Oxygen and Carbon Isotope Variations in a Modern
Rodent Community – Implications for
Palaeoenvironmental Reconstructions
Alexander Gehler1*, Thomas Tu¨tken2, Andreas Pack1
1Georg-August-Universita¨t, Geowissenschaftliches Zentrum, Abteilung Isotopengeologie, Go¨ttingen, Deutschland, 2 Rheinische Friedrich-Wilhelms-Universita¨t,
Steinmann-Institut fu¨r Geologie, Mineralogie und Pala¨ontologie, Emmy Noether-Gruppe Knochengeochemie, Bonn, Deutschland
Abstract
Background: The oxygen (d18O) and carbon (d13C) isotope compositions of bioapatite from skeletal remains of fossil
mammals are well-established proxies for the reconstruction of palaeoenvironmental and palaeoclimatic conditions. Stable
isotope studies of modern analogues are an important prerequisite for such reconstructions from fossil mammal remains.
While numerous studies have investigated modern large- and medium-sized mammals, comparable studies are rare for
small mammals. Due to their high abundance in terrestrial ecosystems, short life spans and small habitat size, small
mammals are good recorders of local environments.
Methodology/Findings: The d18O and d13C values of teeth and bones of seven sympatric modern rodent species collected
from owl pellets at a single locality were measured, and the inter-specific, intra-specific and intra-individual variations were
evaluated. Minimum sample sizes to obtain reproducible population d18O means within one standard deviation were
determined. These parameters are comparable to existing data from large mammals. Additionally, the fractionation
between coexisting carbonate (d18OCO3) and phosphate (d
18OPO4) in rodent bioapatite was determined, and d
18O values
were compared to existing calibration equations between the d18O of rodent bioapatite and local surface water (d18OLW).
Specific calibration equations between d18OPO4 and d
18OLW may be applicable on a taxonomic level higher than the species.
However, a significant bias can occur when bone-based equations are applied to tooth-data and vice versa, which is due to
differences in skeletal tissue formation times. d13C values reflect the rodents’ diet and agree well with field observations of
their nutritional behaviour.
Conclusions/Significance: Rodents have a high potential for the reconstruction of palaeoenvironmental conditions by
means of bioapatite d18O and d13C analysis. No significant disadvantages compared to larger mammals were observed.
However, for refined palaeoenvironmental reconstructions a better understanding of stable isotope signatures in modern
analogous communities and potential biases due to seasonality effects, population dynamics and tissue formation rates is
necessary.
Citation: Gehler A, Tu¨tken T, Pack A (2012) Oxygen and Carbon Isotope Variations in a Modern Rodent Community – Implications for Palaeoenvironmental
Reconstructions. PLoS ONE 7(11): e49531. doi:10.1371/journal.pone.0049531
Editor: Andrew A. Farke, Raymond M. Alf Museum of Paleontology, United States of America
Received May 21, 2012; Accepted October 10, 2012; Published November 19, 2012
Copyright:  2012 Gehler et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Funding was provided by the German National Science Foundation DFG grant PA909/5-1 (A.P.) and DFG grant TU148/2-1 (TT, Emmy Noether-Group
‘‘BoneGeochemistry’’). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: agehler@gwdg.de
Introduction
Stable isotope compositions of mammalian bioapatite are widely
used proxies in palaeoenvironmental and palaeodietary studies.
Starting with pioneering research on carbon [1] and oxygen
isotopes [2–5] in the 1970s and 1980s, stable isotope analysis of
bioapatite from fossil mammals rapidly became an established
method for the reconstruction of palaeoclimate and palaeodiet.
The oxygen isotope composition of bioapatite from terrestrial
mammals can be used to infer air temperature, climate seasonality,
relative humidity or aridity of palaeoenvironments as well as
mobility, birth seasonality and drinking behaviour of specific
mammal taxa (e.g. [6] and references therein; [7–11]). Carbon
isotopes can be used to infer the palaeovegetation, vegetation
cover, dietary strategies, resource partitioning and habitat use (e.g.
[6] and references therein; [8,9,12–15]).
For various reasons, especially to allow easier sampling and
acquisition of sufficient sample material, most studies have focused
predominantly on the isotopic analysis of skeletal remains from
large mammals. New and improved mass spectrometric tech-
niques allow oxygen and carbon isotope analysis of (sub)milligram
sample amounts of bioapatite, bringing small mammal taxa such
as small rodents (with a body mass below 1 kg) into the focus of
interest.
So far only a few oxygen and/or carbon isotope studies are
based exclusively or partly on fossil small rodents [8,16–27].
However, for an improved understanding of oxygen and carbon
isotope compositions in bioapatite of fossil small rodents,
communities of modern analogues have to be studied extensively
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in order to investigate inter- and intra-specific variabilities. This
has been done repeatedly for selected large mammals (e.g., [28–
35]) but no comparable study has been conducted on small
mammals so far. It is the aim of the present study to investigate
such variations for small mammals. Such studies are fundamental
in order to evaluate the number of individuals needed for
statistically significant results and to ascertain whether species-
specific calibrations or calibrations on a higher taxonomic level are
suitable for the reconstruction of the oxygen isotope composition
of local surface water. Furthermore, the intra-individual variability
between bones and permanently growing teeth as well as the intra-
jaw variations need to be determined in order to investigate if
these skeletal tissues record comparable isotope signatures or are
seasonally biased.
We analysed the oxygen (d18O) and carbon isotope (d13C)
composition (for definitions see Materials and Methods section) of
bones and teeth from seven rodent species in multiple individuals.
The samples derive from owl pellets of a locality in northwestern
Germany, which were accumulated over a 4-year period (1991–
1995). Inter-specific, intra-specific, and intra-jaw variations were
investigated, as well as variations between teeth and bones from
the same individuals. These data were compared to published
stable isotope data for large mammals.
Additionally, oxygen isotope analyses of coexisting carbonate
(d18OCO3) and phosphate (d
18OPO4) in rodent bioapatite were
conducted on water voles (Arvicola terrestris). In order to determine if
it is possible to make a correct estimation of the d18OLW from the
d18OCO3 values, the data from the other species were converted to
d18OPO4 equivalent values by using the average offset between
d18OCO3 and d
18OPO4 (D
18OCO3-PO4) obtained for A. terrestris.
Then the data were compared to the three published d18OPO4-
d18OH2O calibration equations determined for different extant
rodent taxa in previous studies [4,16,36].
With the present study, the authors aim to provide a basic
contribution for the enhancement of palaeoenvironmental inter-
pretations of oxygen and carbon isotope data obtained from fossil
rodents.
General Considerations
Oxygen isotopes. The oxygen isotope composition of
mammalian bioapatite is determined by that of body water,
which in turn is controlled by the oxygen isotope compositions of
the different oxygen input sources (drinking water, food water, air
oxygen, organic food compounds and water vapour in air) and
oxygen output fluxes (exhaled CO2, liquid water in sweat, urine
and feces as well as orally, nasally and transcutaneously released
water vapour) [(e.g., [4,33,37]). The oxygen isotope composition of
mammalian body water is linearly related to that of their drinking
water (i.e., local surface water) for those mammals with an obligate
drinking behaviour (e.g., [2,38]). Thus, empirical specific calibra-
tion equations relating d18OPO4/CO3 and d
18OLW can be
developed. Because the oxygen isotope composition of local
surface water varies with air temperature and also with the amount
of local precipitation and evapotranspiration (e.g., [39–42]),
(palaeo-)climatic conditions can be inferred from the oxygen
isotope composition of the skeletal remains of fossil mammals (e.g.,
[9] and references therein).
In bioapatite, oxygen is present in the phosphate, carbonate and
the hydroxyl groups [6]. The average offset between d18OPO4 and
d18OCO3 in skeletal apatite of different mammal taxa is around
9%. This offset ranges from 7.5 to 11.4% in the previously
investigated modern taxa [43–49]. Both, d18OPO4 and d
18OCO3,
can be used to track the oxygen isotope composition of ingested
drinking water. For the offset between d18OPO4 and oxygen bound
in the hydroxyl group, only a single calculated value of 216.6% is
known [50].
Carbon isotopes. The carbon isotope composition of mam-
malian bioapatite is controlled by that of ingested food (i.e., plant
material in herbivores) [1] and can therefore be used to investigate
dietary preferences. Furthermore, the knowledge of the carbon
isotope composition of ingested plant material can mirror specific
habitats, type of vegetation cover (i.e., C3 versus C4 plants) and
density, as well as ecological niches,feeding behaviour and seasonal
changes of diet (e.g., [9] and references therein).
Significant differences in the carbon isotope composition of
plants are induced by different carbon fixation strategies in plants,
i.e., the C3, C4 or CAM (Crassulacean Acid Metabolism)
photosynthetic pathways (e.g. [51–53]). C3 plants, which represent
about 95% of all terrestrial plant species (e.g., [54]), have typical
d13C values between -36 and 222% with an average value of
227%, whereas the d13C values of C4 plants (mostly warm season
grasses and sedges) have 215 to 210%, with an average value of
213% (e.g. [51,52,55,56]). CAM plants have highly variable d13C
values within and between specific taxa, overlapping with both C3
and C4 plants (e.g., [55,57,58]). However, CAM plants represent
only about 4% of all terrestrial plant species [54] and are mostly
succulents [59], playing a minor role in the nutrition of most
herbivorous mammals. Thus, a distinction between browsing and
grazing herbivores based on bioapatite d13C values is possible, as
shown by numerous palaeodietary studies (e.g. [60–65]). However,
this approach is limited to ecosystems with coexisting C3 and C4
plants; the latter were not globally abundant until the late Miocene
[66].
Even in a pure C3 ecosystem, significant differences in the
carbon isotopic composition of plants are caused by varying light-,
nutrient-, water-, CO2- and temperature-settings (e.g., [51,67]).
Subcanopy plants in humid, shaded environments assimilating
CO2, depleted in
13C by soil respiration, have very low d13C
values ranging from 236 to 232% [12,51,68–71]. C3 plants in
arid, open environments have the highest d13C values, up to
221% [51,52,72]. Those variations are also visible in the carbon
isotope composition of bioapatite from modern and fossil
mammals living in C3-environments and reflect differences in
habitat use and/or resource partitioning (e.g., [9,12,13,15,73–78]).
Terrestrial C3 plant ecosystems date back to the Palaeozoic and
are typical for most parts of the modern northern hemisphere (e.g.,
[79]).
The carbon isotope composition of mammalian bioapatite is
enriched by several per mil compared to the respective diet.
Reported enrichment factor values range from 9 to 15%,
depending on the digestive physiology of the investigated taxa
(e.g., [80] and references therein).
Stable carbon and oxygen isotopes from bioapatite of
fossil small rodents and case studies on modern
material. Mammals evolved in the Late Triassic and retained
relatively small body size until their radiation after the Cretaceous-
Palaeogene transition [81]. Rodents first appear in the Palaeogene
fossil record, and fossil rodent remains are widespread in Cenozoic
terrestrial deposits, often in high numbers, accumulated predom-
inantly by ancient avian predators. As reviewed by Grimes et al.
[82], one of the main advantages of stable isotope studies on small
mammals relative to large taxa is the higher abundance of small
mammals in the fossil record, which enhances their availability.
Furthermore, small mammals display a rapid evolution of their
morphology, and hence small mammal remains are often index
fossils for Cenozoic strata that enable a good biostratigraphic
resolution. Additionally, most small mammals occupy a very
restricted habitat, lacking long distance migratory behaviour, thus
Oxygen and Carbon Isotope Variations in Rodents
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reflecting local palaeoenvironmental conditions more precisely
than large mammals. Possible disadvantages compared to large
mammals concerning the oxygen isotope composition may be a
smaller proportion of drinking water in the overall oxygen intake,
a more variable physiology (i.e., body temperature), and a possible
stronger susceptibility to diagenetic alteration due to smaller, more
fragile skeletal elements [82].
Specific calibration equations between d18OPO4 and d
18OLW
have been developed in the past for laboratory rats [4], wild
murids [36,38] and wild arvicolids [16,36]. During the last decade,
the oxygen isotope composition of bioapatite of fossil small rodents
has been used in an increasing number of studies, targeting
Palaeogene [20–22,27], Neogene [8,26,27] and Pleistocene [16–
19] climate change. Carbon isotope records of fossil small rodents
also were used to reconstruct vegetational change and palaeodiets
in the Cenozoic [18,19,23–25].
To date, very little is known about the inter- and intra-
population variability between individuals from the same locality,
as well as about variations between different teeth and bone
material within a single individual. Only one study [83] has
investigated intra-population and intra-jaw variations (d18OPO4) in
small rodents based on a small number of samples of fat dormice
(Glis glis, n = 3) from Great Missenden (Buckinghamshire, UK) and
wood mice (Apodemus sylvaticus, n = 5) from Dungeness (Kent, UK).
From these results, Lindars et al. [83] concluded that a quantity
of.5 different post-weaning teeth should be used for isotope
palaeo-thermometry. Further intra-population d18OPO4 data of
multiple bioapatite analyses from small rodents (Apodemus flavicollis,
Apodemus sylvaticus, Arvicola terrestris, Microtus arvalis and Pitymus sp.)
are presented in a small quantity (n = 3–8) by D’Angela and
Longinelli [38] and Longinelli et al. [36], but no data for distinct
sympatric species from the same locality are available so far.
Materials and Methods
Material
The samples originate from fresh barn owl (Tyto alba) pellets
accumulated over a maximum of four years, collected in April
1995 at ‘‘Hof Gu¨lker’’, located in the nature reserve Rhader
Wiesen, about 9 km north of Dorsten, North Rhine-Westphalia,
Germany (Fig. 1) and are part of the original material from Bu¨low
[84]. Long term mean annual local temperatures in this region
(IAEA-GNIP station Emmerich) average 10uC, and the mean
annual precipitation is 744 mm. The long term weighted mean of
the monthly d18O record of local precipitation between 1980 and
2005 is 27.3%. Considering only the period from April 1991 to
April 1995 of owl pellet deposition, and hence the most likely time
interval when the analysed rodents lived and mineralised their
bones and teeth, the respective d18O value is 27.7. This is very
close to the long term record [85]. The seasonal change in
monthly precipitation of the sampling area is illustrated in Fig. 2.
The rodent skeletal material belongs to the four arvicolid species
Arvicola terrestris, Myodes glareolus, Microtus agrestis and Microtus arvalis
as well as to the three murid species Apodemus sylvaticus, Mus
musculus, and Rattus norvegicus. From the latter species, the owl
pellets contained only juvenile individuals. The analysed teeth
were permanent growing (arvicolid and murid incisors and most
arvicolid molars), thus reflecting the last four to twelve weeks in the
life of the respective individual prior to death [86,87]. Bone
material comprises stable isotope compositions over a longer time
period, approaching the life span of the individual [e.g. 88], which
in the present case is the time until predation by barn owls. None
of the analysed taxa hibernate.
Figure 1. Map showing the sampling locality Rhader Wiesen
near Dorsten, North Rhine-Westphalia, Germany (red star) and
the closest IAEA-GNIP station to it (blue dot).
doi:10.1371/journal.pone.0049531.g001
Figure 2. Mean long-term monthly d18O record (1980–2005) of
local precipitation from the IAEA-GNIP station Emmerich.
doi:10.1371/journal.pone.0049531.g002
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The isotope signatures of the skeletal tissue samples are likely to
be seasonally biased, because of the different tissue formation and
turnover periods and due to the seasonal population dynamics of
the rodents and their predators. Population sizes of arvicolids and
murids in temperate regions typically increase after a minimum at
the end of the winter with the first breed in spring and reach a
maximum (partly up to more than one order of magnitude larger
than the early spring population) in late summer (e.g., [89–91]).
Avian predator populations, responsible for pellet accumulations,
are positively correlated to populations of their small mammal
prey (e.g., [92–95]), causing a warm-season biased pellet
deposition in modern ecosystems and probably in fossil ecosystems
as well. Analogous conditions for modern and fossil samples are an
important prerequisite for the development of d18OPO4/CO3-
d18OLW calibration equations and their application for palaeocli-
mate reconstructions [16].
No specific permits were required for the described field studies.
Methods
Oxygen and carbon isotope analyses of the carbonate in
the bioapatite (d18OCO3, d
13C). Only teeth from taxonomi-
cally identified jaw specimens were used for stable isotope analysis.
Teeth from each species, both upper incisors (in Arvicola terrestris
and Rattus norvegicus only the left upper incisor) of ten individuals,
were extracted from the jaw and inspected to ensure that no signs
of digestive etching were present. Further d18OCO3 and d
13C
analyses were conducted on jaw bone material (derived from the
zygomatic region) of five of the ten individuals of each species. In
Arvicola terrestris, the left upper molars (M1–M3) of the ten
specimens were also analysed.
Bulk tooth and bone material was crushed and ground to a fine
powder using an agate mortar and pestle. The chemical
pretreatment procedure to remove organic matter and adherent
carbonates followed Koch et al. [96]. Approximately 10 mg of
sample powder were soaked with 30% H2O2 (0.1 ml mg
21) for
24 h, rinsed five times with millipore water, soaked for another
24 h with an acetic acid - calcium acetate buffer solution (1 M,
pH=5, 0.05 ml mg21) and rinsed again five times with millipore
water, followed by drying overnight at 50uC. Typically, 1 to
1.3 mg of pretreated sample powder were reacted for 15 min. with
100% H3PO4 at 70uC in a Thermo Scientific KIEL IV automated
carbonate device. Released CO2 was measured in dual inlet mode
with a Finnigan Delta plus isotope ratio gas mass spectrometer at
the stable isotope laboratory of the Geoscience Center at the
University of Go¨ttingen. The measured isotope compositions were
normalised to the NBS 19 calcite standard, measured in the same
runs together with the bioapatite samples. Data are reported in the
d-notation in per mil (%), relative to the international isotope
reference standards Vienna Standard Mean Ocean Water
(VSMOW) for d18O and Vienna Pee Dee Belemnite (VPDB) for
d13C [97].
d18O or d13C ðÞ~ Rsample=Rstandard
 
{1
 
| 1000,
where Rsample and Rstandard are the
18O/16O and 13C/12C ratios in
sample and standard, respectively.
The analytical precision for the NBS 19 was 60.1% (1s) in
d18O and 60.04% (1s) in d13C (n= 232, analysed between
November 2010 and February 2012). For the NBS 120c Florida
phosphate rock standard (pretreated as described above), we
obtained a d18O value of 30.060.2% (1s) and a d13C value of
26.460.04% (1s) (n = 10). Our internal bioapatite standard
AG-Lox (African elephant enamel) had a d18O value of
30.060.08% (1s) and a d13C value of -12.060.03% (1s)
(n = 11). For inter-laboratory comparison, material of our internal
AG-Lox enamel standard will be supplied upon request by the
authors.
Oxygen analyses of the phosphate (d18OPO4). Analyses of
the phosphate moiety of the bioapatite (d18OPO4) were conducted
on the incisors, M1 and bone material from five of the Arvicola
terrestris specimens.
Ag3PO4 was precipitated from about 4 mg of the pretreated
sample powder (see section above), using a method slightly
modified after Dettmann et al. [98] and described in detail by
Tu¨tken et al. [8]. For dissolution of the samples, 0.8 ml of HF
(2 M) were added, and the sample vials were put on a vibrating
table for 12 h. This was followed by centrifugation and transfer
to new vials of the supernatant sample solutions, leaving behind
the CaF solid residue. After neutralising the HF solution with
NH4OH (25%) in the presence of bromothymol blue as pH-
indicator, Ag3PO4 was precipitated rapidly by adding 0.8 ml of
2 M silver nitrate (AgNO3) solution. After settling of the small
Ag3PO4 crystals and centrifugation, the supernatant solution
was pipetted off and the Ag3PO4 was rinsed twice with 1.8 ml
millipore water. The Ag3PO4 was then dried overnight in an
oven at 50uC.
Ag3PO4 aliquots of 0.5 mg were placed into silver capsules and
analysed in triplicate by means of high temperature reduction
Finnigan TC-EA coupled via a Conflo III to a Finnigan Delta Plus
XL GC-IRMS, according to the method of Vennemann et al.
[99].
Statistical Methods
The statistical analyses were performed using Wolfram
Mathematica 7.0 and 8.0. To evaluate if the differences in the
mean isotope values among multiple taxa are statistically
significant, one-factor analyses of variance (ANOVA) were
conducted, followed by posthoc-tests (Tukey) for pairwise com-
parisons. In one case, the assumptions of the parametric ANOVA
were violated (non-normal distribution), therefore we additionally
performed a Kruskal-Wallis non-parametric ANOVA that was not
in disagreement with the parametric ANOVA results.
Minimum sample sizes to represent the population mean in
d18O by 95% degree of confidence within 61s were
determined by a standard bootstrapping approach according
to the method presented by Fox-Dobbs et al. [100]. From the
bulk incisor data sets of the seven analysed species (n = 10 each),
1,000 randomly selected subsamples (nsub) were generated for
every nsub between 2 and n-1. Then, the proportion of
subsamples within all random replicates for a given nsub that
range between 61s of the mean of the original datasets was
determined, using the average value from 100 repeated
evaluations. If the mean values of $950 out of 1000 subsamples
are in a range between 61s of the analysed dataset, it is
indicated that the respective nsub is adequate to estimate the
population mean at least by a 95% confidence level.
Results
Oxygen and Carbon Isotope Data of the Carbonate in the
Bioapatite (d18OCO3, d
13C)
The data, which are summarised in Table S1, include 135
individual d18OCO3 and d
13C analyses of bulk incisor and molar
teeth, as well as d18OCO3 and d
13C values of bone material from
seven modern arvicolid and murid species from a single locality in
NW Germany (see Materials and Methods section).
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Oxygen Isotope Composition of the Incisors
Intra-population variations in d18OCO3 from 2.0 to 3.9% have
been observed in the bulk incisor samples (n = 10 of each species),
with the exception of the arvicolid M. glareolus, which has a
significantly higher range of 6.7%. However, if the most extreme
value is excluded and considered as an outlier, M. glareolus has an
intra-population variation of 3.5% that falls within the range of
variation of the other species (Table 1). The mean d18OCO3 values
of the arvicolid incisors are 26.861.4% (A. terrestris), 27.261.8%
(M. glareolus), 27.361.1% (M. agrestis) and 27.361.2% (M. arvalis).
The murid incisors have mean d18OCO3 values of 27.961.0% (A.
sylvaticus), 28.361.0% (M. musculus) and 29.060.7% (R. norvegicus)
(Fig. 3, Table 1). Between the mean d18OCO3 values of these
species, statistically significant differences (one-way ANOVA,
F= 3.638, P,0.01) were observed, pair-wise comparison revealed
that only A. terrestris and R. norvegicus show significantly different
values (Tukey test, P,0.01). Within arvicolids, no statistically
significant differences in d18OCO3 were detected (one-way
ANOVA, F= 0.304, P = 0.82). Within murids, mean d18OCO3
values differed significantly (one-way ANOVA, F= 3.654, P,0.05)
but only between A. sylvaticus and R. norvegicus were significant
differences detected by pair-wise comparisons (Tukey test,
P,0.05).
Carbon Isotope Composition of the Incisors
The d13C values of the incisor samples (n = 10 of each species) in
three of the four modern arvicolids (A. terrestris, M. glareolus and M.
agrestis) have very narrow intra-population variations of ,2.2%,
with mean values of 217.760.6%, 216.260.7% and
219.760.6%, respectively. The intra-population d13C variations
of M. arvalis and the murid A. sylvaticus are much larger, with
ranges of 4.0% and 5.4% and mean values of 218.861.4% and
215.961.7%, respectively. Very large variations were observed in
the two murids, M. musculus and R. norvegicus, that have an intra-
population d13C range of 12.7% and 10.2% with mean values of -
12.165.3% and -9.663.4%, respectively (Fig. 3, Table 1). Mean
d13C values differed significantly between the analysed species
(one-way ANOVA, F= 20.632, P,0.01). By pair-wise compari-
son, mean d13C values of two murids (M. musculus and R. norvegicus)
differed significantly from those of all arvicolids as well as from A.
sylvaticus. The mean d13C value of M. agrestis showed significant
differences relative to those of A. sylvaticus and M. glareolus (Tukey
test, P,0.05). Considering the arvicolids separately, differences in
the mean d13C values between taxa (one-way ANOVA,
F= 28.813, P,0.01) were observed. By pair-wise comparisons,
significant differences between M. agrestis and M. glareolus
themselves and with both other Arvicolidae were detected (Tukey
test, P,0.05). Within murids, significant differences among taxa
were present as well (one-way ANOVA, F=7.230, P,0.01), but
only A. sylvaticus and R. norvegicus show significant differences by
pair-wise comparison (Tukey test, P,0.05).
Oxygen and Carbon Isotope Compositions of the Bones
The bone d18OCO3 values (n = 5 of each species) deviate from
the incisor values of of the same individuals by 24.2 to+1.5%.
85% (30 out of 35 specimens) have higher d18OCO3 values in their
incisors than in their bones (Fig. 4, Table S1). Mean values are
25.661.2% (A. terrestris), 25.761.4% (M. glareolus), 24.661.3% (M.
agrestis) and 26.661.3% (M. arvalis). The murid bones have mean
d18OCO3 values of 25.860.8% (A. sylvaticus), 28.261.6% (M.
musculus) and 25.861.0% (R. norvegicus) (Table 2).
Figure 3. Mean d18OCO3 and d
13C values of bulk incisors of the
seven analysed rodent species (n=10 in each species) with 1s
error bars. 1: A. terrestris, 2: M. glareolus, 3: M. agrestis, 4: M. arvalis, 5:
A. sylvaticus, 6: M. musculus, 7: R. norvegicus.
doi:10.1371/journal.pone.0049531.g003
Table 1. Mean d18OCO3 and d
13C values of bulk incisors of the seven analysed rodent species (n = 10 in each species) with 1s error
and range of variation (lowest and highest analytical value of the respective species).
Species Material Mean d
18OCO3 Range of variation Mean d
13C Range of variation n
(% vs. VSMOW) in d18OCO3 values (% vs. VPDB) in d
13C values
Arvicola terrestris I1 sin. 26.861.4 24.9 to 28.8 217.760.6 218.5 to 216.7 10
Myodes glareolus I1 sin. u. dex.1 27.261.8 24.7 to 28.2 (31.4)2 216.260.7 217.3 to 215.1 10
Microtus agrestis I1 sin. u. dex. 27.361.1 26.0 to 28.8 219.760.6 220.5 to 218.6 10
Microtus arvalis I1 sin. u. dex. 27.361.2 25.4 to 29.2 218.861.4 220.8 to 216.8 10
Apodemus sylvaticus I1 sin. u. dex. 27.961.0 26.0 to 29.1 215.961.7 217.8 to 212.4 10
Mus musculus I1 sin. u. dex. 28.361.0 26.4 to 29.7 212.165.3 217.4 to 24.7 10
Rattus norvegicus I1 sin. 29.060.7 27.8 to 29.9 29.663.4 214.7 to 24.5 10
doi:10.1371/journal.pone.0049531.t001
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The different bone-incisor offsets have standard deviations
between 0.8 and 1.8% within each analysed species, except for the
juvenile R. norvegicus specimen with a standard deviation of only
0.3%.
The d13C of bone material deviates by+1.9 to 21.6% from that
of the incisors, with the exceptions of one A. sylvaticus (22.9%) and
one M. musculus (+4.3%). Mean values are 217.860.9% (A.
terrestris),217.760.4% (M. glareolus),219.560.6% (M. agrestis) and
218.460.8% (M. arvalis). The murid bones have higher mean
d13C values of 216.461.3% (A. sylvaticus), 211.665.5% (M.
musculus) and 27.962.2% (R. norvegicus). No systematic differences
between incisor and bone material can be observed, as is the case
for d18OCO3 (Fig. 5, Table 2).
Oxygen and Carbon Isotope Compositions of the Molars
of A. terrestris (Intra-jaw Variations)
In A. terrestris the intra-jaw range (n= 10) between the molars
(M1–M3) of the same individual is 0.2 to 1.0% in d18OCO3 and
0.1 to 0.6% in d13C. The overall intra-jaw range (I1, M1–M3)
varies from 0.3 to 1.9% in d18OCO3, while in most cases the
incisors have higher values than the molars (Fig. 6, Table S1). In
d13C, nearly all incisors have lower values than the corresponding
molars, with overall intra-jaw variations between 0.3 to 1.5%
(Fig. 7, Table S1). Mean values for M1, M2, and M3 are
26.161.6%, 26.161.5% and 26.261.3% in d18OCO3 and
216.960.6%, 216.860.6% and 216.760.5%, respectively
(n = 10 of each tooth type).
Oxygen Isotope Analyses of the Phosphate in the
Bioapatite (d18OPO4) of A. terrestris
The mean d18OPO4 of skeletal apatite from A. terrestris are
15.461.1% (bulk incisors, n = 5), 14.961.3% (bulk M1, n= 5)
and 14.960.8% (bone, n = 5). The corresponding d18OCO3 means
are 26.561.2%, 25.861.1% and 25.661.2%, leading to an
D18OCO3-PO4 of 11.261.0% (incisors), 10.960.4% (M1) and
10.760.9% (bone), respectively. The overall average D18OCO3-
PO4 is 10.960.8% (Fig. 8, Table 3).
Minimum Sample Size Calculations
Minimum sample sizes, calculated with the bootstrapping
approach used by Fox-Dobbs et al. [100] are in the same range
or smaller than previously published data from large mammals
(e.g., [28–30,32]; Table 4). From all randomly chosen subsamples
with nsub $4, 95% or more range within 1s of the mean of the
complete data. In the case of a subsample size of nsub $7, even a
99% confidence is reached for a range within 1s of the mean of
the whole dataset.
Discussion
Inter- and Intra-specific Variations in d18OCO3
Mean d18OCO3 values of the analysed species (Fig. 3) show no,
or only minor, differences. This suggests that probably all taxa
drank from isotopically similar water sources. The higher d18OCO3
in R. norvegicus compared to the other rodent species may be
attributed to the juvenile status of the individuals and might reflect
a remnant suckling signal.
Figure 4. Comparison of incisor and bone d18OCO3 values from
the individuals where both tissues were analysed (n=5 in each
species). 1: A. terrestris, 2: M. glareolus, 3: M. agrestis, 4: M. arvalis, 5: A.
sylvaticus, 6: M. musculus, 7: R. norvegicus. The dashed lines represent
the deviation of incisor mean values from bone mean values in% from
the 1:1 line (solid line).
doi:10.1371/journal.pone.0049531.g004
Table 2. Mean d18OCO3 and d
13C values of bone material of the seven analysed rodent species (n = 5 in each species) with 1s error
and range of variation (lowest and highest analytical value of the respective species).
Species Material Mean d
18OCO3 Range of variation Mean d
13C Range of variation n
(% vs. VSMOW) in d18OCO3 values (% vs. VPDB) in d
13C values
Arvicola terrestris bone 25.661.2 24.6 to 27.7 217.860.9 219.0 to 216.8 5
Myodes glareolus bone 25.761.4 24.0 to 27.3 217.760.4 218.3 to 217.3 5
Microtus agrestis bone 24.661.3 23.1 to 26.4 219.560.6 220.0 to 218.5 5
Microtus arvalis bone 26.661.3 24.8 to 27.9 218.460.8 219.5 to 217.6 5
Apodemus sylvaticus bone 25.860.8 24.6 to 26.6 216.461.3 217.3 to 214.1 5
Mus musculus bone 28.261.6 25.5 to 29.5 211.665.5 216.8 to 25.0 5
Rattus norvegicus bone 25.861.0 24.7 to 26.7 27.962.2 210.7 to 25.8 5
1sin. = left, dex. = right.
2value in brackets excluded as an outlier, see results section.
doi:10.1371/journal.pone.0049531.t002
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Due to the lack of published small mammal d18OCO3 data, the
intra-population variability of the analysed species can only be
compared to published d18OPO4 values of rodents. The ranges
from 2.0 to 3.9% (excluding one individual of M. glareolus, see
results section) for the incisor teeth are much larger than the range
in d18OPO4 of 0.7 to 0.8% for fat dormice (Glis glis). However, only
three specimens were investigated by Lindars et al. [83]. Bones of
Pitymus sp., M. arvalis and A. terrestris had d18OPO4 intra-population
variations of 0.8 to 2.2%, 0.4 % and 1.3 to 2.3%, respectively
[36]. These ranges are lower than or reach the lower limit of the
rodent d18OCO3 variability in the present study. However, the
lowest value of the aforementioned ranges is again based on three
to four individuals only and derives mainly from mixed bone
material.
Compared to large mammals, the observed variations of 2.0 to
3.9% are in the same range as previously reported d18OCO3 values
for North American bison (Bison bison) with 2.4 to 3.0% [29],
Baird’s tapir (Tapirus bairdii) with 1.4 to 2.6% [101], feral and
domestic horses (Equus caballus) with 2.0 to 6.5% [28,30,35], and
Figure 5. Comparison of incisor and bone d13C values from the
individuals where both tissues were analysed (n=5 in each
species). 1: A. terrestris, 2: M. glareolus, 3: M. agrestis, 4: M. arvalis, 5: A.
sylvaticus, 6: M. musculus, 7: R. norvegicus. The dashed lines represent
the deviation of incisor mean values from bone mean values in% from
the 1:1 accordance (solid line).
doi:10.1371/journal.pone.0049531.g005
Figure 6. Intra-jaw variations in d18OCO3 of Arvicola terrestris.
Filled triangles = incisors, open diamonds=M1, open squares =M2,
open down triangles =M3.
doi:10.1371/journal.pone.0049531.g006
Figure 7. Intra-jaw variations in d13C of Arvicola terrestris. Filled
triangles = incisors, open diamonds =M1, open squares =M2, open
down triangles =M3.
doi:10.1371/journal.pone.0049531.g007
Figure 8. D18O CO3-PO4 of incisors, first molars and bone in A.
terrestris 1 to 5. Triangles represent the incisors, diamonds the first
molars and circles the bones. The average value for all samples is
10.960.8% (solid and dashed lines).
doi:10.1371/journal.pone.0049531.g008
Oxygen and Carbon Isotope Variations in Rodents
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bobcats (Lynx rufus) with 3.1% intra-population variability at a
single locality [32]. Other published intra-population variabilities
including three or more individuals for large terrestrial mammals
are significantly larger than those observed in the present study, as
is the case for mule deer (Odocoileus hemionus) with a range of 6.2%,
coyotes (Canis latrans) with 6.3% [32], domestic yaks (Bos grunniens)
with 3.7 to 8.5% and domestic goats (Capra aegagrus hircus) with 5.4
to 10.6% intra-population variability [30].
Inter- and Intra-specific Variations in d13C
Mean d13C values of the analysed species are in part
significantly distinct from each other, notably between arvicolids
and murids, indicating dietary differences (Fig. 3). The intra-
population variations in d13C have a wide range from 1.8 to
12.7%, reflecting variations in the d13C values of their diet and in
the nutritional behaviour (i.e., dietary specialists vs. dietary
opportunists). Previously published data on intra-population
variations from large mammals range from 0.7 to 8.5%
[29,30,32,101].
The average carbon isotope fractionation between diet and
bioapatite in rodents (determined on bone material) is+9.9%
[1,23,102]. This suggests average diet d13C values between 229.6
and 226.1% for the arvicolids. For the murids, the average diet
d13C values are higher: 225.8% in A. sylvaticus, 222.0% in M.
musculus and 219.5% in R. norvegicus.
The low intra-population variations of A. terrestris, M. glareolus
and M. agrestis (,2.2%) indicate little dietary variations, which is
supported for A. terrestris and M. agrestis by the observation of nearly
exclusively herbivorous diets [103,104]. An exclusively herbivo-
rous diet is also known for M. glareolus in various regions [105].
Table 3. d18OCO3, d
18OPO4 and D
18O CO3-PO4 of incisors, first molars and bone material of A. terrestris (n = 5).
Species Material d
18OCO3 d
18OPO4 D
18O CO3-PO4
(% vs. VSMOW) (% vs. VSMOW) (%)
Arvicola terrestris 01 I1 sin. 28.4 15.9 12.5
Arvicola terrestris 01 M1 sin. 26.6 16.1 10.5
Arvicola terrestris 01 bone 27.7 15.4 12.3
Arvicola terrestris 02 I1 sin. 25.6 15.6 10.0
Arvicola terrestris 02 M1 sin. 26.1 15.2 10.9
Arvicola terrestris 02 bone 25.5 15.7 9.8
Arvicola terrestris 03 I1 sin. 26.4 15.4 11.0
Arvicola terrestris 03 M1 sin. 25.6 15.2 10.4
Arvicola terrestris 03 bone 25.2 15.0 10.2
Arvicola terrestris 04 I1 sin. 26.7 16.3 10.4
Arvicola terrestris 04 M1 sin. 26.6 15.3 11.3
Arvicola terrestris 04 bone 24.6 13.8 10.8
Arvicola terrestris 05 I1 sin. 25.4 13.6 11.8
Arvicola terrestris 05 M1 sin. 23.9 12.7 11.2
Arvicola terrestris 05 bone 25.2 14.6 10.6
Mean (I1) 26.5±1.2 15.4±1.0 11.2±1.0
Mean (M1) 25.8±1.1 14.9±1.3 10.9±0.4
Mean (bone) 25.6±1.2 14.9±0.7 10.7±1.0
Overall mean 10.9±0.8
doi:10.1371/journal.pone.0049531.t003
Table 4. Results of the bootstrap analysis of d18OCO3 for rodent incisors (n = 10 in each species).
Number of subsamples out of 1000 random replicates of nsub within 1s
Arvicolids (incisors) Murids (incisors)
nsub A. terrestris M. glareolus M. agrestis M. arvalis A. sylvaticus M. musculus R. norvegicus
2 801 819 831 802 829 842 860
3 918 918 911 909 894 897 936
4 963 951 957 950 955 950 969
5 984 970 982 971 978 968 985
6 .990 981 990 983 986 982 .990
7 to 9 .990 .990 .990 .990 .990 .990 .990
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The diet of M. arvalis and A. sylvaticus consists of a considerable but
varying amount of invertebrates [106,107] explaining the higher
variability of 4.0 and 5.3%, respectively. Mean values of these
species correspond to a typical C3 diet. The highest variabilities in
d13C within the individuals from a single population were
observed in R. norvegicus and M. musculus, with 10.2 and 12.7%,
respectively. Both are synanthropic species with a very opportu-
nistic diet that commonly includes human food supplies and food
waste [108–110]. Thus, the high variability in d13C is likely caused
by the presence or absence of C4-sugars and/or products
containing tissues from animals fed on a C4 diet in the rodent diet.
Variations in d18OCO3 and d
13C between Incisor and Bone
Material
The d18OCO3 values of the incisors differ by up to ,4% from
that of bone material (Table S1), which can be explained by
different time intervals in the mineralisation of both tissues.
Rodent incisors (and also the molars of most arvicolids) are
permanently growing and reflect a time span of a few weeks
[86,87], whereas no pronounced remodeling of bone tissue occurs
in rodents. Thus, bones archive a nearly life-long record [88]. This
may also explain the phenomenon of why nearly 90% of the
analysed individuals have higher d18OCO3 values in the incisors
than in the bone material (Fig. 4). Taking into account the general
population dynamics of small rodents in temperate regions with
population maxima in late summer (see also materials and
methods section), the incisor d18OCO3 of a large number of
individuals are likely biased towards the 18O-enriched precipita-
tion of the warm summer months (Fig. 2). In contrast, bone
material was formed over a longer time span, including the colder
spring season or even the winter months in case of adult
individuals that survived this period. This is further supported
by the standard deviations of the bone-incisor offsets in each
species (0.8 to 1.8%), which reflects the different times of
mineralisation of both tissues in relation to a variable age structure
of the individual specimens. One exception is the juvenile R.
norvegicus specimens that have a relatively uniform bone-incisor
offset within the five analysed individuals (,3%), with a small
standard deviation of only 0.3%. This likely reflects their more or
less identical age and hence similar time intervals recorded in bone
and incisor d18OCO3 values.
The d13C values of the bulk incisor teeth, with two exceptions
(Table S1), deviate less than 2% from the d13C results of the
respective bone material, and no general trend to a skewed
variation, as is the case for d18OCO3, can be observed (Fig. 5).
These variations can be explained by variations in the carbon
isotope composition of the consumed food during the different
mineralisation times of incisor and bone material.
Intra-jaw Variations in d18OCO3 and d
13C of A. terrestris
The d18OCO3 of the molars of A. terrestris deviates from the
incisor oxygen isotope composition of the same individual between
0.3 and 1.9%, indicating slight differences in the time periods of
mineralisation of both tooth types. For other arvicolids (Microtus) a
complete renewal of incisors within four to seven weeks is reported
[87], whereas first and second molars of Microtus underly a
complete renewal in a time interval of eight to twelve weeks [86].
Considering this, as well as the fact that the molars mostly have
lower d18OCO3 values than incisors (Table S1; analogous to the
incisor-bone offset), a small bias by seasonally driven population
dynamics is likely the cause for the inter-tooth offset.
The difference in d13C between molar teeth and incisors of all
analysed individuals is less than 2%, comparable to the bone-
incisor variations and reflecting varying carbon isotope composi-
tions of the ingested food (Table S1). Given that the owl pellets
represent a seasonally biased taphocoenosis, the lower d13C values
in nearly all incisors compared to the molars of the same
individuals can result either from a seasonal change in availability
of food plants or from seasonal changes in the carbon isotope
composition of the same plant species, which can be in the range
of several per mil within one plant species (e.g., [67,111]).
Variations among M1 to M3 of the same animal are generally
lower, both in d18OCO3 (0.2 to 1%) as well as in d
13C (0.1 to
0.6%), indicating a more or less contemporaneous growth and
mineralisation of these molars.
The D18OCO3-PO4 in A. terrestris
Comparing incisors, molars and bone, the differences between
d18OCO3 and d
18OPO4 of each element overlap within error
(Table 3). The overall D18OCO3-PO4 of 10.960.8% is high and at
the upper limit of previously reported values for large and
medium-sized mammals (7.5 to 10.6%; [43–45,47,49,112]) that
include perissodactyls, artiodactyls and carnivorans. The only
reported data for rodents (laboratory rats, Rattus norvegicus) from
two different settings show a highly variable D18OCO3-PO4 of 8.5
and 11.4%, respectively [48]. Further studies are needed to
evaluate a possible species or body temperature dependence as
suggested by Martin et al. [45] or a connection to seasonal
variations in the d18O of drinking water and/or a food source of
phosphate as supposed by Kirsanow and Tuross [48].
Minimum Sample Size Calculations
The bootstrapping results of our d18OCO3 data from rodents
(more than 95% of the subsamples are within 61s of the mean if
nsub $4, and even more than 99% if nsub $7) agree to the
minimum sample size recommendations for most large mammals
given in previous studies [28,29,32] as well as for the horses from
the study of Wang et al. [30]. For goats and yaks, a considerably
higher minimum sample size for reliable estimates of the
population mean d18O within a 95% confidence level has been
determined [30], presumably related to differences in physiology
and/or a different nutritional or rather drinking behaviour. Our
data for rodents indicate that the minimum sample size
recommendations, determined for large mammals, potentially
can be extended to small mammal species (i.e., rodents). Further
taxon-specific studies, however, are needed to confirm if the
present finding is valid for small mammals in general.
Suitability for the d18O Reconstruction of Local Water
The d18OCO3 variability of different sympatric rodent taxa from
the studied locality is low. This suggests the reliability of a
d18OCO3/PO4-d
18OLW calibration equation based on a generic,
family or even higher taxonomic level. However, factors that can
affect the oxygen isotope composition of biogenic apatite (e.g.
habitat, drinking behaviour, population dynamics, body size and
thermophysiology) should be considered carefully before inferring
drinking water d18O values from existing calibration equations or
the development of new ones.
To assess if calibration equations developed on bone material
can be unrestrictedly applied to tooth data and vice versa, incisor
and bone mean values were compared to each of the three existing
d18OPO4-d
18OLW calibration equations for different rodent taxa
(Table 5). The d18OPO4 values were calculated from the measured
d18OCO3 values, applying the D
18OCO3-PO4 of 10.960.8%
determined for Arvicola terrestris. Thus, combining the data of all
seven analysed species, the calculated mean d18OPO4 values are
16.860.8% (n = 70) for incisor teeth and 15.161.1% (n = 35) for
bones. These values were plotted versus the long-term local mean
Oxygen and Carbon Isotope Variations in Rodents
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d18OLW of 27.3% in relation to existing d
18OPO4-d
18OLW
regression lines for rodents [4,16,36] (Fig. 9).
The regression line of Luz and Kolodny [4] is based on bone
material from laboratory rats, and that of Longinelli et al. [36] is
based nearly exclusively on bones of wild murids and arvicolids
(including the genera Apodemus, Arvicola, Microtus and Pitymus). The
regression of Navarro et al. [16] was primarily derived using molar
and incisor teeth from wild small arvicolids of the genera Myodes
and Microtus.
As the calibration equation of Luz & Kolodny [4] covers only a
d18OLW range outside the d
18O of local precipitation in our
sample area, a direct comparison to our data is precarious.
However, extrapolation of the regression line reveals that the
bone-derived d18O values from the present study are much closer
to this line than the tooth values (Fig. 9).
A much better fit is obtained if the bone-based equation of
Longinelli et al. [36] is compared with our bone data, which
directly plot on the respective calibration line. A similar
concordance exists by comparing our tooth-data with the tooth-
based equation of Navarro et al. [16] (Fig. 9).
This indicates that the choice of the skeletal element used for the
calibration between the d18O of biogenic apatite and d18OLW has
an important influence, which can be assigned to different
mineralisation periods and seasonally driven population dynamics
in rodents as discussed above. In the case of the study by Luz and
Kolodny [4], an additional explanation for differences from our
data could be the fact that the respective equation is based on
laboratory animals that often differ in their physiology from field
populations, which can have an impact on the oxygen isotope
composition of their biogenic apatite (e.g., [82]). Furthermore, as
laboratory rats were raised under controlled conditions with a
constant diet and water source, this precludes the effect of any
seasonal bias.
Previous studies have shown that tooth enamel should be the
material of choice for oxygen isotope studies in fossil mammals,
because it is less prone to diagenetic alteration than dentine or
bone material [6,113]. Therefore, future d18OPO4-d
18OLW cali-
bration equations of modern taxa that aim to reconstruct
palaeoenvironmental conditions should be based on tooth material
instead of bone. Furthermore, the same tooth type available also
from the fossil taxa should be used, in order to avoid seasonal bias
due to different tooth formation patterns.
Conclusions
The observed inter-specific variations in d18OCO3 within
arvicolid and murid incisors are low. Intra-specific variations in
the d18O of the investigated rodent bioapatite samples are in the
same range as most previously observed values from large
mammals. Statistical evaluations show that minimum sample size
recommendations from existing studies on large mammals agree
well with the results from the present study, indicating at least 95%
confidence that from a subsample size of $4 mean values range
Figure 9. Tooth (triangle) and bone (circle) mean values of all analysed samples converted from d18OCO3 to d
18OPO4 and plotted
versus the long term d18O average of local precipitation (-7.3%). Dashed lines show the existing d18OPO4-d
18OLW regression lines for rodents
developed mainly on bone material by Luz & Kolodny (1985) and Longinelli et al. (2003) [4,36]. The solid line shows the d18OPO4-d
18OLW calibration
equation from Navarro et al. (2004) [16], which is mainly based on tooth material.
doi:10.1371/journal.pone.0049531.g009
Table 5. Existing calibration equations between d18OLW and d
18OPO4 for rodents.
Study developed calibration equation skeletal tissue used included genera
Luz and Kolodny (1985) d18OLW = (d
18OPO4-17.88)/0.49 bone Rattus
Longinelli et al. (2003) d18OLW = (d
18OPO4-23.07)/1.14 bone (nearly solely) Apodemus, Pitymus, Arvicola, Microtus
Navarro et al. (2004) d18OLW = (d
18OPO4-20.98)/0.572 molars, incisors Microtus, Myodes, Lemmus
doi:10.1371/journal.pone.0049531.t005
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within 1s of the mean of the sample population. The observed
minor differences in the intra-specific d18O variability of rodents
compared to large and medium-sized mammals corroborate the
applicability of rodents in palaeoclimatic studies. Due to the
differing time intervals recorded in the different skeletal tissues
(incisors, molars, bone), rodents can show considerable variations
in d18O within a single individual, caused by seasonal biased
d18OLW ingested by individuals. This underscores the importance
of using analogous tissue material when d18O data from fossil
rodents are evaluated with d18OPO4-d
18OLW calibration equations
based on modern taxa. Comparisons of the d18O data from the
analysed arvicolids and murids with the existing calibration
equations relative to the d18O data of local precipitation shows
that a significant deviation from the expected value occurs when
bone-based equations are applied to tooth-data and vice versa.
The D18OCO3-PO4 of 10.960.8% determined for A. terrestris is at
the upper limit of data from large mammals and ranges within the
two previously reported values from another rodent species (R.
norvegicus). Within the present study, no significant disadvantages in
the interpretation of d18O data from rodent bioapatite compared
to large- and medium-sized mammals were observed. Neverthe-
less, it has to be taken into account that fossil rodent
taphocoenoses in temperate regions mostly underlie seasonal and
spatial predator-prey population dynamics.
The d13C data mirror the different nutritional strategies of the
analysed species (i.e., dietary specialisation vs. dietary opportun-
ism). The range of variations within a single species can vary
considerably but agrees well with field observations of the
respective nutritional behaviour. Thus, multiple analyses from
different individuals are an important prerequisite to get deeper
insight into specific nutritional adaptions from fossil small
mammals. Seasonally driven variations in the carbon isotope
composition of the ingested food are reflected in different skeletal
tissues, caused by differing time intervals of incisor, molar and
bone mineralisation.
The present study underscores the high potential of stable
isotope signatures of fossil small mammal skeletal remains to
reconstruct past environmental and climatic conditions. However,
further detailed systematic investigations of modern analogous
communities of small mammals are necessary for a refined
understanding of the incorporation of oxygen and carbon isotope
compositions in the bioapatite of their teeth and bones and to
explore the full potential of fossil rodents for palaeoenvironmental
research.
Supporting Information
Table S1 Detailed summary of analytical results
(XLS)
Acknowledgments
The authors are grateful to B. von Bu¨low, Haltern am See, for providing
the sample material. F. Mayer, Berlin is thanked for providing the elephant
molar tooth to produce our internal bioapatite standard ‘‘AG-Lox’’. We
thank W. von Koenigswald, Bonn, as well as L. Maul, Weimar, for
references and advice regarding growth mechanisms in permanent growing
rodent teeth. For assistance during the sample preparation and carbonate
stable isotope analyses, we are grateful to I. Reuber and N. Albrecht. We
thank S. Grimes, Plymouth and an anonymous reviewer for their
comments and suggestions to improve the submitted manuscript as well
as A. A. Farke, Claremont for the editorial handling.
Author Contributions
Conceived and designed the experiments: AG AP TT. Performed the
experiments: AG TT. Analyzed the data: AG. Contributed reagents/
materials/analysis tools: AG TT. Wrote the paper: AG TT AP.
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