Review of Palaeobotany and Palynology 225 (2016) 21–32
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Review of Palaeobotany and Palynology
journal homepage: www.elsevier.com/locate/revpalbo
Bitterfeld amber is not Baltic amber: Three geochemical tests and further
constraints on the botanical affinities of succinite
Alexander P. Wolfe a,⁎, Ryan C. McKellar b, Ralf Tappert c, Rana N.S. Sodhi d, Karlis Muehlenbachs c
a
Department of Biological Sciences, University of Alberta, Edmonton, AB T6G 2E9, Canada
Royal Saskatchewan Museum, 2445 Albert St., Regina, SK S4P 4W7, Canada
Department of Earth and Atmospheric Sciences, University of Alberta, Edmonton, AB T6G 2E3, Canada
d
Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON M5S 3E5, Canada
b
c
a r t i c l e
i n f o
Article history:
Received 10 October 2014
Received in revised form 12 November 2015
Accepted 16 November 2015
Available online 23 November 2015
Keywords:
Amber
Baltic
Bitterfeld
FTIR
ToF-SIMS
Stable isotope geochemistry
a b s t r a c t
Baltic and Bitterfeld ambers are important deposits of polymerized conifer resin that are widely recognized for
their exquisite fossil inclusions, especially insects. Because of over-arching similarities with respect to visual appearance, organic geochemistry, arthropod assemblages, and proximity to forests of the Paleogene North Sea
margin, these two ambers have not yet been differentiated definitively, leading to ongoing debate as to whether
or not they (and their respective inclusions) are truly equivalent. We combine micro-Fourier transform infrared
spectroscopy (FTIR), time of flight-secondary ion mass spectrometry (ToF-SIMS), and stable isotopes (δ13C and δ2H)
to establish that Baltic and Bitterfeld ambers differ consistently in their geochemical properties, and thus capture distinct depositional episodes in space, but not necessarily in time. Baltic amber has more succinic acid, succinic anhydride, and communic acid relative to Bitterfeld amber, but less dehydroabietic acid. Although both ambers produce
overlapping δ13C values, supporting a similar age of formation, δ2H is markedly depleted (by ~20‰) in Baltic amber
relative to Bitterfeld amber. The hydrogen isotopic results confer paleolatitudinal differences in amber provenance,
that is, a clear differentiation between sources originating from the northern (Baltic) and southern (Bitterfeld)
margins of the Paleogene North Sea. We conclude that the two deposits are geologically distinct in origin, but that
similarities in their respective faunal records arise because they are broadly coeval in time. We also present new
ToF-SIMS results that imply only resins from modern conifers of the families Pinaceae and Sciadopityaceae begin
to satisfy the expanded geochemical profiles presented for Baltic and Bitterfeld ambers.
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
Baltic amber is the world's best known deposit of fossil plant resin,
and by far the single largest repository of fossil insects of any age
(Weitschat and Wichard, 2002, 2010). Unlike in situ fossil resins that
are directly associated with lignite, coal, or other plant-rich strata, Baltic
amber is a secondary deposit found mainly in glauconitic marine sediments of middle Eocene age (Lutetian Stage; 41.3–47.8 Ma), deposited
along the paleo-North Sea margin. The blue earth (or Blaue Erde) in
which Baltic amber is principally hosted occurs in Russia (Kaliningrad
Oblast), Poland, and Germany, but detrital Baltic amber, redeposited
by Quaternary glacial and fluvial processes, reaches Scandinavia, the
Baltic republics, and the British isles. Baltic amber has been exploited
for millennia, and is widely disseminated in European archaeological
contexts (Beck et al., 1965). The botanical origin of Baltic amber is
a topic of intense scrutiny and longstanding debate, for which the
only firm conclusion is that source trees were extinct conifers
⁎ Corresponding author. Tel.: +1 587 879 1142; fax: +1 780 492 9457.
E-mail address: awolfe@ualberta.ca (A.P. Wolfe).
http://dx.doi.org/10.1016/j.revpalbo.2015.11.002
0034-6667/© 2015 Elsevier B.V. All rights reserved.
(Mills et al., 1984; Mosini and Samperi, 1985; Wolfe et al., 2009;
Dolezych et al., 2011).
Bitterfeld amber originates from a much more restricted geographical area, the silts and sands, or “Bernsteinschluff”, of the Cottbus Formation near the town of Bitterfeld in Upper Saxony (Sachsen-Anhalt;
hence the synonym Saxonian amber). Although once assigned a
Miocene age (Barthel and Hetzer, 1982), more recent geochronological
efforts (Knuth et al., 2002) place these sediments in the late Oligocene
(Chattian; 23.0–28.1 Ma). As with Baltic amber, Bitterfeld amber is
a secondary deposit that preserves an exceptional record of fossil
arthropods. Bitterfeld amber was actively mined at the site of Goitzsche
between 1975–1993, yielding a gem quality resource and thousands of
arthropod inclusions (Dunlop, 2010). Careful geological mapping of
the Bitterfeld amber complex shows that amber is concentrated
in low-energy lagoonal facies associated with a deltaic system
discharging into the North Sea from the south (Wimmer et al., 2006;
Fuhrmann, 2008).
Bitterfeld amber is similar to Baltic amber with respect to hardness
and visual appearance (Fig. 1), the ubiquitous presence of succinic
acid (both are referred to as succinites; Anderson and Botto, 1993), several elements of their respective arthropod assemblages, and the
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generalized geography of European amber distribution. For this reason, some have argued that they are necessarily coeval, Bitterfeld
amber being merely a younger redeposited fraction of primary
Eocene Baltic amber. In this model, both ambers share a common
botanical origin. This view is supported by similarities between the faunal inclusions of both deposits with respect to Arachnida (harvestmen:
Dunlop and Mitov, 2009; spiders: Wunderlich, 1993, 2004), Coleoptera
(dermestids: Háva and Alekseev, 2015); Diptera (acalyptrates:
von Tschirnhaus and Hoffeins, 2009; anthomyzids: Roháček, 2013;
ceratopogonids: Szadziewski, 1993; Sontag and Szadziewski, 2001;
limoniids: Kopeć and Kania, 2013; nymphomyiids: Wagner et al.,
2000), and Hymenoptera (apoid bees: Engel, 2001; and wasps: Ohl
and Bennett, 2009). Indeed, the viewpoint that Baltic and Bitterfeld
ambers have an identical provenance is held strongly, and has been
particularly well articulated by Weitschat (2008, pp. 94), whose translated statement reads:
“Northern European amber production began during warm conditions of the early Eocene, and terminated by the end of the middle
Eocene. The cooling trend over this interval resulted in irreversible
changes in the flora and fauna of northern Europe: tropical and
subtropical elements were progressively replaced by boreal ‘arctoTertiary’ assemblages. The amber forests recorded this transition,
given that Baltic and Bitterfeld deposits both contain taxa belonging
to tropical as well as boreal ecotypes, at times the very same species.
The case is especially convincing with regard to spiders, suggesting
that Baltic and Bitterfeld ambers both originated from a single forest
ecosystem in western Scandinavia, which persisted for up to 10
million years under a sustained warm climate regime.”
More recently, even stronger statements to the same effect have
been issued from the paleoentomological community (Szwedo and
Sontag, 2013, pp. 380):
“At present, there is no doubt that amber from Bitterfeld (Saxonian
amber) is contemporaneous with Baltic amber, i.e. that it originated
in the Eocene and that it belongs to the Baltic amber group.”
However, a balanced and thorough review of the subject (Dunlop,
2010) leaves unresolved the question as to whether Baltic and Bitterfeld
ambers are truly identical in age and origin. Arguments based on stratigraphy (Knuth et al., 2002) and organic geochemistry (Yamamoto
et al., 2006) challenge the view that Baltic and Bitterfeld ambers are
equivalent, as do paleobiological studies that nuance the rate and
tempo of evolutionary processes among and between organismal
groups (Barthel and Hetzer, 1982; Dunlop and Giribet, 2003; Schmidt
and Dörfeldt, 2007; Dlussky and Rasnitsyn, 2009). The resolution of
this dilemma constitutes the impetus for the present study, in the footsteps of important yet inconclusive regional symposia on this exact
topic (Ganzelewski et al., 1997; Rascher et al., 2008). We report results
from three parallel suites of geochemical analyses that bear directly on
the differences and similarities between Baltic and Bitterfeld ambers,
and conclude that they are compositionally distinct from each other
and do not share the same geographical provenance, while remaining
largely contemporaneous in their age of formation.
2. Materials and methods
Fig. 1. Photographs of Baltic (A–F) and Bitterfeld (G–N) amber specimens. Polished (A–B)
and unpolished (C) clear Baltic ambers (“honey”), the latter with surface desiccation
cracks. (D) Internal zonation between clear and partially opaque “butterscotch” ambers.
(E–F) Outer and internal views of completely opaque “bone” amber with multiple generations of flow lines, or schlaube. (G–L) Bitterfeld amber ranging from clear yellow to dark
reddish-brown. The dark nearly specimen (M) is classified as “glessite”, the name given to
this variant, which occurs in both Baltic and Bitterfeld deposits. (M–N) Bitterfeld bone
amber.
Samples of Baltic and Bitterfeld ambers have been collected,
purchased, and obtained through exchange with colleagues. A sizeable
collection of Baltic amber specimens from Germany, Lithuania, Poland,
Russia, and southern Sweden was amassed during previous investigations (Wolfe et al., 2009). Baltic amber specimens were sub-sampled
from this collection for the geochemical analyses described below.
Bitterfeld amber specimens include samples confirmed to originate
from the Goitzsche mine and offered for study by Alexander Schmidt
A.P. Wolfe et al. / Review of Palaeobotany and Palynology 225 (2016) 21–32
(University of Göttingen). For both ambers, we analyzed in triplicate the
four most common color variants (“honey”, “butterscotch”, “bone”, and
“glessite”; Fig. 1), which range from clear yellow to dark red, with various degrees of opacity.
Fourier transform infrared (FTIR) spectroscopy has been a mainstay
in amber chemical fingerprinting for half a century (Beck et al., 1964;
Langenheim and Beck, 1965). FTIR remains an important tool in
amber research, in part because new technologies coupled to IR
microscopes obviate the need for an embedding medium (typically
KBr, which is hygroscopic), facilitating the analysis of much smaller
specimens (e.g., mg-scale; Tappert et al., 2011; Seyfullah et al., 2015).
We conducted FTIR micro-spectroscopy on untreated amber flakes
chipped from fresh surfaces free of inclusions. We also obtained FTIR
spectra from monomethyl succinate (99%, Sigma-Aldrich) and three
diterpene resin acids that are important constituents of European Paleogene ambers: abietic acid, dehydroabietic acid, and communic acid. The
latter were isolated from natural pinaceous resins to N 95% purity at the
CanSyn Inc. Laboratory, Toronto (http://www.cansyn.com/index.html).
All specimens were mounted on infrared-transparent NaCl discs and kept
to thicknesses ≤ 10 μm in order to minimize oversaturation. Absorption
spectra were collected over the 700–4000 cm− 1 (wavenumber)
interval (i.e., wavelengths of 2.5–14.0 μm) with a Thermo Nicolet
Nexus 470 FTIR spectrometer equipped with a Nicolet Continuum IR
microscope. Spectral resolution was 4 cm−1 and beam size was set between 50 and 100 μm. No additional manipulations, such as continuum
removal or smoothing, were applied to the spectra. Further details of
our FTIR methodology have been presented elsewhere (Wolfe et al.,
2009; Tappert et al., 2011).
Time of flight-secondary ion mass spectrometry (ToF-SIMS) is an
emerging technology in geobiology, largely because it is amenable to organic molecules from a variety of geological contexts, and furthermore
highly effective in capturing a broad range of ionized products with
extremely high mass resolution (Thiel and Sjövall, 2011). In ToF-SIMS
analysis, samples are bombarded with a high energy primary ion
stream, producing secondary ions from the analyte surface that
enter the detector and consequently form a mass spectrum. In applying ToF-SIMS to amber, specimens were cut with a diamond blade
ultracryomicrotome (Leica EM UC6) immediately prior to their introduction into the load lock with no further sample preparation.
Three fragments of each amber type were analyzed in triplicate. In
order to better constrain the ion fragmentation patterns observed in
amber, we also obtained duplicate ToF-SIMS spectra from monomethyl
succinate, abietic acid, dehydroabietic acid, and communic acid. These
standards were dissolved in chloroform, dried under UV and O3 for
several minutes, then spin-coated onto Si wafers immediately prior
to analysis. We also report exploratory ToF-SIMS analyses of modern
resins from exemplar species of the dominant resin-producing conifer
families: Araucariaceae (Agathis australis), Cupressaceae (Metasequoia
glyptostroboides), Pinaceae (Pinus contorta), Podocarpaceae (Podocarpus
totara), and Sciadopityacae (Sciadopitys verticillata). These materials
originate from our extensive collection (Wolfe et al., 2009; Tappert
et al., 2011) and were prepared for ToF-SIMS in much the same way as
amber specimens. It is important not to use chloroform at any stage of
preparation of amber and resin samples, as this induces a range of
artefacts owing to partial solubility in this solvent. ToF-SIMS spectra
were obtained with an ION-TOF GmbH ToF-SIMS IV equipped with a biscluster was used as the primary
muth (Bi) liquid metal ion gun. The Bi++
3
ion beam operated in high mass resolution bunched mode (Sodhi, 2004).
The ion beam was rastered over an area of 500 × 500 μm for 60–120 s in
order to remain below static limits. Charge neutralization was achieved
using low energy (b 20 eV) electrons supplied by the instrument's pulsed
electron flood gun. Although both positive and negative polarity spectra
were obtained, we report only results obtained in negative mode, which
have proven more interpretable in pilot studies (Sodhi et al., 2013,
2014). The reproducibility of ToF-SIMS spectra was excellent for both ambers, resins, and standards. The Illustrated ToF-SIMS spectra are entirely
23
representative of replicated analyses; all features illustrated and discussed
in the text are manifested reproducibly.
Carbon (δ 13 C) and hydrogen (δ2 H) stable isotopic ratios from
amber provide useful ancillary information for understanding the
genesis of amber deposits, in part because little isotopic exchange occurs between polymerized resins and their surrounding environment
after burial (Murray et al., 1989; Nissenbaum and Yakir, 1995). This is
because the isoprene (C5H8) building blocks of terpenoid cyclic hydrocarbons are especially recalcitrant towards diagenetic isotopic exchange with respect to both C and H. In the present study, δ13C was
measured from 77 Baltic amber specimens and an additional 68 specimens of Bitterfeld amber. δ2H was measured from 34 and 33 specimens
of Baltic and Bitterfeld amber, respectively. All samples were fragments
from freshly broken surfaces cleaned with distilled water and air-dried,
but not chemically or thermally pretreated in any other way. Samples of
2–7 mg were combusted at 800 °C for 12 h in vacuum-sealed quartz
glass with CuO (1 g) as the oxygen source. Evolved CO2 was measured
directly for δ13C, whereas H2O was reduced to H2 with Zn (100 mg)
prior to δ2H analysis. Both gases were measured isotopically with a
Finnigan MAT-252 dual-inlet isotope-ratio mass spectrometer. Results
are expressed in δ notation as ‰ relative to Vienna Pee Dee Belemnite
(VPDB) for δ13C and Vienna Standard Mean Ocean Water (VSMOW)
for δ2H. Analytic precision is ±0.1‰ for δ13C and ±3‰ for δ2H.
3. Results and discussion
3.1. Micro-FTIR
The FTIR spectra of Baltic amber are very similar to each other, irrespective of the color or external texture of the specimen in question
(Fig. 2A–B). This remarkable stability has been noted repeatedly since
the pioneering investigations of Beck et al. (1965), and suggests that
all Baltic amber (sensu stricto) shares a common botanical origin
(Wolfe et al., 2009). The characteristic feature of these spectra is the
“Baltic shoulder” situated between 1190–1280 cm−1, and flanked by a
strong absorbance peak at 1170 cm−1. This feature reflects the succinate content of the amber specifically, as it is strongly expressed in
the spectrum of pure monomethyl succinate (Fig. 2D). While Bitterfeld
amber also displays this spectroscopic feature, in keeping with prior
FTIR analyses of European succinites (Kosmowska-Ceranowicz, 1999),
its expression is far more subdued. Furthermore, the ensemble of FTIR
spectra from Bitterfeld amber is more variable than those obtained
from Baltic amber, potentially reflecting greater variability with regards
to either botanical affinity or diagenetic history.
The spectroscopic difference between consensus spectra derived from
both ambers (Fig. 2C) indicates three regions where Baltic amber has
consistently stronger absorbance relative to Bitterfeld amber: (a) the carbonyl (C= O) band associated with COOH and hence total carboxylic
acids (1700–1800 cm−1); (b) the succinate band (1170–1280 cm−1);
and (c) the out-of-plane aromatic C–H band (870–900 m−1). The latter
feature is supported by the secondary C–H band at 3080 cm−1, which
is also better developed in Baltic amber. Of the three diterpene resin
acids analyzed, these two aromatic C–H bands only figure strongly in
the spectrum of communic acid. The bands from the abietic and
dehydroabietic acid standards that are best expressed in amber specimens are those associated with C–H in CH2 and CH3 (1380–1400 cm−1
and 1440–1460 cm−1, Fig. 2), but these do not contribute to any pronounced spectroscopic differences between Baltic and Bitterfeld amber.
From the synthesis of FTIR results, we are able to surmise that Baltic
amber, on the whole, contains more succinate relative to Bitterfeld
amber, and likely more communic acid.
3.2. ToF-SIMS
Building on results from preliminary studies addressing the ToFSIMS spectra of ambers obtained in negative polarity (Sodhi et al.,
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Fig. 2. FTIR spectra of (A) Baltic (red) and (B) Bitterfeld amber (blue) specimens. Illustrated spectra from both ambers have been combined with additional measurements (n = 8 in each
case) to yield consensus spectra (bold lines), averaged from individual spectra rescaled to a common range of 0–1 relative absorbance units. (C) The difference between Baltic and
Bitterfeld consensus spectra is shown with shaded areas indicating one standard deviation from the mean. (D) FTIR spectra obtained from purified monomethyl succinate and diterpene
resin acids. Vertical gray bars indicate salient spectroscopic features discussed in the text.
2013), we focus on two mass intervals of particular interest: the “succinate region” (70–120 u; Fig. 3) and the “diterpene resin acid region”
(250–350 u; Fig. 4). These are addressed sequentially below. Realized
mass spectra in the succinate region deviate by b 0.02 u from the theoretical monoisotopic masses of [M–H]− ions produced by the identified parent molecules, and by b0.05 u in the diterpene resin acid
A.P. Wolfe et al. / Review of Palaeobotany and Palynology 225 (2016) 21–32
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Fig. 3. Negative polarity ToF-SIMS spectra of the 70–120 u region for (A) Baltic amber, (B) Bitterfeld amber, and (C) monomethyl succinate. The entire range is shown to the left, alongside
expansions of the shaded regions that include peaks in the 72.8–73.2 u, 98.8–99.2 u, and 116.8–117.2 u ranges, primarily associated with ions from the illustrated parent molecules. Black
arrows indicate the theoretical monoisotopic mass of negative ions of propanoic (= propionic) acid, immediately adjacent peaks attributed to C6H−, shown in gray.
region. Such levels of accuracy and precision are consistent with
those reported in current geobiological applications of ToF-SIMS
(Leefmann et al., 2013).
Negative ions that originate from succinic acid (C4H5O−
4 ) produce a
clear [M–H]− peak at 117 u in ToF-SIMS spectra from both Baltic and
Bitterfeld ambers (Fig. 3A–B). This peak is much stronger in Baltic
amber relative to Bitterfeld amber, consistent with a greater total
succinate content and the results from FTIR. Not surprisingly, the
117 u peak is even more pronounced in the mass spectrum of pure
monomethyl succinate (Fig. 3C). Related peaks are those associated
with dehydration and decarboxylation of succinic acid, namely the
ions occurring at [(M–H)–H2O]− = 99 u and [(M–H)–CO2]− = 73 u,
which reflect the parent molecules succinic anhydride and propanoic
(= propionic) acid, respectively. The acid anhydride is not well
expressed in the monomethyl succinate mass spectrum, implying that
its presence in amber relates to processes associated with geological
maturation. This is important because the succinic anhydride peak is
greater in both ambers than that of succinic acid, and particularly
intense in the case of Baltic amber (Fig. 3A). We thus consider succinic
anhydride to reflect the degrees of dehydration during maturation of
the two ambers.
The case of propanoic acid is more complicated for several reasons.
Although negative ions of this molecule have been identified in Baltic
amber before (Tonidandel et al., 2009), in nature propanoic acid results
from microbially-assisted decarboxylation of succinic acid esters
(e.g., Whiteley, 1953). Because propanoic acid is equally well represented in the mass spectra of monomethyl succinate as in those of amber
samples, we infer that it originates from secondary fragmentation of
ionized succinic acid during ToF-SIMS analysis, such that our results
provide no conclusive evidence for the presence of native propanoic
acid in amber. Moreover, the ToF-SIMS peaks obtained in the 73 u region are not as clean and unimodal as those attributed to succinic acid
(117 u) and succinic anhydride (99 u), often yielding a second peak at
slightly lower mass (Fig. 3). The exceptional resolution afforded by
ToF-SIMS allows clear differentiation of peaks occurring at 73.02 u and
74.04 u, for which only the latter can be attributed to propanoic acid.
The 73.02 u peak is attributed to C6H−, for which the parent molecule
may be hexatriyne. Because this peak is better expressed in the ambers
relative to the monomethyl succinate standard, it is provisionally associated with photodissociation during resin polymerization. However,
until further study is conducted, we restrict all subsequent considerations to the 73.04 u peak produced by the [M–H]− ion of propanoic
acid.
Despite these complexities, the recognition of three distinct peaks
associated with succinic acid, the acid anhydride, and secondary fragments of parent molecules containing succinate, which may include
an assortment of fenchyl and bornyl succinates known to occur in
both Baltic and Bitterfeld ambers (Yamamoto et al., 2006), should
be considered a powerful diagnostic tool and a novel application of
ToF-SIMS. Moreover, the succinate region of amber ToF-SIMS spectra
also includes peaks at 80, 85 and 97 u (Fig. 3), which correspond to
−
the [M–H]− ions C5H4O−, C4H5O−
2 , and C5H5O2 , respectively. While
the 85 u peak exists in both ambers, and likely represents vinyl acetate,
the 80 and 97 u peaks are much better expressed in the Bitterfeld material. Although the parent molecules for these peaks remain elusive, they
nonetheless assist in differentiating the ambers geochemically.
With respect to the diterpene resin acids, the availability of purified
crystalline standards of key chemical species proves invaluable with
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Fig. 4. Negative polarity ToF-SIMS spectra of the 250–350 u region (left) and expansion of the 295–305 u region (right) for (A) Baltic amber, (B) Bitterfeld amber, (C) dehydroabietic
acid, (D) abietic acid, and (E) communic acid. Structures are given for parent molecules, whereas black arrows indicate peaks at 317 and 333 u corresponding to oxygen additions to
the [M–H]− = 301 u peak. Shaded zones (left panels) indicate the region magnified at right, in which shaded lines indicate the masses of primary molecular ions associated with diterpene
resin acids.
respect to understanding their distribution in amber (Fig. 4). Furthermore, the tricyclic structure of diterpene resin acids confers considerable stability of these molecules towards fragmentation, implying that
molecular ions are commonly preserved in mass spectra (Dethlefs
et al., 1996; Diefendorf et al., 2012). Negative polarity ToF-SIMS spectra
of Baltic and Bitterfeld ambers both exhibit strong [M–H]− peaks at 299
A.P. Wolfe et al. / Review of Palaeobotany and Palynology 225 (2016) 21–32
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Fig. 5. Stable isotopic ratios of Baltic (red) and Bitterfeld (blue) ambers. Raw data (A and C) and probability density functions (B and D) are shown sequentially for the δ13C and δ2H
values obtained from both materials. In (B), the dashed lines and shaded areas are mean and 2 S.D. ranges of values compiled values from Tappert et al. (2013) for compilations of
Miocene–Oligocene ambers (from German coal, Mexico, Dominican Republic, Malaysia and Borneo) compared to Eocene ambers (from Washington State, the Canadian High Arctic
and kimberlite-hosted sediments, as well as Baltic amber but excluding Bitterfeld). In (D), additional axes have been added for inferred δ2Hplant water using a constant fractionation of
−229‰ between amber and environmental water (Wolfe et al., 2012), and a provisional temperature scale based on the modern relationship between δ2H and air temperature at Kraków,
Poland. The dashed line in (D) separates mesothermal from tropical climates in Köppen–Geiger classification.
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and 301 u. The former corresponds to primary molecular ions of
dehydroabietic acid (C20H28O2), while the latter may be attributable to
either abietic or communic acids (both C20H30O2), or a combination of
both. The peaks observed at 303 u are attributable to C20H32O2 diterpene resin acids having a molecular mass of 304 u, for example
dihydroisopimaric acid, 8-abietenic acid, and 8(14)-abietenic acid
(Sodhi et al., 2014). The 301 u peak also produces corresponding
[(M–H) + O]− and [(M–H) + O2]− peaks observed in both ambers
(i.e., 317 and 333 u). Of the diterpene resin acids considered here,
only abietic acid produces secondary peaks associated with oxygenation, and thus it clearly represents an important component of
both ambers. On the other hand, the 299 u peak associated with
dehydroabietic acid is on average three-fold stronger in Bitterfeld
amber relative to Baltic amber, implying considerably greater concentrations of this diterpene resin acid. Although ToF-SIMS spectra of
abietic and communic acids are indistinguishable from each other in
the 295–305 u range (Fig. 4), we have already shown, on the basis of
FTIR, that communic acid is more abundant in Baltic amber.
What emerges from the combined FTIR and ToF-SIMS results is a
geochemical differentiation between Baltic and Bitterfeld ambers that
is consistent between analytical platforms: Baltic amber has a greater
apportionment of succinic and communic acids, whereas Bitterfeld
amber contains more dehydroabietic acid. The latter observation is
entirely consistent with prior results obtained on comparable materials
using gas chromatography–mass spectrometry (GC–MS; Yamamoto
et al., 2006). Thus, from the perspective of organic geochemistry
alone, Baltic and Bitterfeld ambers appear to be compositionally distinct
in a number of subtle yet reproducible ways. In making this statement,
we advocate strongly the advantages of FTIR and ToF-SIMS as analyses
conducted on amber in the solid state, thereby eliminating issues
associated with these materials being only partially soluble in organic
solvents (Mills et al., 1984). For this reason, we disagree with the comment of Anderson and Botto (1993, pp. 1037) that, with respect to
glessite from Bitterfeld (e.g., Fig. 1J) and genuine Baltic amber: “the extent of similarity of these resinites is such, that the validity of continued
distinction between them is, on chemical grounds at least, unjustified.”
From our perspective, we restrict the compositional similarity between
Baltic and Bitterfeld ambers to stating that both clearly belong to Class Ia
resinites, i.e., those containing diterpenes based on labdanoid skeletal
structures in the presence of succinic acid, and a range of associated
alcohols and esters (Anderson et al., 1992).
3.3. Stable isotopes
The carbon stable isotopic compositions of Baltic and Bitterfeld
ambers are virtually identical, yielding means of δ13C = − 23.6 ±
1.0‰ and −23.9 ± 1.7‰, respectively (Fig. 5A). In a general sense, the
δ13C of amber reflects the localized degree of tree ecophysiological
stress at the time of resin production, with greater stress resulting in
less effective isotopic discrimination against 13C (i.e., higher δ13C values;
McKellar et al., 2011). When probability distribution functions are
applied to the δ13C results from Baltic and Bitterfeld ambers (Fig. 5B),
a principal mode is apparent, flanked by two shoulders that presumably
reflect occasions of resin production under alternately luxuriant (most
depleted δ13C values) and stressed (enriched) environmental conditions that deviate from the central modes expressed in both deposits.
These similarities are such that δ13C values do not differentiate the
two ambers. Indeed, the populations of Baltic and Bitterfeld ambers
δ13C values do not yield statistically significant differences under an unpaired t-test (P = 0.27; t = 1.11; d.f. = 109).
Moreover, at the global scale, amber δ13C is also influenced by atmospheric composition with respect to CO2 and O2 partial pressures, yielding a robust secular trend of ~5‰ towards more depleted values over
the past 50 Ma that is superposed upon the localized influences
discussed above (Tappert et al., 2013). This implies that, if Bitterfeld
amber were truly younger (e.g., Miocene–Oligocene) than Baltic
amber (Eocene) as hypothesized, it should produce mean δ13C values
that are about 2‰ more depleted relative to Baltic amber, which is not
observed in the raw data (Fig. 5B). The simplest interpretation of the
close similarity between Bitterfeld and Baltic amber δ13C values is that
they are equivalent in age, despite the compositional differences
borne out of their organic geochemical characterization.
With respect to amber hydrogen stable isotopes (Fig. 5C–D), the situation differs markedly from the carbon results, noting that all δ2H
values were obtained from a sub-set of the exact same samples analyzed
for δ13C. Baltic amber (mean δ2H = −277 ± 22‰) is consistently more
depleted relative to Bitterfeld amber (mean δ2H = −256 ± 9‰). This
difference is highly significant (P b 0.0001; t = 5.28; d.f. = 99). Because
amber δ2H is ultimately modulated by the isotopic composition of
source waters accessed by trees at the time of resin biosynthesis
(McKellar et al., 2008; Wolfe et al., 2012), this result implies that, on average, forests responsible for the formation of Baltic amber exploited
waters that were 20‰ lighter than those involved in the genesis of
Bitterfeld amber. The most parsimonious interpretation of this difference is that, whereas the drainage source for Bitterfeld amber lay well
to the south of the deposit, that of Baltic amber was situated to the
north in Scandinavia. Such a scenario is readily accommodated by the
paleogeography of regions bordering the Eocene North Sea basin
(Wimmer et al., 2009; Fig. 6).
Modern environments provide additional context with which the
δ2H difference between Baltic and Bitterfeld ambers can be appreciated.
For example, in modern Pinus resins sampled between Scotland and
Cyprus, a 20‰ difference in δ2H corresponds to ~7° of latitude (Stern
et al., 2008). Within the modern isoscape of central Europe, a difference
of 20‰ in leaf-water δ2H represents approximately 800 km of latitude
(West et al., 2008). Both of these examples are entirely consistents
with the envisaged paleogeography at the time of amber formation
(Fig. 6). We conclude from the amber δ2H results that forests responsible for the production of Baltic and Bitterfeld ambers accessed source
waters originating from fundamentally different sectors of the Eocene
North Sea drainage: the Scandinavian highlands in the first case, and
the Paratethyan sector of central Europe in the second.
As perhaps the most salient geochemical difference between Baltic
and Bitterfeld amber, the hydrogen isotopic measurements merit further discussion. First, we note that the range of results from Baltic
amber is considerably larger than that obtained from Bitterfeld amber
(Fig. 5C). This suggests that the climate envelope realized during Baltic
amber formation was broader, and by inference longer, than that associated with Bitterfeld amber. Second, although Bitterfeld amber produces a more enriched mean δ2H signature, Baltic amber nonetheless
produces numerous measurements that are equally enriched, resulting
Fig. 6. A proposed paleogeographic scenario for the provenance of Baltic and Bitterfeld
ambers during the middle Eocene. Shaded areas represent the Eocene landmass, whereas
dashed lines are modern coastlines, following Scotese et al. (1989) and Wimmer et al.
(2009). Dark gray regions approximate the loci of amber deposition.
A.P. Wolfe et al. / Review of Palaeobotany and Palynology 225 (2016) 21–32
in a secondary mode in the probability distribution of isotopic values
(Fig. 5D). This observation is in keeping with the entomological record
of Baltic amber, which contains admixtures of taxa with alternately
tropical and boreal ecological affinities (Larsson, 1978; Weitschat,
1997; Weitschat and Wichard, 2002; Weitschat and Wichard, 2010).
However, if our isotopic results are broadly representative of both deposits, we predict that Bitterfeld amber should contain a greater overall
proportion of warm stenothermous arthropods relative to Baltic amber.
29
We await unbiased, abundance-weighted censuses at sufficient taxonomic resolution to test this hypothesis, acknowledging that several
collections are sufficiently rich to undertake this activity systematically.
We further explore paleoclimatic significance of amber δ2H results
by deriving provisional temperature estimates as follows. First, values
of amber δ2H were converted to δ2Hplant water using a constant fractionation of −229‰ between amber and environmental water (Chikaraishi
et al., 2004; Wolfe et al., 2012). Then, using an appropriate modern
Fig. 7. Negative polarity ToF-SIMS spectra spanning the succinate region (70–120 u) for Baltic and Bitterfeld ambers (A–B) and modern resins representative of various conifer families
(C–G). Vertical shading highlights dominant ions, and arrows indicate ions derived from succinic acid and succinic anhydride peaks, as discussed in the text. Due to between-sample
differences in absolute intensities, all spectra have been rescaled to the maximum peak height in the 70–120 u range.
30
A.P. Wolfe et al. / Review of Palaeobotany and Palynology 225 (2016) 21–32
temperature–δ2H relationship from central Europe, we are able to rescale δ2Hplant water as a first approximation of corresponding ambient
temperatures. We use the strong relationship between air temperature
and precipitation δ2H at Kraków, Poland (50.1°N, 19.9°E), which yields
δ2H = 2.6 (temperature)–91.8 (R2 = 0.59; n = 406). This is among
the most intensely sampled European stations in the Global Network
of Isotopes in Precipitation (GNIP) program (IAEA/WMO, 2014). Results
using other densely sampled stations, such as Leipzig (R2 = 0.35;
n = 341) and Berlin (R2 = 0.32; n = 273), yield similar results but
suffer from lower coefficients of determination between δ 2H and
temperature. Resulting δ2Hplant water and temperature estimates are
portrayed as additional axes on the amber δ2H probability distributions
(Fig. 5D). In this model, the mean temperatures associated with Baltic
and Bitterfeld ambers are 17 °C and 25 °C, respectively, flanking the
18 °C threshold of mean annual temperature that distinguishes tropical
and mesothermal climate regimes (Peel et al., 2007). While both
ambers produce a large number of samples with tropical isotopic
signatures, as discussed above, only Baltic amber produces values consistent with temperatures b 15 °C. Of course, we do not consider these
reconstructions to be definitive; they are presented as an exploratory
tool grounded in the premise that the systematics of isotopes in
precipitation during the Paleogene were mechanistically similar to the
modern world, despite differences in precipitation quantity, ambient
temperature, and seasonality (Wolfe et al., 2012). Nonetheless, the
results appear realistic in the sense of their consistency with the
paleoentomological record, particularly that of Baltic amber. Having
now established on geochemical grounds numerous distinctions between Baltic and Bitterfeld amber, the largest remaining question pertains to the botanical origin of these deposits.
4. ToF-SIMS spectra of modern resins and the botanical origin of
succinites
Negative polarity ToF-SIMS spectra of modern conifer resins assist in
elucidating the botanical provenance of Baltic and Bitterfeld amber.
Of the modern taxa analyzed, only Pinus contorta and Sciadopitys
verticillata preserve pronounced peaks associated with negative ions
from succinic acid and its acid anhydride (Fig. 7). As succinic acid is
the defining biomarker for succinite amber varietals including both
Baltic and Bitterfeld ambers (i.e., Class Ia resinites, Anderson and
Botto, 1993), this is an important confirmatory result. Indeed, conifers
of the family Sciadopityaceae have been proposed as a potential source
for Baltic amber based on extensive comparative analyses of FTIR spectra (Wolfe et al., 2009), whereas various Pinaceae have been invoked in
this same regard for well over a century, largely on the basis of the anatomy of woody inclusions (Goeppert and Menge, 1883; Dolezych et al.,
2011). Fossils belonging to both families are present in Baltic amber, although pinaceous elements appear more conspicuous (Weitschat and
Wichard, 2002). Additional ToF-SIMS [M–H]− peaks that are consistently expressed in the succinate region of modern conifer resins include
−
as
those at 73 u (C3H5O−
2 , which must be differentiated from C6H
−
),
and
97
u
(C
H
O
discussed earlier), 80 u (C5H4O−), 85 u (C4H5O−
2
5 5 2 ).
Two peaks are specific to individual species: 79 u (C5H3O−) in
Sciadopitys verticillata and 112 u (C6H8O−
2 ) in Agathis australis. Further
differentiation between the modern resins can be gleaned from the
diterpene resin acid region of their ToF-SIMS spectra (Table 1).
Although we are only beginning to exploit ToF-SIMS as a strategy in
resin chemotaxonomy, and clearly additional analyses are desirable,
the initial results presented here portend considerable potential for
this approach.
The relationships between amber and modern resin ToF-SIMS
spectra can be formalized by hierarchical cluster analysis of the
dominant recurrent peaks listed in Table 1. We applied clustering by
centroid linkage to matrices of Euclidean distances using Cluster and
TreeView software packages (Page, 1996; de Hoon et al., 2004). Raw
data (i.e., intensities normalized to a range of 0–100) were not transformed in any other way prior to clustering. When peaks restricted to
the succinate region are considered (Fig. 8A), only Sciadopitys verticillata
resin clusters with the amber samples, revealing an especially close similarity to the Bitterfeld material. Resins from Agathis, Pinus, Metasequoia
and Podocarpus form a second high order grouping. In a second and
more inclusive analysis, peaks representing molecular ions of diterpene
resin acids (i.e., 299–303 u) were included in addition to those from the
succinate region, yielding a dendrogram where Pinus joins Sciadopitys as
the modern resins with the highest degree of similarity to the ambers
(Fig. 8B). Collectively, these analyses effectively eliminate conifers of
the families Araucariaceae, Cupressaceae, and Podocarpaceae as potential sources for European succinites, leaving either extinct pinaceous or
sciadopityaceous trees as the most viable candidate source plants. Although the clustering exercises (Fig. 8) illustrate the longstanding
Table 1
Relative intensities of dominant [M–H]− peaks observed in the succinate and diterpene resin acid regions of ToF-SIMS spectra from monomethyl succinate, Baltic and Bitterfeld ambers,
and various modern conifer resins. Values are scaled to the maximum peak intensity within each region.
Succinate region
Relative intensities of dominant peaks (maximum peak in 70–120 u range = 100)
Analyte
n
73.04 u
S.D.
85.09 u
S.D.
Monomethyl succinate
Baltic amber
Bitterfeld amber
Agathis australis resin
Metasequoia glyprostroboides resin
Pinus contorta resin
Podocarpus totara resin
Sciadopitys verticillata resin
2
3
3
3
3
3
3
3
25.94
41.83
19.40
73.06
25.07
59.59
25.15
24.46
3.43
9.10
7.02
0.03
5.66
3.62
2.23
2.53
2.28
22.71
24.47
79.81
100.00
95.01
100.00
20.14
0.52
0.93
3.32
17.63
0.00
8.65
0.00
8.02
Analyte
n
299.21 u
S.D.
300.17 u
Monomethyl succinate
Baltic amber
Bitterfeld amber
Agathis australis resin
Metasequoia glyprostroboides resin
Pinus contorta resin
Podocarpus totara resin
Sciadopitys verticillata resin
2
3
3
3
3
3
3
3
5.19
35.76
98.83
12.12
7.49
90.75
4.17
95.96
0.58
9.12
2.03
7.26
1.17
9.46
0.59
7.00
1.84
8.79
26.52
4.42
3.06
25.24
1.85
50.37
97.04 u
7.90
14.93
78.84
27.79
18.06
36.10
17.03
46.19
S.D.
99.02 u
S.D.
117.03 u
3.19
4.20
25.63
6.52
3.12
4.40
6.03
13.10
9.59
100.00
39.14
14.24
7.50
24.31
11.72
26.83
1.33
0.00
9.34
6.21
1.11
5.40
3.74
8.64
100.00
25.00
6.48
5.58
1.72
8.28
1.99
18.12
302.22 u
S.D.
303.24 u
9.10
28.42
29.20
25.84
22.68
21.68
22.67
45.70
0.05
3.96
2.70
2.09
2.40
3.37
3.16
3.24
4.17
46.34
56.50
11.89
14.19
50.87
10.70
91.54
S.D.
0.00
3.29
0.64
1.56
0.20
0.51
0.54
9.51
Diterpene region
Relative intensities of dominant peaks (maximum peak in 298–304 u range = 100)
S.D.
0.69
0.53
2.37
1.91
0.44
1.25
0.57
2.28
301.22 u
S.D.
100.00
100.00
94.63
100.00
100.00
88.02
100.00
79.69
0.00
0.00
5.83
0.00
0.00
20.74
0.00
2.32
S.D.
1.21
14.30
3.29
6.54
3.95
7.69
3.83
12.18
A.P. Wolfe et al. / Review of Palaeobotany and Palynology 225 (2016) 21–32
31
Fig. 8. Cluster analysis dendrograms produced from selected negative polarity ToF-SIMS peaks from amber and modern conifer resins spanning the succinate region (A), and the combined
dominant peaks of the succinate and diterpene resin acid regions (B). Values used in the analysis were first normalized to the highest peak within each region (Table 1), but otherwise
untransformed. Gray insets show distances between the [M–H]− values used in each analysis.
challenge of identifying modern conifer resins that produce an exact
geochemical match to Baltic amber, they nonetheless narrow the
range of potential source trees to those with resin chemistries resembling those of modern Pinaceae and Sciadopityaceae. More importantly
to the primary objectives of the present study, the cluster analysis
supports the conclusion that Baltic and Bitterfeld ambers are compositionally distinct.
5. Conclusions
While there is no doubt that Baltic and Bitterfeld ambers share many
similarities, most notably the presence of succinic acid and a considerable overlap in their faunal assemblages, closer examination using a
multi-pronged geochemical approach has revealed that they differ
consistently from each other with respect to both composition (FTIR
and ToF-SIMS) and paleogeographic provenance (δ2H signatures). At
the same time, the nearly identical δ13C values obtained from large
populations of both ambers strongly suggest that they are coeval in
age, both having been formed in the middle Eocene. The first implication is that the 15–20 Ma age difference between Bitterfeld and Baltic
amber suggested by stratigraphy and geochronology (Ritzkowski,
1997; Knuth et al., 2002) pertains to host strata only and not the
amber itself. Thus, Bitterfeld amber has been pervasively reworked
prior to deposition in the Cottbus Formation at the type locality. Such
reworking should not be viewed as exceptional, given how frequently
amber is encountered in much younger Quaternary sediments across
the region depicted in Fig. 6, albeit in far lesser quantities than the
Bitterfeld deposit. The second implication of our reconstruction concerns the paleoentomological record. It had previously been problematic to reconcile the suggested age difference between Baltic
and Bitterfeld ambers with the 147 arthropod species (arachnids and
insects) tallied by Weitschat (2008) as co-occurrent in both deposits.
This is because the average persistence of insect species is 3–10 million
years (Grimaldi and Engel, 2005). If the proposed synchronization of
both ambers, based on correlation between their respective δ13C values,
is valid, then the degree of faunal overlap becomes trivial, perhaps even
expected. At the same time, while the number of co-occurrent taxa
testifies to decades of dedicated research, it nonetheless represents a
minute fraction of the many thousand species identified from Baltic
and Bitterfeld ambers collectively. Once again, this is entirely in keeping
with our new interpretation: while we consider both ambers as coeval
in age, we suggest they originated from climatically distinct ecosystems
supporting distinct arthropod communities that produced only a partial
degree of faunal overlap (Fig. 5D).
In conclusion, we surmise that Baltic and Bitterfeld ambers are
compositionally distinct and that each originates from different sectors of the Eocene North Sea drainage: Baltic amber from the north
and Bitterfeld from the south. However, on the basis of identical carbon isotopic signatures, we argue that both ambers are broadly contemporaneous with each other, thus facilitating the interpretation of
their paleobiological records. We suggest that the combined geochemical strategies presented here may provide important clues to
the genesis, in both space and time, of additional European succinite
deposits including, but not limited to, those of northern Ukraine,
southern Belarus, and the Scandianvian Peninsula.
Acknowledgments
We thank Alexander Schmidt (University of Göttingen) and Leif
Brost (Swedish Amber Museum, Höllviken) for samples and discussion,
Thomas Stachel (University of Alberta) for access to FTIR facilities, Bruce
McKague (CanSym, Toronto) for diterpene resin acid standards, Robyn
Goacher and Charles Mims (University of Toronto) for assistance with
ToF-SIMS analysis, the Natural Sciences and Engineering Research
Council of Canada for funding our research (Discovery awards to APW
and KM, postdoctoral fellowship to RCM), and two journal reviewers
for insightful comments that substantially improved the paper.
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