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n, G
The d13C values discriminate between different types ofBones
Bones and teeth are composed of both organic and mineral
fractions which are synthesized during the lifetime of a verte-
brate (Figure 1). The organic fraction of bone and dentine is
Broadmeadow et al., 1992; Gebauer and Schultze, 1991; van
der Merwe and Medina, 1991). As herbivore teeth and bones
record the d13C values of their plant food, it is possible to identifywhich kind of plant was consumed by an herbivore, and there-
fore the type of environment in which it lived (Figure 3).Since the 1970s, stable isotopes in terrestrial teeth and bones
have been providing paleoenvironmental and paleobiological
information on the Quaternary period. Indeed, the skeletal
tissues of an animal through its diet and drinking water yield
paleoenvironmental information in the form of isotopic ratios.
This information is preserved in molecules and minerals found
in fossil teeth and bones.
The most commonly used pairs of isotopes are 13C/12C,15N/14N, and 18O/16O. Isotopes correspond to different types
of atoms for a given chemical element, meaning that two iso-
topes of the same element have the same number of protons,
but differ in the number of neutrons. For instance, an atom of12C (carbon-12) has six protons and six neutrons in its core,
while an atom of 13C (carbon-13) has six protons and seven
neutrons. Therefore, 13C exhibits a higher atomic weight than12C and is thus referred to as the heavy isotope (contrary to12C, which is referred to as the light isotope). Nevertheless,
both isotopes enter in the same molecules with the same types
of chemical bonds. The relative proportion of 13C and 12C in a
given molecule is not random but depends on the relative
content of both isotopes in the source of carbon used to
synthesize this molecule and on the differences of behavior
between both isotopes during chemical reactions.
The relative abundances of the different isotopes are much
higher for light isotopes than for heavy isotopes (e.g., Koch,
2007). The variations of isotopic abundances in natural sam-
ples are very small. They are measured using an isotopic ratio
mass spectrometer, which separates and quantifies the number
of heavy and light isotopes of a given element. In order to
ensure accuracy, measurements are performed simultaneously
on the test sample and on a standard, which allows corrections
that make all results comparable to one another. Due to this
measurement strategy, the results are expressed as relative
abundances, known as delta (d) values, which are defined asfollows:
dEX ERsampleERsample
ERsample 1000%
where EX stands for 13C, 15N, or 18O, respectively. International
reference standards have been established for d13C values (ma-rine carbon PeeDee Belemnite (PDB)), for d15N values (atmo-spheric dinitrogen), and for d18O values (an average mixture ofoceanic waters called Standard Mean Ocean Water (SMOW)).
Paleobiological Tracking by Isotopes in Teeth andTerrestrial Teeth and BonesH Bocherens and D G Drucker, Universitat Tubingen, Tubinge
2013 Elsevier B.V. All rights reserved.
Introduction304plants, principally between marine and terrestrial plants.
Among terrestrial plants, the carbon isotope signature varies
between plants using the two main photosynthetic pathways,
calledC3 andC4 (C3 plants and C4 plants). C4 plants are absent
or very limited in environments with a mild or cold growing
season, as in Europe, Northern latitudes, and high altitudes
(Ehleringer et al., 1997). When present, C4 plants are grasses
and forbs. In environments where all plants use the C3 photo-
synthetic pathway, an isotopic distinction can be seen between
plants growing under a closed canopy and those at the top of the
canopy or growing in an open environment. The possible causes
of the so-called canopy effect are the concentration of recycled
CO2 due to poor ventilation, the light attenuation, and the
relative high water availability in closed canopy forest (e.g.,ermany
mainly formed of a protein, collagen, which contains carbon
(around 40%) and nitrogen (around 15%), while the mineral
fraction contains carbon and oxygen. Enamel is formed of a
very small organic fraction devoid of collagen. In bone, den-
tine, and enamel, the mineral fraction is composed of a cal-
cium phosphate (apatite) with many impurities, including
35% carbonate. The crystal size of apatite is much larger in
enamel than in bone and dentine. The isotopic signatures of
nitrogen are measured in the organic fraction, while oxygen
isotopic signatures are measured in the mineral fraction, both
in phosphate and carbonate. The isotopic signatures of carbon
can be measured in the organic and the mineral phases of bone
and dentine, and in the mineral fraction of enamel.
Carbon
All the carbon of an organism comes from its dietary intake, in
the form of proteins, carbohydrates, and lipids. Some of these
nutrients are incorporated directly by the organism and seques-
tered in different tissues, while other molecules are synthesized
by the organism from dietary nutrients. Due to these different
biochemical characteristics and isotopic fractionations, the av-
erage carbon isotopic abundance of a vertebrate is close to that
of its average diet, but those recorded in a given tissue or
molecule exhibit specific differences (Figure 2). The d13Cvalues of collagen are typically 5% more positive than thoseof the average diet, while the d13C values of the carbonatefraction of bone and tooth mineral fraction is 914% morepositive than that of the average diet (Cerling and Harris, 1999;
Passey et al., 2005). Therefore, the d13C values of collagen andcarbonate apatite are typically used to track the type of plant
food consumed by herbivores and the type of plants at the base
of the food web to which predators belong.
n: ~
ge-co
: ~
ho ~5%
car
CARBONATE STABLE ISOTOPES | Terrestrial Teeth and Bones 305Bone
Organic fractio
Organic fraction: ~30%90% colla10% non
Mineral fraction
Calcium p(includes
Mineral fraction: ~70%
(includes ~3% Nitrogen
Nitrogen is incorporated through dietary intake in the organic
molecules of an organisms tissues. It is usually measured in
the collagen preserved in fossil bones. Contrarily to carbon, the
isotopic signature of nitrogen is significantly enriched in verte-
brate tissues relative to its average diet, typically by 35%(Figure 2; review in Robbins et al., 2005). Therefore, the nitro-
gen isotopic signature of a given individual depends on the
isotopic signature at the base of the food web to which it
belongs (in the plants), and on the position of the specimen
within the food web (herbivore or predator). Not all herbi-
vores present the same d15N values in a given ecosystem be-cause different plants may use nitrogen under different forms,
leading to varying isotopic fractionation and d15N values. For
Figure 1 Summary of the composition of bones and teeth, with emphasis oenamel.
Carbon(d13C)
Inorganicprecursors
Producer(Plants)
C3 Plants(~ 28C4 Plants(~ 13
Marine Pla(~ 20
Non N2 fixing P(~ -5 to +5
H2O in leaO in nutrie
N2 fixing Pla(~ 0)
(variable accoto taxon)
O2
CO2(~ 8)
HCO3-
(~ 0)
(variable)N2 (~0)
H2O(variable
according toenvironment)
NO2NO3NH4
O2
Nitrogen(d15N)
Oxygen(d18O)
Figure 2 Summary of the isotopic fractionation factors associated with thehydrological cycle, with emphasis on bone and tooth tissues. Bold arrows indTooth
Dentine
nllagenous proteins and lipids
~22%
sphate d18Od13C, d18O carbonate)
~78%
d13C, d15NEnamel1%99% calcium phosphate d18O
d13C, d18Obonate)instance, grass and graminoids typically exhibit more positive
d15N values than shrubs and trees, as the latter obtain theirnitrogen through symbiotic fungal mycorrhizae (e.g., Michel-
sen et al., 1998; Schulze et al., 1994). Global climatic factors
such as aridity and temperature lead to increased d15N valuesof plants (Amundson et al., 2003), while d15N of plants tendsto decrease with increasing altitude (e.g., Mannel et al., 2007).
Fire also tends to increase d15N values of plants growing aftersuch an event (Hogberg, 1997).
Oxygen
Oxygen isotopic signatures are measured in the mineral phase
of enamel, dentine, or bone, in the phosphate or the carbonate
n the isotopic signatures that can be retrieved from bone, dentine, and
s Consumers(body average)
Consumers(bone/tooth)
)
)nts)
lants)
vesnts
nts
rding
CO2
H2O
+3 to +5
+ 01+5 in collagen
+18 in PO4
+26 in CO3
Collagen
+9 to +14 incarbonate apatite
transfer of carbon, nitrogen, and oxygen within food webs and theicate steps where significant isotopic fractionation occurs.
--
-
-
d1
d1
d1
Sa
osite d
306 CARBONATE STABLE ISOTOPES | Terrestrial Teeth and Bones-40 -35
-35
-30
-30 -25
-25
-30 -25
-30 -25d13C
Browsers (C3)
Zoo (C3)
Mongolia (C3)
Figure 3 An example of the reconstruction of the carbon isotopic compon their habitat. The d13C of diet has been calculated by adding 14% to thC3 - Grass13C = -26.7 2.3fractions (Kohn and Cerling, 2002). For both fractions, the
d18O values are related to those of environmental water,through drinking water. However, there are complications
due to the incorporation of water from food, and mixture
with oxygen from respiration (Figure 2). This leads to varying
species-dependent relationships between d18O values in car-bonate or phosphate and d18O values in environmental watertaken by the organisms. However, both d18O values are linkedby a clear relationship, independent of the organism.
Variation in oxygen isotope ratios studied from terrestrial
environments is ascribed to environmental temperature changes,
with warmer weather resulting in more positive d18O values andcoolerweather resulting inmorenegative d18O values inmeteoricwaters. However, the pattern is different in warm environments,
where the temperature is higher than 20 C. In such contexts, thed18O values decrease when the amount of precipitation increases.Local parameters, such as evaporation in ponds or streams orig-
inating at high altitudes, may provide drinking water to terrestrial
vertebrates with d18O values different from those of local precip-itation, and thus complicate the interpretation of the oxygen
isotopic composition of fossil teeth and bones.
Chronological Resolution of Skeletal Tissues
Different tissues record the isotopic signature of food or drinking
water during the period of their synthesis. Typically, bone grows
during the first stages of ontogeny, but in higher vertebrates such
Cerling and Harris, 1999). The reconstructed diet of zebras (Equus burchelli)grasses, while those living in a zoo and fed with C3 plants track this differenteating C3 plants in a Mongolian grassland.20 -15 -10
20 -15 -10
20 -15 -10 -53C
3C
3C
vannah
Kenya (C4)
Zebra(Equus burchelli)
Horse(Equus caballus)
Grazers (C4)
ion of the diet consumed by modern equids (horses and allies) based13C values measured in carbonate hydroxylapatite (data from20 -15 -10 -5
C4 - Grass13C = 12.5 1.1as mammals and birds, it continues to remodel and therefore
incorporates newer carbon, nitrogen, and oxygen atoms. An
adult mammal thus dies with its bone isotopic signature reflect-
ing the last years of its lifetime. In contrast to this, mammalian
dental tissues such as dentine and enamel form during a limited
period of an individuals lifetime, and once formed, do not
remodel. In an adult mammal, teeth exhibit isotopic signatures
corresponding to the early years of the individual, depending on
the chronology of tooth development for a given species. Cross
sectionsof dentine and enamel retain their isotopic signature in a
chronological order reflecting time of assimilation (e.g., Drucker
et al., 2010; Fraser et al., 2008). Tooth enamel may thus record
seasonal variations of d18O values (Figure 4). The level of reso-lution in such tissues, especially tooth enamel, is still under
debate as each volume of tissue forms over a significant amount
of time (e.g., Kohn and Cerling, 2002).
Physiological Effects (Suckling, Hibernation, Starvation,and Water Stress)
Some events in the life of a vertebrate can complicate the
record of dietary isotopic signatures. In mammals, diet changes
dramatically between the nursing and adult stages, following
weaning. Maternal milk exhibits isotopic signatures different
from those of the average adult diet, especially with more
positive d15N values and more positive d18O values (Wrightand Schwarcz, 1998). Therefore, suckling mammals exhibit
living in a Kenyan savanna exhibit d13C values similar to those of C4diet, and exhibit d13C values similar to those of horse (Equus caballus)
assessed by the nitrogen content of bulk sample as a proxy
10
09 10 11 12 1
40
n spto
CARBONATE STABLE ISOTOPES | Terrestrial Teeth and Bones 307isotopic differences relative to their adult counterparts, and
tissues formed during this formative period of life, such as
deciduous teeth and first molars, usually exhibit isotopic shifts
relative to other teeth and bone tissues formed after weaning.
In hibernating mammals, such as bears, carbon and nitrogen
isotopic signatures shift in bloodduring thewinter sleep and these
shifts are recorded in tooth dentine (Bocherens, 2004). Starvation
and water stress is also a phenomenon that has been suggested
to induce 15N shifts in vertebrate tissue (Ambrose, 1991), but
recent research on captive and wild mammals suggests that the
18OPO4
Figure 4 Variations of d18O in deer enamel reflect seasonal variations, iThe differences in amplitudes and absolute values of d18O values are dueWINTER
10
20
30
Hei
ght
(mm
)Wyoming
50
60changes in 15N abundances coincident with harsh environments
are most likely due to isotopic changes in the plants, while the
fractionation between diet and animals remains relatively con-
stant (e.g., Hartman, 2010; Murphy and Bowman, 2006).
Diagenetic Alteration
The chemical composition of bone and tooth is modified after
an individuals death, through physical, chemical, and bio-
chemical mechanisms called diagenesis. The intensity of dia-
genetic alteration depends on the time elapsed since death, but
also on sedimentary conditions. High temperatures and hu-
midity as well as high levels of microbial activity will increase
the intensity of diagenetic transformation, increasing the
chances of alteration of the biogenic isotopic signatures.
Organic Fraction
Collagen in fossil bones and dentine can survive for tens of
thousands of years under favorable conditions. Cold and dry
climatic conditions are more favorable than warm and humid
ones, while cave deposits are more favorable than open-air
sites. Collagen preservation in fossil skeletal remains can be(e.g., Bocherens et al., 2005a).
Fossil collagen can be purified through several methods.
The reliability of the isotopic signatures of ancient collagen is
assessed through its chemical similarity to collagen from fresh
bone. Fossil organic extracts with atomic C/N ratios lower than
2.9 or higher than 3.6 are considered unreliable, as well as
those containing less than 5% nitrogen (e.g., Ambrose, 1990).0
3 14 15
14 15 16 17
SUMMER
ecimens from Wyoming and Croatia (based on Fricke et al., 1998).different climatic regimen in both areas.20
Croatia
SUMMERMineral Fraction
While it is almost always possible tomeasure carbon and oxygen
signatures from fossil bones and teeth, the key question is to
determine whether the measured values correspond to those
recorded in the organisms tissues when it was alive. Evaluating
the extent of diagenetic alteration can be done either using the
pattern of isotopic variations observed in the fossil samples as
compared to equivalent modern ones, or using indirect tracers
of modifications, such as crystallinity indexes and uptake of
trace elements (e.g., Kohn and Cerling, 2002). It is commonly
assumed that the isotopic signatures of enamel are much more
stable through time than those of dentine and bone, while the
phosphate fraction is more stable than the carbonate fraction.
However, the phosphate fraction can be also affected under
special circumstances. The best approach is to assess the extent
of diagenetic alteration in each studied site using as many dif-
ferent proxies as possible (e.g., Kohn et al., 1999).
Reconstruction of Terrestrial Paleoenvironments
Tropical Environments
Paleoenvironments where C3 and C4 plants coexist are partic-
ularly favorable for isotopic tracking with carbon in teeth and
brates document these changes in the proportions of con-
Studies based on late Quaternary herbivore tooth enamel
isotopic fractionation is transferred to herbivores consuming
such plants in dense canopy forests. It is thus possible to track
308 CARBONATE STABLE ISOTOPES | Terrestrial Teeth and Bonesshowed that the proportions of C3 and C4 plants in the North-
ern Cape Province, South Africa, did not reflect the predictions
of climatic models about winter and summer rainfall regimes
(Lee-Thorp and Beaumont, 1995) and documented episodes
of wetter and drier conditions from 16000 years ago (Smith
et al., 2002).
AridityIn tropical environments, aridity has been recognized as
a factor influencing the d15N of herbivore collagen, withmore positive d15N values under more arid conditions (e.g.,Ambrose, 1991; Hartman, 2010; Murphy and Bowman, 2006;
Schwarcz et al., 1999). This led to the possibility of quantifying
past annual rainfall based on the d15N of herbivorous taxa, forinstance, macropods (kangaroos) in Australia (Grocke et al.,
1997).
Decreasing humidity has an effect on bone because of
increasing evapotranspiration in leaves, leading to d18O in-crease in leaf water, which is recorded in bone phosphate
(e.g., Ayliffe and Chivas, 1990; Luz et al., 1990). In combina-
tion with high d13C values, the high d18O values exhibitedby middle Pleistocene representatives of marsupial species
that became extinct in the late Pleistocene demonstrate that
these fauna could survive arid episodes, which weakens the
hypothesis that increasing aridity may have led to their final
extinction around 50000 years ago (Prideaux et al., 2007).
Ecological Flexibility of Large MammalsIsotopic tracking of habitat through the plants consumed by
ancient herbivores is a taxon-free approach, and therefore can
be used to document possible diet and habitat change ofsumed plants. However, other factors such as the atmospheric
pressure of CO2 also play a role in this equation (Ehleringer
et al., 1997; Koch et al., 2004). The main climatic parameter in
these environments is the amount of annual rainfall, and
nitrogen isotopic signatures of herbivores can be used to
track past variations in aridity. Some examples are given in
the following section.
Development of C4 biomes during the late QuaternaryThe open environments of the southern half of the North
American continent are a place of competition between C3and C4 herbaceous plants. Today, there is a clear gradient of
decreasing proportions of C4 plants toward the north, with
about 10% of C4 plants at 48N. Carbon and oxygen isotopic
studies of late Pleistocene herbivore tooth enamel from south-
western United States demonstrated the role of low atmo-
spheric CO2 in the expansion of C4 plants during the Last
Glacial Maximum, an expansion that could not be predicted
based on temperature and precipitation changes alone (e.g.,
Koch et al., 2004).bones. Indeed, C4 plants increase in proportion as climatic
conditions become drier and warmer, and C3 plants increase
in proportion as moisture increases. As the difference between
d13C values of these plants relative to C3 plants is around 12%,the d13C value recorded in the skeletal tissues of fossil verte-the changes of habitat for large herbivores at the beginning
of the Holocene in Europe (Figure 6; e.g., Drucker et al.,
2003a, 2008).
Variations in d15N values in ungulate bone collagen seem torelate to changes in soil microbial activity during the climatic
oscillations that occurred in Europe since 30000 years ago
(Drucker et al., 2003a,b; Hedges et al., 2004; Richards and
Hedges, 2003): low temperatures led to decreased microbial
activity and thus reduced nitrogen isotopic fractionation that is
transferred in plants consumed by herbivores. Indeed, a de-
crease in d15N values is recorded during the cold peaks of theLast Glacial Maximum and of the Younger Dryas, while an
increase in d15N values is documented during the warming ofthe BollingAllerod Interstadial and that of the early Holocene
(Preboreal and Boreal). The role of climate fluctuations to
explain these trends in d15N values was confirmed by thed18O values of the phosphate of the same bones that trackstemperature changes, and the observed correlation between
increasing d15N and d18O values, and hence temperature, dem-onstrates the relationship between increasing d15N values inred deer bone collagen and warming in this context (Drucker
et al., 2009). These nitrogen isotopic excursions present geo-
graphic variations according to the intensity of the temperature
changes.some species in relationship with climate change or anthro-
pogenic pressure. For instance, some large herbivores from
the Pleistocene in Florida exhibit diet and presumably habitat
stability between glacial and interglacial periods, such as tapir
and mastodon that remain browsers (C3 diet), while horses
remain grazers (C4 diet). In contrast, other species such as
white-tailed deer exhibit less negative d13C values during theinterglacial period, but still within the C3 diet values, while
extinct camel Hemiauchenia and extinct peccary Platygonus
exhibit a clear shift from C3 to C4 diet between glacial and
interglacial periods (DeSantis et al., 2009; Figure 5). By com-
paring the d13C values of modern mammals in southeasternAsia with those of the same species or related taxa in a
middle Pleistocene glacial site in Thailand, it was possible
to establish that some species were real forest dwellers, such
as Java rhinoceros and orangutan, while others that are now-
adays restricted to forest environments were dwelling in C4savanna, such as the small bovid Capricornis, a likely conse-
quence of increasing anthropogenic pressure (Pushkina et al.,
2010; Figure 5). These examples illustrate how isotopic
tracking can be used to document possible habitat changes,
even for species with modern relatives living in a restricted
environment or with a morphology seemingly adapted to a
given diet and habitat.
Temperate and Boreal Environments
In terrestrial environments, where all plants use the C3 photo-
synthetic pathway, plant d13C values exhibit some differencesrelated to environmental parameters (e.g., Heaton, 1999). In
particular, plants growing under a closed canopy exhibit sig-
nificantly more negative d13C values relative to those growingat the top of the canopy or in open environments. This carbon
Atlantic
-19
-20
-21
-22
d13 C
coll
()
-23
-24
-25
-266000 7000 8000 9000 10000 11000
Age cal BP (years)
12000 13000
French Jura
French Alps
14000 15000 16000
+
Pollen record in French Jura
-
CA
NO
PY
EFF
EC
T
Boreal PreborealYoungerDryas
OldestDryas
Allerd Blling
Figure 6 Evolution of collagen d13C values in red deer (Cervus elaphus) bones from French Jura and French Alps (data from Drucker et al., 2003a,2008). The late Pleistocene samples exhibit d13C values indicative of open environments, while Holocene specimens present more negative d13C valuesin Jura, similar to those exhibited by modern large herbivores dwelling in a closed canopy forest. In the Alps, red deer remain in open environments,probably at higher altitudes.
Denser forest
Florida
SE Asia
C3
-20 -15 -10 -5 0 5
Inglis 1A (Glacial)
Leysey 1A (interglacial)
Thum Wiman Nakin (Saalian)
Modern
-20 -15 -10
d13C-5 0 5
C4C3+C4
Figure 5 d13C values of tooth enamel illustrate stability or changes in diet between glacial and interglacial periods in Florida (top, data from DeSantiset al., 2009) and between Saalian (glacial) and modern (interglacial) periods in southeastern Asia (bottom, data from Pushkina et al., 2010).
CARBONATE STABLE ISOTOPES | Terrestrial Teeth and Bones 309
hominids in Africa. Indeed, modern African apes are restricted
rounding environment, it seems that the most likely explana-
tion for this C4 diet should include a mixture of underground
thals had diets that did not differ significantly from those of
from North America were vegetarian when they coexisted with
310 CARBONATE STABLE ISOTOPES | Terrestrial Teeth and Bonesplant storage organs and animals resources (Lee-Thorp et al.,
2010). In East Africa, high d13Cmeasured in some Paranthropusboisei seems to reflect the consumption of abundant C4 sedges
(Figure 7; Lee-Thorp et al., 2010). In contrast, an older hom-
inid from the Pliocene, Ardipithecus ramidus, did not incorpo-
rate C4 resources in its diet although these resources were
present in the environment (Lee-Thorp et al., 2010). This
supports the view that hominid evolution is linked to savanna,to forested environments, and savanna environments require
special skills for exploitation by primates, such as a bipedal gait
and strong social bonds. The d13C values of tooth enamelof fossil mammals, including hominids, from South Africa,
ranging in age from 1.8 to 1.5 million years old, indicate a
significant C4 component in the environment, as well as sig-
nificant C4 dietary inputs for hominids of the species Para-
nthropus robustus, and Homo ergaster (Figure 7; Lee-Thorp
et al., 2000, 2003, 2010). This C4 dietary input could be linked
to the consumption of C4 plant parts, but also to the consump-
tion of animals feeding on C4 plants, such as invertebrates,
small vertebrates, or grazer meat. Based on other evidence such
as tooth microwear and abundance of C4 plants in the sur-Seasonality
Using carbon and oxygen isotopic variations in hypsodont tooth
enamel, as in horse and bison, allowed the reconstruction of
seasonal changes in consumed plant food and precipitation
isotopic signatures, thus leading to a better understanding of
past climatic regimen and events preceding the death of ancient
animals (e.g., Gadbury et al., 2000; Higgins and MacFadden,
2004, 2009; Kohn andCerling, 2002; Passey and Cerling, 2002).
Reconstruction of the Paleobiology of Extinct Species
Using skeletal material from which collagen can be extracted,
it is possible to reconstruct some aspects of animal diet using
carbon and nitrogen isotopic signatures, because different
dietary items exhibit different isotopic signatures, such as
C3 or C4 plants, animal flesh, or food resources of marine
origin. With material too old or too altered to yield collagen,
the carbon isotopic signatures of tooth enamel can be used
to test dietary hypotheses in contexts where different food
webs start with plants exhibiting different d13C values, suchas tropical environments where C4 grasses coexist with C3tree leaves.
Subsistence Patterns of Ancient Hominids
Environmental and dietary changes are often linked to key
stages of the evolution of hominids. In some cases, the possible
impact of such changes can be tested using the isotopic signa-
ture of fossil bones and teeth.
The importance of savanna for African PliocenePleistocenehominidsA hotly debated issue in the study of Quaternary terrestrial
paleoenvironments is the role of savanna in the evolution ofthe carnivorous short-faced bears, but they became much more
carnivorous after the extinction of these meat-eating bears
(Barnes et al., 2002).early anatomically modern humans (Drucker and Bocherens,
2004). This supports the hypothesis of dietary competition
between Neanderthals and anatomically modern humans
(Bocherens and Drucker, 2006).
Paleodiet of Ancient Bears
Bears, as omnivorous carnivores, form an interesting group to
study using the isotopic approach as their diet can be quite
variable, and sometimes difficult to determine based on their
morphological features. They are all themore interesting because
they have an abundant fossil record, for instance, for cave
bears, and cover a dietary spectrum similar to that of humans.
Detecting changes in bear diet may yield direct information on
the availability of food resources relevant for human diet.
Moreover, the paleogenetics of ancient bears is intensively stud-
ied and combining genetic and paleodietary data through the
evolution of bear lineages provides direct evidence about their
evolutionarybiology.Using carbon- andnitrogen-stable isotopic
signatures, cave bears (Ursus spelaeus) were shown to be essen-
tially vegetarian animals (Bocherens et al., 1994, 2006, 2011a,b),
while the extinct giant short-faced bear (Artodus simus) from
North America has been demonstrated to be a meat eater
(Figure 8; Barnes et al., 2002). Interestingly, the diet of ancient
brown bears living at the same time as extinct bears from spe-
cialized species shifts when the dietary competition stops with
the extinction of their competitor. For instance, brown bearsat least since about 3 Ma, but also contradicts the hypothesis
that different hominids used savanna resources in differing
amounts.
The Diet of Neanderthals and Early Anatomically ModernHumansDuring the Upper Pleistocene, Europe was populated by a
distinctive hominid form, the Neanderthals. The demise of
this hominid around 30000 years ago coincides with the ap-
pearance in Europe of anatomically modern humans, seem-
ingly using a more sophisticated culture. Several Neanderthal
fossils have yielded preserved collagen that could be analyzed
for carbon- and nitrogen-stable isotopic compositions, and
compared to those of Upper Paleolithic anatomically modern
humans (e.g., Bocherens et al., 1999, 2005b, in press; Richards
et al., 2000, 2001, 2008). Comparing Neanderthals with pred-
ators such as hyenas suggests that Neanderthals consumed
mainly large herbivore meat, including a large proportion of
megaherbivores such as mammoth and woolly rhinoceros
(Bocherens et al., 2005b). It appears also that Neanderthals
living under different environmental conditions and at differ-
ent periods had similar diets. Although the absolute isotopic
nitrogen values of early modern humans are higher than those
of the last Neanderthals (Richards and Trinkaus, 2009), possi-
ble variations in the d15N values of the whole terrestrial foodwebs at the same time suggest that, finally, the last Neander-
herbivorous and carnivorous species (data from Fox-Dobbs et al., 2008).These isotopic data of short-faced bears plot together with carnivorous
CARBONATE STABLE ISOTOPES | Terrestrial Teeth and Bones 311-12 -10 -8 -6 -4d13C
-2 0 2 4
-12 -10 -8 -6 -4d13C
-2 0 2 4
Paranthropusrobustus
Homo ergaster
South Africa
Figure 7 Reconstruction of the paleoenvironment and paleodiet offossil hominids from South Africa (Swartkrans Members 1 and 2, 1.5to 1.8 Ma) and East Africa (Olduvai East and Pening, 1.5 to 1.8 Ma).The d13C values of Paranthropus robustus, and Homo ergaster in SouthAfrica show that a significant amount of food resources coming from C4environments (most likely savanna) were consumed by these threehominid species (data from Lee-Thorp et al., 2003). In contrast toParanthropus robustus in South Africa, Paranthropus boisei from EastAfrica exhibits a diet almost completely composed of C4 food resources,while Homo habilis had a diet with d13C values similar to Homo ergasterC3 C4
East AfricaParanthropusboisei
Homo habilis
C3 + C4Paleodiet of Ancient Ungulates
Large herbivorous mammals often exhibit specialized diets, in
connection with their tooth morphology and digestive physi-
ology. The occurrence of some herbivorous mammals in Qua-
ternary fossil and archaeological localities can be used to infer
the paleoenvironments around the site. Isotopic analyses of
fossil herbivores have sometimes yielded additional informa-
tion about the ancient environments in which they used to
dwell, and thus allow more precise reconstructions of the
actual diet of extinct species. For instance, isotopic investiga-
tions of tooth enamel have shown that in southern North
America, horses were mixed feeders consuming C4 grasses
and C3 shrubs, while bisons were grazers, eating only C4grasses (Koch et al., 2004). Horses have hypsodont (high-
crowned) teeth and are traditionally thought to be specialized
grazers. Another example of hypsodont ungulates with diverse
diets are extinct camelids from North America (Feranec, 2003).
Therefore, the specialized morphology of some herbivore teeth
is not always indicative of specialized diet, but rather indicates
dietary flexibility.
Implications for Late Pleistocene Extinctions
The possibility to reconstruct dietary and/or habitat changes
through time for a given species opens the possibility to doc-
ument possible ecological disruption linked to extinctions,
in South Africa.10
9
8
7
6
5
4
d15 N
3
2
1
0
-1
-2-22 -21 -20 -19
d13C
-18 -17 -16
MammuthusEquusBisonBosRangiferPanthera leoCanis lupusBootheriumHomotheriumArctodus
Figure 8 d13C and d15N values of short-faced bears (Arctodus simus)collagen from Alaska and Yukon, compared to those of coevalespecially in the case of the late Pleistocene megafaunal extinc-
tions. Two main explanations are usually given for these ex-
tinctions: climate change and human impact (e.g., Koch and
Barnosky, 2006). Using the isotopic signatures of extinct spe-
cies until the moment they become extinct and those of sur-
viving species before, during, and after the extinction event
may provide information about the factors that could have
changed at this time and therefore help to test different hy-
potheses. In the case of Australia, the stable isotopic tracking of
megafauna paleoecology has yielded very interesting results.
For instance, aridity could be ruled out as a significant factor in
the extinction of megafauna as the extinct species exhibited
carbon and oxygen isotopic signatures that demonstrated that
they did live under arid conditions well before the time of their
extinction (Prideaux et al., 2007). If humans were involved in
the megafaunal extinction in Australia, the question remains
whether their action was mostly direct, through overhunting,
or rather indirect, through environmental disruption, in in-
creasing wildfire frequency for instance. Isotopic tracking, to-
gether with tooth microwear analysis, did provide some
answers to this question. In the case of an extinct giant short-
faced kangaroo Procoptodon, these data suggest a diet including
C4 chenopodiaceae from dry areas and obligate drinking
(Prideaux et al., 2009). Aridity would therefore not be a
problem for this species, but having to go to water holes
would make this species vulnerable to human predation, in
contrast with other kangaroos which survived the extinction
species, such as lion, scimitar-toothed cat, and wolf, clearly indicatingthese that short-faced bears were carnivorous.
additional chemical elements. For instance, the amount of
deuterium, the stable heavy isotope of hydrogen, could be
245: 249261.Bocherens H, Fizet M, and Mariotti A (1994) Diet, physiology and ecology of fossil
mammals as inferred by stable carbon and nitrogen isotopes biogeochemistry:
312 CARBONATE STABLE ISOTOPES | Terrestrial Teeth and Bonesused to study paleoclimates and possible migrations, as it
varies in a way similar to oxygen in relationship with temper-
atures (Bowen et al., 2005; Cormie et al., 1994). The isotopic
signatures of sulfur in collagen may be used to improve paleo-
dietary reconstructions and to provide identification of geo-
graphic origin (Richards et al., 2003).
Improving biomolecular technologies will allow the anal-
ysis of isotopic signatures of other organic compounds, such
as osteocalcin, single amino acids, cholesterol, and fatty acids,
in fossil bones and teeth (e.g., Corr et al., 2005; Smith et al.,
2005, 2009). This is expected to lead to breakthroughs in
retrieving paleobiological information at different timescales
within a single individual, and in obtaining isotopic paleobi-
ological signals in fossil material older than around 100000
years.event, and which can sustain themselves with the water con-
tained in their plant food and do not need to drink at water
holes. An additional contribution of stable isotopic tracking in
the debate on megafaunal extinctions in Australia was to dem-
onstrate that species with dietary specialization were much
more affected by a collapse of ecosystem and that a critical
factor for surviving was the possibility to shift diet (Miller et al.,
2005). A more widespread use of this approach to megafaunal
extinction on other continents should also yield valuable in-
formation. For instance, the carbon- and nitrogen-stable iso-
topes of the last cave lions in Western Europe dated to around
12000 years BP suggest that they have relied mainly on rein-
deer as their preferred prey (Bocherens et al., 2011a). The
extirpation of this predatory species in this region could then
correspond to the coeval extirpation of its main prey.
Perspectives
Stable isotopes in terrestrial teeth and bones have already
yielded invaluable information on Quaternary ecosystems,
and this approach is expected to develop further during the
coming years.
A better knowledge of isotopic variations in modern eco-
systems and modern animals will most probably allow more
accurate paleoenvironmental and paleodietary reconstructions
to be performed based on the stable isotopic signatures of teeth
and bones of Quaternary terrestrial vertebrates. Our under-
standing of the fractionation factors in tissues prone to fossil-
ization still needs improvement, based on controlled diet
experiments of species closely related to the fossil taxa under
study, and on investigations of large mammals in monitored
wild contexts. Also the chronology of isotopic records in incre-
mentally grown tissues, such as tooth enamel, needs more
accurate quantification in the species found in Quaternary
fossil assemblages.
The impact of diagenetic alteration on the isotopic signa-
tures of teeth and bones needs to be better understood, espe-
cially when dealing with the mineral fraction of fossil
vertebrates.
Collagen is currently used for its carbon and nitrogen iso-
topic signatures, but it could yield the isotopic signatures ofImplications for Pleistocene bears. Palaeogeography, Palaeoclimatology,Palaeoecology 107: 213225.
Bocherens H, Germonpre M, Toussaint M, and Semal P (in press) Stable isotopes.In: Semal P and Hauzeur A (eds.) Spy Cave: State of 120 years of PluridisciplinaryResearch on the Betche-aux-Rotches from Spy.
Bocherens H, Stiller M, Hobson KA, et al. (2011b) Niche partitioning between twosympatric genetically distinct cave bears (Ursus spelaeus and Ursus ingressus) andbrown bear (Ursus arctos) from Austria: Isotopic evidence from fossil bones.Quaternary International 245: 238248.
Bowen GJ, Wassenaar LI, and Hobson KA (2005) Global application of stable hydrogenand oxygen isotopes to wildlife forensics. Oecologia 143: 337348.See also: Vertebrate Overview. Archaeological Records: HumanEvolution in the Quaternary; Neanderthal Demise. Carbonate StableIsotopes: Lake Sediments; Nonmarine Biogenic Carbonates;Overview; Speleothems; Terrestrial Organic Materials. Ice CoreRecords: Antarctic Stable Isotopes; Greenland Stable Isotopes.Vertebrate Records: Late Pleistocene Megafaunal Extinctions; LatePleistocene of Southeast Asia. Vertebrate Studies: Ancient DNA;Interactions with Hominids; Speciation and Evolutionary Trends inQuaternary Vertebrates.
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314 CARBONATE STABLE ISOTOPES | Terrestrial Teeth and Bones
Terrestrial Teeth and BonesIntroductionPaleobiological Tracking by Isotopes in Teeth and BonesCarbonNitrogenOxygenChronological Resolution of Skeletal TissuesPhysiological Effects (Suckling, Hibernation, Starvation, and Water Stress)
Diagenetic AlterationOrganic FractionMineral Fraction
Reconstruction of Terrestrial PaleoenvironmentsTropical EnvironmentsDevelopment of C4 biomes during the late QuaternaryAridityEcological Flexibility of Large Mammals
Temperate and Boreal EnvironmentsSeasonality
Reconstruction of the Paleobiology of Extinct SpeciesSubsistence Patterns of Ancient HominidsThe importance of savanna for African Pliocene-Pleistocene hominidsThe Diet of Neanderthals and Early Anatomically Modern Humans
Paleodiet of Ancient BearsPaleodiet of Ancient Ungulates
Implications for Late Pleistocene ExtinctionsPerspectivesReferences