Content uploaded by Thomas Tütken
Author content
All content in this area was uploaded by Thomas Tütken on Oct 21, 2015
Content may be subject to copyright.
ARTICLE
Received 19 Oct 2014 |Accepted 7 Aug 2014 |Published 13 Oct 2015
Isotopic ordering in eggshells reflects body
temperatures and suggests differing
thermophysiology in two Cretaceous dinosaurs
Robert A. Eagle1,2,3,4, Marcus Enriquez1, Gerald Grellet-Tinner5,6, Alberto Pe
´rez-Huerta7, David Hu2,
Thomas Tu¨tken8, Shaena Montanari9, Sean J. Loyd1,10, Pedro Ramirez11, Aradhna K. Tripati1,3,4,12,
Matthew J. Kohn13, Thure E. Cerling14, Luis M. Chiappe15 & John M. Eiler2
Our understanding of the evolutionary transitions leading to the modern endothermic state of
birds and mammals is incomplete, partly because tools available to study the thermo-
physiology of extinct vertebrates are limited. Here we show that clumped isotope analysis of
eggshells can be used to determine body temperatures of females during periods of ovulation.
Late Cretaceous titanosaurid eggshells yield temperatures similar to large modern endo-
therms. In contrast, oviraptorid eggshells yield temperatures lower than most modern
endotherms but B6°C higher than co-occurring abiogenic carbonates, implying that this
taxon did not have thermoregulation comparable to modern birds, but was able to elevate its
body temperature above environmental temperatures. Therefore, we observe no strong
evidence for end-member ectothermy or endothermy in the species examined. Body tem-
peratures for these two species indicate that variable thermoregulation likely existed among
the non-avian dinosaurs and that not all dinosaurs had body temperatures in the range of that
seen in modern birds.
DOI: 10.1038/ncomms9296
1Department of Earth, Planetary, and Space Sciences, University of California, Los Angeles, California 90095, USA. 2Division of Geological and Planetary
Sciences, California Institute of Technology, Pasadena, California 91125, USA. 3European Institute of Marine Sciences (IUEM), Universite
´de Brest, UMR 6539,
Rue Dumont D’Urville, 29280 Plouzane
´, France. 4Natural History Museum of Denmark, University of Copenhagen, Copenhagen K DK-1350, Denmark.
5Orcas Island Historical Museums, Eastsound, Washington 98245, USA. 6CONCIET, Anillaco, Argentina. 7Department of Geological Sciences, University of
Alabama, Tuscaloosa, Alabama 35487, USA. 8Institute of Geosciences, University of Mainz, Johann-Joachim-Becherweg 21, Mainz 55128, Germany.
9Department of Ecology, Evolution, and Environmental Biology, Columbia University, New York, New York 10027, USA. 10 Department of Geological Sciences,
California State University, Fullerton, California 92831, USA. 11 Department of Geosciences and Environment, California State University, Los Angeles,
California 90032, USA. 12 Department of Atmospheric and Oceanic Sciences, Institute of the Environment and Sustainability, California NanoSystems
Institute, University of California, Los Angeles, California 90095, USA. 13 Department of Geosciences, Boise State University, Boise, Idaho 83725, USA.
14 Department of Geology and Geophysics, University of Utah, Salt Lake City, Utah 84112, USA. 15 Natural History Museum of Los Angeles County, Los
Angeles, California 90007 USA. Correspondence and requests for materials should be addressed to R.A.E. (email: rob.eagle@gmail.com).
NATURE COMMUNICATIONS | 6:8296 | DOI: 10.1038/ncomms9296 | www.nature.com/naturecommunications 1
&2015 Macmillan Publishers Limited. All rights reserved.
Was non-avian dinosaur physiology similar to modern
reptiles, which are ectotherms having body temperatures
controlled by exchange of heat with the environment, or
to modern endotherms such as mammals and birds that maintain
high and stable body temperatures through metabolic heat
production and strict thermoregulation1–9?Endothermsare
organisms that utilize internally generated heat to maintain their
body at metabolically favourable temperatures. Ectotherms are
organisms that have small or negligible internal heat production
and rely on heat from the environment to reach metabolically
favourable body temperatures. Alternatively, did some non-avian
dinosaurs have physiological states intermediate between
ectotherms and modern birds, conditions that have been termed
basoendothermy or mesothermy10,11 and have similarities to extant
speciessuchaslamnidsharksandmonotremes
11? Basoendothermy
has been defined as an endotherm with a body temperature of
o35 °C (ref. 10). Mesotherms have been described as organisms
that rely on metabolic heat to raise their body temperatures above
ambient temperature, but that do not defend a thermal set point as
do endotherms11. An intermediate thermophysiology has also been
hypothesized for basal, pre-modern birds12,13.
Body temperatures are not an unambiguous determinate of
physiology as, for example, animals with low metabolic rate can
have elevated body temperature as a result of high body mass
and increased capacity to retain heat; a feature that is sometimes
termed gigantothermy, as seen in extant species such as
leatherback turtles14. Even within endotherms a wide range of
physiological states exists, which can be reflected in body
temperature10,15,16. Mesothermy in dinosaurs is supported by a
recent theoretical study11 and highlights that the debate on
dinosaur thermophysiology is not a question of a simple ‘warm-
blooded’ versus ‘cold-blooded’ dichotomy. Despite these
complexities, body temperature determinations for extinct
species place new constraints on physiology that were not
previously possible, and allow us to test the predictions made by
physiological models and growth rate analysis17,18.
Here we constrain the body temperatures of extinct vertebrates
from the analysis of proportions of multiply-substituted
(13C–18O-bearing; ‘clumped’) carbonate in fossil eggshells. The
analysis of abundances of multiply-substituted, 13C–18O-bearing
molecules in carbonates is an emerging approach to constrain
mineral formation temperatures19,20. Eggshells mineralize in the
lower oviduct and so their isotopic composition should reflect
body core temperatures of females during ovulation. In practice,
the parameter measured is the D
47
value, which refers to the
abundance of mass-47 CO
2
(13C18O16O) in gas liberated by
phosphoric acid digestion of carbonates compared with the
abundance in reference gases of known isotopic composition CO
2
(refs 20–22). We have previously shown that D
47
measurements
on CO
2
derived from carbonate moieties in biogenic phosphate
minerals are correlated with the body temperature of the host
animal16. Therefore, it is possible to reconstruct the body
temperature of extinct vertebrates based on the measurement of
isotopic ordering in fossil tooth enamel, but less reliably in the
more readily diagenetically altered bone and tooth dentin17,18.
CaCO
3
eggshells are another target for body temperature
reconstructions and potentially allow determinations of body
core temperatures in animals where enough well-preserved
bioapatite material is not available. In addition, the stable
isotope composition of eggshells may also record information
on the environments in which an animal lived and their diet23–25.
Results
Clumped isotope data for modern eggshells. Measurements
were carried out on a suite of modern calcite and aragonite
eggshells comprising specimens from 13 different bird species and
9 reptiles to establish the ability of D
47
measurements to distin-
guish between the body temperature of known endothermic and
ectothermic animals. The data are presented in Table 1 and Fig. 1
show that this is clearly achievable, with D
47
-derived temperatures
from bird and reptile eggshells mirroring known body tempera-
ture differences. In reptiles with small fluctuations in body tem-
perature (for example, Galapagos tortoise), we also find that the
average body temperature of the organism is accurately recorded
by the eggshell. In organisms such as crocodilians that can exhibit
more variability in body temperatures, we assumed ovulation
occurred during the warm season given what is reported for
survivorship of hatchlings in crocodilians and some other reptiles
(Supplementary Tables 1, 5 and 6). A caveat is that reptile body
temperatures fluctuate daily and also vary depending on size and
environment and thus the timing and temperature of eggshell
mineralization may not be well constrained. Nevertheless, D
47
temperature determinations for reptiles are plausible average body
temperatures for adults based on instrumental measurements
(Table 1; Supplementary Table 1; Fig. 1) and are clearly resolved
from isotopic temperatures derived from bird eggshells (Table 1).
Eggshells from smaller bird species known to have elevated
metabolic rates and body temperatures also yield higher D
47
-
derived temperatures compared with large flightless birds that
tend to have lower body temperatures (Table 1).
The application of a range of different temperature calibrations
to modern eggshells produces results that match well with
instrumental data on body temperatures. Using the calibration of
Ghosh et al.19, the eggshell D
47
-derived temperatures ranging
from 37 to 45 °C for different bird species are similar to directly
determined bird body temperatures of 38–43 °C (Table 1;
Supplementary Note 2). Similar values of 36–45 °C are obtained
using a calibration derived from a compilation of data generated
from various biogenic carbonates in the Caltech lab (Table 1;
Supplementary Note 2)26, as well as other calibrations as
described in the Supplementary Note 2.
We show in Fig. 1 and Supplementary Note 2 that the slopes of
a regression through the eggshell data cannot be statistically
distinguished from both the inorganic calcite calibration and
biogenic data compilation generated at Caltech19,26. For
completeness, D
47
-based temperatures are estimated using four
different calibrations (Supplementary Tables 1–3), although in
the main text and figures we favour application of the Caltech
biogenic calibration because it is based on the largest data set and
therefore has lower uncertainty26. While it would be possible to
use the eggshell-specific calibration, it is less data-rich and has
uncertainties associated with not knowing precisely the expected
body temperatures at which ectotherms mineralize their eggshells,
and as a result is less constrained than the Caltech biogenic
calibration (Supplementary Note 2).
Preservation of fossil eggshells. Fossil eggshells likely have a
higher potential for preservation than dentin and bone; high
porosity, small crystal sizes and high protein and water contents
(c. 50%) in the latter two materials/tissues tend to lead to massive
recrystallization and isotopic resetting during fossilization27. This
contrasts with the rigid CaCO
3
-dominated eggshells that many
organisms produce. For example, chicken eggshells are 95–97%
CaCO
3
and therefore comprise much lower levels of organic
matter than bone and dentin28. As geochemical preservation
represents a key uncertainty in our analysis, here we have taken
the approach of extensively characterizing each specimen using a
hierarchy of approaches. First, a comparative analysis of the
stable isotope and trace-element composition of the eggshell and
associated carbonates, including diagenetically altered bone and
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9296
2NATURE COMMUNICATIONS | 6:8296 | DOI: 10.1038/ncomms9296 | www.nature.com/naturecommunications
&2015 Macmillan Publishers Limited. All rights reserved.
authigenically formed spar calcite, is used with the reasoning that
unaltered primary eggshells will usually be different in
composition from coexisiting carbonates from other origins
(Fig. 2; Supplementary Figs 4–6). Second, we examined thin
sections of specimens using transmitted light microscopy and
eggshell fragments using scanning electron microscopy to
confirm the retention of primary growth textures and to look
for diagenetic features (Fig. 3; Supplementary Note 3;
Supplementary Figs 1–3). We also carried out electron
backscatter diffraction (EBSD) analysis to examine specimens
for changes in calcite crystallographic orientation associated with
eggshell recrystallization (Figs 4–5; Supplementary Fig. 7).
On the basis of these analyses, we found specimens exhibited a
range of preservation states. We characterized specimens to be
‘apparently well preserved’ if we had no reason to reject them as
diagenetically altered, ‘moderately preserved’ if geochemical
evidence suggested they are well preserved but petrographic
analysis suggested some evidence of alteration, ‘poorly preserved’
when clear evidence of alteration existed by any criteria and,
finally, ‘of uncertain preservation’ where there was not enough
information to judge, for example, because it was not possible to
compare diagenetic phases and potentially preserved fossil
material due to the availability of samples (Table 2;
Supplementary Note 3).
The fossil eggshells that we examined were collected from
Upper Cretaceous formations from three main regions: (1) the
Campanian and Maastrichtian Djadokhta (Ukhaa Tolgod and
Bayn Dzak sites) and Nemegt (Bugin Tsav site) Formations,
Nemegt Basin, Mongolia29; (2) the Upper Cretaceous Anacleto
Formation at Auca Mahuevo, Neuque
´n Province, Argentina30; (3)
the Maastrichtian Rousset and Roques Hautes sections, Provence
Basin, France31,32. Eggshells from the Late Cretaceous of
Montana, Spain, Rio Negro in Argentina and a second eggshell
morphotype from the Nemegt Basin of Mongolia were less
constrained taxonomically and so are not discussed in detail here
but are described in Supplementary Note 3. The taxonomic
identity of the oviraptorid theropod and titanosaurid sauropod
eggshells from Mongolia and Argentina, respectively, was
determined on the basis of in situ embryos30,33, whereas the
taxonomic identity of French eggshells is inferred based on
eggshell morphology and proximity to other fossil remains in the
same strata31. Therefore, the eggshells from the Provence basin
are designated ‘putative titanosaurid’.
Oviraptorid eggshell specimens from the Djadokhta and
Nemegt Formations showed generally good preservation based
on petrographic observations, with most confirmed oviraptorid
eggshells showing little or no evidence for alteration by inspection
of thin sections or eggshell fragments (Supplementary Note 3;
Supplementary Fig. 1). EBSD analysis showed no evidence for
recrystallization of these specimens and also showed the preferred
crystallographic orientation of the calcite caxis is present, but it
did suggest some deformation of calcite crystals, possibly due to
compression of the eggshells on burial (Fig. 5). It is not clearly
known what impact this type of crystal deformation could have
Table 1 | Stable isotope data from modern eggshells. Data are compared with published calibrations in Fig. 1.
Species Common name # n
analyses*
d13C
eggshell
%, V-PDB
d18O
eggshell
%, V-PDB
D
47
%, ARFw
D
47
1 s.e.
D
47
Te m p .
(oC)
inorganic
eq.z
D
47
Temp.
(oC)
biogenic
eq.y
1 s.e. Temp.
(oC)
expected||
d18O
water
%, V-SMOWz
Pipilo erythrophtalmus Eastern towhee 2 13.9 4.6 0.622 0.002 45.4 45.3 0.6 43.3 1.6
Sayornis phoebe Eastern phoebe 1 13.6 1.1 0.625 0.012 44.7 44.4 3.4 43.3 5.0
Vanellus miles Masked lapwing 3 3.9 2.8 0.650 0.005 38.5 37.5 1.3 42.0 2.0
Gallus gallus
domesticus
Domestic
chicken
2 3.3 4.6 0.636 0.008 41.9 41.3 2.2 41.5 0.9
Odontophorus
melanotis
Wood quail 2 3.1 1.2 0.638 0.005 41.4 40.8 1.4 41.5 4.2
Irena puella Asian fairy
bluebird
28.7 0.6 0.640 0.003 40.9 40.2 0.8 41.0 4.7
Columba livia Rock pigeon 4 3.0 1.7 0.637 0.003 41.7 41.1 0.8 41.0 3.8
Cathartes aura Turkey vulture 2 9.5 4.5 0.650 0.011 38.5 37.5 2.9 39.9 0.3
Struthio camelus Common ostrich 3 12.6 3.9 0.643 0.002 40.2 39.4 0.5 39.1 1.3
Cygnus atratus Black swan 4 5.1 11.3 0.646 0.013 39.5 38.6 3.5 39.0 6.3
Tyto alba Barn owl 4 16.1 3.1 0.638 0.012 41.4 40.8 3.3 38.7 2.3
Dromaius
novaehollandiae
Emu 3 8.3 14.3 0.655 0.007 37.3 36.2 1.8 38.0 9.8
Spheniscus
magellanicus
Magellanic
penguin
511.4 7.5 0.643 0.011 40.2 39.4 3.0 37.8 2.4
Varanus gouldii Monitor lizard 2 13.6 3.0 0.674 0.046 33.0 31.3 11.0 33.5–36.3 0.6
Geochelone radiata Radiated
tortoise
314.7 5.4 0.666 0.010 34.8 33.3 2.5 31–34 1.4
Alligator
mississippiensis
American
alligator
313.4 16.6 0.681 0.001 31.4 29.5 0.2 29–33 13.4
Crocodylus acutus American
crocodile
412.5 3.8 0.674 0.007 33.0 31.3 1.8 29–33 0.2
Chelonoidis nigra Galapagos giant
tortoise
216.5 8.6 0.668 0.007 34.3 32.8 1.8 28–32 4.7
Podocnemis sp. Turtle 4 14.5 5.4 0.680 0.007 31.6 29.8 1.7 28–32 2.1
Pantherophis guttatus Corn snake 4 10.4 4.6 0.679 0.009 31.8 30.0 2.2 27–29 1.2
Crocodylus niloticus Nile crocodile 3 9.2 2.5 0.689 0.007 29.6 27.6 1.7 27–28 0.4
Heloderma horridum
exasperatum
Beaded lizard 1 8.9 0.2 0.673 0.009 33.2 31.5 2.3 26–30 2.3
*Represents the number of distinct extractions of CO
2
from a sample that is then purified and analysed. In each case just one eggshell specimen was analysed. s.d. is reported if only two analyses were
made.
wValues given on the ‘absolute reference frame’ or ARF20.
zCalculated using the inorganic calibration of Ghosh et al.19 (Equation (2); Supplementary Note 2).
yCalculated using a compilation of published biogenic calibration (Equation (3), Supplementary Note 2) data collected in the Caltech laboratories and synthesized by Eagle et al.26. See Supplementary
Note 2 for more information on how this calibration was derived.
||From observations of modern animals—sources listed in Supplementary Note 2 and Supplementary Tables 5 and 6.
zd18O of body waters calculated using published mineral-specific equations and using the D
47
(Biogenic) temperature. All eggshells are calcite, except for turtle and tortoise eggshells that are aragonite.
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9296 ARTICLE
NATURE COMMUNICATIONS | 6:8296 | DOI: 10.1038/ncomms9296 | www.nature.com/naturecommunications 3
&2015 Macmillan Publishers Limited. All rights reserved.
on clumped isotope signatures. Although this feature is wide-
spread in the oviraptorid eggshells examined here, measured
clumped isotope values do not show evidence for extensive bond
reordering, which would be expected to yield significantly lower
D
47
values than would be compatible with plausible body
temperatures and the values measured (Table 2). Therefore, we
continued to consider these specimens apparently well preserved,
with the caveat that the impact of compression is not fully
understood. The majority of eggshells from the Djadokhta
Formation at Ukhaa Tolgod are geochemically distinct from
abiogenic nodular carbonates, bone and spar calcites recovered
from the same site, indicating that pervasive diagenesis of
carbonates had not occurred. Two eggshells from Ukhaa Tolgod
were considered poorly preserved, as their isotopic composition
approached that of known diagenetic phases such as sparry
calcites and altered bone (Supplementary Table 3; Supplementary
Fig. 4). Ukhaa Tolgod morphotype 2 eggshells were considered of
uncertain preservation, as petrographic analysis suggested that
they had areas of dissolution in the main eggshell structure that
was much more extensive than in the oviraptorid specimens from
the same site, but geochemically they were also distinct from
diagenetic phases (Fig. 2; Supplementary Fig. 1; Supplementary
Note 3). Four specimens from the Bayn Dzak and Bugin Tsav
localities were considered of uncertain preservation as they had
similar isotopic composition as diagenetic phases from Ukhaa
Tolgod, although we note that, as they are from a different
locality, this comparison may be less instructive. It is possible that
primary and diagenetic carbonate phases could be fortuitously
similar to one another in composition, and therefore our
approach to excluding data is a conservative one.
Auca Mahuevo titanosaurid specimens from different strati-
graphic horizons are variably preserved based on geochemical,
petrographic and EBSD analysis (Figs 2–4; Supplementary Note
3; Supplementary Figs 2 and 5). Notably, EBSD analysis revealed
complete recrystallization of some layer-2 eggshells and good
preservation of layer-4 eggshells (Fig. 5). Layer-4 eggshells are
therefore considered apparently well preserved by our criteria, but
layer-2 specimens of poor preservation.
Rousset level-A eggshells are geochemically distinct from soil
carbonates and the other eggshells from this basin, indicating
differential preservation (Supplementary Figs 3 and 6). In this
case EBSD analysis was instructive, as Rousset level-A eggshells
were found to be completely recrystallized with little of the
normal biological control of crystallographic orientation obser-
vable (Fig. 5; Supplementary Fig. 7). Eggshell specimens from
Roques Hautes in the Provence Basin are considered to be of poor
preservation as microcharacterization showed evidence for
secondary carbonate infilling of pore canals, and geochemically
the eggshells had similar composition to contemporaneous soil
carbonate nodules (Fig. 2; Supplementary Figs 3 and 6). Rousset
B–D eggshells also show overlap in isotopic composition with
Roques Hautes soil carbonates (Fig. 2). Petrographic and EBSD
analysis of Rousset B–D eggshells yielded a mixed picture with
some eggshells showing clear evidence of alteration, while others
looking well preserved (Fig. 3; Supplementary Figs 3 and 7). This
is an indication that eggshells from these strata are variably
preserved and may even have areas of better and worse
preservation within the same individual specimen. For this
reason, we considered these specimens of uncertain preservation.
One clear finding from our study is that eggshell specimens from
different stratigraphic layers of the same formation can show very
different states of preservation, and that variability can also exist
within a stratigraphic layer. Full justifications of our assessment
of the preservation of each specimen including geochemical data
and representative petrographic and scanning electron micro-
scopy images can be found in Supplementary Note 2.
Body temperature determinations from fossil eggshells. The
apparently well-preserved Mongolian oviraptorid eggshells yield
D
47
-derived temperatures of 31.9±2.9 °C (2 s.e.), and the Auca
Mahuevo layer-4 titanosaur eggshells yield temperatures of
37.6±1.9 °C (Table 2), supporting differing thermoregulation
between these species. Rousset level-B–D specimens yielded D
47
temperatures of 39.6±1.4 °C (Table 2) and so also supporting
high body temperatures for some dinosaurs, although these data
should be regarded as less reliable than data from Auca Mahuevo
layer-4. Nevertheless, our confidence in the finding of relatively
high body temperatures in sauropod dinosaurs is increased by
their close agreement with previous results of carbonate-clumped
isotope thermometry of sauropod teeth18. Geochemical data for
soil carbonates, poorly preserved eggshells and known diagenetic
phases (altered bone and spar calcites) are also presented for
comparison (Fig. 2; Table 2; Supplementary Figs 1–6).
Discussion
We have shown that when applied to eggshells, carbonate
‘clumped isotope’ paleothermometry can be used to reconstruct
body temperatures. While uncertainties regarding preservation of
clumped isotopic signals in fossil eggshells remain, through a
hierarchy of approaches we identify a number of dinosaur
eggshell specimens that we have no reason to reject as being
altered. Below we discuss the compatibility of D
47
temperatures
from the apparently well-preserved specimens with other data
and with previous inferences on the nature of non-avian dinosaur
physiology.
0.72
40
T °C
30
0.70
0.68
0.66
0.64
Δ47 (‰, ARF)
0.62
0.60
9.5 10.0
106/T2 (T in Kelvin)
10.5 11.0
Bird eggshells
Reptile eggshells
All eggshell data (this study)
Tang et al., 2014 (synthetic)
Ghosh et al., 2006 (synthetic)
Eagle et al., 2013 (all biogenics)
11.5
Figure 1 | D
47
measurements in modern eggshells. Data from modern bird
and ectotherm eggshells are plotted against published inorganic calcite
calibrations. Data are given in Table 1 and temperature calibrations in
Supplemental Note 2. By convention temperature is plotted as 106/T2(T in
Kelvin) on the xaxis, but we indicate T in °C at the top of the plot. Linear
regressions on the absolute reference frame were calculated as described
previously28. Eggshell data and linear regression match more closely with
the calibrations produced in the Caltech of Ghosh et al.19, and the Caltech
all biogenics calibration of Eagle et al.26, than with that of Tang et al.41
(Supplemental Note 2). The slope of the regression through eggshell data is
described as: D
47
¼0.0493±0.0043 (106T2)þ0.1393±0.0447.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9296
4NATURE COMMUNICATIONS | 6:8296 | DOI: 10.1038/ncomms9296 | www.nature.com/naturecommunications
&2015 Macmillan Publishers Limited. All rights reserved.
The comparison between oviraptorid eggshell D
47
temperatures
and those measured from contemporaneous nodular carbonates
recovered from the same strata is important (31.9 versus 26.3 °C
respectively; Table 2). D
47
temperatures from nodular paleosol
carbonates have been shown to reflect warm month average
temperatures34.Therefore,theB6°C higher temperatures derived
from eggshells compared with nodular carbonates at Uhkaa Tolgod
likely indicates that the oviraptorid dinosaurs were able to elevate
body temperatures above environmental temperatures even when
considering the warmest time of year. This interpretation would be
supported even if partial alteration of 13C–18O signatures had
influenced the absolute value of the oviraptorid body temperature
determinations, because measurements on spar calcite and nodular
carbonates from this site suggests that if diagenesis of oviraptorid
eggshells had occurred it would likely have driven D
47
temperatures towards colder values, not hotter (Table 2).
High body temperatures for large sauropod dinosaurs found here
and in a previous study of sauropod teeth18 could be a result of
–1
–1
1
3
–1
–2
ab
cd
ef
–2
–2
–3
–3
–3
–4
–4
–5
–5
–5
δ
13
C (V-PDB)
δ
18
O
mineral
(V-PDB)
δ
18
O
H
2
O
(V-SMOW)
δ
18
O
H
2
O
(V-SMOW)
δ
18
O
H
2
O
(V-SMOW)
–6
–6
–7
–7
–7
–8
–8
8
8
8
9
6
6
6
7
4
4
4
5
2
2
0
0
0
0
0.0 0.5 1.0 1.5 2.0 2.5
–9
–9
–10
–11
–11
10
10
10
10
15 20
20
Li/Ca (μmol mol
–1
)
Sr/Ca (mmol mol
–1
)
Li/Ca (μmol mol
–1
)Mg/Ca (mmol mol
–1
)
Δ
47
Temperature (°C)
Δ
47
Temperature (°C)
Δ
47
Temperature (°C)
25 30
30
30
30
35
35
35
40
40
40
40
45
45
45
50
50 55
50
50
100
150
200
60
Offset between
carbonate nodules
and ‘apparently
well-preserved’
eggshells
Auca Mahuevo layer 4 eggshell
(apparently well preserved)
Auca Mahuevo layer 2 eggshells
(poor preservation)
Auca Mahuevo layer 2 sediment
Roques Hautes soil carbonate
Rousset A eggshell
(poor preservation)
Roques Hautes eggshell
(poor preservation)
Rousset C-D eggshell
(uncertain preservation)
Average 1 s.e. of clumped
isotope temperature determination
Layer 2 eggshells have
higher levels of trace elements
such as lithium and magnesium
Ukhaa Tolgod bone
Ukhaa Tolgod spar calcite
Ukhaa Tolgod nodular carbonate
Ukhaa Tolgod oviraptorid eggshell
(apparently well-preserved)
Ukhaa Tolgod oviraptorid eggshell
(poor preservation)
Ukhaa Tolgod eggshell morphotype 2
(uncertain preservation)
Bayn Dzak oviraptorid eggshell
(uncertain preservation)
Bugin Tsav oviraptorid eggshell
(uncertain preservation)
Increasing lithium but lower strontium
compared with Rousset A eggshells
–12
–13
Figure 2 | Examples of isotopic and trace-element data used to assess preservation. Supplementary Tables 1–3 contain raw data. Additional data plots are
given in Supplementary Figs 4–6. (a,b) Data from Mongolian samples. (b) Illustration of the large offset between the calculated water d18O for three
apparently well-preserved eggshells from Ukhaa Tolgod, and nodular and spar carbonates recovered from the same strata. Two other eggshells from Ukhaa
Tolgod do not show this offset and so were considered of poor preservation. Detailed description of the other Mongolian eggshell types and their preservation
can be found in the main text and Supplementary Notes 1 and 3, but all were considered of uncertain preservation due to either petrographic analysis or the
fact that they were from a different site and so the comparison between them and other carbonate phases was not possible. (c,d)DatafromAucaMahuevo
samples. D
47
temperatures are noticeably higher for layer-2 specimens, compared with layer-4 specimens; layer-2 specimens are also higher in contents of
elements such as lithium and magnesium which, in addition to petrographic and scanning electron microscopy analysis, lead to us considering layer-4
specimens to be better preserved. (e,f) Data from Provence Basin samples. Eggshells from the Rousset locality, stratigraphic layer A, are notably distinct in
both carbonate d18O (not shown) and calculated d18O of mineral formation water. Rousset A eggshells are also comparatively enriched in strontium, but lower
in lithium compared with eggshells from other stratigraphic layers and eggshells and soil carbonate nodules from the nearby Roques Hautes site. Rousset A
are distinct geochemically and eggshells were found to be most altered by EBSD analysis (Fig. 5; Supplementary Fig. 7).
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9296 ARTICLE
NATURE COMMUNICATIONS | 6:8296 | DOI: 10.1038/ncomms9296 | www.nature.com/naturecommunications 5
&2015 Macmillan Publishers Limited. All rights reserved.
endothermy comparable to large extant mammals and birds, but
could also reflect gigantothermy. Our finding in this study of lower
temperatures in oviraptorid eggshells extends the range of inferred
body temperatures in dinosaurs. Our oviraptorid data coupled with
the sauropod data suggest that non-avian dinosaurs could be
significantly variable in their thermoregulation and did all not have
uniformly high (B36–43 °C) body temperatures as modern birds
do (Fig. 6). These data are potentially consistent with a relationship
between dinosaur body mass and body temperature, but if such a
relationship exists it would need more data to accurately constrain.
Regardless, the fact that oviraptorid dinosaurs apparently could
maintain body temperatures above those of the environment
suggests that either they were ectotherms having thermoregulatory
behaviours or other adaptations such as insulation, or that they had
a metabolism and thermal physiology intermediate between
ectotherms and extant birds (Fig. 4). Additional evidence
supporting an intermediate physiology includes analysis of growth
rates and sexual maturation in oviraptorids, which showed that
sexual maturation occurred well before adult size was reached—a
feature that is shared with reptiles and basal pre-modern birds12,13,
but not extant birds35. In addition, recent theoretical work based on
growth rate analysis has provided evidence for mesothermy in a
range of non-avian dinosaurs11.
The results presented here are striking given what is considered
a close relationship between Oviraptoridae and birds, suggesting
the endothermy of modern birds was not present in at least this
a
ef
ghi
d
bc
Figure 3 | Petrographic and scanning electron microscopy characterization of dinosaur eggshells. Representative images are shown. (a,b) Light and
polarized light images of a thin section of an apparently well-preserved Oviraptorid eggshell from Ukhaa Tolgod, Mongolia. (c) scanning electron
microscopy (SEM) image of a cross-section of the eggshell. Pores such as the one indicated are small and rare compared with Titanosaurid eggshells. As
pores are often foci for dissolution and secondary mineral formation, this may be a reason for the relatively good preservation of the Oviraptorid eggshells.
(d,e) Light and cathodoluminescence images of an Auca Mahuevo titanosaur layer-2 eggshell showing localized areas of secondary carbonate on the upper
and lower surface.(f) SEM image of an Auca Mahuevo layer-4 eggshell. Importantly, energy-dispersive X-ray analysis (EDS) of material infilling Auca
Mahuevo eggshell pores found that it was silica-rich clay rather than secondary calcite (Supplementary Note 3). (g–i) Light, polarized light and
cathodoluminescence images of typical eggshells from the Rousset site in the Provence Basin. Secondary carbonate is often found on the exterior surfaces
and infilling the pores of eggshells from this site, as shown in the cathodoluminescence image. In some cases, Rousset eggshells were found to have a
strong cathodoluminescence signal from within the main eggshell structure, a clear indication of alteration (Supplementary Fig. 3). Scale bar (c)
B100 mm); (f,i) 200 mm. More examples of this type of analysis and more in-depth interpretations can be found in the Supplementary Note 3 and
Supplementary Figs 1–7.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9296
6NATURE COMMUNICATIONS | 6:8296 | DOI: 10.1038/ncomms9296 | www.nature.com/naturecommunications
&2015 Macmillan Publishers Limited. All rights reserved.
one species of non-avian theropod dinosaurs. This proposition is
also congruent with interpretations based on histological analysis
of pre-modern birds, where it is proposed that basal birds were
also not fully endothermic in comparison with extant species and
that full endothermic homoeothermy evolved later and after the
evolution of feathers and flight12,13.
Our novel data therefore provide independent evidence to
support inferences from histology and physiological modelling
that some dinosaurs were not fully endothermic as are modern
birds. Although we cannot extrapolate how representative
oviraptorid body temperatures are of theropods, or non-avian
dinosaurs more broadly, at least in this one case our data
a
d
gh
ef
i
bc
0001
300 μm300 μm
300 μm
300 μm
400 μm
400 μm
1100
0110
1210
–
–
–
Figure 4 | EBSD analysis of Auca Mahuevo eggshells. Images are representative of multiple specimens examined. (a,d,g) Grayscale diffraction maps in
which lighter regions represent areas where greater diffraction is observed. (b,e,h) Colours represent different crystallographic planes of calcite, as shown
in the colour-coded legend given in the bottom of the figure. (c,f,i) The corresponding pole figures of the planes projected on the {0001} plane of calcite.
(a–c) Analysis of the prismatic layer (exterior portion, or palisade layer) of a titanosaur eggshell from Auca Mahuevo layer 4. This section of the eggshell
exhibits excellent preservation on EBSD analysis, with the alternation {0–110} and {1–100} planes of calcite in the cross-section and the caxis parallel to
the elongation of calcite crystals and perpendicular to the shell exterior, and seen in some modern avian eggshells42 and consistent with previous studies
on titanosaur eggshells43.(d–f) Mammilla (inner) layers of the same eggshell, also exhibiting excellent preservation in the regions where calcite crystals
fan out from the cones and the caxis parallel to their elongation, but have a less constrained distribution of poles as shown in the pole figure (f). Areas
between the cones were likely originally organic rich, but are now partially replaced by secondary calcite crystals (isolated orange–yellow–red poles in f). A
similar feature can often be observed in both layer-2 and layer-4 Auca Mahuevo eggshells by cathodoluminescence, for example Fig. 3e. As described in
the Methods, this area of secondary calcite on the interior surface is likely removed by drilling during the preparation stage. (g–i) Auca Maheuvo layer-2
eggshell that is heavily recrystallized. Each calcite crystal has a different orientation and without a preferred orientation of the caxis of calcite, indicative of
recrystallization. The diffraction map shows regular edges of calcite crystals, defining prisms, which would suggest that the recrystallization is due to a
closed-system replacement rather than open-system recrystallization involving dissolution and precipitation of secondary calcite.
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9296 ARTICLE
NATURE COMMUNICATIONS | 6:8296 | DOI: 10.1038/ncomms9296 | www.nature.com/naturecommunications 7
&2015 Macmillan Publishers Limited. All rights reserved.
combined with published growth rate analyses35 are consistent
with a reduced form of endothermy described elsewhere as
‘mesothermy’ or ‘basoendothermy’10,11.
Methods
Specimens.Modern eggshell specimens were obtained from a number of different
sources including the Los Angeles Zoo, the San Francisco Zoo, the Zurich Zoo, the
Western Foundation of Vertebrate Zoology, the South Carolina Zoo, private farms
and collections. Full details on sources can be found in Supplementary Note 1. The
principle localities of fossil material were the Cretaceous strata in the Nemegt Basin,
Mongolia, Auca Mahuevo in Neuque
´n Province, Argentina and the Provence Basin in
France. Fossil specimens were sourced from the American Natural History Museum
in New York, the Natural History Museum of Los Angeles County and the Goldfu-
Museum in Bonn. Full details regarding the specimens and their geological setting can
be found in Supplementary Note 1.
Stable isotope analysis.As described in more detail below, even eggshells with
apparently good preservation as revealed by petrographic examination frequently
show some evidence for dissolution and replacement on their outer surfaces and
around pore canal openings. Therefore, we routinely drilled off and discarded the
outer surface of eggshell specimens. After this initial preparation, a section of
eggshell was broken off and powdered with a pestle and mortar. Where possible,
soil carbonate nodules, spar calcites and bone samples were also drilled out and
analysed but were not subject to any sample pretreatment. A total of 8–10 mg of
carbonate and 120 mg of phosphate was reacted for 20 min in a common phos-
phoric aci d bath heated to 90 °C. Product CO
2
was then purified using the auto-
mated online system, described previously34. This automated purification system
includes passing sample gas through a Porapak Q 120/80 mesh GC column at
20 °C to remove potential organic contaminants and silver wool (Sigma-
Aldrich) to remove sulfur compounds. Purified sample gases were then analysed on
a Thermo Scientific MAT253 dual-inlet gas source mass spectrometer at Caltech
using a published configuration and methods22.
Reference gases of different compositions that had been heated to 1,000 °Cto
approximate a stochastic ‘random’ distribution of isotopes were also analysed to
provide a reference frame for sample D
47
values. Carbonate standards were
analysed every 5–6 analyses. The average external reproducibility (one s.e.) of D
47
measurements presented here is 0.008%. Supplementary Table 7 contains the
heated gas slope and intercepts, and secondary transfer function slope and
intercepts.
abb
f
ed
gh
i
500 μm500 μm
300 μm
300 μm
500 μm
500 μm
Figure 5 | EBSD analysis of Mongolian and French eggshells. Images are representative of multiple specimens examined. Colour coding is as described in
Fig. 4. (a–c,d–f) Two different individual Mongolian Oviraptorid eggshells, respectively. Both eggshells show an overall good preservation, with no evidence
for recrystallization and calcite crystals displaying a preferred crystallographic orientation of the caxis perpendicular to the shell exterior, as shown in pole
figures. In this respect, data are similar to Auca Mahuevo layer-4 eggshells in Fig. 4. There are some indications of deformation of calcite crystals, possibly
due to eggshells being compressed on burial, as indicated discontinuity and deformation of crystal boundaries as seen in diffraction maps and a larger
spreading of poles on the projection of the {0001} plane of calcite than typically observed in modern eggshells. For comparison, the bottom panels (g–i)
show data from a Rousset level-A eggshell that is completely altered and recrystallized, with none of the original microstructure typical of eggshells
observed. Instead, the eggshell has been replaced by microcrystalline calcite with low diffraction, and the pole figure (i) shows the absence of biological
control over crystallographic orientation.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9296
8NATURE COMMUNICATIONS | 6:8296 | DOI: 10.1038/ncomms9296 | www.nature.com/naturecommunications
&2015 Macmillan Publishers Limited. All rights reserved.
Data processing and calculation.D
47
Values are defined as follows:
D47 ¼R47=R47 1
R46=R46 1
R45=R45 1
1
Where Rirepresents mass i/mass 44 and R* represents the abundance of iso-
topologues in a sample with the same bulk isotopic composition, but that conforms
to a random (stochastic) distribution24.
For this study, an acid digestion correction of 0.081%was applied as described
previously34. The vast majority of data for this study was collected before the
proposition of an ‘absolute reference frame’ (ARF) for clumped isotope studies of
CO
2
based on the analysis of water-equilibrated CO
2
gases20. Therefore, we
converted D
47
values into the ARF by applying a secondary transfer function
described previously20, which uses an accepted value of the standards NBS-19 and
Caltech Carrara Marble of 0.392%, and TV03 of 0.713%.
For calcite d18O calculations, an acid digestion fractionation factor of 1.007954
was used36. For aragonite d18O calculations an acid digestion fractionation factor of
1.00854126 was used, calculated by extrapolation from a published calibration37.
Temperatures estimated using sample D
47
values were used to calculate the d18Oof
water from which the mineral precipitated using paired measurements of carbonate
d18O and the following published equation38.
1;000 ln aCalcite H2OðÞ¼18:03103
=T32:42
For turtle and tortoise eggshells made of aragonite, the following equation was
used from Kim et al. (2007)37:
1;000 ln aAragonite H2OðÞ¼17:88103
=T31:14
Propagated errors in water d18O calculations were calculated as follows:
Ed18Owater ¼E2
d18Ocarbonate þE2
T
q
Where Eis one s.e. To derive the E2
T
term for calculating the propagated error in
d18O, the uncertainty in the D
47
-derived temperature is converted into a per mil
value using the appropriate equation.
Elemental analysis and microscopy.Element to calcium ratios (Mg/Ca, Sr/Ca,
Mn/Ca, Fe/Ca and Li/Ca) on splits of the same powders analysed for stable isotopes
were determined on a Jobin-Yvon Ultima-C Inductively Coupled Atomic Emission
Table 2 | Stable isotope data from Cretaceous eggshells and associated phases.
Species or
material*
Locality n
specimens
n
analysesw
d13C
CO3
%, V-PDBz
d18O
CO3
%, V-PDBz
D
47
%, ARFyD
47
Temp. (°C)
inorganic eq.||
D
47
Temp.
(°C) biogenic
eq.z
d18O
water
%,
V-SMOW#
Apparently well-preserved eggshells:
Oviraptorid Ukhaa
Tolgod
395.7±0.3 2.1±0.2 0.671±0.012 30.3±2.6 31.9±2.9 1.6±0.2
Titanosaurid Auca
Mahuevo,
level 4
3712.7±0.0 0.4±0.0 0.650±0.006 35.3±1.4 37.6±1.9 5.2±0.2
Eggshells of uncertain preservation:
Putative
titanosaurid
Rousset,
layers B–D
679.2±0.3 4.8±0.1 0.642±0.005 37.0±1.2 39.6±1.4 0.4±0.3
Oviraptorid Bugin Tsav 3 7 5.5±0.3 9.7±1.3 0.674±0.005 29.7±1.0 31.3±1.2 6.4±0.5
Oviraptorid Bayn Dzak 1 2 3.6±0.0 11.5±0.1 0.684±0.014 27.5±2.9 28.8±3.3 8.4±1.3
Morphotype 2 Ukhaa
Tolgod
4135.7±0.2 3.0±0.4 0.667±0.003 31.2±0.8 32.9±0.8 1.0±0.3
Poorly preserved eggshells:
Oviraptorid Ukhaa
Tolgod
286.6±0.3 8.1±0.2 0.681±0.009 28.2±1.9 29.6±2.2 4.8±0.8
Titanosaurid Auca
Mahuevo,
level 2
4812.6±0.1 0.2±0.1 0.624±0.004 41.6±0.9 44.8±1.0 6.0±0.1
Putative
titanosaurid
Roques
Hautes
3610.5±0.4 3.4±0.7 0.616±0.013 43.6±3.3 47.0±3.8 3.2±1.1
Putative
titanosaurid
Rousset,
layer A
3613.2±0.2 1.2±0.6 0.633±0.001 39.4±0.4 42.3±0.4 6.9±0.6
Associated phases:
Nodular
carbonates
Ukhaa
Tolgod
99
3.3±0.2 11.0±0.3 0.694±0.011 25.2±2.3 26.3±2.6 7.9±0.3
Spar calcite Ukhaa
Tolgod
116.4±0.0 12.2±0.0 0.689±0.007 26.4±1.4 27.6±1.6 8.9±0.4
Bone Ukhaa
Tolgod
113.6±0.0 11.7±0.0 0.668±0.010 31.0±2.1 32.8±2.4 7.5±0.6
Nodular
carbonates
Roques
Hautes
778.9±0.1 4.9±0.1 0.641±0.006 37.4±1.5 40.0±1.7 0.0±0.2
Bulk sediment Auca
Mahuevo,
level 2
119.8±0.0 4.6±0.0 0.630±0.007 40.0±1.8 43.0±2.0 1.2±0.5
±Values are one s.e., or in the case of water isotope values, the propagated s.e.
*Samples were considered to be of uncertain preservation if not enough information from multiple sources was available to make an informed assessment or if different types of analyses were not
conclusive. See Supplementary Note 3 for full description of steps taken to assess preservation, and Supplementary Table 4 for justification for classification of individual specimens.
wRepresents the number of distinct extractions of CO
2
from a sample that were separately purified and analysed. See Supplementary Table 3 for additional data for samples.
zStable isotope values of the carbonate mineral.
yValues given on the ‘absolute reference frame’20.
||Calculated using the inorganic calibration of Ghosh et al.19 (Equation (2); Supplementary Note 2).
zCalculated using a compilation of published biogenic calibration (Equation (3), Supplementary Note 2) data collected in the Caltech laboratories and synthesized by Eagle et al.26. See Supplementary
Note 2 text for more information on how this calibration was derived.
#d18O of mineral formation waters calculated using the equation of Kim and O’Neil38, and using the D
47
(Biogenic) temperature). All eggshells are calcite. Error represents propagated one s.e.
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9296 ARTICLE
NATURE COMMUNICATIONS | 6:8296 | DOI: 10.1038/ncomms9296 | www.nature.com/naturecommunications 9
&2015 Macmillan Publishers Limited. All rights reserved.
Spectrometer39. Eggshell fragments were also inspected for diagenetic features and
by scanning electron microscopy and energy-dispersive spectroscopy using a
Tescan Vega-3 XMU variable-pressure scanning electron microscope. Petrographic
analysis of thin sections of eggshells was carried out by polarized light and
cathodoluminescence. EBSD analysis was carried out using a Hikari camera
mounted on a TESCAN LYRA FIB-FESEM (field emission scanning electron
microscope)40. Also see Supplementary Note 3 and Supplementary Figs 1–7 for
the results and discussion of this analysis.
Modern taxa body temperature data.Stable isotope data for modern eggshells
are in Table 1 and Supplementary Tables 1 and 2. Modern taxa body temperature
data and associated references are given in Table 1, and Supplementary Tables 5
and 6. Expected temperatures for birds have an uncertainty of ±0.2–0.5 °C
associated with data loggers used; if no error was reported in the study, then we
assumed a conservative value for the temperature uncertainty. In reptiles with
small fluctuations in body temperature (for example, Galapagos tortoise), the
average body temperature is accurately recorded by the eggshell. In organisms such
as crocodilians that are from environments with more variability in body tem-
peratures, we used warm season temperatures given what is reported for survi-
vorship in crocodilians and some other reptiles: extremely high mortality rates in
hatchlings of up to 100% are reported when environmental or incubation tem-
peratures are below 27 °C. Thus crocodilian body temperatures associated with
eggshell formation are likely to reflect warm season conditions and not growth
during winter in environments with moderate seasonality. In Supplementary
Note 2 we discuss whether published calibrations reproduce these temperatures,
and then discuss a D
47
-temperature calibration produced using the modern
eggshell data.
D
47
-Temperature calibrations.The D
47
-temperature calibrations utilized in the
main text are the initial inorganic calcite calibration produced in the Caltech lab
converted to the absolute reference frame (D
47
¼0.0636 (106T2)–0.0047)19,20
and the ‘all biogenic materials’ compilation of data from the Caltech lab
(D
47
¼0.0559 (106T2)þ0.0708)26. Temperature is in Kelvin (K). A growing
number of alternative calibrations have been published, many of which we find do
not yield plausible D
47
-derived body temperatures from our eggshell data, as
described in more detail in Supplementary Note 2. Therefore, while D
47
calibrations remain largely empirical, the eggshells studied here appear to be
equivalent to the majority of biogenic material studied to date in the Caltech lab
(Fig. 1) and suggest that alternative calibrations with shallower slopes
(Supplementary Note 2) may not apply to all biogenic materials analysed at Caltech
with the same experimental set-up and during similar analytical time periods
(2008–2015).
References
1. Russell, L. S. Body temperature of dinosaurs and its relationships to their
extinction. J. Paleontol. 39, 497–501 (1965).
2. Bakker, R. Anatomical and ecological evidence of endothermy in dinosaurs.
Nature 238, 81–85 (1972).
3. de Ricqle
`s, A. J. Evolution of endothermy: histological evidence. Evol. Theory 1,
51–80 (1974).
4. Farlow, J. in The Dinosauria (eds Weishampel, D., Dodson, P. & Osmolska, H.)
43–55 (University of California Press, 1990).
5. Padian, K. & Horner, J. R. in The Dinosauria 2nd edn (eds Weishampel, D,
Dodson, P. & Osmolska, H.) 660–671 (University of California Press, 2004).
6. Chinsamy, A. & Hillenius, W. in The Dinosauria 2nd edn (eds Weishampel, D.,
Dodson, P. & Osmolska, H.) 643–659 (University of California Press, 2004).
7. Gillooly, J. F., Allen, A. P. & Charnov, E. L. Dinosaur fossils predict body
temperatures. PLoS Biol. 4, e248 (2006).
8. Amiot, R. et al. Oxygen isotopes from biogenic apatites suggest widespread
endothermy in Cretaceous dinosaurs. Earth Planet. Sci. Lett. 246, 41–54 (2006).
9. Griebeler, E. M. Body temperatures in dinosaurs: what can growth curves tell
us? PLoS ONE 8, e74317 (2013).
10. Lovegrove, B. G. The evolution of endothermy in Cenozoic mammals: a
plesiomorphic-apomorphic continuum. Biol. Rev. Camb. Philos. Soc. 87,
128–162 (2012).
11. Grady, J. M., Enquist, B. J., Dettweiler-Robinson, E., Wright, N. A. &
Smith, F. A. Evidence for mesothermy in dinosaurs. Science 344, 1268–1272
(2014).
12. Chinsamy, A., Chiappe, L. M. & Dodson, P. Growth rings in Mesozoic birds.
Nature 368, 196–197 (1994).
13. Chinsamy, A., Chiappe, L. M. & Dodson, P. Mesozoic avian bone
microstructure: physiological implications. Paleobiology 21, 561–574 (1995).
14. Paladino, F., O’Connor, M. & Spotila, J. Metabolism of leatherback turtles,
gigantothermy, and thermoregulation. Nature 344, 858–860 (1990).
15. Prinzinger, R., Premar, A. & Schleucher, E. Body temperature in birds. Comp.
Biochem. Physiol. A Physiol. 99, 499–506 (1991).
16. Clarke, A. & Rothery, P. Scaling of body temperature in mammals and birds.
Funct. Ecol. 22, 58–67 (2008).
45
40
a
b
35
30
25
15
0.001
Extant animals
(instrumentally measured):
Dinosaur taxa
(clumped isotope T):
0.01
Modern birds Oviraptorid eggshells
Titanosaurid eggshells
Giraffatitan teeth
Camarasaurus teeth
Modern mammals
Modern ectotherms
(mean with observed high and low)
Extant animals (clumped isotope T): Dinosaur taxa (clumped isotope T):
Modern bird eggshells Oviraptorid eggshells
Titanosaurid eggshells
Giraffatitan teeth
Camarasaurus teeth
Modern ectotherm eggshells
0.1 1 10
Body mass (kg)
Body mass (kg)
100 1,000 10,000 100,000
0.010.001 0.1 1 10 100 1,000 10,000 100,000
20
45
40
35
30
25
15
20
Body temperature (°C)
Body temperature (°C)
Figure 6 | Isotope-derived dinosaur body temperatures in context with
modern species data. (a) Isotopic dinosaur body temperature determinations
plotted versus compilations of modern mammal, bird and ectotherm body
temperatures and body masses (Supplementary Tables 5 and 6). Error bars on
dinosaur body temperature estimates represent two s.e. Clumped isotope-based
temperature determinations from teeth of Jurassic Camarasaurus and
Giraffatitan18 plotted alongside determinations from titanosaurid eggshells from
Auca Mahuevo, Argentina, and oviraptorid eggshells from Ukhaa Tolgod,
Mongolia. Modern ectotherm data points are calculated mean values with
measured upper and lower limits indicated by grey bars (Supplementary
Table 6). Clumped isotope estimates of oviraptorid dinosaur body temperatures
are lower than all modern birds and most modern mammals, but are comparable
to a minority of modern mammals. Isotopic estimates of oviraptorid dinosaur
body temperatures are higher than most calculated mean ectotherm body
temperatures but fall within the range exhibited by many ectotherms, including
those of similar mass. Sauropod dinosaur (that is, titanosaurid, Camarasaurus,
Giraffatitan) body temperatures are higher than the mean and range exhibited by
the majority modern reptiles in this compilation. (b) Clumped isotope-based
temperature determinations for both modern eggshells and dinosaur fossils.
Monitor lizard eggshell isotope data with high error is excluded from this figure.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9296
10 NATURE COMMUNICATIONS | 6:8296 | DOI: 10.1038/ncomms9296 | www.nature.com/naturecommunications
&2015 Macmillan Publishers Limited. All rights reserved.
17. Eagle, R. A. et al. Body temperatures of modern and extinct vertebrates
from 13C-18O bond abundances in bioapatite. Proc. Natl Acad. Sci USA 107,
10377–10382 (2010).
18. Eagle, R. A. et al. Dinosaur body temperatures determined from isotopic
(13C-18O) ordering in fossil biominerals. Science 333, 443–445 (2011).
19. Ghosh, P. et al. 13C–18O bonds in carbonate minerals: a new kind of
paleothermometer. Geochim. Cosmochim. Acta 70, 1439–1456 (2006).
20. Dennis, K. J., Affek, H. P., Passey, B. H., Schrag, D. P. & Eiler, J. M. Defining an
absolute reference frame for ‘‘clumped’’ isotope studies of CO
2
.Geochim.
Cosmochim. Acta 75, 7117–7131 (2011).
21. Eiler, J. M. & Schauble, E. 18O13C16O in Earth’s atmosphere. Geochim.
Cosmochim. Acta 68, 4767–4777 (2004).
22. Huntington, K. W. et al. Methods and limitations of ‘‘clumped’’ CO
2
isotope
(D
47
) analysis by gas-source isotope ratio mass spectrometry. J. Mass. Spectrom.
44, 1318–1329 (2009).
23. Folinsbee, R. E., Fritz, P., Krouse, H. R. & Robblee, A. R. Carbon-13 and
oxygen-18 in dinosaur, crocodile, and bird eggshells indicate environmental
conditions. Science 168, 1353–1356 (1970).
24. Von Schirnding, Y., Van Der Merwe, N. J. & Vogel, J. C. Influence of diet and
age on carbon isotope ratios in ostrich eggshell. Archaeometry 24, 3–20 (1982).
25. Schaffner, F. C. & Swart, P. K. Influence of diet and environmental water on the
carbon and oxygen isotopic signatures of seabird eggshell carbonate. Bull. Mar.
Sci. 48, 23–38 (1991).
26. Eagle, R. A. et al. The influence of temperature and seawater carbonate
saturation state on 13C–18O bond ordering in bivalve mollusks. Biogeosciences
10, 4591–4606 (2013).
27. Ayliffe, L. K., Chivas, A. R. & Leakey, M. G. The retention of primary oxygen
isotope compositions of fossil elephant skeletal phosphate. Geochim.
Cosmochim. Acta 58, 5291–5298 (1994).
28. Arias, J. L. & Fernandez, M. S. Role of extracellular matrix molecules in shell
formation and structure. Worlds Poult. Sci. J. 57, 349–357 (2001).
29. Montanari, S., Higgins, P. & Norell, M. A. Dinosaur eggshell and tooth enamel
geochemistry as an indicator of Mongolian Late Cretaceous paleoenvironments.
Palaeogeogr. Palaeoclimatol. Palaeoecol. 370, 158–166 (2013).
30. Chiappe, L. M., Salgado, L. & Coria, R. A. Embryonic skulls of titanosaur
sauropod dinosaurs. Science 293, 2444–2446 (2001).
31. Erben, H. K., Hoefs, J. & Wedepohl, K. H. Paleobiological and isotopic studies
of eggshells from a declining dinosaur species. Paleobiology 5, 380–414 (1979).
32. Cojan, I., Renard, M. & Emmanuel, L. Palaeoenvironmental reconstruction of
dinosaur nesting sites based on a geochemical approach to eggshells and
associated palaeosols (Maastrichtian, Provence Basin, France). Palaeogeogr.
Palaeoclimatol. Palaeoecol. 191, 111–138 (2003).
33. Norell, M. A. et al. A theropod dinosaur embryo and the affinities of the
flaming cliffs dinosaur eggs. Science 266, 779–782 (1994).
34. Passey, B. H., Levin, N. E., Cerling, T. E., Brown, F. H. & Eiler, J. M. High-
temperature environments of human evolution in East Africa based on bond
ordering in paleosol carbonates. Proc. Natl Acad. Sci. USA 107, 11245–11249
(2010).
35. Erickson, G. M., Rogers, K. C., Varricchio, D. J., Norell, M. A. & Xu, X. Growth
patterns in brooding dinosaurs reveals the timing of sexual maturity in non-
avian dinosaurs and genesis of the avian condition. Biol. Lett. 3, 558–561
(2007).
36. Swart, P. K., Burns, S. J. & Leder, J. J. Fractionation of the stable isotopes of
oxygen and carbon in carbon dioxide during the reaction of calcite with
phosphoric acid as a function of temperature and technique. Chem. Geol. Isot.
Geosci. Sect. 86, 89–96 (1991).
37. Kim, S. T., Mucci, A. & Taylor, B. E. Phosphoric acid fractionation factors for
calcite and aragonite between 25 and 75 C: revisited. Chem. Geol. 246, 135–146
(2007).
38. Kim, S.-T. & O’Neil, J. R. Equilibrium and nonequilibrium oxygen isotope
effects in synthetic carbonates. Geochim. Cosmochim. Acta 61, 3461–3475
(1997).
39. Saikku, R., Stott, L. & Thunell, R. A bi-polar signal recorded in the western
tropical Pacific: Northern and Southern Hemisphere climate records from the
Pacific warm pool during the last Ice Age. Quat. Sci. Rev. 28, 2374–2385 (2009).
40. Pe
´rez-Huerta, A., Dauphin, Y., Cuif, J. P. & Cusack, M. High resolution
electronic backscatter diffraction (EBSD) data from calcite biominerals in
recent gastropod shells. Micron 42, 246–251 (2011).
41. Tang, J., Dietzel, M., Fernandez, A., Tripati, A. K. & Rosenheim, B. Evaluation
of kinetic effects on clumped isotope fractionation (D
47
) during inorganic
calcite precipitation. Geochim. Cosmochim. Acta 134, 120–136 (2014).
42. Dalbeck, P. & Cusak, M. Crystallography (electron backscatter diffraction) and
chemistry (electron probe microanalysis) of the avian eggshell. Cryst. Growth
Des. 6, 2558–2562 (2006).
43. Timby, P. & Grellet-Tinner, G. The hidden secrets of dinosaur eggs revealed
using analytical scanning electron microscopy. Infocus Mag. (RSM) 24, 4–21
(2011).
Acknowledgements
R.A.E. was supported by a Caltech Chancellors Postdoctoral Fellowship and by National
Science Foundation grants EAR-1024929 to J.M.E. and R.A.E., ARC-1215551 to
R.A.E. and A.K.T., a LabEx International Research Chair funded by the ‘Laboratoire
d’Excellence’ LabexMER(ANR-10-LABX-19) and co-funded by a grant from the French
government under the programme ‘Investissements d’Avenir’, and a funded visiting
Associate Professor position at the Natural History Museum of Denmark. A.K.T. was
supported by a Hellman Fellowship, and National Science Foundation grant EAR-
0949191. T.T. was supported by DFG grants TU 148/2-1 and TU 148/4-1. M.J.K.
acknowledges NSF grant EAR-1251443. We thank the Los Angeles Zoo, the Zurich Zoo,
San Francisco Zoo, South Carolina Zoo, Gemarkenhof Farm in Germany, The Western
Foundation of Vertebrate Zoology, and Colorado Gators for provision of modern
eggshells. We thank the American Natural History Museum, the Natural History
Museum of Los Angeles County and the GoldfuMuseum of the University of Bonn
for provision of specimens for this study. Eggshell specimens from Montana that were
not considered in the main text were from the Museum of the Rockies and Montana
State and were kindly provided by Frankie Jackson. We also thank Marcus Clauss
(University of Zurich) for provision of the compilations of ectotherm body temperatures
and Frank Corsetti (University of Southern California) for use of petrographic micro-
scopes. Miguel Rincon and Lowell Stott (University of Southern California) assisted with
trace-element analysis.
Author contributions
R.A.E. designed the experiments, carried out isotopic analysis, analysed the data and
wrote the manuscript. M.E. performed petrographic examinations with input from
G.G.-T., S.J.L. and P.R. A.P.-H. carried out EBSD analysis and contributed to data
interpretation. Geochemical analysis was carried out in the laboratories of J.M.E.
and A.K.T. M.K. provided input on design of a modern eggshell sample set. D.H. per-
formed some of the analysis. G.G.-T., S.M., L.M.C., T.T. and T.E.C. provided specimens
for analysis and input into data interpretation. All authors provided input on the
manuscript text.
Additional information
Supplementary Information accompanies this paper at http://www.nature.com/
naturecommunications
Competing financial interests: The authors declare no competing financial interests.
Reprints and permission information is available online at http://npg.nature.com/
reprintsandpermissions/
How to cite this article: Eagle, R. A. et al. Isotopic ordering in eggshells reflects body
temperatures and suggests differing thermophysiology in two Cretaceous dinosaurs.
Nat. Commun. 6:8296 doi: 10.1038/ncomms9296 (2015).
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9296 ARTICLE
NATURE COMMUNICATIONS | 6:8296 | DOI: 10.1038/ncomms9296 | www.nature.com/naturecommunications 11
&2015 Macmillan Publishers Limited. All rights reserved.
