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Quaternary Science Reviews 325 (2024) 108481
Available online 4 January 2024
0277-3791/© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-
nc/4.0/).
A novel ontogeny-related sampling of dental tissues for stable isotopes
interpretation of the paleobiology of the cave bear
U. Kastelic Kovaˇ
ciˇ
c
a
,
*
, I. Debeljak
a
, D. Potoˇ
cnik
b
, N. Ogrinc
b
,
c
, N. Zupanˇ
ciˇ
c
a
,
d
a
Ivan Rakovec Institute of Palaeontology, ZRC SAZU, Novi trg 2, Ljubljana, Slovenia
b
Department of Environmental Science, Joˇ
zef Stefan Institute, Jamova cesta 39, Ljubljana, Slovenia
c
Joˇ
zef Stefan International Postgraduate School, Jamova cesta 39, 1000, Ljubljana, Slovenia
d
University of Ljubljana, Faculty of Natural Sciences and Engineering, Aˇ
skerˇ
ceva cesta 12, Ljubljana, Slovenia
ARTICLE INFO
Handling Editor: Danielle Schreve
Keywords:
Paleobiology
Ontogenetic stages
Diet
Hibernation
δ
13
C and δ
15
N
Life period
Dentin
ABSTRACT
In cave bear studies, stable isotope analysis of carbon and nitrogen is typically conducted on bone collagen.
However, challenges arise due to biases in the classication of bones into age groups, which can be inuenced by
various factors that affect bone size. To overcome such issues, we present and evaluate a novel approach that
involves sampling dental collagen from different ontogenetic stages of 45 fossil teeth, where only a portion of
each tooth formed during the periods of interest was sampled. This approach enabled us to obtain a carbon and
nitrogen ngerprint specic to a particular period in a cave bear’s life without interference from earlier stages.
Stable isotopic analysis revealed a rapid decrease in δ
15
N values from 3 to 15 months of age. After that, the δ
15
N
values remain stable. Based on the nitrogen isotope composition (
15
N/
14
N), cubs aged 0–3 months exclusively
consumed milk, while the gradual introduction of solid plant food occurs at 5–10 months, resulting in a shift in
δ
15
N values. Based on similar δ
15
N values in older age groups, it can be assumed that juveniles did not resume
milk suckling after the rst hibernation. The δ
13
C values increase until 15 months, followed by a gradual
decrease until adulthood, potentially attributable to hibernation. The δ
13
C values also seems to indicate isotopic
differences between yearlings with low δ
13
C values that successfully established hibernation and those with high
δ
13
C values that failed to hibernate and starved. This novel observation, i.e., decreasing δ
13
C values from the
second year of life onward, agrees with predicted isotopic skeletal collagen δ
13
C values calculated from modern
brown bear blood plasma. In summary, the ontogenetic sampling strategy facilitated the acquisition of further
paleobiological insights into the distinct life periods of cave bears.
1. Introduction
The cave bear (Ursus spelaeus sensu lato) is one of the prominent
representatives of the last ice age. During the late Pleistocene, its terri-
tory extended throughout most of Europe (Pacher and Stuart, 2009), and
it is one of the most studied extinct species due to the abundance of fossil
remains in caves. The prevailing opinion is that cave bears died there of
natural causes because they did not successfully survive hibernation
(Kurt´
en, 1976; Andrews and Turner, 1992; Stiner et al., 1998; Debeljak,
2002, 2004, 2007; Grandal-d’Anglade et al., 2019). The cave bear
became extinct during the Last Glacial Maximum as the climate cooled
(Hilderbrand et al., 1996; Pacher and Stuart, 2009).
The availability of a new analytical tool – stable isotope analysis has
contributed to the lively interest in understanding and studying the
paleoecology of extinct animals. The stable carbon (δ
13
C) and nitrogen
(δ
15
N) isotopic composition in the collagen of skeletal tissues primarily
offers valuable insights into dietary patterns, enabling a better under-
standing of and exploration of the lives of past animals (Keeling and
Nelson, 2001). Much research has been focused on the diet of cave bears.
Interestingly, the obtained low δ
15
N and δ
13
C values in cave bears are
comparable to or even lower than those obtained in herbivores
(Bocherens et al., 1994, 1997; Nelson et al., 1998; Münzel et al., 2014;
Bocherens, 2015, Bocherens, 2019). This phenomenon, however, is not
yet clearly understood, but some authors suggest that hibernation may
be a contributing factor (Bocherens et al., 1994; Nelson et al., 1998;
Lid´
en and Angerbj¨
orn, 1999; Fern´
andez-Mosquera et al., 2001; Boche-
rens, 2004), while others propose the environment and diet as possible
factors (DeNiro and Epstein, 1981; Bocherens, 2004).
* Corresponding author.
E-mail address: ursa.kastelic-kovacic@zrc-sazu.si (U. Kastelic Kovaˇ
ciˇ
c).
Contents lists available at ScienceDirect
Quaternary Science Reviews
journal homepage: www.elsevier.com/locate/quascirev
https://doi.org/10.1016/j.quascirev.2023.108481
Received 16 June 2023; Received in revised form 15 December 2023; Accepted 18 December 2023
Quaternary Science Reviews 325 (2024) 108481
2
Nelson et al. (1998) found a clear correlation between animal age
and δ
13
C values by conducting one of the rst isotopic analyses of
collagen samples from cave bear bones from Divje babe I based on
different age groups. Similar analyses were performed on cave bear
bones from other sites (Bocherens, 2004; P´
erez-Rama et al., 2011a;
Bocherens, 2015; Robu et al., 2018; Grandal-d’Anglade et al., 2019), as
well as isotopic analyses of teeth according to the ontogenetic pattern
(Bocherens et al., 1994). It was assumed that juvenile bones could be
easily assigned to age groups (e.g., the 1
st
and 2
nd
winter) based on size
(Kurt´
en, 1976; Lid´
en and Angerbjoern, 1999). However, the problem is
that no precise critical bone size interval exists for a single age group
rendering any classication subjective (P´
erez-Rama et al., 2011b). Also,
sexual dimorphism, different sizes of individuals according to litter size
or maternal experience, all affect the bone size of individuals (Gran-
dal-d’Anglade and L´
opez-Gonz´
alez, 2005; P´
erez-Rama et al., 2011b;
Robbins et al., 2012). For example, Robu et al. (2018) classify specimens
into neonates, juveniles, subadults, and adults without providing an
approximate bone size and age range for a given group. The same is true
for Nelson et al. (1998), where the approximate age range for the group
was added by Keeling and Nelson (2001). Meanwhile, P´
erez-Rama et al.
(2011a) and Grandal-d’Anglade et al. (2019) used the percentage of
adult bones to classify specimens. Consequently, such differences make
it challenging to compare results across different sites. The collagen in
bones is formed during the growth of the individual and is remodeled
throughout life at different rates in different anatomical parts. Bone
remodelation results in the loss of isotopic information from previous
periods. Furthermore, collagen obtained from adult bones represents an
average diet over the last several years (Hindelang et al., 2002; Huja and
Beck, 2007).
An alternative approach to bone sampling would be to sample teeth,
given that a shorter life period of interest can be obtained. For example,
Czernielewski et al. (2020) used an ontogenetic sampling of tooth
enamel, the most resistant to diagenetic changes, and determined the
stable carbon (δ
13
C) and oxygen (δ
18
O) isotopic composition to study
seasonal changes. The enamel develops rapidly, taking approximately
one year, which limits its isotopic record. Alternatively, dentin forms
over a longer period, allowing for dietary changes to be tracked over a
longer period. This prolonged formation period of dentin enables the
acquisition of a broader range of δ
15
N values, offering enhanced insights
into a bear’s diet. Also, dentin has a more resistant structure to
diagenetic changes than bone (Bocherens et al., 1997), and dental tis-
sues do not remodel. Therefore, isotopic records formed during their
lifetime remain preserved. So far, only a limited number of studies have
investigated the stable isotopic compositions of carbon and nitrogen in
cave bear tooth dentin utilizing the entire tooth to record average iso-
topic signals from the period when cubs were still drinking milk until
adulthood (Bocherens et al., 1994, 1997).
Our study aims to gain new data on the nutrition and behaviour of
the cave bear during different life periods. For this purpose, we intro-
duce and evaluate a novel approach to sampling dental collagen based
on the ontogenetic development of dentition in cave bears. This
approach allows us to obtain a δ
15
N and δ
13
C values of a specic life
period of the cave bear without interfering with signals from earlier
periods. Such insights into juvenile life could provide information on
lactation, transition to solid food, rst hibernation, mortality rates and
later independence of (juvenile or) subadult individuals. Understanding
the different life periods, especially the juvenile period, is extremely
important for the species’ survival.
2. Materials and methods
The researched site Divje babe I is a cave located in the Idrijca Valley
in western Slovenia at an altitude of 450 m (Fig. 1). The cave is formed in
Cordevolian dolomite and is lled almost exclusively by autochthonous
clastic sediments (Skaberne et al., 2015). The remains of over 60 animal
species have been found in the cave, among which fossils of the cave
bear predominate (Turk, 2007). This study investigated 45 teeth from
layers 8 to 10, which date to approximately 55–75,000 years old
(Blackwell et al., 2007; Turk, 2007).
2.1. Sampling method
To obtain isotopic record of dental tissues formed during specic life
periods, different teeth or parts of teeth were selected based on our
previous studies on the ontogenetic development of dentition in cave
bear, as well as detailed microscopic analyses of increments in dental
cementum and dentin that can be used for age determinations (e.g.,
Debeljak, 1996, 2002, 2004, 2007, 2011). For the earliest life periods, i.
e., from 0 to 15 months of age, deciduous d
4
teeth, deciduous canines,
and rst lower permanent molars were selected. To study isotopic record
Fig. 1. Location of Divje babe I cave (46
◦06
′
46
″
N 13◦54
′
56
″
E).
U. Kastelic Kovaˇ
ciˇ
c et al.
Quaternary Science Reviews 325 (2024) 108481
3
from life periods from the second year onward, permanent canines were
analysed (Figs. 2–8). Canines are particularly suitable because they are
the largest teeth and take the longest to form, which means that they
contain the isotopic record of many life periods. Furthermore, sex can be
determined based on the dimensions of the canines (e.g., Koby, 1949;
Kurt´
en, 1955; Debeljak, 2002). X-ray images of all the studied teeth
were taken to determine the stage of tooth formation and the degree of
lling of the crown and root with dentin, which is characteristically
age-related. Except for the d
4
teeth, only the lowest parts of the roots
were used for analyses, and all tooth crowns were left intact. We selected
samples which represent the following specic life periods:
2.1.1. The rst three months of life
Six whole deciduous last lower premolars (d
4
) teeth (Fig. 2),
belonging to 2-3 month-old cubs that died between March and April, i.e.,
1–2 months before leaving the den with their mother. These teeth had
just erupted or were just at the end of erupting before death. There are
no wear facets on the crown, the roots are wide open at the ends and
very fragile. According to data for present-day bears (Dittrich, 1960),
the d
4
teeth began to form in the rst month of life. Therefore, we as-
sume that none of the tissue analysed is from the prenatal period. It was
formed in the rst, second and third month of life. During this time, the
cubs only consumed their mother’s milk. The cubs did not hibernate
during their rst winter, but their lactating mother did.
2.1.2. At the age of 5-10 months
The lower half of the roots of six deciduous canines (Fig. 3) of
approximately 10 month-old cubs that died presumably shortly after
entering the cave for hibernation, probably in October or November,
were analysed. The crown of these teeth is worn, the root is fully formed,
closed at the apex, and intact. We assume that resorption of the root and
shedding of the deciduous canines usually occurred later in winter and
that by selecting specimens without signs of resorption, we avoided the
effect of a prolonged hibernation period on the isotopic record. The
crown and the upper half of the root were removed to analyse only the
dental tissues that formed after the fourth month of life when the cubs
already left the den with their mother and were feeding on both plants
and milk. The analysed tissues were formed from May to November
during the rst year of life, at the age of 5–10 months.
2.1.3. At the age of 5–15 months
Dental tissue that formed basically in the same period as group 2 was
sampled, but this time with the addition of the winter months to study
the possible effect of hibernation on isotopic values. The lower two-
thirds of the roots of six permanent rst lower molars (M
1
) of year-
lings (Fig. 4) that died during their second winter were analysed. The
particularly high mortality at this age indicates that the second winter
was a rather critical period in the life of a cave bear (e.g. Debeljak,
2002). The analysed teeth have a slightly worn crown, the roots are still
somewhat open at the apexes, and their wall is 1–1.25 mm thick when
measured about 0.75 cm below the crown (see X-ray image). The crown
and the rst third of the root, which was partially formed already in the
rst 4 months of life, were removed, and only that part of the root that
formed at the age of 5–15 months was sampled.
2.1.4. At the age of 15–27 months
Part of the root (Fig. 5) from eight permanent canines (of four fe-
males and four males) of approximately 2-year-old juveniles that died
during their third winter was analysed. At this stage, one-third to one-
half of the canine root is formed. Here the crown and about 1 cm of
the posterior part of the root, which had already formed in the rst 15
months of life, were removed, and the part that formed from the second
spring to fall and possibly, in a lesser degree, during the third winter, i.
e., at 15–27 months of age was analysed. Nutrition at this period of life is
of special interest. A comparison of the isotopic records of this age group
with those of the previous and next groups could give us some evidence
whether juveniles were able to resume suckling milk after their second
winter.
2.1.5. At the age of 27–39 months
Seven permanent canines (ve belonging to females and two to
males) of approximately 3-year-old subadults that died presumably in
their 4
th
winter were analysed (Fig. 6). About two-thirds to three-fourths
of the canine root is formed at this age, depending on size or sex. The
root is wide open at the apex. The lowest part of the root that formed in
Fig. 2. Deciduous lower fourth premolar (d
4
), belonging to 2-3 month-old cub.
The dashed rectangle indicates that the whole tooth was analysed, i.e. the tissue
from the rst 3 months of life. Right: X-ray image of the same tooth.
Fig. 3. Deciduous canine of approximately 10 month-old cub. The dashed
rectangle indicates that only the lower half of the root was analysed; i.e. the
tissue that formed at the age of 5–10 months. Right: X-ray image of the
same tooth.
Fig. 4. Permanent rst lower molar (M
1
) of a yearling that died in its 2
nd
winter. The dashed rectangle indicates which portion of the root was analysed;
i.e. the tissue that formed in the age interval of 5–15 months. Right: X-ray
image of the same tooth.
U. Kastelic Kovaˇ
ciˇ
c et al.
Quaternary Science Reviews 325 (2024) 108481
4
the 3
rd
year is slightly oblique to the rest of the root. For sampling, only
the lowermost part of the root was collected to the height where a
narrow band of growth arrest from the third winter is often observed
(Fig. 6). Therefore, the sampled tissue is from the age interval of 27–39
months. Third year was presumably another vulnerable period of life
when most young subadult bears, especially males, had to become in-
dependent and nd their own territory.
2.1.6. At the age of 5–6 years
The lowest 0.5–1 cm long part of the root (Fig. 7) of eight permanent
canines (four female and four male) of 5-6-year-old bears (young adults)
that formed after the age of 4 years was analysed. The roots of these
canines were already formed along their entire length but are still
Fig. 5. Permanent canine of approximately 2 year-old male juvenile that died
in its 3
rd
winter. The dashed rectangle indicates which portion of the root was
analysed; i.e. only the tissue that formed in the last year of life, after the 2
nd
winter. Right: X-ray image of the same tooth.
Fig. 6. Permanent canine of approximately 3 year-old male subadult. The
dashed rectangle indicates which portion of the root was analysed; i.e. the
tissue that formed in the last year of life, after the 3
rd
winter. Right: X-ray image
of the same tooth.
Fig. 7. Permanent canine of approximately 6 year-old cave bear (young adult).
The dashed rectangle indicates which portion of the root was analysed; i.e. the
tissue that formed in the last 2 years of life. Right: X-ray image of the
same tooth.
Fig. 8. Permanent canine of approximately 20 year-old male. The dashed
rectangle indicates which portion of the root was analysed. Right: X-ray image
of the same tooth.
U. Kastelic Kovaˇ
ciˇ
c et al.
Quaternary Science Reviews 325 (2024) 108481
5
hollow and open at the apex (Debeljak, 2002, 2007).
2.1.7. From the age of 5 years onwards
Approximately 1 cm of the lowest part of the root (Fig. 8) of four
permanent canines (two female and two male) of older adults was
collected for analysis. The roots of these canines are closed and lled
with dentin, which formed mainly between 5 and 15 years of age until
only a narrow root canal remained. To a lesser extent, the samples also
consist of dental cementum deposited on the root’s surface until the end
of life.
2.2. Sample preparation
A Dremel rotary tool with a round diamond-coated blade was used to
cut the teeth, and collagen extraction and purication were performed
according to previously described techniques (Longin, 1971; Ogrinc and
Budja, 2005). Each individual tooth section was cleaned in an ultrasonic
bath with distilled water and dried at 25 ◦C for 24 h. The dried samples
were then weighed and demineralized in 0.5 M HCl at 4 ◦C until they
became translucent and exible due to the loss of the mineral phase (one
to ve days). The acid was replaced daily. After demineralization, each
sample was washed with distilled water and placed in 0.001 M HCl (pH
3) at 70 ◦C for about 48 h to produce fusible collagen. Each sample was
ltered through a glass-microber disc and a cellulose nitrate lter (5
μ
m). The ltrate was then freeze-dried and weighed. The percentage of
collagen was then calculated based on the initial mass of the sample and
the mass of lyophilized collagen. The dry collagen was then subjected to
isotopic analysis.
2.3. Isotope and elemental analysis of collagen
Stable isotope ratio measurements were performed using isotope
ratio mass spectrometry (IRMS) and reported using the δ notation in ‰
using the following equation (1) (Brand et al., 2014):
(i/jE) = i/jRP−i/jRRef
i/jRRef
(1)
where superscripts i and j denote the highest and the lowest atomic mass
number of element E, and R
P
and R
Ref
indicate the ratio between the
heavier and the lighter isotope (
13
C/
12
C,
15
N/
14
N) in the sample (R
P
)
and reference material (R
Ref
), respectively. The δ
13
C values are reported
relative to the V-PDB (Vienna-Pee Dee Belemnite) standard, and the
δ
15
N values are reported relative to the AIR standard.
Determination of δ
13
C and δ
15
N values in collagen samples (δ
13
C
coll
and δ
15
N
coll
) were performed simultaneously on an IsoPrime100 – Vario
PYRO Cube (OH/CNS Pyrolyser/Elemental Analyzer) (IsoPrime, Chea-
dle, Hulme, UK). Approximately 8 mg of collagen sample was weighed
into a tin capsule, and the same amount of WO
3
was added. The capsule
was then closed with tweezers and placed into the automatic sampler of
the elemental analyzer. The results for carbon and nitrogen were nor-
malised against the following international reference materials: USGS61
with δ
13
C = − 35.05 ±0.04‰ and δ
15
N = − 2.87 ±0.04‰, USGS88 with
δ
13
C = − 16.06 ±0.07‰ and δ
15
N = +14.96 ±0.14‰ and USGS89 with
δ
13
C = − 18.13 ±0.11‰ and δ
15
N =6.25 ±0.12‰. For independent
control, a laboratory reference material CRP-IAEA casein with δ
13
C =
−20.3 ±0.09‰ and δ
15
N = +5.62 ±0.19‰ was used. The analytical
precision, expressed as the standard deviation of the control material,
was ±0.2‰ for δ
13
C and δ
15
N.
The elemental compositions of C and N in the collagen sample were
determined on a Vario PYRO Cube (OH/CNS Pyrolyser/Elemental
Analyzer, IsoPrime, Cheadle, Hulme, UK) tted with a thermal con-
ductivity detector (TCD). The element contents (expressed in %) are
computed from the absolute element contents and the sample weight
according to formula (2):
c=a×100 ×f
w(2)
where c is the elemental concentration (%), a is absolute elemental
content (mg), f is the daily factor, and w is the sample weight (mg). For
the calculation of daily factor f, the following reference materials were
used: USGS61 with 49.48% of C and 28.85% of N, USGS88 with 44.09 ±
0.71% of C and 16.66 ±0.45% of N, USGS89 with 43.03 ±0.77% of C
and 15.78 ±0.19% of N, and USGS42 with 45.7% of C and 15.3% of N.
The analytical precision for C and N is ±1%.
3. Results
Table 1 shows the results of the stable isotope, C and N elemental
analysis, and dental collagen from specic life periods.
Collagen was successfully extracted from all samples. The C/N
atomic ratio ranged from 2.9 to 3.2, except for CyaM-4, which has a ratio
of 2.8. The C
coll
content ranges from 29.6 to 41.8%, and the N
coll
content
from 11.2 to 15.6%. The observed C/N ratios and the percentage of N
coll
and C
coll
indicate that the extracted material is well preserved, providing
reliable isotopic measurements (DeNiro, 1985; Ambrose, 1990; Van
Klinken, 1999).
The measured δ
13
C
coll
values range from −23.1 to −21.0‰, while the
mean value in the 0–3-month group is −22.6‰, −22.3‰ in the 5–10-
month group, −21.3‰ in the 5–15-month group, −21.8‰ in the 2nd
year group, −22.0‰ in the 3rd year group; −22.3‰ in the young adult
group and 22.4‰ in the adult group. When examining the life period
groups, it becomes evident that the δ
13
C
coll
values reach their lowest
point in the 0–3-month group, then the values gradually rise until the
5–15-month group, at which point they gradually decline with each
successive life period group (Fig. 9).
In contrast, the δ
15
N
coll
values decrease rapidly from the 0–3-month
group to the 5–15-month group. A slight decrease is observed until the
3
rd
year group, after which the δ
15
N
coll
values remain relatively stable
(Fig. 10). The measured δ
15
N
coll
values range from 6.4 to 2.2‰. The
mean δ
15
N
coll
value in the 0–3-month group is 6.1‰, 5.1‰ in the 5–10-
month group, 3.4‰ in the 5–15-month group, 3.1‰ in the 2
nd
year
group and 2.8‰ in the remaining three groups. Although no statistically
signicant correlation was found between δ
13
C
coll
and δ
15
N
coll
values,
the clustering of groups and a trend between rst three groups is evident
(Fig. 11).
4. Discussion
The range of δ
13
C values for all groups is from −23.1 and −21.0‰,
indicating a diet of C3 plant origin (Bocherens et al., 1994, 1995; Fizet
et al., 1995). Also, the range for δ
15
N values (2.0–3.4‰) conrms the
herbivorous diet of older juveniles, subadults and adults, whereas higher
δ
15
N values for early life periods indicate a more protein-rich diet due to
milk consumption (Bocherens et al., 1994; Fizet et al., 1995; Bocherens,
2004; Ogrinc and Budja, 2005; P´
erez-Rama et al., 2011a).
The 0-3 month-old group shows the lowest δ
13
C values and the
highest δ
15
N values, which gradually change to those of adults with
increasing age. According to data for present-day bears (Dittrich, 1960),
the d
4
teeth form in the rst month of life. The analysed d
4
teeth
belonged to cubs that consumed only their mother’s milk. The cubs did
not hibernate during their rst winter, but their lactating mother did.
Therefore, the lower δ
13
C and the higher δ
15
N values are a consequence
of the continued suckling of the fat and protein-rich milk of the hiber-
nating mother. The isotopic composition of the hibernating mother’s
milk is different to non-hibernating due to the use of fat stores depleted
in
13
C (Tieszen and Boutton, 1989) and body nitrogen recycling (Nelson
et al., 1998; Bocherens, 2004, 2015; P´
erez-Rama et al., 2011a,b), which
affects the isotopic values of the cub as well (Fogel et al., 1989). The
narrow range of observed δ
13
C and δ
15
N values in this life period can be
U. Kastelic Kovaˇ
ciˇ
c et al.
Quaternary Science Reviews 325 (2024) 108481
6
interpreted as further evidence of homogenous food sources, i.e., milk
from the hibernating mother was the only source of nutrition at this age.
Our results for the youngest cubs are in general agreement with those of
other authors (Bocherens et al., 1994; Nelson et al., 1998; Bocherens,
2004; P´
erez-Rama et al., 2011b; Bocherens, 2015; Robu et al., 2018;
Grandal-d’Anglade et al., 2019). However, proving a direct correlation
is difcult since the age group denitions of other authors are not always
well-dened and do not refer to the ontogenetic stage.
In the second age group (5–10 months), we obtained isotopic signals
from dentin that had already formed after the cubs had left the den with
their mother but continued consuming their mother’s milk. Based on the
study of Jenkins et al. (2001), we assume that the milk’s isotopic
composition would change after the mother stopped hibernating and
resumed feeding, i.e., δ
15
N values would decrease, and δ
13
C values in-
crease, with the consumption of plant food. This change in the mother’s
nutrition is also reected in the isotopic composition of the cub’s
collagen. Also, the observed isotopic shift during this period can, in part,
be also attributed to the gradual inclusion of plant foods into the cub’s
diet (Nelson et al., 1998; Lid´
en and Angerbjoern, 1999; Gran-
dal-d’Anglade et al., 2019), especially in summer and early fall. The
range of isotopic values in this group is broader than in the previous age
group, indicating a more heterogenous food source or more variability
in the proportion of milk vs plant food in the cubs’ diets.
The 5–15 month age group includes the late fall and winter months
as well compared to the previous age group (5–10 months), and the
inclusion of the winter months means that the difference between these
two groups is revealed here for the rst time (Fig. 11). The data shows a
pronounced and rapid δ
15
N and δ
13
C shift in the 5–15 month age group
than in the previous group. We assume that the diet in the 5–15 month
age group was the same as in the 5–10 month age group. However, the
signicant differences in δ
15
N and δ
13
C values between the two groups
may be due to the effects of winter starvation or specic physiology
caused by hibernation. Both mechanisms have different effects on the
isotopic ratios, as observed in the division of observations in two sub-
groups in the 5–15 month age group (Fig. 5). We assume that the two
specimens: M1-1 and M1-2, were able to establish hibernation due to the
signicantly lower δ
13
C values, which are consistent with those of the
permanent canines of individuals that survived at least one hibernation.
The low δ
13
C values (Fig. 11) are consistent with the assumption of
predicted skeletal tissue that was calculated from modern brown bear’s
Table 1
Results of δ
13
C and δ
15
N values, collagen preservation (C%, N%) and atomic carbon on nitrogen ratio (C: N) by life periods of cave bear samples from Divje babe I cave.
Sample Individual age Age interval of sampled tissue Sex Tooth Collagen
Content (%)
δ
13
C
coll
(‰) δ
15
N
coll
(‰) C (wt.%) N (wt.%) C/N
d-1 2–3 m m 0–3 d
4
12.36 −22.6 6.1 35.4 14.1 2.9
d-2 2–3 m m 0–3 d
4
12.14 −22.4 6.4 33.7 13.2 3.0
d-3 2–3 m m 0–3 d
4
11.34 −22.6 6.2 32.9 12.9 3.0
d-4 2–3 m m 0–3 d
4
– −23.1 6.0 40.3 14.7 3.2
d-5 2–3 m m 0–3 d
4
7.96 −22.7 6.0 36.1 14.1 3.0
d-6 2–3 m m 0–3 d
4
11.40 −23.0 5.8 36.3 14.2 3.0
dc-1 ~10 m m 5–10 dc 12.75 −22.1 4.9 38.1 15.4 2.9
dc-2 ~10 m m 5–10 dc 13.56 −22.9 4.9 36.9 14.6 2.9
dc-3 ~10 m m 5–10 dc 10.75 −22.5 5.6 33.1 12.9 3.0
dc-4 ~10 m m 5–10 dc 16.05 −22.1 4.3 34.0 13.4 3.0
dc-5 ~10 m m 5–10 dc 9.08 −22.8 5.7 34.2 13.4 3.0
dc-6 ~10 m m 5–10 dc 11.58 −22.1 5.4 38.5 15.6 2.9
M1-1 ~1 y m 5–15 M
1
– −22.1 2.6 39.2 14.5 3.2
M1-2 ~1 y m 5–15 M
1
– −22.2 3.3 34.4 13.2 3.0
M1-3 ~1 y m 5–15 M
1
13.31 −21.1 3.1 36.1 14.4 2.9
M1-4 ~1 y m 5–15 M
1
13.98 −21.5 3.8 36.7 14.6 2.9
M1-5 ~1 y m 5–15 M
1
16.21 −21.0 4.1 36.8 14.7 2.9
M1-6 ~1 y m 5–15 M
1
15.73 −21.0 3.4 37.5 15.0 2.9
C2F-1 ~2 y m 15–27 (JUV) F C – −21.5 2.4 33.1 12.0 3.2
C2F-2 ~2 y m 15–27 (JUV) F C 14.16 −21.5 4.5 38.4 15.4 2.9
C2F-3 ~2 y m 15–27 (JUV) F C 14.11 −21.9 3.1 32.9 12.7 3.0
C2F-4 ~2 y m 15–27 (JUV) F C 13.00 −21.7 3.0 34.9 13.7 3.0
C2M-1 ~2 y m 15–27 (JUV) M C – −21.5 2.8 39.2 14.4 3.2
C2M-2 ~2 y m 15–27 (JUV) M C 13.11 −22.7 3.4 35.7 14.0 3.0
C2M-3 ~2 y m 15–27 (JUV) M C 16.09 −22.5 3.0 34.3 13.4 3.0
C2M-4 ~2 y m 15–27 (JUV) M C 13.99 −22.0 3.4 37.1 15.1 2.9
C3F-1 ~3 y m 27–39 (SUBAD) F C – −22.5 3.4 40.7 14.8 3.2
C3F-2 ~3 y m 27–39 (SUBAD) F C 15.84 −22.0 2.2 34.5 13.3 3.0
C3F-3 ~3 y m 27–39 (SUBAD) F C 10.21 −22.0 3.2 33.5 13.2 3.0
C3F-4 ~3 y m 27–39 (SUBAD) F C 13.79 −22.7 2.1 35.5 14.0 3.0
C3F-5 ~3 y m 27–39 (SUBAD) F C 11.63 −21.6 2.6 35.9 14.4 2.9
C3M-1 ~3 y m 27–39 (SUBAD) M C 12.53 −21.4 3.3 36.9 14.8 2.9
C3M-2 ~3 y m 27–39 (SUBAD) M C 9.81 −22.5 2.9 34.0 13.5 2.9
CyaF-1 ~5–6 y y 5–6 (YAD) F C 12.29 −22.5 2.9 36.5 14.7 2.9
CyaF-2 ~5–6 y y 5–6 (YAD) F C – −22.0 2.8 41.6 15.3 3.2
CyaF-3 ~5–6 y y 5–6 (YAD) F C 10.71 −22.2 2.7 35.3 14.1 2.9
CyaF-4 ~5–6 y y 5–6 (YAD) F C 10.33 −22.4 2.6 30.6 11.7 3.0
CyaM-1 ~5–6 y y 5–6 (YAD) M C 13.07 −21.6 2.8 29.6 11.2 3.1
CyaM-2 ~5–6 y y 5–6 (YAD) M C – −22.9 2.8 35.0 12.9 3.2
CyaM-3 ~5–6 y y 5–6 (YAD) M C 11.40 −22.5 2.7 37.1 15.1 2.9
CyaM-4 ~5–6 y y 5–6 (YAD) M C 11.51 −22.4 3.5 34.8 14.3 2.8
CadF-1 ~15–20 y y 5–15+(AD) F C – −22.9 2.9 41.8 15.4 3.2
CadF-2 ~15–20 y y 5–15+(AD) F C 12.37 −22.4 2.4 35.0 14.0 2.9
CadM-1 ~15–20 y y 5–15+(AD) M C – −22.5 2.8 41.6 15.3 3.2
CadM-2 ~15–20 y y 5–15+(AD) M C 10.78 −21.8 3.0 29.6 11.5 3.0
*Legend: Individual age - approximate age at death; d
4
– fourth lower deciduous premolar; dc - deciduous canine, M
1
- rst lower permanent molar, C- permanent
canine; m – month(s); y-year(s); M – male; F – female; Simplied notations for age intervals within which the sampled tissues were formed: JUV - juvenile, SUBAD -
subadult, YAD - young adult, AD – adult.
U. Kastelic Kovaˇ
ciˇ
c et al.
Quaternary Science Reviews 325 (2024) 108481
7
blood plasma during hibernation (data from Jenkins et al., 2001;
calculated in Bocherens, 2004) and the use of fat tissues depleted of
heavy isotopes (Tieszen and Boutton, 1989). Alternatively, the higher
δ
13
C values in M1-3, M1-4, M1-5 and M1-6 suggest that these bears were
unable to establish hibernation successfully for a variety of reasons, e.g.,
lack of fat reserves, continued sucking of milk, and continued feeding
until all resources were depleted (Lid´
en and Angerbjoern, 1999;
Debeljak, 2002). Therefore, they were probably awake and starving
since the mechanism of starvation leads to a reduction in carbon intake
and an increase in the use of
13
C-depleted fat reserves in the body that is
metabolized to meet energy needs (DeNiro and Epstein, 1977; Oelber-
mann and Scheu, 2002). We assume that the four observed specimens
(M1-3, M1-4, M1-5, M1-6) with higher δ
13
C values used their
13
C-depleted fat reserves while not hibernating, resulting in an overall
13
C enrichment in animal tissues, as reported in studies addressing the
effects of starvation on stable isotope ratios (Gaye-Siessegger et al.,
2004a, 2004b, 2007; Engel et al., 2023).
The overall δ
13
C trend is consistent with predicted δ
13
C values of
Fig. 9. Box-whisker plot of δ
13
C
coll
values of cave bears for different life periods (Legend in Table 1). *Note: the width of the box indicates the time range of
each group.
Fig. 10. Box-whisker plot of δ
15
N values of cave bears for different life periods (Legend in Table 1). *Note: the width of the box indicates the time range of
each group.
U. Kastelic Kovaˇ
ciˇ
c et al.
Quaternary Science Reviews 325 (2024) 108481
8
skeletal tissue for modern bears (Fig. 12), where δ
13
C values in cubs
increase with the age of the individual till the rst hibernation (Jenkins
et al., 2001; Bocherens, 2004). In the cave bear canines and modern
bears (Ursus arctos) (Jenkins et al., 2001; Bocherens, 2004), a noticeable
trend of decreasing δ
13
C values is observed in the samples of dental
tissues formed after the rst year of life.
For the cave bear specimens in their 2
nd
year of life, the δ
13
C values
are already lower due to two periods of hibernation. Nutrition during
this period of life is of particular interest because there are some ques-
tions regarding possible continued milk consumption (Lid´
en and
Angerbj¨
orn, 1999). By comparing the N isotopic record of this age group
with those of the previous and following groups, we nd evidence that
the juveniles did not suckle milk again after their second winter, during
which they did not consume food or water for six months or more.
In specimens in the 3
rd
year of life, δ
13
C values are even lower,
probably due to better utilization of fats and, consequently, the
Fig. 11. A scatter plot of δ
13
C and δ
15
N values in cave bear teeth grouped according to life period (Legend in Table 1).
Fig. 12. Comparison of δ
13
C and δ
15
N trend of values from Nelson et al. (1998) and results of this study with calculated skeletal tissue collagen isotopic values of a
modern brown bear from Bocherens (2004).
U. Kastelic Kovaˇ
ciˇ
c et al.
Quaternary Science Reviews 325 (2024) 108481
9
incorporation of lighter isotopes (
12
C) into tissues during hibernation
(Tieszen and Boutton, 1989). The third year was probably another
vulnerable period when the youngest subadult bears, especially males,
had to become independent and nd their own territory (Debeljak,
2002). Concerning the N isotopic composition, they seemed to have the
same diet as adults but with a wider isotopic range.
In young adults and adults where ve years and older was recorded,
the δ
13
C values are slightly lower (Fig. 9). This decrease is probably the
effect of better physiological tness and the ability to nd food and store
sufcient fat for hibernation. Multiple hibernation periods in the
sampled tissue could also account for the more negative δ
13
C values
during hibernation and the use of fat stores (Tieszen and Boutton, 1989;
Bocherens et al., 1997). The δ
15
N values in canines formed after the 5
th
year are more consistent than in earlier life periods but still decrease
slightly and are characteristically low. This consistency of δ
15
N values
indicates a uniform plant-based diet, with some exceptions (Fig. 10).
The change in nitrogen (δ
15
N) and carbon (δ
13
C) trends between the
rst three groups (0–3 months, 5–10 months, and 5–15 months) and the
last four groups (2
nd
year, 3
rd
year, young adults, and adults) suggests
signicant changes in cave bear physiology and behaviour (Fig. 12). We
assume that the change of the slope of the trend of δ
15
N values, which is
steep for the rst three life periods and at for the older four groups
indicate cessation of consumption of mother’s milk before the second
winter, while the change in the trend of δ
13
C values indicates the in-
uence of hibernation. Overall low δ
15
N values could be interpreted as a
consequence of hibernation or starvation.
Comparison of our results with other studies is hindered due to dif-
ferences in sampling approach and the different criteria for determining
the age groups. Bocherens et al. (1994) sampled different teeth to
observe and simulate different ontogenetic stages of dentition. A prob-
lem arose with whole permanent teeth, where the measured isotopic
composition also included the isotopic record from the early life stage of
suckling. Other studies (Nelson et al., 1998; Bocherens, 2004;
P´
erez-Rama et al., 2011a; Bocherens, 2015; Grandal-d’Anglade et al.,
2019) also did not use a comparative sampling approach or sample
material, i.e., each study used a different number of age groups, mostly
dening the age of the individual at the time of death, which is not
explicitly dened, rather than a specic part of the lifespan. When only a
few age groups were used, a general, simplied decreasing trend in δ
15
N
values and a general increasing trend in δ
13
C values were often
observed.
Only the age groups of Nelson et al. (1998) and Keeling and Nelson
(2001) can be roughly compared with the groups in this study, although
bones and not teeth were sampled. In addition, Keeling and Nelson’s
(2001) estimated age group for each age range covers a slightly different
life span than our classication (Fig. 12). The additional advantage of
this comparison is that the material comes from the same site Divje babe
I, which makes the comparison more meaningful and reliable. The re-
sults show that the general trends for up to the 5–15 month-old groups
are similar in both studies. However, in our case, there is an apparent
downward shift in δ
13
C values after the rst hibernation, consistent with
the predicted isotopic shift calculated using modern brown bear blood
plasma (Fig. 12, Jenkins et al., 2001; Bocherens, 2004). As Nelson et al.
(1998) sampled whole bone with a slower turn-over rate, their observed
isotopic composition represents a longer life span, averaging the isotopic
composition of several life periods. Also, fewer outliers of δ
15
N values
were detected compared to Nelson et al. (1998).
Comparing our results with the predicted collagen of the skeletal
tissue of the modern brown bear (Jenkins et al., 2001; Bocherens, 2004),
there is a great match between the results. In both cases, the δ
13
C values
show an increasing trend until the rst hibernation and then decrease
during hibernation. Such a trend has not been observed yet in the fossil
material of cave bears. A good t of the results with modern brown bears
is also observed for δ
15
N values in the rst year until the rst hiberna-
tion. In later periods there is a discrepancy between modern bear and
cave bear δ
15
N values. In modern bears, increased δ
15
N values have been
observed during hibernation attributed to nitrogen recycling (Jenkins
et al., 2001). Neither in this study nor in other studies of cave bears is an
increase in δ
15
N values observed during hibernation (Bocherens et al.,
1994; Nelson et al., 1998; Bocherens, 2004; P´
erez-Rama et al., 2011a;
Bocherens, 2015; Grandal-d’Anglade et al., 2019).
5. Conclusions
A novel ontogeny-based sampling approach for carbon (δ
13
C) and
nitrogen (δ
15
N) isotope analyses of tooth collagen provided new
paleobiological insights into cave bear life during different life periods.
With this method, it is possible to exclude tissues formed before the life
period of interest, which is impossible using bone samples. Furthermore,
including additional groups, each representing different life periods,
signicantly enhanced the isotopic resolution of the ndings, yielding
unprecedented insight into the cave bear’s rst year of life, period of
independence, and adult life. The isotopic composition and narrow
range of observed δ
13
C and δ
15
N values for 0-3-month-old cubs
conrmed they fed exclusively on milk from a hibernating mother. The
δ
15
N values obtained from tissues formed during the 5–10 month age
interval, after the cubs had left the den accompanied by their mother
and before their second winter, showed a marked decrease. This rst
isotopic shift may be attributed in part to the altered isotopic values of
milk after the mother ended hibernation and resumed feeding on plants,
as well as to the gradual inclusion of solid plant foods into the diet of the
cubs during the rst summer and fall. The observed δ
13
C and δ
15
N values
in the rst 10 months of life agree with isotopic trends in modern-day
brown bears. The second isotopic shift in δ
15
N and δ
13
C values of sam-
ples belonging to yearlings that died in their second winter is even more
pronounced and abrupt. We interpret the low δ
13
C values in yearlings as
an effect of hibernation and attribute high δ
13
C values to yearlings that
failed to establish hibernation and likely starved to death. Based on
similar δ
15
N values in older age groups, we can assume that juveniles did
not return to suckle milk again after hibernation. The typically low δ
15
N
values of tissues from older juveniles, subadults, and adults may be
strongly inuenced by specic hibernation physiology or starvation
during winter. The isotopic composition of carbon in our study differs
from that of other studies by showing decreasing δ
13
C values after the
rst hibernation but agrees well with the predicted isotopic composition
of carbon in skeletal tissues of modern brown bears calculated from
blood plasma.
This study is the rst time stable isotopes of carbon and nitrogen of
specic age groups of cave bears have been studied. They revealed
nutritional and behavioural changes in the rst year of life that are
critical for survival. They also answer important questions regarding the
diet of juveniles after the rst hibernation, periods of subadult inde-
pendence, and the behavioural patterns of adult cave bears.
CRediT authorship contribution statement
U. Kastelic Kovaˇ
ciˇ
c: Conceptualization, Methodology, Data cura-
tion, Formal analysis, Writing. I. Debeljak: Conceptualization, Meth-
odology, Formal analysis, Writing, Reviewing. D. Potoˇ
cnik:
Methodology, Formal analysis, Writing, Reviewing. N. Ogrinc: Meth-
odology, Formal analysis, Reviewing. N. Zupanˇ
ciˇ
c: Conceptualization,
Methodology, Data curation, Formal analysis, Supervision, Writing,
Reviewing.
Declaration of competing interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this paper.
U. Kastelic Kovaˇ
ciˇ
c et al.
Quaternary Science Reviews 325 (2024) 108481
10
Data availability
Data will be made available on request.
Acknowledgements
We would like to thank Ivan Turk, Matija Turk, Peter Turk and
Natural History Museum of Slovenia for providing the dental material
used in this study. We thank David Heath for proofreading, Sara Raspor
and Animalia veterinary clinic in Zagorje for x-ray images, ˇ
Spela Gor-
iˇ
can for critical reading of the article and Filip Litera for technical
support. Many thanks to both reviewers for their help in improving the
paper. This work was supported by the Slovenian Research Agency
within Programmes P1-0143, P2-0424 and P1-0008, project N7-0194,
and the Young Researcher grant programme (no. 54760).
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