Available online at www.sciencedirect.com
Geochimica et Cosmochimica Acta 117 (2013) 161–179
www.elsevier.com/locate/gca
Leaf wax n-alkane distributions in and across modern
plants: Implications for paleoecology and chemotaxonomy
Rosemary T. Bush a,⇑, Francesca A. McInerney a,b
a
Department of Earth and Planetary Sciences, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208-3130, USA
Sprigg Geobiology Centre, Environment Institute and School of Earth and Environmental Sciences, University of Adelaide,
Mawson Laboratories, Adelaide, South Australia 5005, Australia
b
Received 20 June 2012; accepted in revised form 18 April 2013; available online 29 April 2013
Abstract
Long chain (C21 to C37) n-alkanes are among the most long-lived and widely utilized terrestrial plant biomarkers. Dozens
of studies have examined the range and variation of n-alkane chain-length abundances in modern plants from around the
world, and n-alkane distributions have been used for a variety of purposes in paleoclimatology and paleoecology as well
as chemotaxonomy. However, most of the paleoecological applications of n-alkane distributions have been based on a narrow
set of modern data that cannot address intra- and inter-plant variability. Here, we present the results of a study using trees
from near Chicago, IL, USA, as well as a meta-analysis of published data on modern plant n-alkane distributions. First, we
test the conformity of n-alkane distributions in mature leaves across the canopy of 38 individual plants from 24 species as well
as across a single growing season and find no significant differences for either canopy position or time of leaf collection. Second, we compile 2093 observations from 86 sources, including the new data here, to examine the generalities of n-alkane
parameters such as carbon preference index (CPI), average chain length (ACL), and chain-length ratios for different plant
groups. We show that angiosperms generally produce more n-alkanes than do gymnosperms, supporting previous observations, and furthermore that CPI values show such variation in modern plants that it is prudent to discard the use of CPI
as a quantitative indicator of n-alkane degradation in sediments. We also test the hypotheses that certain n-alkane chain
lengths predominate in and therefore can be representative of particular plant groups, namely, C23 and C25 in Sphagnum
mosses, C27 and C29 in woody plants, and C31 in graminoids (grasses). We find that chain-length distributions are highly variable within plant groups, such that chemotaxonomic distinctions between grasses and woody plants are difficult to make
based on n-alkane abundances. In contrast, Sphagnum mosses are marked by their predominance of C23 and C25, chain
lengths which are largely absent in terrestrial vascular plants. The results here support the use of C23 as a robust proxy
for Sphagnum mosses in paleoecological studies, but not the use of C27, C29, and C31 to separate graminoids and woody plants
from one another, as both groups produce highly variable but significant amounts of all three chain lengths. In Africa, C33
and C35 chain lengths appear to distinguish graminoids from some woody plants, but this may be a reflection of the differences
in rainforest and savanna environments. Indeed, variation in the abundances of long n-alkane chain lengths may be responding in part to local environmental conditions, and this calls for a more directed examination of the effects of temperature and
aridity on plant n-alkane distributions in natural environments.
Ó 2013 Elsevier Ltd. All rights reserved.
1. INTRODUCTION
⇑ Corresponding author. Tel.: +1 832 588 7290; fax: +1 847 491
8060.
E-mail address: rbush@earth.northwestern.edu (R.T. Bush).
0016-7037/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.gca.2013.04.016
Long chain normal alkanes (n-alkanes), C21–C37, are
synthesized as part of the epicuticular leaf wax of terrestrial
plants and are among the most recognizable and widely
used plant biomarkers, with a history of research stretching
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back almost a century (Chibnall et al., 1934). Plants typically produce a range of n-alkanes, commonly with a strong
odd-over-even predominance and one or two dominant
chain lengths (Eglinton and Hamilton, 1963, 1967). Because
they are straight-chain hydrocarbons lacking functional
groups, n-alkanes are especially stable and long-lived molecules that can survive in the fossil record for tens of millions
of years (Eglinton and Logan, 1991; Peters et al., 2005). nAlkanes occur in both modern and fossil leaves (e.g. Huang
et al., 1995; Otto et al., 2005); in soils, paleosols, and fluvial
sediments (e.g. Quenea et al., 2004; Smith et al., 2007); and
in both lacustrine and marine sediments (Schefuß et al.,
2003; Sachse et al., 2004; Handley et al., 2008). The stable
isotopic compositions (d13C and dD) of n-alkanes and their
applications in paleoecology and paleoclimatology have
been studied extensively (see Castañeda and Schouten
(2011) and Sachse et al. (2012) for review). Long chain n-alkanes have great potential to inform us on past terrestrial
ecosystems and environments, but their interpretation as
paleo-proxies requires a strong understanding of variations
in n-alkane production both within and between modern
plants.
n-Alkanes contribute to the hydrophobic properties of
leaf wax and serve as part of the plant’s first barrier from
the external environment, protecting the leaf from water
loss via evaporation (Post-Beittenmiller, 1996; Jetter
et al., 2006). Early studies suggest that leaf wax n-alkanes
accumulate rapidly during leaf maturation in spring and
early summer, and that hydrocarbon amounts remain relatively constant through the remainder of the growing season (Eglinton and Hamilton, 1967; Avato et al., 1984;
Gülz and Müller, 1992; Tipple et al., 2013), although this
may not necessarily mean that n-alkane proportions are
maintained (Stránský et al., 1967). Sachse et al. (2010)
and Tipple et al. (2013) demonstrate that isotopic values
of leaf wax n-alkanes reflect the isotopic values of leaf water
at the time of leaf formation, but other studies found that
the isotopic composition of leaf wax n-alkanes changes
through the growing season (Lockheart et al., 1997;
Chikaraishi et al., 2004; Sachse et al., 2009), suggesting that
turnover of surface leaf wax may depend on plant type or
environmental conditions (e.g. wind). The rate of wax turnover and variations in hydrocarbon production across species due to environmental pressures, e.g. temperature,
aridity, or ablation by wind, are relatively unknown (Jetter
et al., 2006). Furthermore, within an individual tree, leaf
physiology can vary from sun-exposed to shaded canopy
positions, and spatial variation in overall n-alkane production across the canopy has been observed for some tree species (Dyson and Herbin, 1968; Lockheart et al., 1997).
Similarly, variation in n-alkane distribution across a tree’s
canopy or across a growing season could undermine the
power of comparisons between modern leaves, typically
collected during the height of the growing season, and sedimentary n-alkane profiles derived from leaves that were
likely, although not necessarily, naturally abscised. Most
leaves in the fossil record and the leaves which would have
contributed n-alkanes to the sedimentary record likely grew
on the part of the canopy with the greatest amount of light
exposure (Greenwood, 1991), but shaded leaves also would
have contributed to fossilized soil and sedimentary organic
matter. Understanding intra-plant variability in n-alkane
production is critical to applying n-alkanes to paleoecological interpretations, as it is often difficult to assess the canopy position and age of leaves that have contributed to
sedimentary n-alkane records.
A great deal of research has been devoted to identifying,
quantifying, and interpreting naturally occurring leaf wax
n-alkanes in modern plants, often with the goal of using
them as taxon-specific chemical fingerprints. Chemotaxonomic studies often use one or a few plants from a single
location to represent a species (e.g. Maffei et al., 2004);
however, n-alkane distributions can vary within a species
across its range (Dodd and Afzal-Rafii, 2000; Dodd and
Poveda, 2003). Geochemical studies of sedimentary longchain n-alkanes have focused on the application of ratios
of particular chain lengths in an effort to reconstruct past
ecosystems. The ratio of longer chain lengths (e.g. C29 or
C31) to C17 has been used as a proxy for relative inputs of
terrestrial plants versus aquatic algae and phytoplankton
in lake sediments (Cranwell et al., 1987; Meyers and Ishiwatari, 1993). n-Alkanes with more intermediate chain
lengths—C23, and to a lesser extent C25—have been utilized
to model Sphagnum peat moss (Nott et al., 2000; Pancost
et al., 2002) and aquatic plants (Ficken et al., 2000; Mügler
et al., 2008). Longer chain lengths have also been used as
identifiers for different vascular plant groups. For example,
in studies of lake sediments it has been postulated that C31
represents input from grasses while C27 and C29 represent
input from trees and shrubs (Meyers and Ishiwatari, 1993;
Meyers, 2003). This reasoning has been used to interpret ratios constructed from these n-alkanes in lake cores and loess
sequences as changes in ecosystem structure over time corresponding with climate or land use change (Brincat et al.,
2000; Schwark et al., 2002; Hanisch et al., 2003; Zhang
et al., 2006). These n-alkane ratios often can be correlated
with other evidence of plant community change, such as
pollen records, and are compelling in such context. However, the supposition that single n-alkanes represent such
large plant groups as grasses and woody plants is based
on relatively sparse original data (Wakeham, 1976;
Cranwell, 1984; Kawamura and Ishiwatari, 1984; Cranwell
et al., 1987), and although widely cited, Cranwell (1973)
does not present any data on the distribution of n-alkanes
in grasses in contrast to trees.
In addition to ratios of individual n-alkane abundances,
several other methods for characterizing a given n-alkane
distribution have been developed. The two most common
are average chain length (ACL) and carbon preference index (CPI). ACL is the weighted average of the various carbon chain lengths, usually defined as
ACL ¼ RðCn nÞ=RðCn Þ;
where Cn is the concentration of each n-alkane with n carbon atoms. Carbon preference index measures the relative
abundance of odd over even carbon chain lengths, where
CPI ¼ ½Rodd ðC21–33 Þ þ Rodd ðC23–35 Þ=ð2Reven C22–34 Þ;
and captures the degree to which odd carbon number
n-alkanes dominate over even carbon numbers
R.T. Bush, F.A. McInerney / Geochimica et Cosmochimica Acta 117 (2013) 161–179
(see Marzi et al., 1993). CPI values greater than 1 mean a
predominance of odd over even chain lengths. In sediments,
CPI > 1 is used to indicate a terrestrial plant source and
thermal immaturity of the source rock (Bray and Evans,
1961; Eglinton and Hamilton, 1967). Other numerical
parameters have also been used to describe and distinguish
n-alkane distributions, including the location-specific matrix model developed by Jansen and coworkers (Jansen
et al., 2006, 2010).
The robust application of n-alkane distributions as
paleoecological biomarkers requires the systematic survey
of variation among different modern plants (Diefendorf
et al., 2011). With the wealth of studies now published, a
more comprehensive analysis of n-alkanes in modern plants
is now possible and can inform their use as paleoecological
indicators. Therefore, this study uses a combination of new
data on n-alkane distributions from plants growing at the
Chicago Botanic Garden and the Lincoln Conservatory
(Chicago, IL, USA) as well as a survey of the published
n-alkane literature (totaling 2093 n-alkane measurements
from 86 sources) to examine the following sets of questions:
(1) Do n-alkane distributions vary within a plant with
canopy position or leaf age? To test this, leaves were
simultaneously collected from sun-exposed and
shaded portions of a tree canopy during the summer
and autumn across 38 individual plants: 22 different
outdoor angiosperm and gymnosperm tree species
and two ferns from the Chicago Botanic Garden.
(2) How do n-alkane chain-length distributions vary
among plant types? How does the absolute abundance of n-alkanes differ between angiosperms and
gymnosperms? Compiled literature data on n-alkane
distributions and abundances from locations on
every continent except Antarctica, as well as new
measurements from the Chicago Botanic Garden
and Lincoln Conservatory, are analyzed to address
these questions.
(3) Can the abundance of different chain lengths be used
to reconstruct plant types? How well do C23 and C25,
C27 and C29, and C31 actually distinguish Sphagnum
mosses, woody plants, and grasses, respectively?
These questions were similarly addressed using the
compilation of new and published data.
The purpose of this work is to address questions concerning the use of n-alkane chain lengths as a proxy for
plant types, and the answers to these questions are fundamental to paleoecological interpretations of sedimentary
n-alkane distributions.
2. METHODS
2.1. Sample species and collection
Leaf samples were collected from the grounds and tropical greenhouse of the Chicago Botanic Garden (CBG) in
Glencoe, IL, and from the tropical greenhouse at the Lincoln Conservatory (LC), in Lincoln Park, Chicago (Table 1).
Chicago Botanic Garden and Lincoln Park greenhouses had
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Table 1
Species collected for this study from the Lincoln Conservatory
(LC), Chicago, IL and from the Chicago Botanic Garden (CBG),
Glencoe, IL. Species marked in bold were collected from greenhouse plants; all other plants were grown outdoors.
Species
Family
Location
Pteridophytes
Cyathea cooperi
Matteuccia pensylvanica
Osmunda regalis
Cyatheaceae
Polypodiaceae
Osmundaceae
CBG
CBG
CBG
Gymnosperms
Cycas circinalis
Ginkgo biloba
Larix decidua
Metasequoia glyptostroboides
Picea abies
Pinus sylvestris
Taxodium distichum
Taxus cuspidata
Thuja occidentalis
Cycadaceae
Ginkgoaceae
Pinaceae
Cupressaceae
Pinaceae
Pinaceae
Cupressaceae
Taxaceae
Cupressaceae
CBG
CBG
CBG
CBG
CBG
CBG
CBG
CBG
CBG
Angiosperms
Alnus glutinosa
Annona muricata
Artocarpus altilis
Asimina triloba
Carpinus caroliniana
Celtis occidentalis
Fagus sylvatica
Gleditsia triacanthos
Koelreuteria paniculata
Lindera benzoin
Platanus orientalis
Populus deltoides
Pterocarya stenoptera
Rhus typhina
Salix alba
Tamarindus indica
Tilia cordata
Washingtonia robusta
Betulaceae
Annonaceae
Moraceae
Annonaceae
Betulaceae
Ulmaceae
Fagaceae
Fabaceae
Sapindaceae
Lauraceae
Platanaceae
Salicaceae
Juglandaceae
Anacardiaceae
Salicaceae
Fabaceae
Tiliaceae
Arecaceae
CBG
LC
LC
CBG
CBG
CBG
CBG
CBG
CBG
CBG
CBG
CBG
CBG
CBG
CBG
CBG
CBG
CBG
similar climates, with windows open to the outside for air
circulation and daytime temperatures 27–32 °C in summer. One to several leaves (depending on leaf size) were collected from at least one individual per species, and five
individuals each for the species Alnus glutinosa, Gleditsia triacanthos, Pinus sylvestris, and Taxodium distichum. To ensure equal and full light exposure, only isolated trees that
stood free from surrounding trees were selected when possible. ‘Sun’ leaves were gathered from the outer edge of the
canopy, on the southern side of each plant, and typically
at the maximum extent of the pole pruners (3 m height).
‘Shade’ leaves were taken from the bottom, northern side,
and as near to the trunk as possible. Leaf samples were collected in both the summer (July 2007) and fall (October–
November 2007). Fall samples were collected as close to
the time of abscission from the tree as possible, although
for a subset of species, multiple leaf samples were collected—either at two different dates in the fall or of two or
more colors of foliage representing differing levels of leaf decay on the tree (green, yellow, or brown). Shade leaves were
not collected for all outdoor species in fall because with
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attenuating canopy cover, shadiness of samples could not be
verified. Each leaf sample was stored in a paper bag and allowed to dry completely before further preparation.
2.2. n-Alkane identification and quantification
Leaves from coniferous species were separated and
stored in clean glass jars by age cohort. Evergreen and
deciduous leaves of the same age were compared to one another, to avoid effects of age variability (Brooks et al.,
1997). Dried leaves were homogenized by grinding in liquid
N2 with mortar and pestle. Lipids were extracted from 0.2–
0.4 g of leaf sample in 20–30 ml dichloromethane:methanol
(9:1 v/v) using a Microwave Accelerated Extraction System
(MarsX) with a ramp to 100 °C over 5 min, a hold time at
100 °C for 15 min, and cooling time of 30 min. Total lipid
extracts were concentrated under a stream of dry N2.
Non-polar lipids, including n-alkanes, were separated from
the polar lipids via short column silica gel chromatography
(Eglinton and Hamilton, 1967), using 1 g of activated silica gel in a Pasteur pipette plugged with glass wool and
4 mL hexanes. Once isolated, non-polar lipids were again
concentrated by evaporation with dry N2 and analyzed by
gas chromatography/mass spectrometery (GC–MS)
(Medeiros and Simoneit, 2007). Samples were passed
through the gas chromatograph (Thermo Scientific Trace
GC Ultra, with 15 m, 0.25 mm ID Thermo TR-5ms SQC
column) for separation and then to a quadrapole mass spectrometer for identification (Thermo Scientific DSQII). Samples were simultaneously injected into a separate column
(with the same specifications) in the same instrument and
analyzed by a flame ionization detector (FID) for assessment of relative abundance. The GC oven temperature
method is as follows: initial temperature at 100 °C, hold
for 2 min, then ramp at 11 °C/min to 320 °C, and hold at
320 °C for 5 min. All samples were compared to a standard
homologous series of n-alkanes from C21 to C40, inclusive
(Fluka, Sigma–Aldrich).
2.3. Literature analysis
Data for comparisons of n-alkane distributions among
plant groups were collected from the published literature,
which reported either tables or graphs of n-alkane
amounts—either relative (% of total) or absolute (mass
unit) amounts, both for odd and even chain lengths or solely odd chain lengths. Publications reporting only derived
parameters such as n-alkane ratios, ACL, or CPI, without
reporting chain-length abundances or amounts, were not
included in the analysis. The total number of observations
compiled was 2093. A supplementary spreadsheet is provided with all literature data and their source references
as well as all new data from the Chicago Botanic Garden
and Lincoln Conservatory. The spreadsheet includes only
the chain lengths that were reported in the source publications, with unreported chain lengths left blank; in order to
assimilate diverse data, subsequent analyses assume that
unreported chain lengths reflect an absence of that chain
length (i.e. calculations assume 0% C35 where the source
reported abundances for C27–C33 only), although this
may not be true in all cases. (Note that data from outdoor
plants at the Chicago Botanic Garden included in the literature compilation are for summer, sun leaf samples
only.) All statistical analyses were performed with Stata
IC 11.1.
3. RESULTS
3.1. Average chain length
Using both the new data from the Chicago Botanic Garden and Lincoln Conservatory as well as the collected literature data, average chain length was calculated twice: first
using odd chain lengths only (ACLodd, n = 1964), and then
again using both odd and even chain lengths (ACLall,
n = 1736). The two calculated ACL values were then regressed across all data and found to be strongly and significantly
correlated
(linear
regression,
r2 = 0.937,
p < 0.00005, Fig. A-1). In a Student’s t-test, the two groups
are significantly different (p < 0.00005), but the difference is
small (0.166). And when the cycad family Zamiaceae
(Osborne et al., 1989, 1993) is excluded, the correlation
becomes slightly stronger (linear regression, n = 1654,
r2 = 0.939, p < 0.00005) and the difference between the
means smaller (0.159). Because 15 among 86 of the literature sources published distribution values for odd chain
lengths only, ACLodd was used in all subsequent analyses
and is referred to only as ACL in subsequent text.
3.2. n-Alkane distributions within trees
There was no significant difference in ACL values between sun and shade leaves of outdoor plants from the Chicago Botanic Garden, controlling for collection date, in
either angiosperms or gymnosperms in a paired Student’s
t-test (two-tailed, p = 0.2836). In linear regression analysis,
sun and shade ACL values were strongly correlated in both
angiosperms (r2 = 0.914, p < 0.00005) and gymnosperms
(both evergreen and deciduous, r2 = 0.742, p < 0.00005,
Fig. 1A). For one species of gymnosperm, T. distichum,
sun leaves had higher ACL values than shade leaves, but
with a sample size of only 3, the results were not statistically
significant; other deciduous gymnosperms do not show the
same pattern as T. distichum. Similarly, there was no significant difference in ACL values between summer and fall
leaves, controlling for canopy position, in both angiosperms and gymnosperms in paired Student’s t-test (twotailed, p = 0.6923). Summer and fall ACL values were
strongly correlated in linear regression in angiosperms
(r2 = 0.976, p < 0.00005) and gymnosperms (r2 = 0.817,
p < 0.00005, Fig. 1B). In Thuja occidentalis, even as sun
leaves go from green to yellow to brown in the fall, shortly
preceding abscission, ACL values remained constant
(ACL = 34.53, 34.54, and 34.42 for green, yellow, and
brown leaf samples, respectively).
3.3. n-Alkanes across plant functional types
Plants were assigned categories based on phylogenetic domain and growth habit: mosses, ferns, woody gymnosperms
R.T. Bush, F.A. McInerney / Geochimica et Cosmochimica Acta 117 (2013) 161–179
165
Fig. 1. (A) Average chain length (ACL) of sun leaves versus shade leaves collected on the same day within the canopy of angiosperm trees
(blue diamonds) and gymnosperm trees (filled red circles are evergreen, open circles deciduous). (B) ACL of summer leaves versus fall leaves
collected at the same tree canopy location, as well as summer and fall ACL values for two outdoor ferns (green crosses). Dashed lines
represent 1:1 lines. All data from the Chicago Botanic Garden. (For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article.)
(conifers, cycads, and ginkgo), woody angiosperms, forbs
(herbaceous, non-woody dicots), graminoids (grasses, sedges,
and rushes), aquatic plants, and succulents. Fig. 2 shows the
distributions of ACL, CPI, and selected n-alkane ratios across
these groups. (Note that Fig. 2D–H report moss values for
Sphagnum only, excluding other moss species, as discussed below.) Fig. 3 shows ACL distributions for woody angiosperms
and gymnosperms from temperate latitudes (40°–60°) separated into evergreen and deciduous taxa. The evergreen/deciduous comparison is limited to temperate latitudes where leaf
deciduousness can be defined based on whether leaves
over-winter on the plant or are dropped in autumn, as deciduousness in this region is largely based on temperature and
light-based winter seasonality. For tropical and sub-tropical
plants, assessing a binary deciduous/evergreen trait is more
difficult because deciduousness can be driven by other factors
(e.g. drought) and leaf life span exhibits more of a continuum.
Temperate gymnosperms show a similar distribution of ACL
values for deciduous and evergreen taxa, despite the difference
in sample size between the two groups (Fig. 3A). By contrast,
in temperate woody angiosperms, evergreen species have higher ACL values on average than do deciduous species, although
both groups cover a similar range of ACL values (Fig. 3B).
However, almost all of the evergreen angiosperm species (97
of 101) were drawn from a single plant family, Ericaceae,
and thus this group may suffer from sampling bias. Overall,
the distribution of ACL values is similar across all groups
(Fig. 2A), except mosses, which have lower values. It should
be noted that the relatively low ACL values in mosses are
found predominantly in Sphagnum species, and not other
moss genera (Nichols et al., 2006). The group with the smallest
range of values, aquatic plants, is also the group with the
smallest number of data; all other groups demonstrate a large
range of ACL values.
Similarly to ACL, there is a large range of CPI values
across plant groups (Fig. 2B). Of 1722 total observations,
the highest CPI value is 99 (woody angiosperms, Velloziaceae), while the lowest CPI value is 0.039 (graminoid, Poaceae). 96.0% of all CPI values are greater than or equal to 1;
81.2% greater than or equal to 2; and 60.7% greater than or
equal to 5. Median CPI for all observations is 7.06; mean
CPI is 10.69. Lastly, the large number of outlier CPI values
for some plant groups demonstrates the large range in CPI
values and the asymmetrical group skew towards relatively
lower values for all groups and across plant families. Dominance, or the relative percent abundance of the most dominant n-alkane in each distribution, also has a large range,
from 100% (woody gymnosperms, woody angiosperms,
succulents, multiple families) to 15.7% (succulents, Cactaceae) at the lowest (Fig. 2C).
Measurements of absolute n-alkane amounts are recorded in the literature less frequently than relative
amounts. Most sources report measurements as lg
n-alkanes/g dry leaf material. A few sources report mg
n-alkanes/g wax, but this is more difficult to measure and
less meaningful for applications where comparisons between
n-alkane amounts and biomass production are important.
Angiosperms have on average 506 lg n-alkanes/g dry leaf
(n = 282, std. dev. = 497). In contrast, gymnosperms have
an average of 46 lg n-alkanes/g dry leaf material (n = 120,
std. dev. = 335, Fig. 4); excluding one outlier (Podocarpus
latifolius) with reported 3607 lg n-alkanes/g dry leaf (Ficken
et al., 2000), the average for gymnosperms drops to 16 lg nalkanes/g dry leaf (n = 119, std. dev. = 69). n-Alkane quantities for only three Sphagnum moss species, reported as lg
n-alkanes/g dry plant material, were included in this study
(Pancost et al., 2002), and their values (72, 182, and 192)
are more similar to the range of angiosperm values than to
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Fig. 2. Box plots divided by plant type for (A) average chain length (ACL), (B) carbon preference index (CPI), (C) relative percentage of the
most dominant chain length of each n-alkane distribution (Dominance), (D) C31/(C29 + C31), (E) C31/(C27 + C31), (F) C23/(C23 + C29), (G)
C33/(C29 + C33), (H) C35/(C29 + C35). (A–C) Show results for all moss data; (D–H) Report data for Sphagnum mosses only. Sample size of
each type in parentheses. Each box represents range of middle 50% of group values, where center line is group mean. Whiskers are outside
25% each, and points are outliers.
R.T. Bush, F.A. McInerney / Geochimica et Cosmochimica Acta 117 (2013) 161–179
Fig. 3. Box plots of average chain length (ACL) for temperate
(40°–60° latitude) woody plants: (A) Temperate deciduous and
evergreen woody gymnosperms, (B) Temperate deciduous and
evergreen woody angiosperms. Note that 97 of 101 evergreen
woody angiosperms are from the Ericaceae family. Sample size of
each group in parentheses.
gymnosperms. Fig. 4 also demonstrates that gymnosperms
have a significantly lower average CPI value than angiosperms: 5.20 (n = 296, std. dev. = 6.33) and 11.76
(n = 1380, std. dev. = 12.25), respectively (Student’s t-test,
p < 0.00005).
3.4. n-Alkane chain lengths and ratios as plant proxies
In addition to plotting ratios by plant group (Fig. 2D–
H), two other approaches were employed to examine the
applicability of previously posited generalities that C31 predominates in grasses and C27 and C29 predominate in woody plants, i.e. trees and shrubs (Meyers and Ishiwatari,
1993; Meyers, 2003), and similarly that C23 and C25 predominate in Sphagnum mosses (Corrigan et al., 1973; Nott
167
et al., 2000). Comparisons were made first between woody
plants and graminoids (a group which includes grasses as
well as grass-like sedges and rushes: Poaceae, Cyperaceae,
and Juncaceae families, respectively) and then secondly between woody plants and Sphagnum mosses. The interpretation of n-alkane chain-length ratios in terms of plant
community rests on a foundational assumption of equivalent n-alkane production by different plant groups. Within
the angiosperms, using observations with published n-alkane masses, we found that woody angiosperms (n = 85)
and graminoids (n = 138) produce statistically equivalent
amounts of n-alkanes in their leaf tissues (Student’s twotailed t-test p = 0.0141; Fig. A-2). n-Alkane production appears to be much higher in angiosperms than gymnosperms
(Fig. 4), and thus we included only woody angiosperms in
the comparisons, but results for conifers (woody gymnosperms) were similar. First, we plotted groups on ternary
diagrams, using either C27, C29, and C31 or C23, C27, and
C29 as vertices (Fig. 5). Second, we performed discriminant
analyses for each set of plant groups, using the percentage
of two or more chain lengths as the determinant variables
(Tables 2 and 3, A-1). Average group chain-length profiles
are also presented (Fig. 6).
3.4.1. Woody plants and graminoids: C27, C29, and C31
In order to approximate a geographically realistic
assessment of plant groups, the data were divided into
broad geographic distributions. One such geographic division includes all temperate zone plants, 40–60° latitude,
from both hemispheres. Because of the large number of
measurements made on temperate species in the Ericaceae
(n = 128, of 188 temperate woody angiosperms), largely
due to the work of Salasoo (1981, 1983, 1987a, 1987b,
1987c), this family was separated from all other woody
angiosperms. Fig. 5A shows the ternary diagram of C27,
C29, and C31 abundances for temperate woody angiosperms, excluding Ericaceae, alongside C3 and C4 graminoids and the high degree of overlap between all three
groups. Sub-Saharan Africa has the most n-alkane measurements of all tropical and sub-tropical areas, and so
Fig. 4. Log–log plot of CPI values by n-alkane amounts for both angiosperms (filled diamonds, n = 196) and gymnosperms (open diamonds,
n = 109).
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Fig. 5. Ternary diagrams of n-alkane chain-length abundances by plant group. All temperate woody angiosperms exclude the family
Ericaceae, except in 5G, where they are plotted separately. (A) Temperate woody angiosperms and C3 and C4 graminoids by C27, C29, and
C31. (B) African woody angiosperms and C4 graminoids by C27, C29, and C31. (C) Temperate woody angiosperms and C3 and C4 graminoids
by C27, C29, and C33. (D) African woody angiosperms and C4 graminoids by C27, C29, and C33. (E) Temperate woody angiosperms and C3 and
C4 graminoids by C27, C29, and C35. (F) African woody angiosperms and C4 graminoids by C27, C29, and C35. (G) Temperate woody
angiosperms, Ericaceae, and Sphagnum by C23, C27, and C29 n-alkanes.
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Table 2
Results of discriminant analyses for graminoids and woody angiosperms using C29
and C31. True plants are actual group assignments, while determined (Det.) plants
are statistically assigned. The integer indicates the number of observations for each
category, and the number in parentheses indicates the percentage for each category
from the row total. The percentage in parentheses for the grand total indicates the
percentage of correctly assigned plants for both groups.
Det. Graminoids
(A) All plants, classified
True Graminoids
True Woody Ang.
Total
Total
298
591
889 (58%)
(B) Temperate plants (40°–60° latitude)
True Graminoids
93 (51%)
True Woody Ang.
100 (53%)
Total
193 (52%)
91 (49%)
88 (47%)
179 (48%)
184
188
372 (49%)
(C) Sub-Saharan African
True Graminoids
True Woody Ang.
Total
4 (6%)
54 (62%)
58 (38%)
64
87
151 (75%)
plants
60 (94%)
33 (38%)
93 (62%)
the same comparisons between plant groups were also made
using African data only (Fig. 5B). Fig. 5C–F show additional ternary diagrams constructed for the same temperate
and African plant groups using chain lengths C27, C29, and
C33 as well as C27, C29, and C35. These plots (Fig. 5C–F)
show less overlap between woody angiosperms and graminoids than the C31-based plots (Fig. 5A and B) based
mostly on the absence of the longer C33 and C35 chain
lengths in many woody angiosperms. Nonetheless, there is
still a wide distribution of woody angiosperm values.
To test the predictive power of the C29 and C31 chain
lengths among plant groups, discriminant analyses were
performed (Table 2). Relative abundances of chain lengths
C29 and C31 were used as discriminant variables to classify
plants as woody angiosperms and graminoids (both C3 and
C4). To do this, plants were treated as unknowns and statistically assigned to either woody angiosperms or to graminoids based on the proximity of their abundances of C29
and C31 to the group means. Table 2A shows the results
of this analysis for all plants regardless of geographic locaTable 3
Results of discriminant analyses for Sphagnum mosses and
temperate woody angiosperms. True plants are actual group
assignments, while determined (Det.) plants are statistically
assigned. The integer indicates the number of observations for
each category, and the number in parentheses indicates the
percentage for each category from the row total. The percentage
in parentheses for the grand total indicates the percentage of
correctly assigned plants for both groups.
All plants, classified using C23 and C29 as discriminants
Det. Sphagnum Det. Woody Ang. Total
True Sphagnum
39 (83%)
True Woody Ang. 6 (3%)
Total
45 (19%)
Det. Woody Ang.
using C29 and C31 as discriminants
200 (67%)
98 (33%)
273 (46%)
318 (54%)
473 (53%)
416 (47%)
8 (17%)
182 (97%)
190 (81%)
47
188
235 (94%)
tion: out of 298 total “true” graminoids, 200 (67%) were
correctly determined to be graminoids while 98 (33%) were
statistically assigned to “woody angiosperms.” Similarly, of
591 total woody angiosperms, 318 (54%) were correctly
identified and 273 (46%) were incorrectly identified as
“graminoids” by the discriminant analysis. The same analysis was done for temperate plants from 40° to 60° latitude
(Table 2B) and also for plants from sub-Saharan Africa
(Table 2C). The discriminant chain lengths are capable of
determining sub-Saharan African graminoids with 94%
accuracy but also incorrectly identify 38% of woody angiosperms as “graminoids”. In the real world, the true identities are not known, and therefore the success of the
approach must be measured by the proportion of both
groups that are correctly identified. Thus, when both woody angiosperm and graminoid groups are considered together, the discriminant analyses are never better than
75% accurate for any of the geographic areas. The amount
of variability in chain-length abundances is always higher
for woody angiosperms than for graminoids (Fig. 2C),
and therefore the discriminant analysis is less accurate for
woody angiosperms in every case (Table 2). Discriminant
analyses utilizing C27 fared no better than those using C29
and C31 alone with the total correct assignments ranging
from 53% (temperate) to 76% (sub-Saharan Africa)
(Table A-1). Fig. 6A shows the average profiles for temperate woody angiosperms (with Ericaceae excluded) as well as
temperate C3 and C4 graminoids, and Fig. 6B shows the
average profiles for African woody angiosperms and C4
graminoids. C31/(C29 + C31) and C31/(C27 + C31) ratio values for all plants, temperate plants, and plants from Africa
as well as North America are provided in Tables A-2 and
A-3.
To explore whether the apparent separation between
African woody angiosperms and African C4 graminoids
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Fig. 6. Average n-alkane distributions. (A) Temperate woody angiosperms (Ericaceae excluded), temperate C3 graminoids, and temperate C4
graminoids. (B) African woody angiosperms and C4 graminoids. (C) Temperate Sphagnum mosses, woody angiosperms (Ericaceae excluded),
and Ericaceae species. Numbers in parentheses are sample sizes. Error bars are ±1 standard deviation.
(Figs. 5B, D, F and 6) was related to growing environment
in Africa, woody angiosperms published in Vogts et al.
(2009) were separated into either rainforest or savanna biomes (following the categories listed by the authors) and
plotted on ternary diagrams alongside savanna forbs and
C4 graminoids from eastern or southern Africa (Fig. A-3).
Fig. A-3A shows that for C31, there is still a large amount
of overlap among all groups, including both rainforest
and savanna plants, but for chain lengths C33 and C35
(Fig. A-3B and C, respectively) rainforest woody angiosperms as a group have lower amounts of these longer chain
lengths than any of the savanna plants. Discriminant analysis was also performed for the rainforest versus savanna
woody angiosperms only, using C29 and C31. The analysis
correctly identified 22 of 24 rainforest woody plants and
37 of 45 savanna woody plants, for a total accuracy of
86% (Table A-4). Including C33 and C35 in the discriminant
analysis also produced 86% total accuracy.
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3.4.2. Sphagnum mosses and woody plants: C23, C27, and C29
Fig. 2F demonstrates the differentiation between Sphagnum and other plant groups based on a C23/(C23 + C29) ratio. The ternary diagram in Fig. 5G also visually
demonstrates the ability of C23, C27, and C29 chain-length
abundances to separate Sphagnum mosses from woody
angiosperms. Because the Ericaceae plant family is an
important component of the bog wetland ecosystems where
Sphagnum species predominate, we have plotted woody
angiosperms as two groups, one excluding Ericaceae, similar to the comparison with graminoids, and the other the
Ericaceae family itself (Fig. 5G). The two groups of woody
angiosperms overlap one another, and the Sphagnum
mosses are distinguished from woody angiosperms almost
entirely by their high proportion of C23. Table 3 shows
the results of a discriminant analysis using relative abundances of C23 and C29 to differentiate temperate woody
angiosperms from Sphagnum. Because Ericaceae and other
woody angiosperms were so similar in their distributions of
C23, C27, and C29, the two groups were not separated from
one another in the discriminant analysis. Results of discriminant analyses using all three chain-length abundances (C23,
C27, and C29) and of only C23 and C27 are not shown because they are exactly the same as for C23 and C29. The
chain lengths can be used to separate the two plant groups
from one another with overall 94% accuracy (Table 3).
n-Alkane ratios were also calculated for woody plants and
Sphagnum mosses, with C23 and C25 used to represent
Sphagnum and C27 and C29 to represent woody plants. Ratios
using C23 (Table A-4) provided a better proxy for separating
mosses from woody plants than did ratios using C25 (Tables
A-5 and A-6). Sphagnum mosses had C23/(C23 + C27) and
C23/(C23 + C29) ratios of 0.773 and 0.728, respectively, while
those for woody angiosperms were 0.089 and 0.071. Data for
Sphagnum came only from temperate regions and so were
compared only with temperate woody plants. Fig. 6C shows
the average profiles for Sphagnum mosses, woody angiosperms excluding Ericaceae, and Ericaceae.
4. DISCUSSION
4.1. n-Alkane distributions within trees
n-Alkane distributions for temperate trees from the
Chicago Botanic Garden do not appear to significantly vary
with either canopy position or with sampling date, for both
angiosperms and gymnosperms (Fig. 1). The stability of
n-alkane distributions over a growing season shown here
is in apparent contrast to the findings of other researchers,
who describe changes in n-alkane amounts as leaves mature
across a growing season (Piasentier et al., 2000). However,
Piasentier et al. (2000) show that the greatest changes in relative n-alkane amounts occur during the growth and expansion of the young leaf, and they note that the n-alkane
patterns of the mature and senescing leaves (which correspond to the leaves sampled in this study) remain markedly
similar, in agreement with the findings presented here. Similarly, although Sachse et al. (2009) report variability in leaf
n-alkane amounts across multiple dates in a single growing
season, suggesting ablation and turnover of surface waxes,
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the relative chain-length proportions remain fairly steady,
and Tipple et al. (2013) show that both ACL and isotopic
values reflect the time of leaf formation and remain steady
through the rest of the growing season. Sampling of immature leaves may also explain the variation observed by
Stránský et al. (1967). Lastly, Avato et al. (1984) also found
that once leaves mature, n-alkane quantities remain relatively constant. The results presented here suggest that for
fully developed leaves, there is no significant difference in
relative n-alkane distributions either around a tree or within
a growing season. It should be noted that Maffei et al.
(1993) found that n-alkanes in the evergreen Rosmarinus
officinalis correlated with temperature when sampled across
summer and winter, suggesting that for evergreen leaves nalkane composition shifts to longer chain lengths in response to lower winter temperatures in overwintering
leaves. However, for the outdoor trees measured in this
study across a summer growing season, n-alkane distribution does not significantly change from at least the point
of leaf maturation to leaf abscission in the autumn
(Fig. 1B). Although these data come from a limited number
of species from a non-natural (although outdoor) environment, the strong correlation between sun and shade as well
as summer and fall n-alkane distributions supports
comparisons of the distributions of modern plants with
fossil n-alkanes of unknown canopy position and season
of abscission, and in this sense makes them well suited for
use as paleoecological proxies.
4.2. n-Alkane distributions across plant functional types
Average chain length is a broad-brush parameter that
does little to differentiate large-scale plant groups for any
group except Sphagnum mosses (Fig. 2A). Some species exhibit a high degree of genetic control over their n-alkane
distributions, such that different plants growing in different
locations can have relatively similar patterns of n-alkane
distribution, e.g. the almost exclusive production of C27
by Fagus sylvatica leaves (Gülz et al., 1989; Lockheart
et al., 1997; Tu et al., 2007; Sachse et al., 2009; this study).
However, other species have been shown to have variable nalkane distributions across different environments, limiting
the usefulness of n-alkane profiles in chemotaxonomy
(Dodd and Poveda, 2003; Vogts et al., 2009). Because the
leaf wax is a plant’s first barrier to the external environment, it is reasonable to hypothesize that the composition
of that wax, including the n-alkanes, would be controlled
to some degree by environmental adaptation and therefore
plastic in response to external conditions (Shepherd and
Griffiths, 2006). This plasticity would contribute to the observed variation and lack of differentiation among plant
groups.
Regarding CPI, the vast majority (but not all) of measured modern plants have values greater than 1 (Fig. 2B),
meaning that odd chain lengths are more abundant than
even chain lengths. Perhaps not surprisingly, a reasonable
cut-off for determining a relatively unmodified terrestrial
plant source for sediment n-alkanes appears to be a CPI
of 1 or greater (Douglas and Eglinton, 1966), and a more
rigorous threshold might be 2 or greater, as the majority
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of modern plants (81% of 1723 measurements) fall above 2.
However, in light of the tremendous diversity of CPI values
in modern plants, it seems prudent to use CPI as an indicator of terrestrial plant origin in a more binary (either the nalkanes are faithful to the original plant source or they are
not) sense rather than interpreting along a spectrum of values. It seems especially difficult to apply sedimentary CPI
values in any quantitative way towards determining the degree of n-alkane degradation as in Buggle et al. (2010) and
Zech et al. (2013), although they may still qualitatively indicate thermal maturity in oils and petroleum source rocks
(Bray and Evans, 1961). The use of CPI in combination
with n-alkane ratios to estimate the n-alkane contributions
of graminoids and trees (discussed in Section 4.3), e.g. following Zech et al. (2009), should be viewed with particular
caution or even be disregarded due to the extreme variability and overlap in these parameters in modern plants
(Fig. 2).
Lastly, both deciduous and evergreen gymnosperms
produce, on average, much smaller quantities of n-alkanes
per unit leaf mass than do angiosperms. It was observed
previously that in some individuals of the gymnosperms
Pinaceae and Cupressaceae, n-alkanes constituted only
1% of leaf wax (Herbin and Robins, 1968; Chikaraishi
et al., 2004), while leaf waxes of some angiosperm species
were over 90% n-alkanes (Herbin and Robins, 1969; Dove,
1992). Diefendorf et al. (2011) also found in their measurements of leaf n-alkanes from Pennsylvania, Wyoming, and
Panama that angiosperms consistently produced more n-alkanes than gymnosperms. Here we expand on these findings by demonstrating this difference across 274 species
and a range of plant families and locations (from Africa,
Europe, Australia, and South America) (Fig. 4). This finding in modern plants fits with the observation by Lockheart
et al. (2000) and Otto et al. (2005) that gymnosperm fossils
had smaller amounts of n-alkanes than did angiosperm fossils from the same localities. For paleoecological applications, this difference in n-alkane production means that
even for an ecosystem with a minority of angiosperms
and a majority of gymnosperms, angiosperms would likely
be the dominant source of in situ sediment n-alkanes in the
local soil organic matter. For example, if we consider an
ecosystem that is 90% gymnosperm and only 10% angiosperm and we use the average amounts of n-alkanes reported for each group here (510 lg n-alkane/g dry leaf in
angiosperms and 16 lg in gymnosperms, excluding the outlying Podocarpus measurement, Fig. 4), the mixed sedimentary n-alkanes would be roughly 22% gymnosperm and 78%
angiosperm in origin. If we consider an ecosystem that is an
even mix of angiosperms and gymnosperms, angiosperms
would produce 97% of the total n-alkane signal. The difference in n-alkane production between angiosperms and gymnosperms observed here for total n-alkanes is of a similar
magnitude to that reported by Diefendorf et al. (2011).
Sphagnum mosses appear to produce n-alkanes in their tissues in greater quantities than most gymnosperms, but still
less than those of angiosperm leaves (Pancost et al., 2002).
Inferences of plant cover from distributions and stable isotope ratios of n-alkanes often implicitly assume that plants
contribute n-alkanes in direct proportion to their biomass
(e.g. Nichols et al., 2006; Smith et al., 2007; Diefendorf
et al., 2010). However, the demonstrated difference in n-alkane production by angiosperms and gymnosperms requires careful interpretation of n-alkanes from
sedimentary archives (Fig. 4; Diefendorf et al., 2011).
Although n-alkane production in Sphagnum and angiosperms appears to be more similar, additional data on absolute amounts of n-alkane chain lengths in modern plants
(lg n-alkane/g dry leaf) in woody, herbaceous, and Sphagnum-dominated ecosystems are needed.
4.3. n-Alkane chain lengths as grass and woody plant proxies
The initial studies that are often cited in support of the
C31/C29 grass/woody plant comparison make no mention
of grasses separate from other terrestrial plants, but rather
focus on terrestrial plants as a group in comparison with
aquatic plants or peat (Cranwell, 1973, 1984; Cranwell
et al., 1987). The compilation of published data and new
analyses presented here demonstrate that both graminoids
and woody angiosperms produce C29 and C31 in abundance
relative to other n-alkane chain lengths (Fig. 6A), and the ratios of these two compounds are highly variable and overlapping between these groups (Fig. 2D). Therefore, C29
and C31 cannot serve as generalized proxies for separating
grasses and woody plants. The ternary diagrams demonstrate visually the large amount of variation within the
groups, especially in woody angiosperms, as well as the
overlap of graminoids and woody angiosperms (Fig. 5A
and B). Discriminant analyses using C29 and C31, with or
without C27, are at worst little better than random guessing
at correctly identifying graminoids and woody angiosperms
by their chain-length abundances (49% for temperate plants,
58% correct globally) and are at best 75% accurate for tropical regions (Table 2), mostly because of the large amount of
variation in woody angiosperm n-alkane abundances.
A possible exception is the regional grouping of SubSaharan graminoids, which appear to be distinguishable
based on C29 and C31 relative abundances (Table 2C,
Fig. 5B). However, this apparent association between chain
lengths and plant groups may instead reflect the influence of
environment concealed by inadvertent sampling bias. Most
of the sub-Saharan graminoids are C4 plants that are
adapted to hot, dry environments (Rommerskirchen
et al., 2006). By comparison, many of the woody angiosperm observations are drawn from a greater diversity of
environments (hot and dry as well as cooler and wetter,
e.g. Vogts et al., 2009). It seems plausible that the greater
predominance of longer chain lengths (C31, C33, and C35)
in graminoids than in woody angiosperms in Africa
(Figs. 5B, D, F and 6) could be a more direct reflection
of local climatic influences such as aridity than of the plant
groups per se (Rommerskirchen et al., 2006). To evaluate
this possibility, we examined the n-alkanes from woody
angiosperms sampled from both African rainforest and savanna biomes in the study by Vogts et al. (2009). Savanna
woody angiosperms have relative abundances of the longer
chain lengths (C31, C33, and C35 relative to C29 and C27)
similar to the C4 graminoids and forbs (Fig. A-3). In
contrast, rainforest woody plants group separately from
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the savanna woody angiosperms, graminoids, and forbs,
suggesting that climate could be the controlling factor
rather than woody versus herbaceous life habit (Fig. A-3).
Furthermore, when considering only woody angiosperms
(i.e. controlling for plant type), a discriminant analysis
using C29 and C31 is able to correctly identify plants from
the two separate biomes (rainforest and savanna) with
86% accuracy (Table A-4). Interestingly, C35 relative to
C29 and C27 appears to largely separate graminoids and forbs from savanna woody angiosperms (Fig. A-3C), but further investigation is required to determine whether this
pattern reflects plant type or differences in microclimates
within the savanna (e.g. woody species sampled from riparian environments). African graminoids may occupy a more
restricted range of chain-length distributions (Fig. 5), similar to savanna forbs and woody plants and in contrast to
rainforest woody plants (Fig. A-3), because their environmental range is more restricted (i.e. to warmer, more arid
environments) than woody plants, which can be found in
some capacity in almost any environment.
Other studies have also suggested correlations between
n-alkane chain lengths and environment (Kawamura
et al., 2003; Rommerskirchen et al., 2003; Sachse et al.,
2006). For example, Castañeda et al. (2009) examined several molecular proxies from sediment cores in Lake Malawi
and showed that n-alkane chain length was correlated with
temperature reconstructions and did not correlate with
reconstructions of grass and woody plant dominance based
on lignin phenolic compounds. Vogts et al. (2012) found
that ACL values from marine sediments off the coast of
western Africa correlated with continental aridity and that
ACL-based reconstructions of C4 plant abundances
matched d13C-based reconstructions. Studies of n-alkane
distributions in living plants (Dodd and Poveda, 2003;
Sachse et al., 2006), as well as studies of aeolian dust and
marine sediments from the Atlantic and Pacific (Rinna
et al., 1999), have found correlations between n-alkane
chain lengths and latitude or altitude, suggesting instead
that there may be a relationship between longer chain
lengths and warmer, drier, or possibly more irradiated
environments.
4.4. n-Alkane chain lengths as a Sphagnum proxy
In contrast to the comparison between graminoids and
woody plants, ratios of the shorter C23 n-alkane to either
C27 or C29 appear to readily distinguish modern Sphagnum
mosses from woody angiosperms. Indeed, it seems reasonable to expand this conclusion to encompass all non-Sphagnum terrestrial plants, including grasses, forbs, and
gymnosperms, because of the lack of distinction among all
of the other plant groups from one another (Fig. 2F). Sphagnum and woody angiosperms are largely distinguished from
one another by the relative abundance of C23 (Fig. 5G):
Sphagnum samples in the ternary diagrams have abundances
greater than 25% C23, compared to C27 and C29, while most
woody angiosperms (including the Ericaceae) have less than
10% C23. The results of the discriminant analyses utilizing
C23 and C29 as well as C23 and C27 both showed 94% overall
accuracy in correctly assigning groups to either Sphagnum
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or woody angiosperm. Most woody angiosperms also have
more C29 than C27, which supports the use of comparisons
between C23 and C29 as a proxy for inputs of Sphagnum
moss and woody plants (Nichols et al., 2006). However, submerged and floating aquatic plants have also been demonstrated to contain a relative abundance of C23 and C25
(Ficken et al., 2000; Mügler et al., 2008), and therefore, as
in any consideration of ecological proxy indicators, the environmental context—e.g. of a Sphagnum-dominated bog or a
lacustrine system lacking in Sphagnum but with aquatic
macrophytes present—is crucial. Nonetheless, the data presented here demonstrate that for bog ecosystems, this is a
useful proxy for estimating the proportion of n-alkanes from
Sphagnum mosses or from woody plants (Nott et al., 2000;
Nichols et al., 2010).
4.5. Implications for paleoecology
From a paleoecological perspective, the consistency of nalkane chain-length patterns within mature leaves over time
and canopy position validates the comparison of n-alkanes
from modern plants with sediment or fossil n-alkanes that
derive from leaves of unknown provenance. However, the
tremendous range of CPI values in living plants makes
any application for sediment CPI values beyond a rough
qualitative assessment of source fidelity particularly problematic. Genetic controls clearly exert some degree of influence over the production of different n-alkane chain lengths,
as demonstrated by chemotaxonomic studies (Vioque et al.,
1994; Maffei et al., 2004; Medina et al., 2006; Bingham et al.,
2010) and biosynthetic studies (Jetter et al., 2006; Kunst
et al., 2006). Similarly, the relative abundances of n-alkanes
appear to be relatively tightly controlled within a single
plant, across both canopy and growing season (Fig. 1). Beyond the individual plant, it remains unclear why one plant
group per se (e.g. graminoids or woody plants growing in the
same environment) would favor production of one chain
length over another. The variation in chain-length abundances within most groups is large, even when accounting
for factors such as region and photosynthetic pathway
(Figs. 2 and 5). This makes it inadvisable to use n-alkane
chain-length abundances as chemotaxonomic indicators
for broad plant functional groups, with the exception of
Sphagnum mosses from peat bog ecosystems. Based on the
modern plant data, ratios of chain lengths C27 or longer,
such as C31/(C29 + C31), do not appear to be a robust proxy
for separating grasses from woody plants. By contrast, C23
appears to generate a generally reliable proxy for Sphagnum
mosses when compared against C27 or C29 as indicators of
input from other terrestrial plants, which is consistent with
the findings of others (Pancost et al., 2002; Nichols et al.,
2006, 2010). With further investigation into the absolute
quantities of n-alkanes produced per plant biomass and cover, it appears that these n-alkane chain lengths could potentially be a semi-quantifiable proxy for plant group cover in
Sphagnum wetlands (Nichols et al., 2010) or for Sphagnum-sourced organic matter in river and marine sediments
(Vonk et al., 2008; Vonk and Gustafsson, 2009).
Environmental factors may play a role in the plasticity
of n-alkane distributions and explain some of the variation
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in large-scale plant groups. It is possible that climate can
independently drive both plant community composition
and n-alkane chain-length distribution, and that the two
phenomena could be correlated without a direct causal relationship. Most sedimentary n-alkane archive studies report
concurrent shifts in local climate and plant communities,
e.g. the large climate shifts associated with glacial/interglacial cycles (Brincat et al., 2000; Zhang et al., 2006; Zech
et al., 2009) and with the Paleocene-Eocene Thermal Maximum (Smith et al., 2007). It is difficult to disentangle
whether the observed changes in n-alkane distributions
through time are due to the direct influence of climate,
based on the plastic response of n-alkanes to temperature
or aridity, or to its indirect influence via climate-driven
shifts in the local plant community. However, given the
range of variation within plant groups, another possibility
remains that shifts in sediment n-alkane distributions may
reflect species turnover in plant communities that is de-coupled both from plant types and from a direct climate driver
(e.g. due to succession, competition, etc.) Considering the
low amounts of n-alkanes synthesized in gymnosperm
leaves, the gymnosperm contribution to sediment n-alkanes
may only rarely be significant (e.g. where pollen or leaf fossil evidence indicates a preponderance of gymnosperms in
the local plant community). Future research would benefit
from a determination of the environmental controls on biosynthesis of n-alkanes and the extent to which climate plays
a role in determining n-alkane chain lengths in modern
plants and sediments.
canopy and growing season, and these findings validate the
comparison between modern plant n-alkanes and n-alkanes
from fossil soils and sediments derived from leaves of unknown provenance. Furthermore, consistent with other recent findings (Diefendorf et al., 2011 and references
therein), the evidence here shows that angiosperms produce
orders of magnitude more n-alkanes in their leaves than do
gymnosperms: typically 100s to 1000s of lg n-alkanes per g
dry leaf matter compared to 10s or less, respectively. We
also demonstrate that n-alkane chain lengths can serve as
useful chemotaxonomic proxies for the identification of
Sphagnum mosses from other plant groups, but are unable
to distinguish graminoids from woody plants, and that
modern plants exhibit a tremendous range of n-alkane ratio
and CPI values. Previous studies have suggested that n-alkane chain-length distributions may be influenced by environment, possibly in addition to genetic controls. Thus, it is
possible that coherent patterns of n-alkanes in sediment records are not artifactual but rather may directly result from
climate forcings, meaning that there is present potential for
n-alkane chain-length distributions to serve as a paleoclimate proxy. However, this possibility must first be directly
tested in modern plants and sediments. Examination into
the biosynthetic controls on n-alkane production and the
manifestation of n-alkane response to climatic influences
will further elucidate the ecological interpretations possible
with these valuable plant biomarkers.
5. CONCLUSIONS
We thank the research and horticultural staff and especially
Nyree Zerega and Celeste VanderMey at the Chicago Botanic Garden for their facilitation in sample collection. We are grateful to
Sarah Feakins for sharing data, and to Ellen Currano, Anna Henderson, Phil Meyers, Scott Wing, and three anonymous reviewers
for comments. Funding was provided by EPA Science to Achieve
Results (STAR) Graduate Fellowship (to R.T.B.), research grants
from the Plant Biology and Conservation graduate program at
Northwestern University and the Chicago Botanic Garden (to
R.T.B.), the Initiative for Sustainability and Energy at Northwestern (ISEN) (to R.T.B. and F.A.M.) and the Australian Research
Council FT110100793 (to F.A.M.).
n-Alkanes have been acknowledged for decades as useful
biomarkers for terrestrial plants, and dozens of studies have
examined the distributions of n-alkane chain lengths across
hundreds of different plant species. Until now, however,
most of these studies have focused on single taxonomic
groups or geographic areas, and broad paleoecological
interpretations have been based on a relatively narrow
and incomplete set of modern data. Here, we find that in
temperate trees n-alkane distributions are consistent across
ACKNOWLEDGMENTS
Table A-1
Results of discriminant analyses for graminoids and woody angiosperms using C27, C29, and C31. True plants are actual group assignments,
while determined (Det.) plants are statistically assigned. The integer indicates the number of observations for each category, and the number
in parentheses indicates the percentage for each category from the row total. The percentage in parentheses for the grand total indicates the
percentage of correctly assigned plants for both groups.
Det. Woody Ang.
Total
(A) All plants, classified using C27, C29, and C31 as discriminants
True Graminoids
196 (66%)
True Woody Ang.
287 (49%)
Total
483 (54%)
Det. Graminoids
102 (34%)
304 (51%)
406 (46%)
298
591
889 (56%)
(B) Temperate plants (40°-60° latitude)
True Graminoids
True Woody Ang.
Total
115 (63%)
104 (55%)
219 (59%)
69 (38%)
84 (45%)
153 (41%)
184
188
372 (53%)
(C) Sub-Saharan African plants
True Graminoids
True Woody Ang.
Total
55 (86%)
27 (31%)
82 (54%)
9 (14%)
60 (69%)
69 (46%)
64
87
151 (76%)
R.T. Bush, F.A. McInerney / Geochimica et Cosmochimica Acta 117 (2013) 161–179
Table A-2
Average C31/(C29 + C31) ratios for plant types, including sample
size (n) and standard deviation (SD).
C31/(C29 + C31)
n
Average
SD
All plants
Conifers
Woody Angiosperms
Graminoids
C3 Graminoids
C4 Graminoids
219
585
298
150
144
0.642
0.479
0.610
0.559
0.660
0.303
0.289
0.194
0.206
0.167
Temperate (40–55° latitude)
Conifers
Woody Angiosperms
Woody Angiosperms (excl. Ericaceae)
Ericaceae
Graminoids
C3 Graminoids
C4 Graminoids
170
188
60
128
184
125
57
0.643
0.484
0.359
0.542
0.555
0.553
0.556
0.314
0.257
0.283
0.223
0.196
0.210
0.164
87
64
1
63
0.399
0.731
0.645
0.732
0.270
0.113
–
0.114
158
83
75
46
18
27
0.418
0.291
0.558
0.601
0.664
0.551
0.292
0.310
0.191
0.177
0.151
0.179
Africa
Woody Angiosperms
Graminoids
C3 Graminoids
C4 Graminoids
North America
Woody Angiosperms
Woody Angiosperms (excl. Ericaceae)
Ericaceae
Graminoids
C3 Graminoids
C4 Graminoids
Table A-3
Average C31/(C27 + C31) ratios for plant types, including sample
size (n) and standard deviation (SD).
C31/(C27 + C31)
n
Average
SD
All plants
Conifers
Woody Angiosperms
Graminoids
C3 Graminoids
C4 Graminoids
219
581
298
150
144
0.689
0.685
0.770
0.790
0.749
0.295
0.341
0.197
0.205
0.186
Temperate (40–55° latitude)
Conifers
Woody Angiosperms
Woody Angiosperms (excl. Ericaceae)
Ericaceae
Graminoids
C3 Graminoids
C4 Graminoids
171
188
60
128
184
125
57
Africa
Woody Angiosperms
Graminoids
C3 Graminoids
C4 Graminoids
North America
Woody Angiosperms
Woody Angiosperms (excl. Ericaceae)
Ericaceae
Graminoids
C3 Graminoids
C4 Graminoids
87
64
1
63
154
79
75
46
18
27
0.684
0.693
0.516
0.776
0.757
0.794
0.680
0.579
0.792
0.826
0.791
0.646
0.498
0.803
0.748
0.896
0.641
0.310
0.319
0.342
0.272
0.217
0.208
0.211
0.338
0.134
–
0.135
0.387
0.435
0.250
0.235
0.125
0.237
175
Table A-4
Results of discriminant analyses for African rainforest versus
savanna woody angiosperms from Vogts et al. (2009). True plants
are actual group assignments, while determined (Det.) plants are
statistically assigned. The integer indicates the number of observations for each category, and the number in parentheses indicates
the percentage for each category from the row total. The
percentage in parentheses for the grand total indicates the
percentage of correctly assigned plants for both groups.
African woody angiosperms, classified using C29 and C31 as
discriminants
True Rainforest
True Savanna
Total
Det. Rainforest
Det. Savanna
Total
22 (92%)
8 (18%)
30 (43%)
2 (8%)
37 (82%)
39 (57%)
24
45
69 (86%)
Table A-5
Average C23/(C23 + C27), C23/(C23 + C29), and C23/(C23 + C31)
ratios for woody angiosperms and Sphagnum mosses, including
sample size (n) and standard deviation (SD).
C23/(C23 + C27)
Temperate (40–55° latitude)
Woody Angiosperms
Woody Angiosperms (excl.
Ericaceae)
Ericaceae
Sphagnum
C23/(C23 + C29)
Temperate (40–55° latitude)
Woody Angiosperms
Woody Angiosperms (excl.
Ericaceae)
Ericaceae
Sphagnum
C23/(C23 + C31)
Temperate (40–55° latitude)
Woody Angiosperms
Woody Angiosperms (excl.
Ericaceae)
Ericaceae
Sphagnum
n
Average
SD
188
60
0.089
0.132
0.157
0.183
128
47
0.069
0.773
0.139
0.120
188
60
0.071
0.099
0.166
0.153
128
47
0.057
0.728
0.170
0.171
185
57
0.112
0.180
0.234
0.240
128
47
0.081
0.608
0.225
0.229
Table A-6
Average C25/(C25 + C27), C25/(C25 + C29), and C25/(C25 + C31)
ratios for woody angiosperms and Sphagnum mosses, including
sample size (n) and standard deviation (SD).
C25/(C25 + C27)
Temperate (40–55° latitude)
Woody Angiosperms
Woody Angiosperms (excl.
Ericaceae)
Ericaceae
Sphagnum
C25/(C25 + C29)
Temperate (40–55° latitude)
Woody Angiosperms
n
Average
SD
188
60
0.210
0.248
0.173
0.199
128
47
0.193
0.747
0.158
0.089
188 0.139
0.203
(continued on next page)
176
R.T. Bush, F.A. McInerney / Geochimica et Cosmochimica Acta 117 (2013) 161–179
APPENDIX A.
Table A-6 (continued)
Woody Angiosperms (excl.
Ericaceae)
Ericaceae
Sphagnum
C25/(C25 + C31)
Temperate (40–55° latitude)
Woody Angiosperms
Woody Angiosperms (excl.
Ericaceae)
Ericaceae
Sphagnum
59
0.341
0.332
128
47
0.105
0.695
0.183
0.151
APPENDIX B. SUPPLEMENTARY DATA
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.gca.2013.04.016.
187
69
0.197
0.350
0.293
0.326
128
47
0.132
0.572
0.249
0.206
Fig. A-1. ACL calculated using only odd chain length n-alkanes (Odd ACL) compared with ACL calculated using both odd and even chain
lengths (Full ACL). Dashed line is 1:1 line. Solid line is linear regression line.
Fig. A-2. Log–log plot of CPI by n-alkane amounts across angiosperm habit groups for which data was available.
R.T. Bush, F.A. McInerney / Geochimica et Cosmochimica Acta 117 (2013) 161–179
177
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Associate editor: Peter Hernes