EPSL

ELSEVIER Earth and Planetary Science Letters 148 (1997) 349-357

A stable isotope study of fossil mammal remains from the

Paglicci cave, Southern Italy. N and C as palaeoenvironmental
indicators
P. Iacumin a.b.*, H. Bocherens b, A. Delgado Huertas a-c,A. Mariotti b,
A. Longinelli a
a Unicersifir di Trieste, Dipartimento di Science della Terra, Via E. Weiss 6. 34100 Trieste. Ital)
h Unirersitk Pierre et Marie Curie, CNRS-UMR 162, Laboratoire de Bioge’ochimie Isotopique. 4 place Jussieu. 75252 Paris. France
’ Estacidn Experimental de1 Z&din. CSIC. Prqf: Albareda I, 18008 Granada, Spain

Received 25 April 1996; revised 27 December 1996; accepted 22 January 1997

Abstract

A set of 102 tooth and bone samples of Pleistocene age (32,600-13.300 yr BP) belonging to the species Cetws eluphus,
Bos primigenius and Equus cabal/us and coming from the Paglicci cave (Southern Italy) was studied for the carbon (6 “C)
and nitrogen (8 15N) isotopic composition of bone and dentine collagen and for the carbon (6 ‘“C,) isotopic composition of
tooth enamel carbonate. The amount of collagen extracted from bone and tooth samples (mg/g) was rather variable,
representing approximately only O&-15% of the collagen present in a fresh bone. However, the loss of an important fraction
of the original collagen during diagenesis did not change the in vivo isotopic composition. In general, when the 6 ‘sC of both
collagen and carbonate and the 6 “N of collagen obtained from each level for the three species are compared, wild ox shows
the most increased values, deer the most decreased values and horse shows intermediate results. These differences are
probably related to distinct diets or to differences in their physiological behaviour. However, the isotopic results suggest that
the three species considered lived in an open environment with a diet based on C, plants. The stratigraphic sequence of light
and heavy nitrogen isotope values between 19,000 and 15.000 may be related to shifts from arid to humid conditions, while
the overall trend shown by 613C toward lighter values may be related to a progressive development of a forest habitat.

Keywords: stable isotopes: C-13/C-12: N-15/N-14: paleoenvironment

1. Introduction and N isotopic signatures. reflecting the dietary pref-

According to previous investigations (e.g.. [l-3])
collagen of fossil bone and dentine as well as the
mineral fraction of tooth enamel should preserve C
Corresponding author. E-mail: iacumen@ccr.jussieu.fr
erences.
The 6 13C and 6 15N of bone and tooth collagen
reflect the integrated mean value of the same dietary
variables during the mean lifetime of collagen in
bone and during the period of tooth growth. The
former values deviate from dietary values by about
+ 5%0 for carbon [4,5] and about + 3%0 for nitrogen

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PII SOO12-821X(97)00015-0
350 P. Iacumin et ul./Earth and Planetary Science Letters 148 (19971 349-357
[2]. Carbonincorporated into biogenic hydroxylap-
atite by herbivorous mammals is consistently 13C-en-
riched by 13-14%0 relative to food web [3,5].
Climate may indirectly affect the 613C and S15N
values of terrestrial mammals through its effect on
plant S13C and soil 6 15N, at the base of the food
chain. A decrease in relative humidity and/or in the
total amount of precipitation and a temperature in-
crease cause a 6 13C enrichment, probably as a result
of water stress on the stomata1 resistance and partial
pressure of plant CO, [6-81. The canopy effect in
densely forested areas drastically decreases the 6 I3C
of the food chain, owing to the recycling of isotopi-
tally light CO, from plant respiration and decompo-
sition of soil litter [9,10]. The total soil S 15N varies
considerably, from about - 7%0 to about + 18%0
[l l-131, this wide range of values probably arising
from the climatically sensitive processes of bacteria1
N, fixation, nitrification and denitrification. N, fixa-
tion is inhibited by high temperatures and soil dry-
ness and higher soil 6 15N might be observed in
Savannah type environments [ 14,151. Additional vari-
ations may occur in plants related to seasonality
and/or to root depth. Trees show lower 6 15N values
compared with herbaceous forms [16]. Plants grow-
ing on acid soils are I5N depleted, their 6 I5N values
being as low as - 5%0 [ 131; even lower values have
been measured in deciduous forests south of Paris
D71.
Climate may also directly affect the 6 15N of
terrestrial mammals. The 615N of mamma1 bone
collagen was found to increase with decreasing pre-
cipitation [18]. This may be related to water conser-
vation in animals in arid areas and excretion of
15N-depleted urine [ 15,191.
The present work deals with the study of fossil
skeletal remains of herbivores from the Paglicci cave
in Southern Italy, from which a set of isotopic data is
reported. The carbon and nitrogen isotopic composi-
tion of bone and tooth collagen (6 13C and S 15N)
and the carbon isotopic composition ( 6 I3C, l of CO,
derived from apatite enamel are reported for a set of
samples ranging in age from 32,600 to 13,300 yr BP
(calibrated 14C ages). The set of bones studied was
considered of interest since the Paglicci deposit spans
the transition between isotopic stages 2 and 1 and the
period of maximum ice volume and because of the
relatively low latitude of the site and its position in
the centre of the Mediterranean basin. The aim of
this study was:
1. to check whether or not collagen and carbonate
arequantitatively and isotopically well preserved
under temperate-warm climatic conditions;
2. to obtain information on the environmental condi-
tions throughout this period;
3. to check the possible presence of C, plants;
4. to check whether the isotopic values recorded the
climatic changes.
2. Materials
The Paglicci cave sedimentary sequence yielded
an abundant micro- and macro-fauna associated with
upper Palaeolithic (Gravettian and Epigravettian)
artefacts [20]. The Paglicci cave is located at about
100 m a.s.1. in southeastern Italy (41”393N, 15”373E)
at the southwestern edge of the Gargano Promontory.
The maximum elevation of the promontory, running
in a west-east direction for some 60 km, is close to
1000 m a.s.1. The 29 conventional radiocarbon dates
available from combusted bone and vegetal carbon
range from 32,600 to 13,300 yr BP [21]. The 102
bone and tooth samples from stratigraphic layers 2a
to 23a belonging to specimens of red deer (Ceruus
eluphus), wild ox (or aurochs - Bos primigenius)
and horse (Equus caballus) were analysed.
The species of micro- and macro-mammals recov-
ered (in the latter case essentially food refuse) sug-
gest local environmental conditions dominated by
prairies and steppes.
3. Methods
Collagen was extracted from about l-2 g of
powdered bone or dentine by decalcification in 1 M
HCI for 20 min at about 20°C. After filtering, the
insoluble residue, containing collagen, was treated at
room temperature with 0.125 N NaOH for 20 h.
Collagen was then filtered again, rinsed with distilled
water and solubilised in lo-’ M HCl (pH 2) in
closed tubes at 100°C for 17 h. After centrifugation,
the supernatant containing solubilised collagen was
freeze dried. Extraction yield (milligram/gram) is
 P. Iacumin et al. /Earth and Planetaq Science Letters 148 (1997) 349-357 351

expressed as the ratio of the freeze dried organic Table 1

matter to the dry weight of the bone or tooth sample. Stratigraphic levels, calibrated 14C ages and yield of collagen
extraction
Collagen was analysed for the isotopic composition
of nitrogen and carbon by means of an EA-IRMS
elemental analyser connected to a VG Optima Mass
Spectrometer, which also allowed the calculation of
the percentage C and percentage N and thus the
C. elaphus B. prinrigenius
C/N ratios. The standard deviation of the measure-
2a 13.355 0.70 9.70*
ments is about +0.1%0 for carbon and + 0.2%0 for 3a 48.20 0.w
4b 13.955 4.70 32.14* 5.10*
nitrogen (1 u 1. The 6 13C values are reported vs. 4c 0.W 31.00 o.oO*
PDB-1 and the S 15N values vs. atmospheric N, 3.20 29.06
:: 16.290 3.00* 15.%* 6.40*
international standards. 5c 1x.-m 12.SO”
6a 9.30' 16.34%
To measure the carbon isotopic composition of :,P 17.105 20.80 6.80" 11.59*
enamel hydroxylapatite carbonate an aliquot of about 12.20 0.00
6d 46.60 17.x9*
100 mg of powdered sample was reacted for 1 day 7a 19.20- 13.90
7b 17.730 0.80* 1.92 15.20
with 2% NaOCl solution, rinsed with distilled water 7c 10.20" 10.20* x.07*
and treated for about 72 h with a 1 M acetic acid- 24.00
ik 37.10 13.08* 26.98
acetate buffer solution (pH 4.75) to remove diage- 9.80* 33.X8
ii: 10.60
netic calcium carbonate 1221. After washing and 9a 1 18.200 39.90
drying, the sample was reacted overnight at 50°C 9d o.OO*
10a 24.02 23.44
with 100% H,PO, and the liberated CO, purified IOb 1.00
IOC 14.60'
and measured by means of a Finnigan Delta E Mass 1Od 18,245 50.80 14.54 8.06*
Spectrometer. The standard deviation of our mea- IOCI 29.82
IOeII 8.54
surements is about + O.l%o (1 al and the isotopic lOelI 32.20 2O.W 21.17
IOelV 27.07 14.20
values are reported vs. PDB-1. Ila 10.17
I Ic 1.47 10.69
12b 24.7Y
12e 24.64
l2f 6.20* 5.00' x.50
4. Results and discussion l2g 7.40
12- 19.83 Y.12
13-14
4.1. Collagen yields in bone and dentine 14b 19.20s 3.20 0.50 0.00
15b 19.302 0.W 6.90
16a 19.365 0.(X1
l7b 21.360 00.:
The amount of collagen (milligram/gram) ex- l8b 23.836 4.00 0.K 60.06
tracted from bone and tooth samples is variable IYa 24.480 3.40
IYb 2.20* 53.70 4.47*
(Table 11. For the majority of samples the yield 20a 1.30 13.30
‘Oh 25.095 4.30 750

1
values range between 1 and 30 mg/g, these values 2oc 26.107 25.60* 9.70
representing approximately only 0.5-15% of the col- 20d 26.650 6.90 10.30 0.2')
2Oe 13.00 11.03
lagen present in a fresh bone [23]. Generally, the 21a 27.130 0.50
2lb 27.600 0.40
average amount of collagen extracted from bones 22a 31.243 13.21
(15.0 f 14.8) is higher than that from teeth (9.7 + 22d 15.22
221 32.844 7.10' 8.22
7.6) (Fig. I), the difference is statistically significant 23a 32.633 0 34
Stars indicate tooth sampIes
(z > 1.961. Samples whose yield ranges between 0
and 1 mg/g are reported only in Fig. 1 and are not
considered in the discussion since they have no
remaining collagen or show a C/N ratio > 3.6 in
their residual collagen. from layers 5a- 16a, the corresponding time interval
Samples of different species from the same strati- being 16,000-19,000 yr BP (calibrated 14C ages).
graphic layer exhibit very different collagen con- There are no samples between 19,300 and 23,600
tents: the majority of the measured samples come and between 28,200 and 31,500 or the existing sam-
352 P. Iacumin et al. /Earth and Planetary Science Letters 148 (1997) 349-357

4.2. Preservation of the C and N isotopic composi-

n bJrJth
tion of collagen
0 bone

The C/N ratios of the extracted organic matter

are of importance to check whether the collagen
preserved its in vivo isotopic value. The C/N values
of isotopically well preserved collagen are thought to
range between 2.9 and 3.6 [23]. Only five out of 93
samples yielded C/N ratios higher than 3.6. These
0 IO 20 50 40 ilo so io samples generally showed the lowest collagen ex-
traction yields and the lowest percentage C and N in
WC4
residue (N < 10%).
Fig. 1. Frequency distribution of tooth and bone samples as a
Another important point to be taken into account
function of collagen yield (mg/g) for the three species studied.
The dashed fields refer to samples with a yield < 1 mg/g. is whether or not the loss of collagen of fossil
samples leads to an alteration in the pristine biogenic
ples show negligible collagen contents. Samples with isotopic signal. The samples from the Paglicci cave
high collagen contents are randomly distributed show very different levels of collagen preservation
among the various layers (Table 1). even in the case of samples coming from the same

0 10 20 30 40 50 60 70

cl

a8 0
2
Y
Q
0 00
Cl
0

l C. etaphus

0 8. primigenius

r~ E. caballus

-23), /,I ,, I,, ,, . , ,,,, ,,I ,I, 1 > 7 /

0 10 20 30 40 50 60
mglg

Fig. 2. 6 “N and 6 13C values of bone and tooth collagen samples vs. collagen yield.
 P. lucumin et al. /Earth and Planeta? Science Letters 148 C1997) 349-357 353

layer. There are no significant trends in 15N and 13C

enrichment or depletion related to the decrease in the
quantity of collagen recovered from bone and tooth
samples (Fig. 2). However, Fig. 2 could suggest a
tendency for Bos samples with low yield to give lo-

high 6 I5N values. It should be pointed out that: the 9-

number of Bos samples with high yield is too small

a86
to be compared to the numbers of samples with %2 7-
yields lower than about 20 mg/g; that it is quite
6-
unlikely to have such an effect only for Bos samples
5-
and not for deer and horse; and that the highest 6 15N
4-
value is shown by a Bos sample with a yield > 20
3-
mg/g. Moreover, there is no correlation between
6 “N and C/N values. 2-
Mmllac
The scattering of isotopic values within the same l-

species is wider for nitrogen than for carbon. How- 0 ; I I I I

-26 -24 -22 -20 -16 -16 4
ever, this is consistent with the variability observed
in modern ecosystems [ 141. b'% %a

The measured 6 ” N and S 13C can be compared
with those obtained from modern European and
African mammals. Most of the 6 13C values of the
Paglicci samples range between - 2 1 and - 19, only
a few values lie outside this range (min. -22.3;
max. - 17.8 [data are available upon request from
the authors and as an EPSL Online Background
Dataset ‘I>. The published 6 13C values from mod-
ern European herbivores range from - 21.5 to - 19.0
[24.25] while those from modern African herbivores
eating C, plants range from - 21 to - 18 [25]. Most
S “N values from temperate European mammals
range from about 2.5%0 to 6100 and the average S I5N
from modem African herbivores [25] is 7.1 f 2.4.
Most of the 6 15N from the Paglicci samples range
between 5 and 9. only a few values being more
positive, up to 11.9 (aurochs samples, Fig. 3 [data
are available upon request from the authors and as an
EPSL Online Background Dataset]). The isotopic
composition of the Paglicci mammals is, therefore,
within the range of values obtained from modem
mammals from temperate and warm areas (Fig. 3).
When the Paglicci values are compared with those
obtained from other Pleistocene herbivores. such as
those from France (Marillac, about 40,000 yr BP
[26]) and Great Britain (Kent’s Cavern about
Fig. 3. 6”N vs. S “C values from the Paglicci mammals com-
pared with the overall range of values from modem European and
African herbivores and with the range of values from Pleistocene
herbivores from Marillac and Kent’s Cavern sites.
28,000-38,000 yr BP [27]) the data are very close in
the case of horse and deer samples (Fig. 3). Aurochs
from Paglicci show somewhat higher S 15N than the
rest of herbivores, as is the case for bison samples of
the Marillac site.
These results show that the loss of an important
fraction of the original collagen during diagenetic
and taphonomic processes does not significantly
change the in vivo isotopic composition.
Unfortunately, we cannot compare the nitrogen
and carbon isotopic composition of herbivores with
that of carnivores since the five samples of Canis
lupus and Vulpes wipes coming from the oldest
layers in the cave. did not yield well preserved
collagen (C/N > 3.6). Also. we have no tooth and
bone samples belonging to the same specimen to
check for higher values in dentine relative to bone
collagen [28].
4.3. Preservation of the C isotopic compositiorz qf
carbonatehydroxylapatite

We analysed only the 6 13Cc of enamel because it

I http://www.elsevier.nl/locate/epsl (mirror site USA. is more resistant to carbon exchange during diagene-
http://www.elsevier.com/locate.epsl) sis than bone and dentine [29,30]. The carbonate
354 P. Iacumin et al. / Earth and Planetary Science kiters 148 (1997) 349-3.57

i313C values (- 13.4 to - 9.3 [data are available
upon request from the authors and as an EPSL
Online Background Dataset]) overlap the range
reported by [24] for recent European and Alaskan
large herbivores (- 13.7 to - 10.3) and by [28] for
Pleistocene herbivores from Kent’s Cavern (- 12.0
to -8.9).
Another way to assess the isotopic preservation of
enamel carbonate is to consider the difference be-
tween the S j3C values of collagen and those from
hydroxylapatite (A13C), which has been related to
digestive physiology and climatic conditions
[5,28,31]. Paglicci herbivores, on which S13C from
both collagen and carbonate has been measured,
have a mean A13C value of 9.0 + 1.04 (n = 28). The
mean A13C values from modem European and
Alaskan herbivores were 8.4 & 1.0 and 8.6 k 0.5 and
10.1 & 0.6 for Pleistocene French and English herbi-
vores, respectively. The three species considered
from Paglicci show similar mean A13C values, even
though Bos primigenius samples show slightly higher
values.
4.4. Palaeobiological and palaeoenvironmental con-
siderations
The 6 13Cvalues of collagen from the three species
considered are characteristic of animals feeding on
plants with a C, photosynthetic pathway. These
values are higher than those of recent mammals
living in a closed-canopied forest and, therefore, the

12-

ll-
,BpMIIBI”yII
lo-

% 1; \

l-

6- 10

5- 9

\ 7%

4
s-

I-
7
\

Fig. 4. Temporal variation in 6 “N collagen values from deer, horse and wild ox.
 P. Iacumin et al. /Earth and Planetary Science Letters 148 (1997) 349-357 355

Paglicci species probably lived in an open environ-

ment. Only a few deer and horse samples show S 13C
values lower than - 21 and this may perhaps be
referred to a canopy effect, and thus a forested
environment during the life of these individuals.
18
However, the mean 6 13C of bone samples is almost
the same in deer and horses, but is about 1%0 higher 19
in auroch samples. If we take into account a 13C
enrichment of collagen of about 5%0, when compared 20 2

with the mean 6 13C of food, the mean carbon

isotope composition of the plants eaten is about
-25.5 for deer, - 25.2 for horse and -24.5
wild ox. The 6 13C values of food that may be
calculated from the carbonate apatite values assum-
ing a 13C enrichment of bone of about 14%0, are
quite similar to the previous ones: -26.1 for deer,
- 25.3 for horse and -24.9 for wild ox. These
values are very close to the mean carbon isotope
composition of C, plants [32,33]. If we compare the
6 13C values obtained from each level for the three
species, aurochs generally show the most 13C en-
riched values, deer show the most 12C enriched
values while horses show intermediate results. A
similar pattern is found in S15N values. This moder-
ate, though systematic, difference may result from
distinct diets, with different isotopic values perhaps
related to different habitats, or, alternatively, to dif-
ferences in the physiological behaviour. Deer browse
on small leaves and shrubs which can have lower
6 I5N than other non-woody plant types [ 161 and they
probably mainly lived in the forests of the Gargano
Promontory. Wild ox and horse are grazers and lived
in the prairies of the plain and, in the case of horse,
also in the lower section of the promontory [34].
The mean bone and tooth S 15N value is close to
6.8 for horse and to 7.9 for wild ox, which is more
similar to the mean value of African herbivores
rather than to that of European herbivores (Fig. 3).
This may suggest that, in the period considered, the
mean environmental conditions were more arid than
at present.
The S 15N and 6 13C of collagen are potential
proxy for palaeoclimatological studies. However, in
this case we could measure only one specimen of
each species from each layer and, consequently,
having no data on the intraspecific variability, we
cannot suggest reliable and detailed palaeoclimato-
logical considerations.
21
for
22
Age BP
Fig. 5. Temporal variation in 6’jC collagen values from deer,
horse and wild ox.
The S *‘N record of the three species displays
marked and rapid variations reaching about 5.8%0,
the standard deviation of the measurements being
about f 0.2-0.4%0 (Fig. 4).
If we consider the time interval between about
13,000 and 19,500 yr BP a succession through time
of light and heavy nitrogen isotope values taking
place alternately f& relatively short time intervals
may be related to shifts from arid to humid condi-
tions, perhaps related to fast-changing climatic
and/or meteorological conditions during the
deglaciation period. The same rapid transitions from
arid to humid conditions were recorded in the Sahara
and Sahel areas of North Africa during the last
deglaciation [35].
The variations in 6 13C values vs. age are reported
graphically in Fig. 5. A mild although general trend
towards lighter values is apparent between 19,500
and 15,800 yr BP, even though, in this case as well,
large and fast oscillations can be observed. This may
be directly related to the interaction between wet
environmental conditions and the development of a
forest habitat. This is also supported by the common
presence in the cave skeletal remains, after 15,800 yr
356 P. Iacumin et al./Earth and Planetar?, Science Letters 148 (1997) 349-357
BP, of typical forest mammals such as wild pig and
deer.
5. Conclusion
The isotopic study carried out on bone and tooth
samples of three species from the Paglicci cave,
suggests the following considerations:
1. in spite of the loss of an important fraction of the
original collagen during diagenesis, it seems that
the isotopic composition of the measured samples
did not change significantly as long as 2.9 < 3.6;
2. the S 13C values of collagen from the three species
are characteristic of animals feeding on C, plants
and living in an open environment;
3. wild ox shows the most enriched 13C (both colla-
gen and carbonate) and 15N values while deer
shows the most depleted ones, probably suggest-
ing a distinct diet related to a distinct habitat or a
difference in the physiological behaviour;
4. the S “N values measured are more similar to the
values of African herbivores (feeding on C,
plants) than to those of European herbivores. This
may suggest that in the period considered the
mean environmental conditions were more arid
than at present;
5. the three species studied yield fairly similar iso-
tope trends along the stratigraphic sequence. The
relatively fast succession of light and heavy nitro-
gen isotope values may be related to shifts from
arid to humid conditions following climatic
and/or meteorological changes during deglacia-
tion .
The S 13C trend towards slightly lighter values
from about 19,500 to 15,800 yr BP is apparent even
though large and fast oscillations take place. This
may be directly related to the progressive establish-
ment of wet environmental conditions and the devel-
opment of a forest habitat.
Acknowledgements
The authors are deeply indebted to Prof. D. Torre
and to Dr. L. Abbazi of the University of Florence
and to Prof. A. Palma di Cesnola of the University of
Siena (Italy) for kindly supplying fossil material
from the Paglicci cave. This research was carried out
with the financial support of the Italian National
Research Council which is here gratefully acknowl-
edged. [FAI
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