Long chain n-alkanes from the leaves of thirteen extant cycad species within the

Cycadacaea, Stangeriaceae, Zamiaceae families were measured by gas chromatographymass

spectrometry (GC-MS). Cycad n-alkane patterns ranged from nC10-nC37, were

unimodal in distribution, maximised at nC27-nC33 and gave average chain length (ACL)

values of 23.9 to 31.0. Low carbon preference indices (CPI) in the range of 0.88 to 2.70

were observed which is atypical of lipids from the leaves of terrestrial plants. Analysis

of variance between the three families of true cycads showed that there were no

significant differences between CPI values. The unusually low CPI values (<1.5) in

nine of the thirteen cycad species analysed suggests that caution needs to be exercised in

the use of n-alkanes distibutions as a chemical marker of terrestrial plant input in

sediments from tropical and sub-tropical regions

Cave, M.R.

Kim, A.W.

Li, N.

Vane, C.H.

NERC Open Research Archive

 Copyright © 2014 Cycad Society of South Africa  This version available http://nora.nerc.ac.uk/509715/   NERC has developed NORA to enable users to access research outputs wholly or partially funded by NERC. Copyright and other rights for material on this site are retained by the rights owners. Users should read the terms and conditions of use of this material at http://nora.nerc.ac.uk/policies.html#access    This is a pre-copy-editing, author-produced PDF of an article accepted for publication in Encephalartos following peer review. The definitive publisher-authenticated version is available online at http://cycadsociety.org/?page_id=623     Article (refereed) - postprint   Vane, C.H.; Kim, A.W.; Cave, M.R.; Li, N.. 2014 Equal abundance of odd and even n-alkanes from cycad leaves: can the carbon preference index (CPI) faithfully record terrestrial organic matter input at low latitudes? Encephalartos (118).                          Contact BGS NORA team at  norabgs@bgs.ac.uk    The NERC and BGS  trademarks and logos (‘the Trademarks’) are registered trademarks of NERC in the UK and other countries, and may not be used without the prior written consent of the Trademark owner.    1 Equal abundance of odd and even n-alkanes from cycad leaves: Can the carbon preference index (CPI) faithfully record terrestrial organic matter input at low latitudes?  C.H. Vane1*, A.W. Kim1, M.R. Cave1 and N. Li2  1British Geological Survey, Environmental Science Centre, Keyworth, Nottingham,  NG12 5GG, Tele.: +44(0)115 936 3017: Email: chv@bgs.ac.uk  2Fairy Lake Botanic Garden, Xianhu Road 160, Liatang Shenzhen, Guangdong, 518004, China Abstract Long chain n-alkanes from the leaves of thirteen extant cycad species within the Cycadacaea, Stangeriaceae, Zamiaceae families were measured by gas chromatography-mass spectrometry (GC-MS). Cycad n-alkane patterns ranged from nC10-nC37, were unimodal in distribution, maximised at nC27-nC33 and gave average chain length (ACL) values of 23.9 to 31.0.  Low carbon preference indices (CPI) in the range of 0.88 to 2.70 were observed which is atypical of lipids from the leaves of terrestrial plants. Analysis of variance between the three families of true cycads showed that there were no significant differences between CPI values.  The unusually low CPI values (<1.5) in nine of the thirteen cycad species analysed suggests that caution needs to be exercised in the use of n-alkanes distibutions as a chemical marker of terrestrial plant input in sediments from tropical and sub-tropical regions.  1. Introduction  2Long chain n-alkanes from the cuticular waxes of  higher plant leaves are characterised by a strong odd  predominance, carbon preference index (CPI) values ranging from ~3  to 34 and average chain lengths (ACL) of 26 to 30 (Eglinton & Hamilton, 1967; Rommerskirchen et al., 2006; Stojanovic et al., 2003). In combination, the n-alkane distribution pattern and dominance of nC27, nC29 and nC31 over lower molecular weight homologues nC15, nC17 and nC19 have been widely applied as a proxy for terrestrial land plant input to lake, river, estuarine and coastal sediments (Jaffé et al., 2001; Meyers, 2003; Wang et al., 2003). Exceptions to the odd dominated n-alkane pattern from higher plants have been shown and on at least one occasion re-examined and corrected (Reddy et al., 2000).  Investigations of the solvent soluble lipid content of Cycadophyta the ancient seed bearing plants found in tropical and subtropical regions reported that n-alkanes from the Zamiaceae  family varied in a non-systematic manner with respect to genus (Osborne et al., 1993). Epicuticular leaf waxes from the Ceratozamia and Encephalartos genera ranged from nC16 to nC33 with maxima at nC18 to nC19. The same study reported that certain species n-alkane distributions were characterised by equal amounts of odd and even alkanes, as well as  an absence of significant concentrations of branched chain isomers (Osborne et al., 1993). The aim of this study was to examine the n-alkane distribution of Cycad foliage in order to improve our understanding of the utility of the n-alkane distributions and associated ratios in ancient sediments.  2. Methods 2.1 Samples and extraction Fresh leaf material was obtained in October, 2006 from Fairy Lake Botanic Gardens, Shenzhen, China. Leaves were washed with 18 M distilled water, freeze  3dried and ground using a freezer mill (SPEX CertiPrep 6850). Powdered leaves ~2.5 g were extracted in an accelerated solvent extraction system ASE 200 (Dionex) using 60 ml dichloromethane (DCM). Extracts were evaporated under a stream of dry nitrogen gas to a constant weight and reconstituted in 3 ml DCM.  2.2 Fractionation and Instrumental Analysis Lipids were isolated by chromatography on columns containing 6 g Silica (deactivated) eluted with 40 ml n-hexane. The hydrocarbons were further fractionated into adduct and non-adduct fractions by urea adduction. Adduct fractions were analysed using a Varian CP3800 series gas chromatograph (GC) directly coupled with a Varian 1200L triple Quadrupole MS/MS system (GC/MS). Peak assignments were made by comparison with mass spectra and retention times of authentic standard compounds.  2. Results and discussion   4   Figure 1.  Gas chromatograms of the saturated hydrocarbon fraction extracted from cycad (Ceratozmaia, Dioon, Macrozamia) and tree fern (Brainea) leaves.   5Alkanes ranging from nC10 to nC37 were identified in the leaves of 16 specimens from six plant families. For all specimens the major n-alkanes were C27, C29, C31 and C33 in contrast n-alkanes in the range C16 to C26 were mainly present at lower relative abundances (Fig. 1). Total amounts of n-alkanes ranged from 0.55 µg/g dry wt for Cycas panzhihuaensis to 6.8 µg/g dry wt for Encephalartos gratus (Table 1). The average chain length (ACL) ranged from 23.9 for Cycas panzhihuaensis to a maximum of 31.0 for Encephalartos gratus, confirming the notion that cycad leaves contain a broad range of n-alkanes (Table 1). By contrast long chain n-alkane distibutions for non-cycads, namely Equisetacea and Blechnaceae plant families, gave a limited distribution range dominated by C27, C28, C29 (Fig 1;Table 1). The carbon preference index (CPI) of the true cyacds (Cycadacaea, Stangeriaceae, Zamiaceae) ranged from as low as 0.88 in Cycas panzhihuaensis to 2.70 for Zamia fischeri Miq. The slight predominance of odd over even n-alkanes was confirmed by the mean CPI of the 12 cycad leaf specimens of 1.47. The cycad CPI values presented here are considerably lower than those usually reported for higher plant sourced n-alkanes which typically vary from about 5 to 30. An earlier study of n-alkane leaf waxes of 28 cycad species from Mexico, Florida and Australia reported that Macrozamia moorei, Zamia fischeri, Zamia furfuracea and Ceratozamia robusta yielded odd over even ratios of ~ 1.5, unimodal in distributions which were maximal at nC17 to nC21 and showed no significant quantities of branched chain isomers (Osborne et al., 1993).  In this current study we report broadly similar CPI values of 1.21 Macrozamia moorei, 1.30  Ceratozamia robusta, 1.32  Zamia furfuracea, but higher CPI values for the cycad Zamia fischeri at 2.70 (Table 1). Overall results differ from that of the earlier study of Osborne et al (1993) in that here the cycad n-alkanes were generally maximal at C29 to C31. Furthermore, branched chain isomers were such a significant component of the  6solvent extracts that urea-adduction was required to remove these components in order to achieve baseline separation and facilitating quantification of individual n-alkane homologues. One plausible explanation for the difference in n-alkane distributions between this and previous studies of the same plant species could be that we analysed the whole leaf as compared to only extracting the surface epicuticular waxes of the leaves. Alternatively it has been speculated that the method of cultivation can modify epicuticular leaf wax n-alkane distributions, although this hypothesis remains untested (Osborne et al., 1993).  Table 1. Summary of n-alkane data for cycad, horsetail and tree fern leaves    The differences between the means of the CPI data (Table 1) for the three true cycad families Cycadacaea, Stangeriaceae, Zamiaceae  were analysed using one-way analysis of variance (ANOVA) in the R statistical programming language (R Development Core Team (2008)). Figure 2 shows a comparative box plot of the CPI values for the three cycad families showing all three have similar median values and  7interquartile ranges with a possible outlier (Zamia fischeri Miq.) in the Zamiaceae family. The analysis of variance gave a non significant p-value of 0.8531 showing that there are no significant differences between the CPI values for the three families. As an additional check, a non-parametric equivalent method to the ANOVA, was also applied (Kruskal-Wallis) which confirmed no significant difference with a p-value of 0.5906.  Figure 2. Boxplot of the CPI values for Cycadacaea (n=4), Stangeriaceae (n=2), Zamiaceae (n=7).   4  Conclusions This study indicates that soils and sediments accumulating significant amounts of cycad foliage may have low CPI values and that the widely applied n-alkane based terrestrial source inference requires refinement particularly in tropical and subtropical soils where cycads grow naturally.   5. Acknowledgements  8C.H. Vane is indebted to Nigel Birch at the British Consulate-General, Guangzhou, for logistical and financial assistance. This paper is published by permission of the Executive Director, British Geological Survey.   6. References Eglinton, G., Hamilton, J., 1967. Leaf epicuticular waxes. Science, 156, 1322-1335. Jaffé, R., Mead, R., Hernandez, M.E., Peralba, M.C., DiGuida, O.A., 2001. Origin and transport of sedimentary organic matter in two subtropical estuaries: a comparative, biomarker-based study. Organic Geochemistry, 32, 507-526. Meyers, P.A., 2003. Applications of organic geochemistry to paleolimnological reconstructions: a summary of examples from the Laurentian Great Lakes. Organic Geochemistry, 34, 261-289. Osborne, R., Salatino, M.L., Sekiya, C.M., Vazquez Torres, M., 1993. Alkanes of foliar epicuticular waxes from five cycad genera in the Zamiaceae. Phytochemistry, 33, 607-609. Reddy, C.M., Eglinton, T.I., Palic, R., Benitez-Nelson, B.C., Stojanovic, G., Palic, I., Djordjevic, S., Eglinton, G., 2000. Even carbon number predominance of plant wax n-alkanes: a correction. Organic Geochemistry, 31, 331-336. Rommerskirchen, F., Plader, A., Eglinton, G., Chikaraishi, Y., Rullkötter, J., 2006. Chemotaxonomic significance of distribution and stable carbon isotopic composition of long-chain alkanes and alkan-1-ols in C4 grass waxes. Organic Geochemistry, 37, 1303-1332. Stojanovic, G., Palic, R., Tarr, C.H., Reddy, C.M., Marinkovic, O., 2003. n-Alkanes and fatty acids of Hypericum perforatum, Hypericum maculatum and Hypericum olympicum. Biochemical Systematics and Ecology, 31, 223-226.  9Wang, X.C., Chen, R.F., Berry, A., 2003. Sources and preservation of organic matter in Plum Island salt marsh sediments (MA, USA): long-chain n-alkanes and stable carbon isotope compositions. Estuarine, Coastal and Shelf Science, 58, 917-928.   10 

Equal abundance of odd and even n-alkanes from cycad leaves: can the carbon preference index (CPI) faithfully record terrestrial organic matter input at low latitudes?

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Equal abundance of odd and even n-alkanes from cycad leaves: can the carbon preference index (CPI) faithfully record terrestrial organic matter input at low latitudes?

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