Organic Geochemistry 67 (2014) 99–102

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Organic Geochemistry

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Leaf wax n-alkanes and d13C values of CAM plants from aridsouthwest Africa

0146-6380/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.orggeochem.2013.12.005

⇑ Corresponding author. Tel.: +44 (0) 1162523832.E-mail address: ab269@le.ac.uk (A. Boom).

A. Boom a,⇑, A.S. Carr a, B.M. Chase b,c, H.L. Grimes a, M.E. Meadows d

a Department of Geography, University of Leicester, University Road, Leicester LE1 7RH, United Kingdomb Institut des Sciences de l’Evolution de Montpellier, Département Environnements, UMR 5554, Université Montpellier 2, Bat. 22, CC061, Place Eugène Bataillon, 34095 MontpellierCedex 5, Francec Department of Archaeology, History, Culture and Religion, University of Bergen, Postbox 7805, 5020 Bergen, Norwayd Department of Environmental and Geographical Sciences, University of Cape Town, Rondebosch, Cape Town 7701, South Africa

a r t i c l e i n f o

Article history:Received 5 September 2013Received in revised form 15 November 2013Accepted 10 December 2013Available online 16 December 2013

a b s t r a c t

We analysed leaves from 42 plants from the South African Succulent Karoo. Whole leaf d13C values clearlydifferentiated 3 different populations, consisting of plants operating under obligate CAM (crassulaceanacid metabolism), facultative CAM and C3 modes. In contrast, the leaf wax n-alkane d13C data from thesemetabolic groups showed a broader overlapping distribution. CAM plants operating under full CAM modeproduced a wide range of apparent 13C fractionation. Succulent/CAM plant wax yield was considerable(up to 23 mg/g in our plants), so its contribution to soil composition and sedimentary leaf wax compo-sition should not be dismissed. The presence of CAM plant wax n-alkanes in sedimentary n-alkanes couldtherefore be a problem for accessing ecosystem scale C3–C4 proportion.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

n-Alkanes are increasingly being used as palaeoenvironmentalproxies, particularly through the application of compound-specificd13C and dD analyses. The proportion of plants using the C3 and C4

photosynthetic pathways can be reconstructed from sedimentaryn-alkanes using established end member d13C values from modernC3 and C4 plants (e.g. Maslin et al., 2012). A significant isotopic frac-tionation of 13C during leaf wax lipid synthesis produces a largeoffset between bulk tissue and leaf wax lipids (de Niro and Epstein,1977). For n-alkanes, this is in the region of 6‰ and 10‰ for C3 andC4 plants, respectively (Collister et al., 1994; Lockheart et al., 1997;Bi et al., 2005; Vogts et al., 2009).

The isotopic composition of leaf wax derived from the thirdmajor photosynthetic pathway, crassulacean acid metabolism(CAM) has received much less attention. Up to 33 families and10,000 angiosperms (ca. 7% of vascular plants) utilise CAM metab-olism (Cushman, 2001). CAM plants are particularly common intropical and arid regions (Ting, 1985).

CAM plant whole leaf d13C values are known to be highlyvariable, ranging between �10 and �20‰ (Mooney et al., 1977;Lüttge, 2004). This reflects flexibility in the CAM photosyntheticmechanism. Some species are obligate CAM plants, while othersare facultative and are able to shift between C3 and CAM. The effectof CAM plant waxes on biomarker-based palaeoecological records,

particularly marine leaf wax records close to arid parts of Africa, isunclear. It is often assumed to be low (e.g. Maslin et al., 2012), butthere is not much basis for this, and it may depend on the geo-graphical region. Only a handful of studies have reported leafwax d13C values for known CAM plants and none were from naturalhabitats (Feakins and Sessions, 2010 and references therein). Withso few data points it is not possible to evaluate the wider signifi-cance of CAM plants for n-alkane proxy records.

In arid western parts of southern Africa CAM plants arecommon and dominate in the Succulent Karoo Biome. Here wepresent n-alkane d13C data for 42 succulent plants from theirnatural habitat across the Succulent Karoo Biome of South Africa.The data are considered in the context of CAM plant ecophysiologyand the implications for palaeoecological reconstruction in the re-gion. More detailed descriptions of sampling and leaf wax n-alkanedistributions are presented in an accompanying study (Carr et al.,2014).

2. Methods

Samples of leaf and stem succulents were selected from plantsobtained from a wider study that encompassed the Succulent Kar-oo and Fynbos Biomes of the Western and Northern Cape Provincesof South Africa during April/May 2010 (Table S1). They were storedin paper bags, air dried at the University of Cape Town and freezedried. Ca. 0.5 g powered photosynthetic tissue was extracted usinga Soxhlet system [24 h, hexane/CH2Cl2/MeOH (1:2:2 v/v/v)];5a-cholestane was added as internal standard. Each extract was

Fig. 1. Frequency distributions showing, (A) whole leaf d13C values, (B) weighted mean average of n-alkane d13C values and (C) Dd13C (whole leaf- WMA alkanes) of 42selected succulent plants from the succulent Karoo in South Africa.

100 A. Boom et al. / Organic Geochemistry 67 (2014) 99–102

purified and separated using an Al2O3 column. Gas chromatogra-phy–mass spectrometry (GC–MS) analysis of each apolar fractionwas carried out with a Perkin Elmer Clarus-500 (column: CP-Sil5CB MS, 30 m � 0.25 mm). The linearity of the GC–MS systemwas tested using an n-alkane standard (C7-C40) at a concentrationrange from 5 lg/ml to 800 lg/ml. High signal linearity wasachieved over the full chain length range and no trends in response

factor with n-alkane chain length were observed. For more detailssee Carr et al. (2014).

The n-alkane d13C values were determined using isotope ratiomonitoring GC–MS (irm-GC–MS), with an Agilent 6890N gas chro-matograph equipped with a PtCu wire combustion reactor, inter-faced to a SerCon GC-CP and 20–20 MS instrument. Isotopereference compounds comprising 5a-cholestane and C19 fatty acid

A. Boom et al. / Organic Geochemistry 67 (2014) 99–102 101

methyl ester were co-injected with every analysis; n-alkanes weremeasured in triplicate with a precision of 0.2‰. Whole leaf d13Cvalues were determined in triplicate using a SerCon ANCA GSLelemental analyser interfaced to the SerCon 20–20 MS, with a pre-cision better than 0.1‰.

3. Results

3.1. 13C stable isotope results for whole leaf tissue

Whole leaf d13C values showed marked similarities withreported values (von Willert et al., 1977; Rundel et al., 1999;Winter and Holtum, 2002), with clusters (Fig. 1) centred at �13to �18‰ (n = 17) representing obligate CAM plants and �20 to�27‰ (n = 18) representing facultative CAM plants. A cluster of 6C3 plants was centred at �25 to �29‰. With the exception ofSalsola tuberculata, a C4 plant (�13‰), the most enriched plantswere almost certainly obligate CAM on the basis of the measure-ments (Winter and Holtum, 2002). They were from three families:Xanthorrhoeaceae (Aloë), Crassulaceae (Tylecodon) and Aizoaceae(Cheiridopsis, Ruschia and Mesembryanthemum). Aloë ramosissimais an obligate CAM plant (Rundel et al., 1999), as is Ruschia stricta(Matimati et al., 2012). The lower d13C cluster (�20 to �27‰)formed a broader and more complex distribution, probably reflect-ing plants capable of CAM flexibility (Rundel et al., 1999) for whichthere is a variation in day and dark fixation (Winter and Holtum,2002).

3.2. n-Alkanes

The n-alkane concentrations and compound specific d13C valuesare shown in the Supplementary material (Table S1). A range fromC21 to C35 was found, with C36 and C37 detected in a few samples,but in low amount (< 1%). Total (C21–C35) concentration rangedfrom 0.02 to 22 mg/g dry wt. (dw). The yield distribution washighly skewed by a small number of outliers and the majority(58%) of plants produced a yield < 10 mg/g dw (see Carr et al.,2014 for a more detailed discussion). All plants had a strong odd/even predominance and a maximum at C31 or C33, although a max-imum at C29 was also observed.

4. Discussion

The whole leaf d13C values clearly indicated three distinctpopulations, one with an obligate CAM photosynthetic pathway,one reflecting a varied CAM-C3 flexibility and the third, C3 plants(Winter and Holtum, 2002). The n-alkane concentration valueswere markedly higher than reported for southern African grasses(Rommerskirchen et al., 2006 – avg. yield 0.3 to 0.4 mg/g dw),savannah vegetation (avg. ca. 0.6 mg/g dw; Vogts et al., 2009), aswell as most woody vegetation from the Succulent Karoo and Fyn-bos biomes (woody shrub avg. 1.1 ± 2.6 mg/g dw; Carr et al., 2014).A high yield from drought adapted plants is perhaps unsurprising.Maffei et al. (1997) reported n-alkane concentration of 1–10 mg/g(fresh weight) for various Cactaceae, further suggesting that lipidconcentration from CAM plants may be substantial.

Average chain length (ACL) values were similar to thosereported for CAM plants (Feakins and Sessions, 2010). In generalthe distributions are comparable to reported values for naturalsouthern African vegetation (Vogts et al., 2009), although therewas an overall tendency for succulent plant functional types toproduce longer and less dispersed distributions in the study area(Carr et al., 2014).

In terms of d13C, the plants showed some interesting patterns.The CAM-C3 flexible plants produced n-alkanes depleted in 13C

vs. whole leaf tissue by on average 6.1‰. This is consistent withearlier studies, which reported Dd13C as being near 6‰ for C3

and some CAM plants (Collister et al., 1994; Feakins and Sessions,2010). However, the obligate CAM plants were highly variable;although their mean Dd13C value was similar at 7.1‰, there wasa greater spread, ranging from 0.5‰ to 12.8‰. This implies largedifferences in C allocation to lipid biosynthesis, presumably toaid water conservation.

As a result of this highly varied fractionation during lipid syn-thesis, the resulting distribution of the n-alkane d13C valuesshowed far less separation of the two modes of photosynthesisthan the plot of bulk tissue data (Fig. 1). This has potential implica-tions for attempts at quantitative C3/C4 vegetation reconstructionin regions where CAM/succulent plants are a component of thelandscape. This may be particularly important in certain regions,such as the west coast of southern Africa, where these plants dom-inate. Our data also demonstrate n-alkane yield similar to, or evenmarkedly higher than, that reported for ordinary C3 and C4 plants(see also Carr et al., 2014).

5. Conclusions

Whole leaf d13C analysis of naturally occurring succulent plantsclearly differentiated obligate CAM and C3/CAM flexible plants. Then-alkane d13C values were less discriminatory, with CAM plantsoperating under full CAM exhibiting a wide range of Dd13C values,obscuring the pattern observed for whole leaf tissue. This impliesthat n-alkane d13C measurement is not a good tool for determiningthe mode of CAM, nor for differentiating some CAM plants from C4

plants. Succulent/CAM plant wax yield can be substantial.Although these plants are not important in most biomes, thereare some regions, such as southern Africa, where this is not thecase and their influence on soil and sedimentary leaf wax compo-sition should not be dismissed.

Acknowledgements

The study was funded by the Leverhulme Trust (Grant # F/00212/AF). B.M.C. is also supported by FP7 ERC Starting Grant‘‘HYRAX’’ (Agreement #258657). We would like to thank twoanonymous reviewers for valuable comments.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.orggeochem.2013.12.005.

Associate Editor – I.D. Bull

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