16]. PLOS ONE | www.plosone.org 1 October 2014 | Volume 9 | Issue 10 | e111303 Terpenoid and Botanical Origins of Chinese Amber Figure 1. Map showing the locations of Xixia and Zhangpu where the studied amber was collected. doi:10.1371/journal.pone.0111303.g001 Chinese fossil collection and mining laws and management grained sandstones, pale sandy conglomerates, purplish- and regulations. brownish-red siltstones and pebble-bearing sandstones intercalated with silty mudstones (Fig. 3A) [20]. It is assigned to Late Cretaceous, most probably Coniacian-Campanian, constrained Localities and geological setting by studies of fossil bivalve [22] and ostracoda assemblages [23]. The Late Cretaceous Xixia amber was collected from the Gaogou Formation at Wuliqiao Town (33u1893999N, Plant macrofossils and pollen have not been reported in the Gaogou Formation. Xixia possesses the currently known largest 111u2894899E), Xixia County, Henan Province, Central China (Fig. 1). The Gaogou Formation is amongst the non-marine Late amber deposits in China [18]. The Xixia amber is preserved as lenses in the fine-grained sandstones in the middle part of the Cretaceous red beds in Xixia Basin that are well-known for abundant well-preserved dinosaur eggs [20,21]. The formation is Gaogou Formation (Fig. 3A). The amber is yellow, brown, composed of brownish red calcareous siltstones, fine to coarse- brownish yellow, brownish red in color, the single amber pieces Figure 2. Photos of Late Cretaceous Xixia amber (A) and middle Miocene Zhangpu amber (B). doi:10.1371/journal.pone.0111303.g002 PLOS ONE | www.plosone.org 2 October 2014 | Volume 9 | Issue 10 | e111303 Terpenoid and Botanical Origins of Chinese Amber Figure 3. Generalized stratigraphic sections of the Gaogou Formation in Xixia (A) and Fotan Group in Zhangpu (B). The stratigraphic position of the amber (indicated by four pointed star) and radiometric age of the basaltic rock samples are shown. doi:10.1371/journal.pone.0111303.g003 PLOS ONE | www.plosone.org 3 October 2014 | Volume 9 | Issue 10 | e111303 Terpenoid and Botanical Origins of Chinese Amber are usually minute in dimension (Fig. 2A). So far no insects have Most of the diterpenoids belong to abietane class and these are been reported from the Xixia amber. 16,17,19-trisnorabietane, bisnorabietane, trisnorabieta-8,11,13-tri- The Miocene Zhangpu amber was collected from the Fotan ene, norabietane and fichtelite. Besides, tricyclic rimuane and Group at Qianting Town (24u1690399N, 117u5990199E), Zhangpu tetracyclic phyllocladane are also abundantly present in the solvent County, Fujian Province, Southeast China (Fig. 1). The Fotan extract of the Xixia amber. Phenolic abietanes such as totarol, Group is mainly distributed in the coastal areas of southeastern ferruginol, sugiol were not detected. The molecular compositions Fujian, as well as in Mingxi and Ninghua County of western suggest that the Xixia amber is most likely derived from the conifer Fujian [24]. It consists of arenaceous conglomerate rocks, family Araucariaceae. sandstones, sandy mudstones, mudstones, lignite and diatomite, Abietane type diterpenoids that are detected in the Xixia amber with interbedded three layers of basaltic rocks derived from several are characteristic biomarkers for gymnosperms, especially conifers episodes of volcanic activity during the Neogene (Fig. 3B) [24]. [5]. These diterpenoids are derived from abietic acid which has 40 39 The Ar 2 Ar dating of the basaltic rocks in the middle part of been reported from almost all conifer families [5]. However, the the Fotan Group rendered an age of 14.7–14.960.6 Ma (Fig. 3B) presence of tetracyclic diterpenoids (e.g. phyllocladane) can [25], belonging to the Langhian Stage (middle Miocene), exclude the contribution of Pinaceae to the Xixia amber since consistent with the study of palynological assemblage of the group resins of extant Pinaceae differ from those of other extant conifer [24]. The Fotan Group in Zhangpu County yields abundant well- families in lacking phenolic abietanes and tetracyclic diterpenoids preserved plant leaf and fruit fossils, including Clusiaceae, [5]. Moreover, the exclusive presence of labdane and abietane Dipterocarpaceae, Fagaceae, Hamamelidaceae, Lauraceae, Legu- diterpenoids is also characteristic for extant Pinaceae [28] whereas minosae and Moraceae. The plant fossil assemblage of the Fotan labdane dericatives are not detected in the Xixia amber. Group indicates a middle Miocene dipterocarp forest and Derivatives of cedrane and cuparane, only reported from resins probable tropical rain forest in Southeast China [26,27]. The of extant Cupressaceae, are characteristic biomarkers for this Zhangpu amber is preserved in blue-grey sandy mudstone, or conifer family [29]. These compounds are, however, not detected sometimes in situ in lignified fossil wood. It is yellow, brown to in the solvent extracts of Xixia amber, making a cupressaceous brownish red in color, the single pieces of Zhangpu amber can be origin of the Xixia amber unlikely. Besides, the phenolic abietanes big but they are extremely fragile (Fig. 2B). Although neither such as totarol, ferruginol, sugiol that are produced only by extant insects nor plant microfossils have been reported from the Cupressaceae and Podocarpaceae [5,28,30], are totally absent in Zhangpu amber, it is considered potential source for paleontolog- the Xixia amber, further supporting the exclusion of a cupressac- ical studies. eous source for Xixia amber. Tricyclic rimuane that is detected in the Xixia amber is derived Repository from the Southern Hemisphere conifers Araucariaceae and Approximately 200 g Xixia amber and 3 kg Zhangpu amber Podocarpaceae [31] whereas phyllocladane is known from were collected and deposited permanently in Nanjing Institute of Araucariaceae, Podocarpaceae and Cupressaceae sensu lato [5]. Geology and Palaeontology, Chinese Academy of Sciences. The The absence of phenolic abietanes limits the possibility of the Xixia amber pieces were preserved in one vial and assigned one podocarpaceous source of the Xixia amber and therefore it is most registered number, PB21517; the Zhangpu amber pieces were likely that the Xixia amber was derived from Araucariaceae. assigned PB21518. However, the contribution from Podocarpaceae cannot be completely ruled out. Gas Chromatography-Mass Spectrometry (GC-MS) Plant macrofossils (leaves, fruits, wood) and microfossils (pollen) Amber fragments were extracted with a dichloromethane and that occur in or associated with amber are usually considered methanol (ratio 9:1) mixture for one hour. The total extracts were potential candidates for the botanical origins of the amber analyzed by gas chromatography-mass spectrometry (GC-MS). [1,6,14,28,32,33]. Unfortunately, neither plant macrofossils nor The GC-MS analysis was performed on an Agilent 5975 mass pollen have been reported from the Gaogou Formation of Xixia spectrometer interfaced to a 7890 gas chromatograph. Extracts Basin, so that it is impossible to explore the botanical source of were analyzed on HP-5 MS fused silica (30 m6 0.25 mm i.d.,6 Xixia amber based on co-occurring fossil plants currently. Further 0.25 mm film thickness) GC column. Helium was used as carrier paleobotanical exploration in the Gaogou Formation may help gas at a flow rate of 1 ml/min. The initial GC oven temperature clarify the origin of Xixia amber. In the following paragraphs we was held at 40uC for 5 minutes and then ramped to 310uCata review briefly the biogeographical history of Araucariaceae and rate of 4uC/min. The mass spectrometer detector was pro- Podocarpaceae. grammed as EI mode with ionization energy 70 eV. The samples Araucariaceae contain three genera that are primarily in the were analyzed in a full scan mode (mass range 50–600 dalton). Southern Hemisphere, with a disjunct distribution in Malaysia, The data processing was taken by Chemstation software and the Indonesia, Philippines, New Guinea, Australia, New Zealand, identification of compounds was carried out based on their elution New Caledonia, Vanuatu, Fiji, Norfolk Island and southern South pattern and the comparison of mass spectra with published America [34]. Paleobotanical evidence indicates, however, the literatures. family had a wide distribution in both the Northern and Southern Hemispheres during the Mesozoic and disappeared in most parts Results and Discussions of the Northern Hemisphere by the latest Cretaceous [35,36]. Modern trees of Araucariaceae, especially the genus Agathis Late Cretaceous Xixia amber (Fig. 4; Table 1) Salisb. are highly resinous [34] and araucariacean trees are The total ion chromatogram from the GC-MS analysis of the suspected as a common source for amber all through the Mesozoic Xixia amber is characterized by the distribution of sesquiterpe- [32,33,37,38]. It is of interest to note that amber had not become noids and diterpenoids (Fig. 4; Table 1). The major sesquiterpe- abundant until Early Cretaceous [16], when the Araucariaceae noids are drimane, homodrimane, 1,1,6-trimethyl-1,2,3,4-tetra- attained their greatest diversity and widest distribution in both the hydronaphthalene, 4b-eudesmane, ionene and some unknown Northern and Southern Hemispheres [35]. C -C sesquiterpenoids with base peak 109 (Fig. 4; Table 1). 13 17 PLOS ONE | www.plosone.org 4 October 2014 | Volume 9 | Issue 10 | e111303 Terpenoid and Botanical Origins of Chinese Amber Figure 4. Total ion chromatogram of the Late Cretaceous Xixia amber from GC-MS analysis. The identified peaks are listed in Table 1. doi:10.1371/journal.pone.0111303.g004 Podocarpaceae consist of 18 extant genera that are distributed family unlikely a potential source for fossil resins. Biogeographi- predominantly in the tropical and subtropical mountains of the cally, Podocarpaceae are considered distributed essentially in Southern Hemisphere, and in the Northern Hemisphere extend- Gondwana, or Gondwana-derived plates during the Cretaceous ing northward to subtropical China, Japan, Mexico and the [39]. This appears not to support the occurrence of Podocarpa- Caribbean Islands [34]. Different from Araucariaceae, living trees ceae in the Cretaceous of East Asia and a podocarpaceous origin of Podocarpaceae are only slightly resinous [34], making the of the Late Cretaceous Xixia amber. Table 1. Major compounds identified from the Late Cretaceous Xixia amber. Peak number Compound Name Base peak Molecular ion 1 Unknown C sesquiterepnoid 109 180 2 Unknown C sesquiterpenoid 109 194 3 Unknown C sesquiterpenoid 109 194 4 Drimene+1,1,6-Trimethyl-1,2,3,4-tetrahydronaphthalene 123,159 208,174 54b-Eudesmane 109 208 6 Ionene 173 188 7C sesquiterpenoid 109 206 8 Homodrimane 123 222 9 Unknown C sesquiterpenoid+ Trimethyl naphthalene 109,155 220,170 10 C sesquiterpenoid 109 236 11 16,17,19-trisnorabietane 109 234 12 Bisnorabietane 109 248 13 Isomer of bisnorabietane 109 248 14 Trisnorabieta-8,11,13-triene 131 228 15 Norabietane 109 262 16 Possibly isomer of norabietane 109 262 17 Unknown C diterpenoid 109 248 18 C Diterpenoid 109 260 19 Fichtelite 109 262 20 Possibly isomer of norabietane 95 262 21 Unknown C diterpenoidpossibly rimuane 109 276 22 a- phyllocladane 123 274 doi:10.1371/journal.pone.0111303.t001 PLOS ONE | www.plosone.org 5 October 2014 | Volume 9 | Issue 10 | e111303 Terpenoid and Botanical Origins of Chinese Amber Cheirolepidiaceae, an extinct Mesozoic conifer family ranging classes based on the molecular compositions [43]. Class I fossil from Late Triassic to Late Cretaceous [40], has been considered resins are based primarily on polymers of labdatriene carboxylic the botanical source of the Late Triassic amber of Italy based on acids, especially communic or ozic acids and are contributed by the presence of in situ fossil resins within plant macrofossils of the Leguminosae. Fossil resins derived from conifers also belong to Cheirolepidiaceae, and the abundant occurrence of pollen and Class I [43]. Class II fossil resins are based on polymer of bicyclic cuticles of this extinct family in the paleosol where the dispersed sesquiterpenoid and triterpenoids hydrocarbons and are contrib- amber was buried [2]. Since that Cheirolepidiaceae was the most uted by the family Dipterocarpaceae and a genus Mastixia Blume dominant conifer family through the Jurassic and Early Creta- (Cornaceae) [44,45]. Class III fossil resins with a polystyrene based ceous, it has been considered potential candidate for the botanical structure are contributed by the family Hamamelidaceae. Class IV origin of some Jurassic and Cretaceous amber [28,41,42]. fossil resins, the botanical affinity of which is uncertain, have a Molecular composition analyses revealed that the amber and the cedrane polysesquiterpenoid based structure. The Zhangpu amber co-occurring fossil foliage compressions of Frenelopsis Schenk with a prominent distribution of cadalene-based sesquiterpenoid (Cheirolepidiaceae) from the Lower Cretaceous of Spain are both clearly belongs to Class II. Although trees of the genus Mastixia characterized by the presence of phenolic abietane [42]. Although also produce resins of Class II, the occurrence of fossil winged the totally absence of phenolic abietane in the Xixia amber may fruits of Dipterocarpaceae in the Fotan Group of Zhangpu [26,27] eliminate a significant contribution of Cheirolepidiaceae, the confirms a Dipterocarpaceae origin of the Zhangpu amber. cheirolepidiaceous affinity cannot be completely excluded since Dipterocarpaceae are a tropical tree family that dominate the compounds might be not extracted thus not detectable in the GC- emergent canopy of most lowland rain forests in the Southeast MS analysis. Asia [46]. The family comprises three subfamilies: Dipterocarpoi- deae in the Asian tropics and Seychelles; Pakaraimoideae restricted to the Guyana and Venezuela of tropical South Middle Miocene Zhangpu amber (Fig. 5; Table 2) America; and Monotoideae in tropical Africa, Madagascar and The total ion chromatogram from the GC-MS analysis of the Southeast Colombia [46]. Molecular phylogenetic and biogeo- middle Miocene Zhangpu amber is given in the figure 5. The total graphic studies indicate that the family had an ancient extract of Zhangpu amber is characterized by sesquiterpenoids Gondwanan origin and arrived in Asia after the establishment of and triterpenoids. The major sesquiternoids are isoledene; 1H- 3a,7-methanoazulene, octahydro-1,4,9,9-tetramethyl-(1a,3aa,4- the land connection between the Indian and Asian plates [14,47,48]. This hypothesis was supported by the earliest fossil b,7a,8a); dehydro-ar-curcumene; d-selinene; calamenene; methyl record of the family which is from the lower Eocene of India [14]. drimane and cadalene (Table 2). Triterpenoids such as a and b- amyrone; a and b-amyrin and hop-22(29)-en-3b-ol are abundantly Among the family only trees of the Asian subfamily Dipterocar- poideae are highly resinous whereas the Pakaraimoideae and present in the sample (Fig. 5; Table 2). Monotoideae lack resin ducts [46]. Amber derived from the The presence of amyrin and amyrone-based triterpenoids in the solvent extract of the Miocene Zhangpu amber indicates a Dipterocarpaceae has been reported from the Eocene and Miocene of India [13,14,15,49] and the Eocene of Vietnam contribution from angiosperms. Fossil resins are divided into four Table 2. Major compounds identified from the middle Miocene Zhangpu amber. Peak number Compound Name Base peak Molecular ion 1 Isoledene 105 204 2 Unknown C sesquiterpenoid 81 206 3 Unknown C sesquiterpenoid 95 206 4 1H-3a,7-Methanoazulene, octahydro-1,4,9,9-tetramethyl- 163 206 (1a,3aa,4b,7a,8ab) 5 Dehydro-ar- curcumene 119 204 6 Unknown C sesquiterpenoid 191 206 7 d-Selinene 161 206 8 Calamenene 159 202 9 Tetramethyl naphthalene 169 184 10 Methyl drimane 137 222 11 Unknown sesquiterpenoid 109 204 12 Methyl drimane+ Unknown sesquiterpenoid 137, 109 222, 204 13 Unknown C sesquiterpenoid 81 206 14 Cadalene 183 198 15 Methyl drimane 137 222 16 b-amyrone 218 424 17 b-amyrin+ a-amyrone 218,218 426,424 18 a-amyrin 218 426 19 Hop-22(29)-en-3b-ol 189 426 doi:10.1371/journal.pone.0111303.t002 PLOS ONE | www.plosone.org 6 October 2014 | Volume 9 | Issue 10 | e111303 Terpenoid and Botanical Origins of Chinese Amber Figure 5. Total ion chromatogram of the middle Miocene Zhangpu amber from GC-MS analysis. The identified peaks are listed in Table 2. doi:10.1371/journal.pone.0111303.g005 [50]. The middle Miocene Zhangpu amber represents the The total extracts of the middle Miocene Zhangpu amber northernmost distribution of Dipterocarpaceae-derived amber contain amyrin and amyrone-based triterpenoids, and cadalene- and this discovery corroborates the existence of a dipterocarp based sesquiterpenoids. These compounds are characteristic for forest in Zhangpu of Southeast China and the northward dammar resins that are produced by trees of the tropical family movement of the tropical family during the Mid-Miocene Climatic Dipterocarpaceae. The Dipterocarpceae origin of the Zhangpu Optimum [26,27]. Today the Dipterocarpaceae is absent in the amber is also corroborated by the co-occurring fossil winged fruit Zhangpu area, which today has a typical subtropical monsoon of this family. All evidence suggests the presence of a dipterocarp climate and subtropical evergreen broadleaved forests [51]. forest in the Southeast China during the middle Miocene. Conclusions Acknowledgments In this paper we studied the terpenoid compositions and We are grateful to academic editor Qi Wang for handling the submission, botanical origins of the Late Cretaceous and middle Miocene to Dany Azar and an anonymous referee and the editor for their helpful comments and suggestion. G. Shi thanks S. Mei, H. Yang for help in amber from China by GC–MS analysis. This work represents the fieldwork. S. Dutta is thankful to Department of Science and Technology first detailed biomarker study for amber from East Asia. The Late (DST), India for providing support to procure GC-MS under FIST Cretaceous Xixia amber is characterized by sesquiterpenoids, Programme. abietane and phyllocladane type diterpenoids, but lacks phenolic abietanes and labdane derivatives. This suggests a significant Author Contributions contribution of Araucariaceae to the Xixia amber. Although no plant fossils have been reported in the Late Cretaceous red bed of Conceived and designed the experiments: GS SD. Performed the Xixia, paleobotanical records indicate that the Araucariaceae, experiments: SD SP. Analyzed the data: SD SP GS BW. Contributed reagents/materials/analysis tools: GS SD FMBJ. Contributed to the which is mainly distributed in the Southern Hemisphere today, did writing of the manuscript: GS SD SP BW. occur in the Late Cretaceous of the mid-latitude regions of the Northern Hemisphere. References 1. Rust J, Singh H, Rana RS, McCann T, Singh L, et al. (2010) Biogeographic and 8. Trapp S, Croteau R (2001) Defensive resin biosynthesis in conifers. Annual evolutionary implications of a diverse paleobiota in amber from the early Eocene Review of Plant Biology 52: 689–724. of India. Proceedings of the National Academy of Sciences U.S.A. 107: 18360– 9. 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PLOS ONE | www.plosone.org 8 October 2014 | Volume 9 | Issue 10 | e111303 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png PLoS ONE Pubmed Central

Terpenoid Compositions and Botanical Origins of Late Cretaceous and Miocene Amber from China

PLoS ONE , Volume 9 (10) – Oct 29, 2014

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Abstract

The terpenoid compositions of the Late Cretaceous Xixia amber from Central China and the middle Miocene Zhangpu amber from Southeast China were analyzed by gas chromatography-mass spectrometry (GC-MS) to elucidate their botanical origins. The Xixia amber is characterized by sesquiterpenoids, abietane and phyllocladane type diterpenoids, but lacks phenolic abietanes and labdane derivatives. The molecular compositions indicate that the Xixia amber is most likely contributed by the conifer family Araucariaceae, which is today distributed primarily in the Southern Hemisphere, but widely occurred in the Northern Hemisphere during the Mesozoic according to paleobotanical evidence. The middle Miocene Zhangpu amber is characterized by amyrin and amyrone-based triterpenoids and cadalene-based sesquiterpe- noids. It is considered derived from the tropical angiosperm family Dipterocarpaceae based on these compounds and the co-occurring fossil winged fruits of the family in Zhangpu. This provides new evidence for the occurrence of a dipterocarp forest in the middle Miocene of Southeast China. It is the first detailed biomarker study for amber from East Asia. Citation: Shi G, Dutta S, Paul S, Wang B, Jacques FMB (2014) Terpenoid Compositions and Botanical Origins of Late Cretaceous and Miocene Amber from China. PLoS ONE 9(10): e111303. doi:10.1371/journal.pone.0111303 Editor: Qi Wang, Institute of Botany, China Received August 12, 2014; Accepted September 30, 2014; Published October 29, 2014 Copyright:  2014 Shi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper. Funding: This work was supported by the 973 Project of China (2012CB821900, http://program.most.gov.cn/), National Natural Science Foundation of China (41206173, http://www.nsfc.gov.cn/), and the Humboldt Research Fellowship for Postdoctoral Researchers to BW (http://www.humboldt-foundation.de/web/ humboldt-fellowship-postdoc.html). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: glshi@nigpas.ac.cn As a kind of traditional Chinese medicine that could calm nerve, Introduction amber has been known and collected for a long history in China. Amber is fossilized natural resin that was produced by secretary Although amber has been reported from several sites in China, cells of ancient plants. It is known as a source for jewelry and also most of these sites were known based on very little amber pieces as an effective preservation medium for fossil insects and soft- [17,18]. Only in three sites amber has been extensively collected bodied microorganisms [1–4]. Resins are polymerized from a by local people, including Fushun in Northeast China, Xixia in broad range of isoprenoid compounds including primarily Central China, and Zhangpu in Southeast China (Fig. 1). The terpenoids, carboxylic acids and associated alcohols [5,6]. The early Eocene Fushun amber is well-known for containing a diverse function of resin is not clearly understood, but is considered to assemblage of fossil insects and plant remains, and has been protect plants from invasion by fungi and insects after injury [7–9]. studied in entomological, gemological and geochemical aspects Terpenoids are amongst the most diverse plant natural products, [4,19]. The terpenoid compositions indicate that the Fushun with about 25,000 known compounds and are often diagnostic for amber was derived from the conifer family Cupressaceae [4]. In certain plant groups [10]. Albeit various chemical transformations contrast to the Fushun amber, little attention has been paid to the during burial, the terpenoids in fossil resins and fossil plant Late Cretaceous Xixia amber (Fig. 2A) and middle Miocene remains often retain their characteristic basic structural skeletons Zhangpu amber (Fig. 2B) probably because they are minute or and can thus be used as biomarkers for botanical origins of the fragile and thus not suitable for making jewelry. In this paper we fossils [5,11]. Based on compositions of terpenoids, for example, investigated the terpenoid compositions of the Xixia and Zhangpu the Early Cretaceous Burmese amber was considered derived from amber and explored its botanical affinities based on biomarkers. the conifer family Pinaceae [12]; the early Eocene Cambay amber of India from the tropical angiosperm family Dipterocarpaceae Materials and Methods [13–15], and the Eocene Baltic amber of Europe from the conifer family Sciadopityaceae [6]. Other plant families from which Ethics Statement amber was probably produced include Araucariaceae, Cupressa- The collection of amber under study was permitted by the local ceae sensu lato (including Cupressaceae sensu stricto and governments of Xixia County, Henan Province and Zhangpu Taxodiaceae), Leguminosae, Burseraceae, Hamamelidaceae, County, Fujian Province. The field trip was done in non-National Combretaceae and the extinct conifer family Cheirolepidiaceae Nature Reserves and non-private areas. We did not violate the [2,16]. PLOS ONE | www.plosone.org 1 October 2014 | Volume 9 | Issue 10 | e111303 Terpenoid and Botanical Origins of Chinese Amber Figure 1. Map showing the locations of Xixia and Zhangpu where the studied amber was collected. doi:10.1371/journal.pone.0111303.g001 Chinese fossil collection and mining laws and management grained sandstones, pale sandy conglomerates, purplish- and regulations. brownish-red siltstones and pebble-bearing sandstones intercalated with silty mudstones (Fig. 3A) [20]. It is assigned to Late Cretaceous, most probably Coniacian-Campanian, constrained Localities and geological setting by studies of fossil bivalve [22] and ostracoda assemblages [23]. The Late Cretaceous Xixia amber was collected from the Gaogou Formation at Wuliqiao Town (33u1893999N, Plant macrofossils and pollen have not been reported in the Gaogou Formation. Xixia possesses the currently known largest 111u2894899E), Xixia County, Henan Province, Central China (Fig. 1). The Gaogou Formation is amongst the non-marine Late amber deposits in China [18]. The Xixia amber is preserved as lenses in the fine-grained sandstones in the middle part of the Cretaceous red beds in Xixia Basin that are well-known for abundant well-preserved dinosaur eggs [20,21]. The formation is Gaogou Formation (Fig. 3A). The amber is yellow, brown, composed of brownish red calcareous siltstones, fine to coarse- brownish yellow, brownish red in color, the single amber pieces Figure 2. Photos of Late Cretaceous Xixia amber (A) and middle Miocene Zhangpu amber (B). doi:10.1371/journal.pone.0111303.g002 PLOS ONE | www.plosone.org 2 October 2014 | Volume 9 | Issue 10 | e111303 Terpenoid and Botanical Origins of Chinese Amber Figure 3. Generalized stratigraphic sections of the Gaogou Formation in Xixia (A) and Fotan Group in Zhangpu (B). The stratigraphic position of the amber (indicated by four pointed star) and radiometric age of the basaltic rock samples are shown. doi:10.1371/journal.pone.0111303.g003 PLOS ONE | www.plosone.org 3 October 2014 | Volume 9 | Issue 10 | e111303 Terpenoid and Botanical Origins of Chinese Amber are usually minute in dimension (Fig. 2A). So far no insects have Most of the diterpenoids belong to abietane class and these are been reported from the Xixia amber. 16,17,19-trisnorabietane, bisnorabietane, trisnorabieta-8,11,13-tri- The Miocene Zhangpu amber was collected from the Fotan ene, norabietane and fichtelite. Besides, tricyclic rimuane and Group at Qianting Town (24u1690399N, 117u5990199E), Zhangpu tetracyclic phyllocladane are also abundantly present in the solvent County, Fujian Province, Southeast China (Fig. 1). The Fotan extract of the Xixia amber. Phenolic abietanes such as totarol, Group is mainly distributed in the coastal areas of southeastern ferruginol, sugiol were not detected. The molecular compositions Fujian, as well as in Mingxi and Ninghua County of western suggest that the Xixia amber is most likely derived from the conifer Fujian [24]. It consists of arenaceous conglomerate rocks, family Araucariaceae. sandstones, sandy mudstones, mudstones, lignite and diatomite, Abietane type diterpenoids that are detected in the Xixia amber with interbedded three layers of basaltic rocks derived from several are characteristic biomarkers for gymnosperms, especially conifers episodes of volcanic activity during the Neogene (Fig. 3B) [24]. [5]. These diterpenoids are derived from abietic acid which has 40 39 The Ar 2 Ar dating of the basaltic rocks in the middle part of been reported from almost all conifer families [5]. However, the the Fotan Group rendered an age of 14.7–14.960.6 Ma (Fig. 3B) presence of tetracyclic diterpenoids (e.g. phyllocladane) can [25], belonging to the Langhian Stage (middle Miocene), exclude the contribution of Pinaceae to the Xixia amber since consistent with the study of palynological assemblage of the group resins of extant Pinaceae differ from those of other extant conifer [24]. The Fotan Group in Zhangpu County yields abundant well- families in lacking phenolic abietanes and tetracyclic diterpenoids preserved plant leaf and fruit fossils, including Clusiaceae, [5]. Moreover, the exclusive presence of labdane and abietane Dipterocarpaceae, Fagaceae, Hamamelidaceae, Lauraceae, Legu- diterpenoids is also characteristic for extant Pinaceae [28] whereas minosae and Moraceae. The plant fossil assemblage of the Fotan labdane dericatives are not detected in the Xixia amber. Group indicates a middle Miocene dipterocarp forest and Derivatives of cedrane and cuparane, only reported from resins probable tropical rain forest in Southeast China [26,27]. The of extant Cupressaceae, are characteristic biomarkers for this Zhangpu amber is preserved in blue-grey sandy mudstone, or conifer family [29]. These compounds are, however, not detected sometimes in situ in lignified fossil wood. It is yellow, brown to in the solvent extracts of Xixia amber, making a cupressaceous brownish red in color, the single pieces of Zhangpu amber can be origin of the Xixia amber unlikely. Besides, the phenolic abietanes big but they are extremely fragile (Fig. 2B). Although neither such as totarol, ferruginol, sugiol that are produced only by extant insects nor plant microfossils have been reported from the Cupressaceae and Podocarpaceae [5,28,30], are totally absent in Zhangpu amber, it is considered potential source for paleontolog- the Xixia amber, further supporting the exclusion of a cupressac- ical studies. eous source for Xixia amber. Tricyclic rimuane that is detected in the Xixia amber is derived Repository from the Southern Hemisphere conifers Araucariaceae and Approximately 200 g Xixia amber and 3 kg Zhangpu amber Podocarpaceae [31] whereas phyllocladane is known from were collected and deposited permanently in Nanjing Institute of Araucariaceae, Podocarpaceae and Cupressaceae sensu lato [5]. Geology and Palaeontology, Chinese Academy of Sciences. The The absence of phenolic abietanes limits the possibility of the Xixia amber pieces were preserved in one vial and assigned one podocarpaceous source of the Xixia amber and therefore it is most registered number, PB21517; the Zhangpu amber pieces were likely that the Xixia amber was derived from Araucariaceae. assigned PB21518. However, the contribution from Podocarpaceae cannot be completely ruled out. Gas Chromatography-Mass Spectrometry (GC-MS) Plant macrofossils (leaves, fruits, wood) and microfossils (pollen) Amber fragments were extracted with a dichloromethane and that occur in or associated with amber are usually considered methanol (ratio 9:1) mixture for one hour. The total extracts were potential candidates for the botanical origins of the amber analyzed by gas chromatography-mass spectrometry (GC-MS). [1,6,14,28,32,33]. Unfortunately, neither plant macrofossils nor The GC-MS analysis was performed on an Agilent 5975 mass pollen have been reported from the Gaogou Formation of Xixia spectrometer interfaced to a 7890 gas chromatograph. Extracts Basin, so that it is impossible to explore the botanical source of were analyzed on HP-5 MS fused silica (30 m6 0.25 mm i.d.,6 Xixia amber based on co-occurring fossil plants currently. Further 0.25 mm film thickness) GC column. Helium was used as carrier paleobotanical exploration in the Gaogou Formation may help gas at a flow rate of 1 ml/min. The initial GC oven temperature clarify the origin of Xixia amber. In the following paragraphs we was held at 40uC for 5 minutes and then ramped to 310uCata review briefly the biogeographical history of Araucariaceae and rate of 4uC/min. The mass spectrometer detector was pro- Podocarpaceae. grammed as EI mode with ionization energy 70 eV. The samples Araucariaceae contain three genera that are primarily in the were analyzed in a full scan mode (mass range 50–600 dalton). Southern Hemisphere, with a disjunct distribution in Malaysia, The data processing was taken by Chemstation software and the Indonesia, Philippines, New Guinea, Australia, New Zealand, identification of compounds was carried out based on their elution New Caledonia, Vanuatu, Fiji, Norfolk Island and southern South pattern and the comparison of mass spectra with published America [34]. Paleobotanical evidence indicates, however, the literatures. family had a wide distribution in both the Northern and Southern Hemispheres during the Mesozoic and disappeared in most parts Results and Discussions of the Northern Hemisphere by the latest Cretaceous [35,36]. Modern trees of Araucariaceae, especially the genus Agathis Late Cretaceous Xixia amber (Fig. 4; Table 1) Salisb. are highly resinous [34] and araucariacean trees are The total ion chromatogram from the GC-MS analysis of the suspected as a common source for amber all through the Mesozoic Xixia amber is characterized by the distribution of sesquiterpe- [32,33,37,38]. It is of interest to note that amber had not become noids and diterpenoids (Fig. 4; Table 1). The major sesquiterpe- abundant until Early Cretaceous [16], when the Araucariaceae noids are drimane, homodrimane, 1,1,6-trimethyl-1,2,3,4-tetra- attained their greatest diversity and widest distribution in both the hydronaphthalene, 4b-eudesmane, ionene and some unknown Northern and Southern Hemispheres [35]. C -C sesquiterpenoids with base peak 109 (Fig. 4; Table 1). 13 17 PLOS ONE | www.plosone.org 4 October 2014 | Volume 9 | Issue 10 | e111303 Terpenoid and Botanical Origins of Chinese Amber Figure 4. Total ion chromatogram of the Late Cretaceous Xixia amber from GC-MS analysis. The identified peaks are listed in Table 1. doi:10.1371/journal.pone.0111303.g004 Podocarpaceae consist of 18 extant genera that are distributed family unlikely a potential source for fossil resins. Biogeographi- predominantly in the tropical and subtropical mountains of the cally, Podocarpaceae are considered distributed essentially in Southern Hemisphere, and in the Northern Hemisphere extend- Gondwana, or Gondwana-derived plates during the Cretaceous ing northward to subtropical China, Japan, Mexico and the [39]. This appears not to support the occurrence of Podocarpa- Caribbean Islands [34]. Different from Araucariaceae, living trees ceae in the Cretaceous of East Asia and a podocarpaceous origin of Podocarpaceae are only slightly resinous [34], making the of the Late Cretaceous Xixia amber. Table 1. Major compounds identified from the Late Cretaceous Xixia amber. Peak number Compound Name Base peak Molecular ion 1 Unknown C sesquiterepnoid 109 180 2 Unknown C sesquiterpenoid 109 194 3 Unknown C sesquiterpenoid 109 194 4 Drimene+1,1,6-Trimethyl-1,2,3,4-tetrahydronaphthalene 123,159 208,174 54b-Eudesmane 109 208 6 Ionene 173 188 7C sesquiterpenoid 109 206 8 Homodrimane 123 222 9 Unknown C sesquiterpenoid+ Trimethyl naphthalene 109,155 220,170 10 C sesquiterpenoid 109 236 11 16,17,19-trisnorabietane 109 234 12 Bisnorabietane 109 248 13 Isomer of bisnorabietane 109 248 14 Trisnorabieta-8,11,13-triene 131 228 15 Norabietane 109 262 16 Possibly isomer of norabietane 109 262 17 Unknown C diterpenoid 109 248 18 C Diterpenoid 109 260 19 Fichtelite 109 262 20 Possibly isomer of norabietane 95 262 21 Unknown C diterpenoidpossibly rimuane 109 276 22 a- phyllocladane 123 274 doi:10.1371/journal.pone.0111303.t001 PLOS ONE | www.plosone.org 5 October 2014 | Volume 9 | Issue 10 | e111303 Terpenoid and Botanical Origins of Chinese Amber Cheirolepidiaceae, an extinct Mesozoic conifer family ranging classes based on the molecular compositions [43]. Class I fossil from Late Triassic to Late Cretaceous [40], has been considered resins are based primarily on polymers of labdatriene carboxylic the botanical source of the Late Triassic amber of Italy based on acids, especially communic or ozic acids and are contributed by the presence of in situ fossil resins within plant macrofossils of the Leguminosae. Fossil resins derived from conifers also belong to Cheirolepidiaceae, and the abundant occurrence of pollen and Class I [43]. Class II fossil resins are based on polymer of bicyclic cuticles of this extinct family in the paleosol where the dispersed sesquiterpenoid and triterpenoids hydrocarbons and are contrib- amber was buried [2]. Since that Cheirolepidiaceae was the most uted by the family Dipterocarpaceae and a genus Mastixia Blume dominant conifer family through the Jurassic and Early Creta- (Cornaceae) [44,45]. Class III fossil resins with a polystyrene based ceous, it has been considered potential candidate for the botanical structure are contributed by the family Hamamelidaceae. Class IV origin of some Jurassic and Cretaceous amber [28,41,42]. fossil resins, the botanical affinity of which is uncertain, have a Molecular composition analyses revealed that the amber and the cedrane polysesquiterpenoid based structure. The Zhangpu amber co-occurring fossil foliage compressions of Frenelopsis Schenk with a prominent distribution of cadalene-based sesquiterpenoid (Cheirolepidiaceae) from the Lower Cretaceous of Spain are both clearly belongs to Class II. Although trees of the genus Mastixia characterized by the presence of phenolic abietane [42]. Although also produce resins of Class II, the occurrence of fossil winged the totally absence of phenolic abietane in the Xixia amber may fruits of Dipterocarpaceae in the Fotan Group of Zhangpu [26,27] eliminate a significant contribution of Cheirolepidiaceae, the confirms a Dipterocarpaceae origin of the Zhangpu amber. cheirolepidiaceous affinity cannot be completely excluded since Dipterocarpaceae are a tropical tree family that dominate the compounds might be not extracted thus not detectable in the GC- emergent canopy of most lowland rain forests in the Southeast MS analysis. Asia [46]. The family comprises three subfamilies: Dipterocarpoi- deae in the Asian tropics and Seychelles; Pakaraimoideae restricted to the Guyana and Venezuela of tropical South Middle Miocene Zhangpu amber (Fig. 5; Table 2) America; and Monotoideae in tropical Africa, Madagascar and The total ion chromatogram from the GC-MS analysis of the Southeast Colombia [46]. Molecular phylogenetic and biogeo- middle Miocene Zhangpu amber is given in the figure 5. The total graphic studies indicate that the family had an ancient extract of Zhangpu amber is characterized by sesquiterpenoids Gondwanan origin and arrived in Asia after the establishment of and triterpenoids. The major sesquiternoids are isoledene; 1H- 3a,7-methanoazulene, octahydro-1,4,9,9-tetramethyl-(1a,3aa,4- the land connection between the Indian and Asian plates [14,47,48]. This hypothesis was supported by the earliest fossil b,7a,8a); dehydro-ar-curcumene; d-selinene; calamenene; methyl record of the family which is from the lower Eocene of India [14]. drimane and cadalene (Table 2). Triterpenoids such as a and b- amyrone; a and b-amyrin and hop-22(29)-en-3b-ol are abundantly Among the family only trees of the Asian subfamily Dipterocar- poideae are highly resinous whereas the Pakaraimoideae and present in the sample (Fig. 5; Table 2). Monotoideae lack resin ducts [46]. Amber derived from the The presence of amyrin and amyrone-based triterpenoids in the solvent extract of the Miocene Zhangpu amber indicates a Dipterocarpaceae has been reported from the Eocene and Miocene of India [13,14,15,49] and the Eocene of Vietnam contribution from angiosperms. Fossil resins are divided into four Table 2. Major compounds identified from the middle Miocene Zhangpu amber. Peak number Compound Name Base peak Molecular ion 1 Isoledene 105 204 2 Unknown C sesquiterpenoid 81 206 3 Unknown C sesquiterpenoid 95 206 4 1H-3a,7-Methanoazulene, octahydro-1,4,9,9-tetramethyl- 163 206 (1a,3aa,4b,7a,8ab) 5 Dehydro-ar- curcumene 119 204 6 Unknown C sesquiterpenoid 191 206 7 d-Selinene 161 206 8 Calamenene 159 202 9 Tetramethyl naphthalene 169 184 10 Methyl drimane 137 222 11 Unknown sesquiterpenoid 109 204 12 Methyl drimane+ Unknown sesquiterpenoid 137, 109 222, 204 13 Unknown C sesquiterpenoid 81 206 14 Cadalene 183 198 15 Methyl drimane 137 222 16 b-amyrone 218 424 17 b-amyrin+ a-amyrone 218,218 426,424 18 a-amyrin 218 426 19 Hop-22(29)-en-3b-ol 189 426 doi:10.1371/journal.pone.0111303.t002 PLOS ONE | www.plosone.org 6 October 2014 | Volume 9 | Issue 10 | e111303 Terpenoid and Botanical Origins of Chinese Amber Figure 5. Total ion chromatogram of the middle Miocene Zhangpu amber from GC-MS analysis. The identified peaks are listed in Table 2. doi:10.1371/journal.pone.0111303.g005 [50]. The middle Miocene Zhangpu amber represents the The total extracts of the middle Miocene Zhangpu amber northernmost distribution of Dipterocarpaceae-derived amber contain amyrin and amyrone-based triterpenoids, and cadalene- and this discovery corroborates the existence of a dipterocarp based sesquiterpenoids. These compounds are characteristic for forest in Zhangpu of Southeast China and the northward dammar resins that are produced by trees of the tropical family movement of the tropical family during the Mid-Miocene Climatic Dipterocarpaceae. The Dipterocarpceae origin of the Zhangpu Optimum [26,27]. Today the Dipterocarpaceae is absent in the amber is also corroborated by the co-occurring fossil winged fruit Zhangpu area, which today has a typical subtropical monsoon of this family. All evidence suggests the presence of a dipterocarp climate and subtropical evergreen broadleaved forests [51]. forest in the Southeast China during the middle Miocene. Conclusions Acknowledgments In this paper we studied the terpenoid compositions and We are grateful to academic editor Qi Wang for handling the submission, botanical origins of the Late Cretaceous and middle Miocene to Dany Azar and an anonymous referee and the editor for their helpful comments and suggestion. G. Shi thanks S. Mei, H. Yang for help in amber from China by GC–MS analysis. This work represents the fieldwork. S. Dutta is thankful to Department of Science and Technology first detailed biomarker study for amber from East Asia. The Late (DST), India for providing support to procure GC-MS under FIST Cretaceous Xixia amber is characterized by sesquiterpenoids, Programme. abietane and phyllocladane type diterpenoids, but lacks phenolic abietanes and labdane derivatives. 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