The stable carbon isotopic composition (δ13C) of organic matter provides key information about fundamental metabolic pathways used by the organism and the environmental conditions during formation. For example, trees in a temperate forest have a different isotopic composition compared to grasses on the African savannah and both have a very different isotopic composition compared to bacterial methane (Fig. 1). As such δ13C is a commonly used tool in organic geochemistry and has been applied to a wide-range of environmental and paleoclimatological problems.
Introduction into stable carbon isotopes
Carbon, the fourth most abundant element in the universe, exists in two stable forms; 12C with a core consisting of 6 neutrons and 6 protons and 13C which has one additional neutron. Of all carbon on earth, 99% is present as 12C and 1% consists as 13C. In addition to these two stable forms, in nature a small fraction of carbon exists as 14C, which is radioactive and decays into 14N with a half-life of ~ 5,700 yrs. In material older than ~ 57,000 years normally all 14C has decayed into 14N and this material is defined as being “radiocarbon dead”.
The ratio of 13C compared to 12C is generally expressed as δ13C, which reflects the ratio of 13C/12C in a natural sample compared to an inorganic standard called the Vienna Pee Dee Belemnite (VPDB).
Organisms generally prefer 12C over 13C because it is a little bit lighter, leading to an enrichment of 12C over 13C in most types of organic matter. As VPDB is rich in 13C, most organic matter is depleted in 13C relative to the standard (Fig. 1). For example, if organic matter has a δ13C value of -25 ‰, this sample has 25 fewer 13C atoms per thousand carbon atoms compared to that of the inorganic standard and is depleted in 13C.
Bulk versus compound specific δ13C
Although bulk δ13C values indicate the fundamental metabolic pathways used by the organism and the environmental conditions during formation, in geological archives, such as sediments, bulk δ13C values are often not very diagnostic. This is because bulk δ13C reflects a mixture of different types of organic matter [Pagani et al. 2000]. As a result changes in the relative contribution of different types of organic matter can have a large impact on bulk δ13C values. To circumvent this complication, organic geochemists use combustion isotope ratio mass spectrometry coupled to gas chromatography to determine the δ13C of individual lipids. If diagnostic lipids are targeted (biomarkers), changes in compound specific δ13C values reflect changes in the δ13C of specific (parts of) organisms and are not thought to be biased by changes in the type of organic matter.
Compound specific δ13C
One of the most widely used biomarkers for paleoclimatological studies are higher plant waxes. These waxes consist of long-chain aliphatic compounds such as n-alkanes (Fig. 2) [Eglinton and Hamilton, 1967].
Plants produce the epicuticular waxes to protect their leaves from the environment and to reduce water loss. Importantly, these biomarkers are easily transported by wind and rivers over thousand of kilometers [Simoneit, 1977], are very resistant to degradation and can be abundant in ancient open marine sediments [Schefuss et al., 2003; Naafs et al. 2012] making these ideal targets for compound specific δ13C studies. However, although most higher plants produce epicuticular waxes, there is a large spread in both their abundance and δ13C value because different higher plants use different metabolic pathways. One of the key differences is the mechanism used to fix carbon during photosynthesis (see below)
C3 and C4 plants
There are three distinct types of higher plants: C3 plants, C4 plants, and CAM plants. CAM plants, which use the crassulacean acid metabolism (CAM), form a minor component of high-plants (7% of total). As such, we will not discuss these in much detail today. The majority (85%) of higher plants are C3 plants. Most shrubs, herbs, trees, and cool-season grasses are C3 plants. In C3 plants CO2 is directly provided to Rubisco, the key-enzyme in the Calvin cycle (Fig. 3). The down-side of this mechanism is that oxygen can also bind to Rubisco in a process that is called photorespiration, which costs energy without fixing carbon. C4 plants differ from C3 plants in that CO2 is first concentrated in the mesophyll cell before it enters the Calvin cycle, ensuring that Rubisco is mainly used to fix carbon (Fig. 3). Typical C4 plants include tropical grasses and sedges and C4 plants are responsible for roughly a third of the global terrestrial carbon fixation. Because of their different pathways to fix carbon the organic matter of C3 and C4 plants, including the epicuticular waxes, have a distinctly different δ13C value. Specifically, C3 plants are generally ~ 10 ‰ lighter compared to C4 plants (Fig. 1).
The paper (Diefendorf et al., 2015; Leaf wax composition and carbon isotopes vary among major conifer groups)
Conifers are an essential component of modern day ecosystems. They are particularly abundant within northern hemisphere high-elevation and high-latitude ecosystems but can also be found in tropical and southern hemisphere ecosystems. Previous studies (e.g. Diefendorf et al., 2011; Bush and McInerney, 2013) suggest that conifers do not produce large quantities of epicuticular plant waxes (e.g. n-alkanes) compared to angiosperm plants. This would suggest that conifers do not form a significant source of epicuticular plant waxes in geological archives such as marine sediments or lignite deposits. However these previous studies were based on a limited number of conifer species. For example, tropical and Southern Hemisphere conifer species had not been explicitly studied for their epicuticular plant waxes chemistry.
In this paper Diefendorf et al. (2015) set out to fill the gap in our understanding of the contribution of conifer-derived plant waxes (predominantly n-alkanes) in natural archives. In total, 43 conifer species were sampled from the Botanical Garden at Berkley during December 2011. Sampling the conifers at the same location and the same time ensured that all plants experienced similar climatic variables and the only variable was plant species. Plant waxes were extracted from the powdered needles by means of solvent extraction and analyzed by means of GC-FID. The δ13C was determined by GC-C-IRMS.
Abundance and δ13C of n-alkanes in conifers
Previous studies (Diefendorf et al. 2011) have shown that the contribution of n-alkanes to the sedimentary pool is fairly insignificant compared to that of the angiosperm contribution (<200x higher). However, these new results indicate that conifers can be an important source of n-alkanes in natural archives. Their abundance in and on the needles of conifer trees rivals that of the angiosperms gram per gram.
Interestingly the concentration of n-alkanes is highly variable among different conifer groups (Fig. 5). In the early diverging taxodioid lineages, the concentrations of n-alkanes is low, while higher concentrations are found in other species. A particularly strong phylogenetic signal can be observed in the n-C29 alkane abundance and in the average chain length (ACL), complicating the use of ACL in natural archives to infer climatic changes.
In this study the authors show that the overall fractionation between bulk conifer leaf and leaf waxes such as n-alkanes is around 4.1‰. This is consistent with previous studies and similar to that observed in angiosperms. There are however distinct differences in the fractionation of 13C across the different analyzed phylogenentic groups. For example Taxaceae is characterized by a particularly large fractionation of 8 ‰ (Diefendorf et al. 2015). The authors suggest that this is a result difference in the metabolic pathways associated with carbon fixation, but acknowledge that further research is required. The large difference in leaf/wax fractionation values observed in some conifer species may therefore complicate our understanding of sedimentary n-alkane δ13C records.
In summary, this paper indicates that conifers should be taken seriously in terms of their input into sedimentary systems. Their contribution to the n-alkane pool in natural archives cannot be neglected, specifically since this study has shown that the concentrations of n-alkanes in some conifers is significant even compared to that of angiosperms. In addition the authors stress the importance of taking conifers into account when interpreting δ13C-values from paleo-archives, because of the difference in fractionation between angiosperms and conifers.
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