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Proc. R. Soc. B
doi:10.1098/rspb.2011.0276
Published online
Insect outbreaks produce distinctive carbon
isotope signatures in defensive resins and
fossiliferous ambers
Ryan C. McKellar1,*, Alexander P. Wolfe1, Karlis Muehlenbachs1,
Ralf Tappert1, Michael S. Engel2, Tao Cheng3
and G. Arturo Sánchez-Azofeifa1
1
Department of Earth and Atmospheric Sciences, University of Alberta, 1-26 Earth Sciences Building,
Edmonton, Alberta, Canada T6G 2E3
2
Division of Entomology, Natural History Museum and Department of Ecology and Evolutionary Biology,
University of Kansas, 1501 Crestline Drive, Suite 140, Lawrence, KS 66049-2811, USA
3
Department of Land, Air and Water Resources, University of California, One Shields Avenue,
Davis, CA 95616, USA
Despite centuries of research addressing amber and its various inclusions, relatively little is known about the
specific events having stimulated the production of geologically relevant volumes of plant resin, ultimately
yielding amber deposits. Although numerous hypotheses have invoked the role of insects, to date these have
proven difficult to test. Here, we use the current mountain pine beetle outbreak in western Canada as an
analogy for the effects of infestation on the stable isotopic composition of carbon in resins. We show that
infestation results in a rapid (approx. 1 year) 13C enrichment of fresh lodgepole pine resins, in a pattern
directly comparable with that observed in resins collected from uninfested trees subjected to water stress.
Furthermore, resin isotopic values are shown to track both the progression of infestation and instances
of recovery. These findings can be extended to fossil resins, including Miocene amber from the Dominican
Republic and Late Cretaceous New Jersey amber, revealing similar carbon-isotopic patterns between visually clean ambers and those associated with the attack of wood-boring insects. Plant exudate d13C values
constitute a sensitive monitor of ecological stress in both modern and ancient forest ecosystems, and provide
considerable insight concerning the genesis of amber in the geological record.
Keywords: mountain pine beetle; Dominican amber; New Jersey amber; stable isotopes
1. INTRODUCTION
The stable isotopic composition of plant carbon has the
capacity to record physiological stress, such as that
induced by insect attack, because 13C discrimination is
reduced under impaired conditions [1]. Although
carbon stable isotopes have been considered from a
range of plant tissues (e.g. [2 –5]), relatively little
attention has been paid to exuded secondary metabolites
[6– 10], despite their excellent potential for preservation
in the fossil record as amber [11,12]. Resin retains its
original stable isotopic composition throughout maturation with a high degree of fidelity [7,10], making
analogies between modern resin and fossil amber tenable.
Within a framework of actualistic palaeontology, we
examined the role of wood-boring insects as a potential
cause for amber production by investigating the isotopic
response of modern resins generated in direct response
to a major insect outbreak event.
Mountain pine beetle (Dendroctonus ponderosae,
Coleoptera: Curculionidae: Scolytinae; hereafter MPB)
is currently devastating western North American forests,
with pronounced economic, ecological and biogeochemical consequences [13]. Climate change (specifically,
warmer winter temperatures) has allowed MPB to infest
extensive tracts of lodgepole pine (Pinus contorta var.
latifolia; figure 1) that are already weakened by multiple
years of drought. MPB larvae over-winter in galleries
excavated in the host tree’s cambium, where they feed
on blue stain fungus (Grosmannia clavigera, Ascomycota).
Adults emerge in late summer, disperse and form mass
attacks guided by combinations of pheromones and volatile
resin constituents released during pioneer attacks [14].
This strategy frequently overcomes the constitutive
(preformed) oleoresin defense system of the host tree
(known as pitch-out), while MPB boring introduces
fungi that act as a food source as galleries develop. Once
galleries and the spread of fungi have girdled the tree,
there is further impairment of both induced (secondary)
resin production and the mechanics of water circulation
in the phloem [15]. Foliage of infested trees first dulls
then yellows and reddens over 1–2 years following a successful MPB attack [13]. By the time foliage of infested
trees becomes visibly impacted on a large scale, MPB
has often dispersed to new hosts.
The example of MPB provides a powerful analogy to
explore d13C patterns in geologically relevant volumes of
plant resins and test hypotheses concerning the origins
* Author for correspondence (rcm1@ualberta.ca).
Electronic supplementary material is available at http://dx.doi.org/10.
1098/rspb.2011.0276 or via http://rspb.royalsocietypublishing.org.
Received 7 February 2011
Accepted 3 March 2011
1
This journal is q 2011 The Royal Society
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2 R. C. McKellar et al. Carbon isotopes in resins and amber
(a)
(b)
Figure 1. Products of infestation in trees and resin. (a) Foliage colour changes owing to MPB attack in west-central Alberta
(photo courtesy J. Cooke, University of Alberta). (b) Pitch-out tubes at base of MPB-infested lodgepole pine trunk (inset:
detail of single tube; scale bar, 1 cm).
of amber deposits. In the course of developing a database
of resin and amber stable isotopic compositions for the
major world deposits, sufficient material was acquired
and analysed to examine in detail two major deposits
that are putatively associated with insect damage:
Dominican and New Jersey (NJ) amber.
Abundant deposits of fossiliferous Miocene [16]
amber from the Santiago region of the Dominican Republic have been attributed with confidence to the
leguminous tree genus Hymenaea [17], for which
modern taxa produce large volumes of resin in response
to injury [11,17]. Dominican amber is considered to
have formed as a result of catastrophic events in an established moist tropical forest [18]. Hurricane damage has
been invoked as the most likely factor leading to the massive production of resin within this forest [19,20],
ultimately producing the amber deposit. The large
number of bark beetles preserved as inclusions within
the amber [18,21] suggests an alternative cause, and
provide a unique opportunity for comparison with
modern resins produced as a direct result of infestation
by scolytine beetles like MPB.
Late Cretaceous (Turonian) amber from NJ has been
associated with both forest fires and the activities of
wood-boring beetles with associated pathogenic fungi
[22]. Although amber from this deposit is chemically
homogeneous and potentially originates from a single
source conifer taxon, three distinct visual categories
emerge from the census of thousands of specimens examined [22]. Approximately 70 per cent of specimens are
turbid, and contain the majority of insect inclusions as
well as copious wood particulate material directly comparable with that produced by insect boring [22]. Most
remaining specimens comprise small droplets that are
optically transparent, but a third category includes rare
specimens of opaque amber having a frothed appearance
from microscopic gas inclusions associated with burning
[22]. Only the opaque amber can be related directly to
fire events given that, beyond its distinctive bubble-rich
appearance, inclusions of fusainized wood are also
found within. The presence of fusainized plant remains
within the surrounding clay has led to the suggestion
Proc. R. Soc. B
that the entire amber deposit resulted from forest fires
[22], but it remains unclear whether this sedimentological
association is the result of taphonomic processes [23,24].
2. MATERIAL AND METHODS
(a) Samples
We sampled lodgepole pine resins in an experimental forest
plot near Grande Prairie, Alberta, Canada (558050 1300 N,
1188120 5400 W), in both 2008 and 2009. Lodgepole pine
resins were collected from infested and uninfested trees in
close proximity in order to rule out the effects of microhabitat variability. Resin was collected from fresh flows on the
lower trunk or from freshly trimmed branches. For NJ and
Dominican amber, isotopic measurements were made from
marginal cuttings of barren and insect-bearing specimens,
avoiding bubbles and all particulate inclusions.
(b) Methods
Established methods were employed for carbon stable isotopic
measurements and amber palaeoentomological investigations
[8,25]. All isotopic data are reported in delta notation
(d13C) relative to VPDB (Vienna PeeDee Belemnite) with a
precision of +0.1 per mille. Modern resins were first heated
(508C) for 12 h under vacuum to remove the most volatile
resin components and environmental water. Supplementary
experiments with modern and subfossil Hymenaea resin
showed no measurable fractionation associated with heating.
All statistical differences between populations of resin and
amber d13C values were assessed using Welch two-sided
t-tests with 95 per cent confidence intervals. Mean values
for each sample population are reported +1 standard deviation. All results and sample descriptions are available online
as electronic supplementary material (table S1).
3. RESULTS AND DISCUSSION
(a) Mountain pine beetle attack and resin carbon
isotopes
During the 2008 MPB outbreak expansion, we observed
d13C enrichment in resins from trees in the earliest stages
of infestation (known as green attack), compared with
adjacent healthy control trees (figure 2). The difference
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Carbon isotopes in resins and amber
δ13C (VPDB) ‰
–30 –29 –28 –27 –26 –25 –24 –23 –22 –21 –20
modern Pinus contorta resins
MPB 2008 control trees
–28.2 ± 0.68‰
MPB 2008 green attack
–27.0 ± 0.91‰
MPB 2009 control, surviving trees
–28.0 ± 0.65‰
MPB 2009 infested, surviving trees
–28.2 ± 0.80‰
MPB 2009
MPB 2009
–23.2‰ red attack
yellowing –26.4 ± 0.22‰
root-bound nursery trees
–26.9 ± 0.81‰
Hymenaea resins, copal and amber
modern Hymenaea courbaril resin
–28.3 ± 0.71‰
sub-recent Colombian copal
R. C. McKellar et al.
3
by MPB attack can thus be induced rapidly by water
stress alone, supporting the notion that phloem interruption is the most likely mechanism mediating isotopic
enrichment in both cases.
Additional lodgepole pine resins from the Grande
Prairie test plot were collected in 2009, following a relatively harsh winter and late spring that slowed MPB
expansion. Surviving infested trees produced new defensive resins that registered a return to pre-infestation
values (mean d13C ¼ 228.2 + 0.80‰), indistinguishable
from either 2008 or 2009 healthy control trees (figure 2).
Both surviving populations are in stark contrast with trees
developing visible yellow foliage (mean d13C ¼ 226.4 +
0.22‰). By far the most enriched sample was from a
tree entering red attack (d13C ¼ 223.2‰), which provides an important end-member because few red attack
specimens retain the capacity to exude new resin. These
analyses demonstrate that the trajectory of MPB outbreak
in lodgepole pine resin is isotopically discernible in real
time: MPB attack induced positive d13C excursions averaging 1.1 per mille that intensify if the tree eventually
succumbs, but are rapidly reversed in cases of rebound.
–27.5 ± 0.55‰
barren Dominican amber
–26.0 ± 1.01‰
Dominican amber,
insect inclusions
–24.9 ± 1.54‰
Dominican amber,
boring-beetle inclusions
–23.6 ± 1.25‰
New Jersey Cretaceous amber
barren
–22.3 ± 1.19‰
turbid
–21.9 ± 0.98‰
opaque
–20.6 ± 0.37‰
–30 –29 –28 –27 –26 –25 –24 –23 –22 –21 –20
δ13C (VPDB) ‰
Figure 2. d13C values of modern resins and amber analogues.
The mean and standard deviation of each sample population
are shown beneath the data. Colours correspond to inferred
levels of tree ecophysiological stress, based on consensus of
the extent of observed insect damage and attendant mean
exudate d13C value within each population, ranging from
healthy (dark green) to near death (red).
between 2008 newly infested green attack trees (mean
d13C ¼ 227.0 + 0.91‰) and uninfested control trees
(mean d13C ¼ 228.2 + 0.68‰) is highly significant (p ¼
0.018), with resins from infested trees consistently
enriched relative to neighbouring healthy trees subjected
to identical microclimatic conditions. In one tree where
superimposed resin flows could be dissected sequentially
to capture conditions prior to and during MPB attack,
resin d13C values shifted from 228.2 (pre-2008) to
226.7 per mille (2008). Freshly produced resins from
uninfested lodgepole pine trees that had been subjected
to water stress for four months since excavation and bagging of the root mass yielded mean d13C ¼ 226.9 + 0.81
per mille. A similar isotopic enrichment to that induced
Proc. R. Soc. B
(b) Dominican amber and Hymenaea resins
Modern
Hymenaea
courbaril
resin
(mean
d13C ¼ 228.3 + 0.71‰) and subfossil Hymenaea copal
(mean d13C ¼ 227.5 + 0.55‰) constrain the isotopic
composition of resins derived from wounding of otherwise healthy trees. Values from Dominican amber are
enriched relative to these baseline values (figure 2), and
progress from specimens barren of insects (mean
d13C ¼ 226.0 + 1.01‰) to those with inclusions of
generalist insects unrelated to infestation (mean
d13C ¼ 224.9 + 1.54‰) and finally to amber containing
obligate tree-boring beetles of the subfamilies Scolytinae
and Platypodinae (Curculionidae, figure 3b; mean
d13C ¼ 223.6 + 1.25‰). The latter population’s isotopic composition is significantly different (p ¼ 0.011)
from that of other Dominican amber specimens and
Hymenaea resins.
When compared with the MPB dataset, the distribution of d13C values observed in Dominican amber
strongly suggests that many of the pieces containing
boring beetles were produced at times of increased tree
stress, probably as a direct result of insect attacks. In a
survey of 2924 Dominican amber arthropod inclusions,
115 platypodine beetles were observed [18], while
others have noted that scolytines comprise approximately
7 per cent of insect inclusions within the deposit [26].
Wood-boring beetles are thus a prominent group within
the entombed fauna, and their activities may have contributed meaningfully to amber production. For
example, remaining barren Dominican amber specimens
present intermediate d13C values relative to Hymenaea
resins produced as a result of mechanical damage and
those containing fossil insects (figure 2). This suggests
that mechanical injury alone was insufficient to produce
the isotopic compositions observed in the latter
population.
(c) New Jersey amber
All three visual classes of NJ amber (figure 3c) were analysed isotopically, revealing a progression of d13C
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4 R. C. McKellar et al. Carbon isotopes in resins and amber
(b)
(a)
(c)(i)
(ii)
(iii)
(d)
Figure 3. Products of infestation in amber. (a) Ophrynopus peritus Engel, an orussid wasp in Dominican amber, which attacks
wood-boring beetles. (b) Cenocephalus quasiexquisitus Davis & Engel, a platypodine boring beetle in Dominican amber with
wood particulates. (c) NJ amber physical classes: (i) clear amber droplet, (ii) turbid amber with multiple flows and (iii)
opaque amber with weathering rind. (d) Boreobythus turonius Engel & Grimaldi, a scolybethid wasp that attacks wood-boring
beetles, shrouded by turbid NJ amber. All scale bars, 1 mm.
enrichment between fragments of transparent (mean
d13C ¼ 222.3 + 1.19‰), turbid (mean d13C ¼ 221.9 +
0.98‰) and opaque (mean d13C ¼ 220.6 + 0.37‰)
amber. This distribution indicates that many amber specimens containing insect inclusions and plant debris were
formed from resins produced under conditions of pronounced ecophysiological stress, thus supporting the
association between insect-mediated damage and defensive
resin production. The further enrichment observed in
opaque NJ amber is consistent with the loss of isotopically
depleted volatile moieties during fire events, as implied
independently by the record of bubble and fusain
inclusions.
Although the direction and amplitude of these isotopic
shifts are consistent with the MPB results, we note that
the baseline d13C of NJ amber is consistently enriched
(by approx. 5‰) relative to modern lodgepole pine
resins. We suggest that combinations of source tree
metabolism, fundamental differences in the Cretaceous
carbon cycle [27] and slight degrees of long-term resin
diagenesis explain these differences, but that the original
fingerprint of tree physiological stress is nonetheless
recorded by amber isotopic compositions. Despite these
potential influences, the distribution of d13C values
retains a clear pattern within the deposit, which is directly
comparable with the pattern produced by MPB
infestation.
One limitation of invoking wood-boring beetles in the
genesis of NJ amber is their relative scarcity as inclusions
within the amber. In a survey of 1032 NJ amber arthropod inclusions [22], 37 identifiable beetles were
recovered, two of which belonged to live-wood-boring
groups (Cupedoidea). Scolytinae and Platypodinae are
known from Early Cretaceous Burmese amber, and Scolytinae are also present in Early Cretaceous Lebanese
amber [26], but both groups have yet to be recovered
Proc. R. Soc. B
from NJ amber. While the presence of obligate beetle
parasitoids (such as wasps of the families Megalyridae,
Orussidae and Scolebythidae [22,28]) provides indirect
evidence for wood-boring beetles, at present, the abundance of wood particulates within turbid amber
provides the strongest independent evidence for woodboring (figure 3d ). These indirect inferences are now
augmented convincingly by the results of our isotopic
analyses.
4. CONCLUSIONS
The direction and magnitude of resin d13C values from
Dominican and NJ amber mirrors that expressed in
MPB-infested pine forests (figure 2). In each instance, a
strong case can be made for the association of insects to
physiological stress in host trees, which in turn has the
potential to become recorded in the d13C of resins synthesized shortly after attack. As a corollary, not all
ambers preserving inclusions of wood-boring insects
have strongly enriched isotopic values, as expected given
opportunities for recovery that may equally be registered
by d13C. For example, although a 1.3 per mille increase
in mean d13C values was observed in Dominican amber
samples containing boring beetles, there is substantial
overlap with specimens lacking direct evidence of insect
attack. We envisage that much of the isotopic variability
documented originates in water stress mediated by insects
and their fungal symbionts, which affects both the quantity and quality of defensive resins. Water stress results in
the progressive enrichment of the carbon-isotopic composition of photosynthetic products by reducing stomatal
conductance, and hence the ability to discriminate against
13
C [1]. We have shown here that this isotopic effect
is propagated to secondary metabolites. Boring insect
attacks induce the same general pattern of isotopic
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Carbon isotopes in resins and amber
enrichment produced by drought alone, because the
attacks interfere with water transport in host trees. As a
corollary, pre-conditioning by drought clearly has been
a factor in the magnitude and severity of current MPB
outbreaks [13].
In modern and ancient ecosystems, d13C values from
tree resins constitute a novel early indicator of insect outbreak events. This tool can be applied to ambers as old
as the Mesozoic, and is expressed in both conifer and
angiosperm resins. More intensive work is required to disambiguate the isotopic effects specific to insects and plant
water stress, but our work addressing MPB suggests that
resin composition responds faster than foliage colour,
and hence can diagnose the severity of an outbreak more
rapidly than satellite remote sensing, with implications
for management. The analogies with both Cretaceous
and Miocene ambers provide new lines of evidence supporting the active participation of insects in the original
production of resins now preserved as amber, and correlate
nicely with the diversity of wood-boring insects and
their associates recovered from these deposits. Carbon
stable isotopic ratios in fossil resins, together with detailed
palaeoentomological
consideration
of
associated
inclusions, can be exploited to better understand this
important dimension of plant–insect coevolution.
The authors thank J. Chacko, B. D. E. Chatterton, S. Gibb, W. O.
Hobbs, L. Leighton, J. Newman, D. Shpeley, F. A. Sperling and
Alberta Sustainable Resource Development for assisting with
our research, and reviewers for their improvements. We thank
M. Bryman, J. Cooke, A. D. McDonnell and Alberta
Sustainable Resource Development for providing access to
photographs, and for providing figure 1a. Funding was
provided by the Natural Sciences and Engineering Research
Council of Canada, the Alberta Ingenuity Fund, and the US
National Science Foundation.
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