Comparative Metabolomic Reveals Chemotaxonomic Markers of Resin Fossils for Identification of Botanical Origins
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摘要:
琥珀中的甾萜化合物是高等植物分泌的次生代谢物[1]。因不同植物分泌出的次生代谢物不同，这些丰富的甾萜化合物可作为解译琥珀古植物来源的化学指纹[2]。但是，现有关于琥珀或树脂化石的化学分类难以区分出琥珀古植物来源的种属，甚至较为粗略层次的分类指标(如生物分类系统中的门、科等)都未见系统研究[3-6]。本研究借用了生物化学领域中常用的比较代谢组学研究思路[7-8]，以顶空固相微萃取与全二维气相色谱-飞行时间质谱联用为技术手段，系统分析了来自古裸子植物(始新世柏科、白垩纪南洋杉科)和古被子植物(中新世龙脑香料、中新世豆科植物)琥珀中的甾萜化合物[9]，鉴定的次生代谢物包括83种单萜、186种倍半萜、84种二萜和43种正构烷烃。通过保留指数校正及与国际标准NIST质谱数据库比对，明确了其中大部分次生代谢物的结构及其化学名称。在化学分类前，我们先测试了样品的镜质体反射率和最大热解峰温(Tmax)，证明本文选取的四类琥珀样品具有非常相似的热成熟度；再利用多元统计法从鉴定的396种甾萜化合物中提取出了四条琥珀古植物来源分类标准：(1)根据单萜和二萜的相对含量可区分裸子植物琥珀和被子植物琥珀；(2)10种化学分类标志物可用于区分出两种裸子植物琥珀，包括柏科和南洋杉科；(3)被子植物琥珀中，豆科琥珀和龙脑香料的化学超类存在差异，豆科琥珀的主要成分是倍半萜和二萜，而龙脑香料的主要成分是单萜和倍半萜；(4)其它特殊的化学成分可将豆科琥珀的古植物源鉴定到属级别(Hymenaea mexicana and Hymenaea protera)。由于古植物次生代谢物成分是由植物的基因组控制，本文揭示的化学分类标志具备遗传基础[10-12]，研究结果全面建立了古树脂植物次生代谢物数据库，为古植物及古环境研究提供数据基础。
Abstract:The terpene compounds in amber are secondary metabolites secreted by higher plants. Due to the different secondary metabolites secreted by various plants, these abundant terpene compounds can serve as chemical fingerprints for interpreting the ancient plant sources of amber[1-2]. However, the existing chemotaxonomy of fossil resins is limited in its ability to distinguish them at phylum, family, and genus levels using small amount of samples[3-6]. Herein an advanced head-space solid phase microextraction-comprehensive two-dimensional gas chromatography- time-of-flight mass spectrometer platform was established to analyze the complex compositions of resin fossils derived from ancient gymnosperms (e.g., Eocene Cupressaceae, Cretaceous Araucariaceae) and angiosperms (Miocene Dipterocarpaceae and Miocene Hymenaea)[7-9]. The secondary metabolites including 83 monoterpenes, 186 sesquiterpenes, 84 diterpenes, and 43 n-alkanes, were annotated and then chemically fingerprinted for the first time. Prior to conducting chemical composition classification, this study tested the vitrinite reflectance of resin-bearing rocks and maximum pyrolysis peak temperature (Tmax) of the ambers to demonstrate their similar thermal maturity. After that, chemotaxonomic markers were disclosed using multivariate statistical analysis to identify the botanical origins of resin fossils, and four identification rules were concluded. Gymnosperm and angiosperm resins can be distinguished by the relative content of monoterpenes and diterpenes; Eocene Cupressaceae and Cretaceous Araucariaceae resins can be identified by ten chemotaxonomic markers which were screened out by orthogonal partial least-squares regression; Miocene Hymenaea and Dipterocarpaceae resins have different combinations of chemical superclasses, with sesqui- and di-terpenes being the major compositions of Hymenaea resins, and mono- and sesqui- terpenes accounting for most of the compositions of Dipterocarpaceae resins; Specific chemotaxonomic markers can identify Hymenaea mexicana and Hymenaea protera resins. As the chemical components of fossil resins were determined by the genes present in their botanical origins, the chemotaxonomic markers revealed in this study have a genetic basis[10-12]. These findings provide a comprehensive database of secondary metabolites from ancient resin plants, which can be utilized for further research on their corresponding paleoenvironment and to clarify their phylogeny.
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Keywords:
- resin fossils /
- terpenes /
- chemotaxonomic markers /
- comparative metabolomics /
- botanica origin /
- resin plants
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图 1 紫绿玛瑙样品QZ-1 (a)和QZ-9 (b)Figure 1. Purple-green agate samples QZ-1 (a) and QZ-9 (b)![]()
图 2 紫绿玛瑙的紫外-可见光谱一阶导图Figure 2. First-order derivatives of the UV-Vis spectra obtained for purple-green agate samples致谢: 该项研究受国家自然科学基金(42307510)，中央高校基础研究经费(CUG2106128)及湖北省珠宝工程研究中心((CIGTXM-03-202103) 资助。感谢南京古生物研究所王博研究员提供了福建漳浦的琥珀样品；感谢宁涛博士和陈品博士在分析测试过程中提供的帮助。 -
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图 1 树脂化石化学指纹图谱分析流程: (a)样品采集; (b)仪器分析和生物信息学分析
Figure 1. Graphic illustration of the workflow to aquire chemical fingerprints of fossil resins, including sample collection(a), and instrument analysis and bioinformatics analysis(b)
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图 2 4类琥珀次生代谢物化学分类(a)和小提琴图对比四组次生代谢物(b)
Figure 2. Chemical composition classification of the secondary metabolites expressed across the four resin plant (a), and violin plots of the secondary metabolites among four comparative groups (b)
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图 3 次级代谢产物相对含量的正交偏最小二乘回归及判别分析(a, b, d, e中红点为化学分类标志物), 箱线图显示南洋杉科和柏科树脂(c)以及豆科树脂(f)化学分类标记的相对含量对比
Figure 3. Orthogonal partial least-squares regression and discriminant analysis of the relative contents of secondary metabolites (a and d); S-plot where points with different colors represent the chemotaxonomic markers, the farther away points the center, the higher contribution of the markers to the separation of the groups (b and e); Boxplots showing relative content levels of chemotaxonomic markers in group Araucariacae and Pinaceae resins and group Hymenaea resins (c and f)
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