Submitted 17 April 2015
Accepted 10 June 2015
Published 2 July 2015
Corresponding author
Leyla J. Seyfullah,
leyla.seyfullah@geo.uni-
goettingen.de
Academic editor
Kenneth De Baets
Additional Information and
Declarations can be found on
page 13
DOI 10.7717/peerj.1067
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2015 Seyfullah et al.
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OPEN ACCESS
Species-level determination of closely
related araucarian resins using FTIR
spectroscopy and its implications for the
provenance of New Zealand amber
Leyla J. Seyfullah, Eva-Maria Sadowski and Alexander R. Schmidt
Department of Geobiology, University of Go¨ttingen, Go¨ttingen, Germany
ABSTRACT
Some higher plants, both angiosperms and gymnosperms, can produce resins and
some of these resins can polymerize and fossilize to form ambers. Various physical
and chemical techniques have been used to identify and profile different plant resins
and have then been applied to fossilized resins (ambers), to try to detect their parent
plant affinities and understand the process of polymerization, with varying levels of
success. Here we focus on resins produced from today’s most resinous conifer family,
the Araucariaceae, which are thought to be the parent plants of some of the Southern
Hemisphere’s fossil resin deposits. Fourier transform infrared (FTIR) spectra of the
resins of closely related Araucariaceae species were examined to test whether they
could be distinguished at genus and species level and whether the results could then
be used to infer the parent plant of a New Zealand amber. The resin FTIR spectra are
distinguishable from each other, and the three Araucaria species sampled produced
similar FTIR spectra, to which Wollemia resin is most similar. Interspecific variability
of the FTIR spectra is greatest in the three Agathis species tested. The New Zealand
amber sample is similar in key shared features with the resin samples, but it does
differ from the extant resin samples in key distinguishing features, nonetheless it is
most similar to the resin of Agathis australis in this dataset. However on comparison
with previously published FTIR spectra of similar aged amber and older (Eocene)
resinites both found in coals from New Zealand and fresh Agathis australis resin,
our amber has some features that imply a relatively immature resin, which was not
expected from an amber of the Miocene age.
Subjects Paleontology, Plant Science
Keywords Araucariaceae, Amber, New Caledonia, FTIR, New Zealand
INTRODUCTION
Resins are secondary metabolites produced in higher plants. Among the gymnosperms
the most resinous plants today are found in the conifers, particularly the Pinaceae and
the Araucariaceae, although some Cupressaceae can also produce some significant resin
amounts (Langenheim, 2003). Tappert et al. (2011) showed that modern conifer resins fall
into two broad categories: pinaceous resins and cupressaceous resins, depending on their
terpenoid (isoprenoid) composition. Pinaceous resins, from the Pinaceae and Torreya
How to cite this article Seyfullah et al. (2015), Species-level determination of closely related araucarian resins using FTIR spectroscopy
and its implications for the provenance of New Zealand amber. PeerJ 3:e1067; DOI 10.7717/peerj.1067
(Taxaceae), are based on abietane or pimarane terpenes, whereas cupressaceous resins,
which include the Araucariaceae, Cupressaceae, Podocarpaceae and Sciadopityaceae, are
based on labdane terpenes. This indicates differences in the terpenoid sythases, which are
genetically controlled, and so are of phylogenetic significance (Tappert et al., 2011).
Resins can become fossilized although their potential to do so is directly linked to their
terpene chemistry and this varies greatly across conifers. Resin chemistry analyses therefore
allow correlations between living plant taxa and can indicate relationships with fossil
resinous plants (Lambert, Santiago-Blay & Anderson, 2008).
Resins become fossilized through maturation; this includes their hardening and burial
in sediment, where temperature, pressure and permeating fluids affect the rate of chemical
transformation (Ragazzi & Schmidt, 2011). Resin maturation is age-related, but it also
depends on the thermal history of the resin (Anderson, Winans & Botto, 1992), as well as its
chemical structure and composition, since resins are a heterogeneous mixture of chemicals
(Langenheim, 2003). Isotopic and chemical changes in amber composition through
maturation are minor, except for polymerisation and the loss of volatile components
(Nissenbaum & Yaker, 1995; Stout, 1995).
Fossil resins classified as Class I (polylabdanoid diterpenoids), based on the polymeric
skeletons of their terpenes (Anderson, 1995; Lambert, Santiago-Blay & Anderson, 2008),
comprise the majority of the world’s major amber deposits and thus can be thought to be
most chemically similar to the cupressaceous conifer resin type of Tappert et al. (2011).
However, the parent plants are still heavily disputed for the largest deposit ever discovered,
the Baltic amber succinite deposit, despite being Class 1 (Class 1a: Anderson, 1995)
ambers, and various pinaceous, araucarian and sciadopitoid affinities have been suggested
(Schubert, 1961; Gough & Mills, 1972; Mosini & Samperi, 1985; Katinas, 1987; Beck, 1993;
Langenheim, 1969; Langenheim, 2003; Wolfe et al., 2009) and as yet, none accepted.
Despite the problems of trying to identify the Baltic amber parent plant(s), advances are
being made in identifying the parent plant(s) of other major world amber deposits (Penney,
2010). The important amber deposits in the Southern Hemisphere were thought to be
mainly araucarian-derived (Lambert, Santiago-Blay & Anderson, 2008) fossil resins (Class
1b: Anderson, 1995). Southern Hemisphere amber has recently been recorded from Peru,
South Africa, and Argentina in very small amounts, with more significant amounts found
in Australia (Hand et al., 2010) and New Zealand (Kaulfuss et al., 2011).
In Australia amber occurs in Miocene coals (Lyons, Masterlerz & Orem, 2009), and
forms the Cape York deposit (post-Jurassic, pre-late Miocene in age: Hand et al., 2010).
Amber that has been washed up on southern Australian beaches is not in situ as it
has been transported across the ocean (Murray et al., 1994). Various sources for these
ambers have been suggested, both an araucarian (Colchester, Webb & Emseis, 2006) and a
dipterocarpacean (Sonibare et al., 2014) origin has been postulated. In New Zealand amber
is found in Eocene, Oligocene and Miocene sediments and generally, an araucarian origin
has been suggested (Thomas, 1969; Lambert et al., 1993; Lyons, Masterlerz & Orem, 2009).
Other Australian ambers tested by Lambert et al. (1993) and Lyons, Masterlerz & Orem
(2009), both from southern Australia; and Sonibare et al. (2014, Cape York, northern
Seyfullah et al. (2015), PeerJ, DOI 10.7717/peerj.1067 2/17
Australia) appear to have a different botanical source, potentially among the Diptero-
carpaceae (Sonibare et al., 2014), although the source area has not yet been identified
(Lambert, Santiago-Blay & Anderson, 2008; Lambert et al., 2012; Lyons, Masterlerz
& Orem, 2009). Members of the Dipterocarpaceae are some of the most resinous
angiosperms (flowering plants) today and their resins can form amber, classified as Class II
(polycadinene) fossil resins (Anderson, 1995; Rust et al., 2010).
No Dipterocarpaceae are known in today’s Australian flora, but they are abundant in
Southeast Asia, and are thought to have originated in Gondwana in the Late Cretaceous
then rafted on the Indian plate and spread out into Asia, based on plant biogeography
(Morley, 2000). The dipterocarps have a fossil resin and pollen record stretching back to
the Eocene of India (Dutta et al., 2009), however, Australia’s fossil record does not include
Dipterocarpaceae, and so the source of the ambers with dipterocarpacean affinities is
questionable. Sonibare et al. (2014) suggest transportation of amber from New Guinea
despite amber not being known there, or from other Southeast Asian amber deposits.
Murray et al. (1994) similarly suggested long distance oceanic transport of dipterocarp
resin on to southern Australian beaches. Dipterocarpaceae is not present in the extant flora
of New Zealand, nor in its fossil record.
A Podocarpaceae conifer origin for a sole mid-Eocene amber from New Zealand was
suggested by Grimalt, Simoneit & Hatcher (1989). This amber was associated with uniden-
tified coalified wood from the Brunner Coal Measures which had the Podocarpaceae pollen
Dacrydiumites mawsonii (now Phyllocladidites mawsonii Cookson 1947 ex Couper 1953)
present. Lyons, Masterlerz & Orem (2009) also tested resins from the Eocene bituminous
coals of the Brunner Coal Measures of the Reefton Coalfield, but inferred that this amber
was more mature Agathis amber than younger New Zealand ambers.
Podocarpaceae are not highly resinous today and the family has not been analyzed
chemically in detail. Resin only notably occurs in leaves but not from the stems of
Podocarpaceae in quantities that would be commercially viable (Langenheim, 2003). There
are representatives of Podocarpaceae in both the Australian and New Zealand floras today,
and there is a fossil record dating back to the Cretaceous in both Australia and New Zealand
(Pole, 1995; Pole, 2000; Parrish et al., 1998).
The majority of New Zealand ambers are thought to have been produced by Agathis
(Lambert et al., 1993; Lyons, Masterlerz & Orem, 2009). The record of araucarian
macrofossils in New Zealand may date back to the Cenomanian (Late Cretaceous, Pole,
2008). Agathis fossils are also known from the Eocene fossil record in Australia (Carpenter
et al., 2004), and araucarian pollen is known from the Cretaceous of both New Zealand and
Australia (Raine, Mildenhall & Kennedy, 2006). However, both Lambert et al. (1993), using
NMR C13 spectroscopy, and Lyons, Masterlerz & Orem (2009) using FTIR spectroscopy,
showed that some Australian ambers tested have a different, but related chemistry to those
of New Zealand, potentially indicating a different parent plant species within Agathis or the
Araucariaceae.
Araucarian conifers today have a Southern Hemisphere distribution and comprise three
genera: Agathis Salisb., with 21 species, Araucaria Juss., with 19 species, and the monotypic
Seyfullah et al. (2015), PeerJ, DOI 10.7717/peerj.1067 3/17
Table 1 Modern Araucariaceae resins sampled.
Genus, species Locality collected, date Location of resin sampled
Agathis australis (D. Don) Loud. Northland New Zealand, 2011 Trunk
Agathis lanceolata Warb. Riviere Bleue, New Caledonia, 2011 Trunk
Agathis ovata (C. Moore ex Veill.) Warburg west of Yate´, New Caledonia, 2011 Trunk
Araucaria heterophylla (Salisb.) Franco cultivated tree, Go¨ttingen, Germany, 2014 Trunk
Araucaria humboldtensis J Buchholz Mt Humboldt, New Caledonia, 2011 Branch tip
Araucaria nemorosa de Laub. Port Boise´, New Caledonia, 2011 Trunk
Wollemia nobilis WG Jones, KD Hill & JM Allen cultivated tree, Go¨ttingen, Germany, 2014 Branch tip
Wollemia nobilis WG Jones, KD Hill & JM Allen. Agathis is the most resinous genus today.
There have been some investigations of the resin chemistry of the Araucariaceae (Lyons,
Masterlerz & Orem, 2009; Wolfe et al., 2009; Tappert et al., 2011), but to date the sampling
within this family has focused on several species of Araucaria, the monospecific Wollemia
and a few Agathis species (see Lyons, Masterlerz & Orem, 2009; Tappert et al., 2011).
Several different solid state spectroscopy methods have been used to analyse the bulk
chemistry of both resins and ambers, including infrared (IR; e.g., Beck, Wilbur & Meret,
1964), Fourier transform infrared (FTIR; Wolfe et al., 2009), Raman (Edwards, Farwell
& Villar, 2007) and 13C nuclear magnetic resonance (13C NMR; Lambert et al., 1999;
Martinez-Richa et al., 2000) spectroscopy. Interestingly, Wolfe et al. (2009) indicated that
infra-specific variability in conifer resins was low, meaning that it could be possible to
identify different species, perhaps despite quite different local ecological conditions.
Here we use Fourier transform infrared spectroscopy (FTIR) for studying the bulk
chemistry of samples of resins across the Araucariaceae following Tappert et al. (2011)
to investigate the resin chemistry variability between selected species of Araucariaceae
(including some species that were not previously sampled), and to test subsequently
whether some Miocene amber from New Zealand, thought to derive from Agathis, can
be compared to or distinguished from the resins of these extant Agathis species that occur
close to present day New Zealand.
MATERIAL & METHODS
Seven resins from across the Araucariaceae were sampled from wild and cultivated
specimens (Table 1). All except the Araucaria heterophylla and the Wollemia nobilis
resins were collected in New Caledonia and New Zealand in 2011, with the two
excepted resins collected in 2014. Fieldwork and collection in southern New Caledonia
were kindly permitted by the Direction de l’Environnement (Province Sud), permit
no. 17778/DENV/SCB delivered in November 2011. Samples were preferentially collected
from trunks, if available, but exudates from branches were used if trunk exudates were
not available (Table 1). Hardened resin was preferred, but in two cases (W. nobilis and
Ar. heterophylla) semi-solidified resin was collected and prepared. A sample of amber from
the early Miocene Idaburn locality in southern New Zealand (Fig. 1) was also tested (see
geological information for the amber specimen below). This single large piece of amber
Seyfullah et al. (2015), PeerJ, DOI 10.7717/peerj.1067 4/17
Figure 1 Map of New Zealand and close-up of Otago with Idaburn amber locality (red dot) indicated.
was collected in October 2011 and is housed at the Geology Museum of the University
of Otago in Dunedin, collection number OU 33159.1. All samples without inclusions or
any observable contaminants were chosen, freshly fractured and reduced to a fine powder
for application to the central measuring point of the FTIR spectrometer, only tens of
micrograms of samples are required per test. Pelletization with KBr was not necessary as
the Attenuated Total Reflectance (ATR) technique was used. The absorption spectra were
collected in the range 4000–650 cm−1 (wavenumbers), equivalent to 2.5–15 µm, using a
Jasco 4,100 Fourier transform infrared (FTIR) spectrometer. Spectral resolution was set
at 4 cm−1. Multiple replicate tests were run per sample until at least three spectra when
overlaid were exactly the same, and each time new portions of the ground samples were
used. The baseline was not corrected. Bands were identified by comparison with previous
reports (e.g., Lyons, Masterlerz & Orem, 2009; Tappert et al., 2011).
Geological information for the Idaburn amber sample
The amber sample was collected from the disused Idaburn Coal Mine (Fig. 2A),
near Oturehua, Central Otago, New Zealand. The GPS coordinates of the site are
44◦58′58.63′′S 169◦58′52.65′′E. The sample (Figs. 2B–2C) was taken from the 4 m thick
Oturehua Seam (Fig. 3), Fiddlers Member, Dunstan Formation, Manuherikia Group
(Douglas, 1986; Lee et al., 2003). The Manuherikia Group consists of fluvial lignite-bearing
Dunstan Formation, and the overlying Bannockburn Formation that consists entirely of
lacustrine sediments (Douglas, 1986; Lee et al., 2003).
The Fiddlers Member of the Dunstan Formation is widespread in the northern
Idaburn district and varies from a few metres to c. 150 m thick. It primarily consists
of a fine-grained non-carbonaceous mud-dominated succession with occasional lignite
beds. The Fiddlers Member is interpreted as a widespread low gradient flood-basin
Seyfullah et al. (2015), PeerJ, DOI 10.7717/peerj.1067 5/17
Figure 2 Amber from the former Idaburn Coal Mine, Otago, southern New Zealand. (A) Overview of
the exposure of the Oturehua Seam in the Fiddlers Member, Dunstan Formation, from which the amber
was collected. (B) In situ amber piece at the exposure of the lignite (Oturehua Seam). (C) Washed amber
sample (shown in B) from the same site. Scale is 5 cm.
dominated plain, peripheral to an enlarging lake (Lake Manuherikia), with relatively few
river channels (Lee et al., 2003).
The lignite of the Oturehua Seam (Fig. 3) was formed in a swamp forest and includes
some beds with relatively high fusain content. There are some horizons with abundant
woody remains (including tree trunks and stumps), and beds composed almost entirely
of fern-like rachis litter. Very fine sand is found as discontinuous laminae in the lignite
(Douglas, 1986; Lee et al., 2003). This means that the amber is considered in situ with no
discernable transportation. Interestingly, the lignite has not been very deeply buried, in
contrast to other lignites from elsewhere in New Zealand. This is something that could be
important in understanding diagenesis (particularly of this amber) and will be investigated
further in future research (DE Lee, pers. comm., 2015).
The age of the lignite is early Miocene (Mildenhall, 1989). The amber sample (OU
33159.1, Geology Department collections, University of Otago) used for FTIR analyses was
a single in situ piece (Fig. 2B) from near the top of the Oturehua Seam, which was sampled
once removed and washed clean (Fig. 2C).
RESULTS
The FTIR results (Fig. 4) show that the seven resins and the Miocene amber from New
Zealand are clearly true plant resins, since they appear to have generally similar spectra,
but they are distinguishable from each other. Analysis of the key features of the spectra
helps to compare and distinguish the resins (Table 2). Moving from left to right across the
spectra, key features are highlighted (Figs. 4 and 5). The first is a shoulder generally found
around 3,400 cm−1 of variable amplitude caused by the stretching of O–H bonds, although
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Figure 3 Diagrammatic representation of the exposure at the former Idaburn Coal Mine, Otago,
southern New Zealand, showing where the amber was collected, with an interpretation of the deposi-
tional environment, redrawn from Lee et al. (2003)with permission.
it is absent in Agathis lanceolata, Agathis ovata and Wollemia. All samples share a small
peak at 3,076 cm−1 caused by the asymmetric C–H stretching of monoalkyl groups and a
more prominent peak at around 2,935 cm−1 represents a doublet produced by methylene
groups, as well as two smaller peaks off the shoulder of the prominent (2,935 cm−1) peak
at 2,870 cm−1 and 2,848 cm−1. These three peaks result from aliphatic stretching of single
C–H bonds. The 2,870 cm−1 peak is associated with methyl groups and the 2,848 cm−1
one is a doublet produced by methylene groups.
The next peak shared by all taxa is at 1,693 cm−1, with a weak shoulder present at
around 1,722 cm−1 for some taxa (amber sample, Agathis australis, Wollemia, Araucaria
Seyfullah et al. (2015), PeerJ, DOI 10.7717/peerj.1067 7/17
Figure 4 Fourier-Transform infrared (FTIR) spectra of araucarian resins and aMiocene New Zealand
amber.
humboldtensis and Agathis lanceolata), both are related to the C=O bonds in the carboxyl
groups of resin acids. A smaller peak shared by all taxa at 1,640 cm−1 is probably related to
O–H bending bond (Tappert et al., 2011) or to exomethylene (Lyons, Masterlerz & Orem,
2009). The next section (between 1,550–650 cm−1), known as the fingerprint region is
shown in detail (Fig. 5), as there are many peaks and troughs here. At 1,460 cm−1 Agathis
australis, Agathis lanceolata, Araucaria humboldtensis and Araucaria nemorosa have a small
trough on the shoulder of the peak at 1,448 cm−1, which is shared by all samples. These
features are related to C–H bending motions of methyl and methylene functional groups.
Seyfullah et al. (2015), PeerJ, DOI 10.7717/peerj.1067 8/17
Figure 5 Close-up of the 1,550–650 cm−1 spectral region of the Fourier-Transform Infrared (FTIR)
spectra of araucarian resins and aMiocene New Zealand amber shown in Fig. 4.
The 1,385 cm−1 peak is shared by all samples, except for the amber sample where this
is a shoulder to a peak at around 1,375 cm−1, Agathis australis which also has a second
equal peak at around 1,365 cm−1, and Araucaria heterophylla, which also has a slightly
stronger second peak at around 1,365 cm−1, the other resins have a small shoulder at
around 1,365 cm−1.
The peaks between 1,300–1,100 cm−1 are features assignable to C-O single bonds, with
the next peak occurring at 1,265 cm−1, except for Agathis australis, Agathis ovata and the
Seyfullah et al. (2015), PeerJ, DOI 10.7717/peerj.1067 9/17
Table 2 Distinctive features of FTIR spectra summarized allowing sample differentiation.
Sample tested Key distinguishing features (cm−1)
3,400 1,722 1,460 1,385 1,365 1,265 1,234 1,178 1,150 1,091 1,030 791
Idaburn amber s s - s s - - - - - off s
Agathis australis s s T p p - s s p p off s
Agathis lanceolata - s T p s p p p p p p p
Agathis ovata - - - p s - - s p - wide p
Araucaria heterophylla s - - p p p p p p s p p
Araucaria humboldtensis s s T p s p p p p p p p
Araucaria nemorosa s - T p s p p p p p p p
Wollemia nobilis - s - p s p p p p p p p
Notes.
p, peak; s, shoulder; -, no feature present; T, trough; wide, relatively wider peak; off, offset peak from measurement.
amber sample. All samples except that of the amber, Agathis australis and Agathis ovata
share a peak at 1,234 cm−1, and all samples except Agathis australis, Agathis ovata and the
amber, have a peak at 1,178 cm−1, whereas both Agathis australis and Agathis ovata have
a shoulder and the amber may be interpreted to have a shoulder to a very small peak. All
samples, except for the amber share a peak at 1,150 cm−1. All samples then have a tiny
peak at 1,091 cm−1, except the amber and a shoulder instead for Araucaria heterophylla.
The next, larger peak is at 1,030 cm−1 present in all samples except those of Agathis ovata,
where it is a wider, shallower peak, and amber, where the peak appears offset, occurring at
around 1,012 cm−1.
The next peak shared by all samples is at 887 cm−1 which is attributed to the out-of-
plane C–H bending motions in terminal methylene groups. Both Araucaria heterophylla
and Araucaria nemorosa have a smaller peak preceding this at around 930 cm−1, on the
shoulder of the peak at 887 cm−1. The final feature of interest is a peak at 791 cm−1, shared
by all samples’ spectra except those spectra for the amber and Agathis australis samples,
which have a shoulder rising to a peak at around 780 cm−1 instead.
The overall picture in terms of spectra from the resins of extant araucarian trees is
that the three Araucaria species are the most similar to each other as they each have nine
distinguishing features in common. Wollemia and the three Araucaria species share eight
distinguishing features, and the greatest variability in distinguishing features is seen within
the three Agathis species (Table 2).
The Idaburn amber sample is similar in key (shared) features with the resin samples
(Figs. 4 and 5), but does differ from the extant resin samples in some of the distinguishing
features (Table 2), particularly at 1,385 cm−1, with a shoulder instead of a peak, and having
no distinctive peaks between 1,265–1,091 cm−1, unlike the resin spectra, which is most
likely due to the different chemistry that has resulted from fossilization/polymerization.
In terms of comparing the amber to the resins tested here, the amber shared most features
with that of Agathis australis (Figs. 4, 5 and Table 2).
Seyfullah et al. (2015), PeerJ, DOI 10.7717/peerj.1067 10/17
DISCUSSION
Comparing our results with that of Tappert et al. (2011) shows that our spectrum for
Agathis australis is highly comparable to theirs, although there are some minor intensity
differences. Our Wollemia spectrum is broadly similar, except that we do not have
the shoulder at around 3,400 cm−1. This is most likely due to a difference in age or
freshness between our sample and that of Tappert et al. (2011), and therefore the degree
of polymerization, as suggested by Tappert et al. (2011). Mustoe (1985) states that while
these hydroxyl groups may be structural components of the resin, they may also be from
water vapour absorbed from the atmosphere. KBr pelletization saturates samples and
can affect this signal, but the variation we see is unlikely to be due to sample preparation
since both we and Tappert et al. (2011) did not need to use KBr pelletization. Diagenetic
alteration can be excluded from our modern resin samples.
Wolfe et al. (2009) stated that the intensity of the C=O absorbance at 1,600–1,800 cm−1
and that of C–H at 1,300–1,500 cm−1 are related and modulated by the samples’ oxidation
history, but they also show that these spectral regions are still potentially useful in
discriminating modern resin samples, where little oxidation would have occurred. Thus
this potential alteration in the C=O absorbance at 1,600–1,800 cm−1 and of C–H at
1,300–1,500 cm−1 spectral regions must be taken in to account when comparing FTIR
spectra of modern resins directly with fossilized ones. Polymerization reduces the number
of hydroxyl (OH) groups, as well as having effects on other groups, and this can be seen to
affect the intensity of the 3,400 cm−1 region. The 3,400 cm−1 region may also be affected
by diagenetic alteration and by use of KBr pelletization (see below for the discussion about
the Idaburn amber).
The differences could also possibly be due to differences in the environment surround-
ing the trees when they produced the resins, which could potentially affect the primary
resin chemistry. The similarities of our spectra with those of Tappert et al. (2011) lead us to
conclude that the spectra for our samples are comparable and expand our knowledge on
araucarian resin FTIR spectra.
The relatively low variability of the Araucaria species’ resins could be due to the low
sample size across this genus of 19 species. Previous work by Tappert et al. (2011) sampled
Araucaria laubenfelsii, and the published spectrum indicates potentially more variability
across this genus. The greater variability of FTIR spectra within Agathis species shown here
may also indicate that further sampling of araucarian species is needed to map the full
FTIR spectral variation of both Araucaria and Agathis species. Additional samples from
other parts of the plants should also be included to understand any potential variation in
an individual of a species. Intraspecific variation of these species has not yet been tested,
but Wolfe et al. (2009) showed that FTIR spectra of Pinus has greater interspecies variation
than intraspecific variation across Canada.
Cupressaceous resins, which derive from Araucariaceae, Podocarpaceae, Sciadopity-
aceae and Cupressaceae, are mainly diterpenoid and have similar FTIR spectra, Tappert
et al. (2011) state that they are not distinguishable, based on a very small sample number
illustrated. This study has however shown that some bulk chemistry differences within
Seyfullah et al. (2015), PeerJ, DOI 10.7717/peerj.1067 11/17
the resins of the Araucariaceae are detectable and that there is much more variation
between species than expected. This means that FTIR can be used as a first step to assess the
similarity/differences of closely related resins and can be helpful in making an assessment
of their interspecific variation.
A second application would be the detection of the first changes denoting polymeriza-
tion of resins (Tappert et al., 2011). This means that resin FTIR spectra could be used to
guide more intensive and expensive subsequent physical and chemical identification work
(e.g., 13C NMR, Pyrolysis gas chromatography mass spectrometry). Here we show that
the New Zealand amber sample is quite distinct in our dataset, and our results support
an Agathis affinity as there was most similarity to Agathis australis resin, which has been
previously suspected of being the parent plant of New Zealand ambers (Lambert et al.,
1993); but the sample size is too small to confirm this and a potentially extinct parent
plant of the Araucariaceae cannot yet be ruled out. Pollen records show that Araucariacites
australis Cookson has been present in Australia and New Zealand since the Cretaceous
(Raine, Mildenhall & Kennedy, 2006), but this pollen could represent extinct species of both
Agathis and Araucaria. Macrofossil evidence supports the presence of both Agathis and
Araucaria in southern New Zealand in the late Oligocene to early Miocene (Lee, Bannister
& Lindqvist, 2007; Jordan et al., 2011). The third Araucariaceae genus, Wollemia may
also have been present in New Zealand from the Jurassic to the early Miocene, based
on distinctive pollen, although this pollen type could also have been produced by other
Agathis species (Jordan et al., 2011; MacPhail & Carpenter, 2013).
Lyons, Masterlerz & Orem (2009) compared modern Agathis australis resin with various
Southern Hemisphere resinites (amber fragments inside coals) of Eocene to Miocene age.
Our amber spectrum has overall similarities to all of theirs, but ours lacks clear peaks
between about 1,265–1,091 cm−1, and we suspect that this reflects an effect of maturation,
a diagenetic alteration of the fossil resin.
Our spectrum of modern Agathis australis is more similar to the New Zealand resinite
samples than to those Australian ones of Lyons, Masterlerz & Orem (2009). They considered
the Australian resinites to have a different botanical origin from the Agathis source of the
New Zealand resinites. Interestingly our amber spectrum has some further features that
imply a relatively immature amber. Fossil resins undergo structural and compositional
changes as a consequence of the effects of increasing degrees of maturation (e.g., Anderson,
Winans & Botto, 1992). In Class 1b fossil resins, to which the Idaburn amber is thought to
belong, this is most apparent by the loss of exomethylene through isomerization (Beck,
Wilbur & Meret, 1964; Beck et al., 1965; Beck, 1986; Anderson, Winans & Botto, 1992;
Anderson, 1995; Clifford & Hatcher, 1995).
The presence of exomethylene groups are peaks at around 3,082 cm−1, 1,644 cm−1 and
887 cm−1 (Anderson, Winans & Botto, 1992; Lyons, Masterlerz & Orem, 2009), seen in all
samples tested here, although slightly less intense in the amber sample. Lyons, Masterlerz &
Orem (2009) showed that exomethylene (C=CH2) amounts in New Zealand fossil resinites
decrease with maturation, with the exomethylene peaks becoming less distinct, eventually
disappearing in more mature material. The Idaburn amber sample shows fairly strong
Seyfullah et al. (2015), PeerJ, DOI 10.7717/peerj.1067 12/17
exomethylene signals (particularly when compared to the Miocene amber FTIR spectra
of Lyons, Masterlerz & Orem, 2009), indicating a relatively immature fossil resin, which is
unexpected since the in situ Idaburn amber is Miocene in age (Mildenhall, 1989).
Other effects of maturation may be seen in FTIR spectra of the Idaburn amber sample
when compared with the resin samples. Wolfe et al. (2009) stated that the absorbance of
C–H at 1,300–1,500 cm−1 are related and modulated by the samples’ oxidation history,
possibly explaining why there is a shoulder at 1,385 cm−1 rather than the peaks seen in all
the resin samples. Absorbance peaks between 1,265 cm−1 and 1,091 cm−1 indicate C–O
bonds, and these are subdued or absent in the amber, but various peaks are seen here in
the resins.
It may be that the relatively shallow burial of the lignites of the Dunstan Formation (DE
Lee, pers. comm.) is being at least partially reflected in the apparent low maturity of the
Idaburn fossil resin, when compared to ambers from other localities in New Zealand where
the lignites are known to have been more deeply buried.
CONCLUSIONS
This is the first study to apply FTIR spectroscopy to resins produced across closely related
members of the Araucariaceae from Agathis and Araucaria plants growing in New Zealand
and New Caledonia, and Wollemia from Australia (but grown in Germany). FTIR spectra
of resins sampled across the Araucariaceae show unexpected variation, despite the small
sample size: environmental variation could be a reason for the variability, but the spectra
also show that the species’ resins are similar in chemical composition. When the resin FTIR
spectra are compared with a Miocene New Zealand amber sample, a clear relationship is
supported showing that the amber is indeed a fossilized Araucariaceae plant resin, but a
contradiction appears since the amber has some features of a (relatively) immature fossil
resin, particularly when compared to other fossil resins of the same age from New Zealand,
perhaps indicating differences in diagenetic histories.
Further investigation is needed to better understand the chemistry of New Zealand
amber. FTIR is a very simple, cheap and efficient method for detecting bulk chemistry of
both resins and ambers, and needs very little preparation and sample size, making it an
excellent first step in the physical and chemical analyses of resins.
ACKNOWLEDGEMENTS
We are grateful to Holm Frauendorf (Go¨ttingen) for his generous support and fruitful
discussions on FTIR analyses. We would like to thank Ralf Gerke (Go¨ttingen) for assistance
with the FTIR spectrometer, Daphne Lee (Dunedin) for her contribution to the geology
of the Idaburn locality, and to both Daphne Lee and Uwe Kaulfuss (Dunedin) for
guidance during field work in New Zealand, and Christina Beimforde (Go¨ttingen), Vincent
Perrichot (Rennes) and Jouko Rikkinen (Helsinki) for assistance during field work in New
Caledonia. We would also like to thank both our reviewers for their helpful comments.
Seyfullah et al. (2015), PeerJ, DOI 10.7717/peerj.1067 13/17
ADDITIONAL INFORMATION AND DECLARATIONS
Funding
The work was supported by a Dorothea Schlo¨zer fellowship for LJS. Field work in New
Zealand was supported by the Marsden Research Grant UOO1115 entitled’ Life in and
beyond maars: a revolution in understanding New Zealand Miocene terrestrial biodiversity
and ecosystems’ that is operated by the Royal Society of New Zealand. The funders had no
role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript.
Grant Disclosures
The following grant information was disclosed by the authors:
Dorothea Schlo¨zer fellowship.
Marsden Research: UOO1115 operated by the Royal Society of New Zealand.
Competing Interests
The authors declare there are no competing interests.
Author Contributions
• Leyla J. Seyfullah conceived and designed the experiments, performed the experiments,
analyzed the data, contributed reagents/materials/analysis tools, wrote the paper,
prepared figures and/or tables, reviewed drafts of the paper.
• Eva-Maria Sadowski performed the experiments, wrote the paper, reviewed drafts of the
paper.
• Alexander R. Schmidt conceived and designed the experiments, contributed
reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables,
reviewed drafts of the paper.
Field Study Permissions
The following information was supplied relating to field study approvals (i.e., approving
body and any reference numbers):
Fieldwork and collection in southern New Caledonia were kindly permitted by the
Direction de l’Environnement (Province Sud), permit no 17778/DENV/SCB delivered in
November 2011.
Supplemental Information
Supplemental information for this article can be found online at http://dx.doi.org/
10.7717/peerj.1067#supplemental-information.
Seyfullah et al. (2015), PeerJ, DOI 10.7717/peerj.1067 14/17
REFERENCES
Anderson KB. 1995. The nature and fate of natural resins in the geosphere. Part V. New evidence
concerning the structure, composition, and maturation of class I (Polylabdanoid) Resinites.
In: Anderson KB, Crelling JC, eds. Amber, resinite, and fossil resins, vol. 617. Washington:
American Chemical Society, 105–129 and errata thereto; Organic Geochemistry 1997, 25 (3/4),
v–vi.
Anderson KB,Winans RE, Botto RE. 1992. The nature and fate of natural resins in the Geosphere.
II. Organic Geochemistry 18:829–841 DOI 10.1016/0146-6380(92)90051-X.
Beck CW. 1993. Der Wissenstand u¨ber chemische Struktur und botanische Herkunft des
Bernsteins. Miscellanea Archeologica Thaddaea Malinowski Dedicata 1993:27–38.
Beck CW. 1986. Spectroscopic investigations of amber. Applied Spectroscopy Reviews 22:57–110
DOI 10.1080/05704928608060438.
Beck CW,Wilbur E, Meret S. 1964. Infrared spectra and the origin of amber. Nature 201:256–257
DOI 10.1038/201256a0.
Beck CW,Wilbur E, Meret S, Kossove D, Kermani K. 1965. The infrared spectra of amber and the
identification of Baltic amber. Archaeometry 8:96–109
DOI 10.1111/j.1475-4754.1965.tb00896.x.
Carpenter RJ, Hill RS, Greenwood DR, Partridge AD, BanksMA. 2004. No snow in the
mountains: early Eocene plant fossils from Hotham Heights, Victoria, Australia. Australian
Journal of Botany 52:685–718 DOI 10.1071/BT04032.
Clifford DJ, Hatcher PG. 1995. Maturation of Class Ib resinites. In: Anderson KB, Crelling JC, eds.
Amber, resinite, and fossil resins, vol. 617. Washington: American Chemical Society, 92–104.
Colchester DM,Webb G, Emseis P. 2006. Amber-like fossil resin from north Queensland,
Australia. Gemmologist 22:378–834.
Douglas BJ. 1986. Lignite resources of Central Otago, Manuherikia Group of Central Otago, New
Zealand: stratigraphy, depositional systems, lignite resource assessment and exploration models.
Auckland: University publication. 2 volumes.
Dutta S, Mallick M, BertramN, Greenwood PF, Mathews RP. 2009. Terpenoid composition
and class of Tertiary resins from India. International Journal of Coal Geology 80:44–50
DOI 10.1016/j.coal.2009.07.006.
Edwards HGM, Farwell DW, Villar SEJ. 2007. Raman microspectroscopic studies of amber resins
with insect inclusions. Spectrochimica Acta Part A 68:1089–1095 DOI 10.1016/j.saa.2006.11.037.
Gough LJ, Mills JS. 1972. The composition of Succinite (Baltic amber). Nature 239:527–528
DOI 10.1038/239527a0.
Grimalt JO, Simoneit BRT, Hatcher PG. 1989. The chemical affinities between the solvent
extractable and the bulk organic matter of fossil resin associated with an extinct Podocarpaceae.
Phytochemistry 28:1167–1171 DOI 10.1016/0031-9422(89)80202-X.
Hand S, Archer M, Bickel D, Creaser P, DettmannM, Godthelp H, Jones A, Norris B, Wicks D.
2010. Australian cape york amber. In: Penney D, ed. Biodiversity of fossil in amber from the major
world deposits. Manchester: Siri Scientific Press, 69–79.
Jordan GJ, Carpenter RJ, Bannister JM, Lee DE, Mildenhall DC, Hill RS. 2011. High conifer
diversity in Oligo-Miocene New Zealand. Australian Systematic Botany 24:121–136
DOI 10.1071/SB11004.
Katinas V. 1987. U¨ber die Genese des Bernsteins. In: Stroganow I, ed. Zum Geheimnis des
Bernsteins, vol. 31. Moscow: Prawda, 10.
Seyfullah et al. (2015), PeerJ, DOI 10.7717/peerj.1067 15/17
Kaulfuss U, Lee D, Bannister J, Lindqvist J, Mildenhall D, Perrichot V, MaraunM, Schmidt AR.
2011. Discovering the New Zealand amber forest biota. Geoscience Society of New Zealand
Newsletter 5:20–25.
Lambert JB, Johnson SC, Poinar GO, Frye JS. 1993. Recent and fossil resins from New Zealand
and Australia. Geoarchaeology 8:141–155 DOI 10.1002/gea.3340080206.
Lambert JB, Santiago-Blay JA, Anderson KA. 2008. Chemical signatures of fossilized resins and
recent plant exudates. Angewandte Chemie 47:9608–9616 DOI 10.1002/anie.200705973.
Lambert JB, Shawl CE, Poinar Jr GO, Santiago-Blay JA. 1999. Classification of modern resins
by solid nuclear magnetic resonance spectroscopy. Bioorganic Chemistry 27:409–433
DOI 10.1006/bioo.1999.1147.
Lambert JB, Tsai CY-H, ShahMC, Hurtley AE, Santiago-Blay JA. 2012. Distinguishing amber
classes by proton magnetic resonance spectroscopy. Archaeometry 54:332–348
DOI 10.1111/j.1475-4754.2011.00625.x.
Langenheim JH. 1969. Amber: a botanical inquiry. Science 163:1157–1169
DOI 10.1126/science.163.3872.1157.
Langenheim JH. 2003. Plant resins. Chemistry, evolution, ecology and ethnobotany. Portland,
Cambridge: Timber Press.
Lee DE, Bannister JM, Lindqvist JK. 2007. Late Oligocene-Early Miocene leaf macrofossils
confirm a long history of Agathis in New Zealand. New Zealand Journal of Botany 45:565–578
DOI 10.1080/00288250709509739.
Lee DE, Lindqvist JK, Bannister JM, Cieraad E. 2003. Paleobotany and sedimentology of Late
Cretaceous-Miocene nonmarine sequences in Otago and Southland. In: Cox S, Smith Lyttle
B, eds. Geological society of New Zealand miscellaneous publication 116B field trip guide. Otago:
Geological Society of New Zealand, 1–48.
Lyons PC, Masterlerz M, OremWH. 2009. Organic geochemistry of resins from modern Agathis
australis and Eocene resins from New Zealand: diagenetic and taxonomic implications.
International Journal of Coal Geology 80:51–62 DOI 10.1016/j.coal.2009.07.015.
MacPhail M, Carpenter RJ. 2013. New potential nearest living relatives for Araucariaceae
producing fossil Wollemi Pine-type pollen (Dilwynites granulatus W.K. Harris, 1965).
Alcheringa 38:135–139 DOI 10.1080/03115518.2014.843145.
Martinez-Richa A, Vera-Graziano R, Rivera A, Joseph-Nathan P. 2000. A solid-state 13C NMR
analysis of ambers. Polymer 41:743–750 DOI 10.1016/S0032-3861(99)00195-0.
Mildenhall DC. 1989. Summary of the age and paleoecology of the Miocene Manuherikia
Group, Central Otago, New Zealand. Journal of the Royal Society of New Zealand 19:19–29
DOI 10.1080/03036758.1989.10426452.
Morley RJ. 2000. Origin and evolution of tropical rain forests. Chichester: John Wiley and Sons.
Mosini V, Samperi R. 1985. Correlations between Baltic amber and Pinus resins. Phytochemistry
24:859–861 DOI 10.1016/S0031-9422(00)84910-9.
Murray AP, Padley D, McKirdy DM, BoothWE, Summons RE. 1994. Oceanic transport of
fossil dammar resin: the chemistry of coastal resinites from South Australia. Geochimica et
Cosmochimica Acta 58:3049–3059 DOI 10.1016/0016-7037(94)90178-3.
Mustoe GE. 1985. Eocene amber from the Pacific Coast of North America. Bulletin of the Geological
Society of America 96:1530–1536 DOI 10.1130/0016-7606(1985)96<1530:EAFTPC>2.0.CO;2.
Nissenbaum A, Yaker D. 1995. Stable isotope composition of amber. In: Anderson KB, Crelling JC,
eds. Amber, resinite, and fossil resins, vol. 617. Washington: American Chemical Society, 32–42.
Seyfullah et al. (2015), PeerJ, DOI 10.7717/peerj.1067 16/17
Parrish JT, Daniel IL, Kennedy EM, Spicer RA. 1998. Paleoclimatic significance of
Mid-Cretaceous floras from the Middle Clarence Valley, New Zealand. Palaios 13:149–159
DOI 10.2307/3515486.
Penney D. 2010. Dominican amber. In: Penney D, ed. Biodiversity of fossils in amber from the major
world deposits. Manchester: Siri Scientific Press, 22–41.
Pole MS. 1995. Late Cretaceous macrofloras of eastern Otago, New Zealand: Gymnosperms.
Australian Systematic Botany 8:1067–1106 DOI 10.1071/SB9951067.
Pole MS. 2000. Mid-Cretaceous conifers from the Eromanga Basin, Australia. Australian Systematic
Botany 13:153–197 DOI 10.1071/SB99001.
Pole MS. 2008. The record of Araucariaceae macrofossils in New Zealand. Alcheringa 32:409–430
DOI 10.1080/03115510802417935.
Ragazzi E, Schmidt AR. 2011. Amber. In: Reitner J, Thiel V, eds. Encyclopedia of Geobiology. Berlin:
Springer, 24–36.
Raine JI, Mildenhall DC, Kennedy EM. 2006. New Zealand fossil spores and pollen: an illustrated
catalogue. GNS science miscellaneous series, 2nd ed. vol. 4. Available at http://www.gns.cri.nz/
what/earthhist/fossils/spore pollen/catalog/index.htm.
Rust J, Singh H, Rana RS, McCanna T, Singh L, Anderson K, Sarkare N, Nascimbene PC,
Stebner F, Thomas JC, Solo´rzano KraemerM,Williams CJ, Engel MS, Sahni A, Grimaldi D.
2010. Biogeographic and evolutionary implications of a diverse paleobiota in amber from
the early Eocene of India. Proceedings of the National Academy of Sciences of the United States of
America 107:18360–18365 DOI 10.1073/pnas.1007407107.
Schubert K. 1961. Neue Untersuchungen ueber Bau und Leben der Bernsteinkiefern (Pinus
succinifera (Conw.) emend.). Ein Beitrag zur Palaeohistologie der Pflanzen. Geologisches
Jahrbuch, Beihefte 45:1–150.
Sonibare OO, Agbaje OB, Jacob DE, Faithfull J, Hoffmann T, Foley SF. 2014. Terpenoid
composition and origin of amber from the Cape York Peninsula, Australia. Australian Journal
of Earth Sciences 61:979–985 DOI 10.1080/08120099.2014.960897.
Stout S. 1995. Resin-derived hydrocarbons in fresh and fossil Dammar resins and Miocene rocks
and oils in the Mahakam delta, Indonesia. In: Anderson KB, Crelling JC, eds. Amber, resinite,
and fossil resins, vol. 617. Washington: American Chemical Society, 43–75.
Tappert R,Wolfe AP, McKellar RC, Tappert MC, Muehlenbachs K. 2011. Characterizing modern
and fossil gymnosperm exudates using micro-Fourier transform infrared spectroscopy.
International Journal of Plant Sciences 172:120–138 DOI 10.1086/657277.
Thomas BR. 1969. Kauri resins—modern and fossil. In: Eglinton G, Murphy MTJ, eds. Organic
geochemistry. Berlin: Springer, 599–618.
Wolfe AP, Tappert R, Muehlenbachs K, BoudreauM,McKellar MC, Basinger JC, Garrett A.
2009. A new proposal concerning the botanical origin of Baltic amber. Proceedings of the Royal
Society, B 276:3403–3412 DOI 10.1098/rspb.2009.0806.
Seyfullah et al. (2015), PeerJ, DOI 10.7717/peerj.1067 17/17