The oxygen (δ18O) and carbon (δ13C) 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.
The δ18O and δ13C 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 δ18O means within one standard deviation were determined. These parameters are comparable to existing data from large mammals. Additionally, the fractionation between coexisting carbonate (δ18OCO3) and phosphate (δ18OPO4) in rodent bioapatite was determined, and δ18O values were compared to existing calibration equations between the δ18O of rodent bioapatite and local surface water (δ18OLW). Specific calibration equations between δ18OPO4 and δ18OLW 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. δ13C values reflect the rodents’ diet and agree well with field observations of their nutritional behaviour.
Rodents have a high potential for the reconstruction of palaeoenvironmental conditions by means of bioapatite δ18O and δ13C 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, Tütken 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.
Stable isotope compositions of mammalian bioapatite are widely used proxies in palaeoenvironmental and palaeodietary studies. Starting with pioneering research on carbon  and oxygen isotopes – 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.  and references therein; –). Carbon isotopes can be used to infer the palaeovegetation, vegetation cover, dietary strategies, resource partitioning and habitat use (e.g.  and references therein; , , –).
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 techniques 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 , –. 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 in order to investigate inter- and intra-specific variabilities. This has been done repeatedly for selected large mammals (e.g., –) 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 (δ18O) and carbon isotope (δ13C) 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 (δ18OCO3) and phosphate (δ18OPO4) 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 δ18OLW from the δ18OCO3 values, the data from the other species were converted to δ18OPO4 equivalent values by using the average offset between δ18OCO3 and δ18OPO4 (Δ18OCO3-PO4) obtained for A. terrestris. Then the data were compared to the three published δ18OPO4-δ18OH2O calibration equations determined for different extant rodent taxa in previous studies , , .
With the present study, the authors aim to provide a basic contribution for the enhancement of palaeoenvironmental interpretations of oxygen and carbon isotope data obtained from fossil rodents.
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., , , ). 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., , ). Thus, empirical specific calibration equations relating δ18OPO4/CO3 and δ18OLW 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., –), (palaeo-)climatic conditions can be inferred from the oxygen isotope composition of the skeletal remains of fossil mammals (e.g.,  and references therein).
In bioapatite, oxygen is present in the phosphate, carbonate and the hydroxyl groups . The average offset between δ18OPO4 and δ18OCO3 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 –. Both, δ18OPO4 and δ18OCO3, can be used to track the oxygen isotope composition of ingested drinking water. For the offset between δ18OPO4 and oxygen bound in the hydroxyl group, only a single calculated value of −16.6‰ is known .
The carbon isotope composition of mammalian bioapatite is controlled by that of ingested food (i.e., plant material in herbivores)  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.,  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. –). C3 plants, which represent about 95% of all terrestrial plant species (e.g., ), have typical δ13C values between -36 and −22‰ with an average value of −27‰, whereas the δ13C values of C4 plants (mostly warm season grasses and sedges) have −15 to −10‰, with an average value of −13‰ (e.g. , , , ). CAM plants have highly variable δ13C values within and between specific taxa, overlapping with both C3 and C4 plants (e.g., , , ). However, CAM plants represent only about 4% of all terrestrial plant species  and are mostly succulents , playing a minor role in the nutrition of most herbivorous mammals. Thus, a distinction between browsing and grazing herbivores based on bioapatite δ13C values is possible, as shown by numerous palaeodietary studies (e.g. –). However, this approach is limited to ecosystems with coexisting C3 and C4 plants; the latter were not globally abundant until the late Miocene .
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., , ). Subcanopy plants in humid, shaded environments assimilating CO2, depleted in 13C by soil respiration, have very low δ13C values ranging from −36 to −32‰ , , –. C3 plants in arid, open environments have the highest δ13C values, up to −21‰ , , . 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., , , , , –). Terrestrial C3 plant ecosystems date back to the Palaeozoic and are typical for most parts of the modern northern hemisphere (e.g., ).
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.,  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 . Rodents first appear in the Palaeogene fossil record, and fossil rodent remains are widespread in Cenozoic terrestrial deposits, often in high numbers, accumulated predominantly by ancient avian predators. As reviewed by Grimes et al. , 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 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 .
Specific calibration equations between δ18OPO4 and δ18OLW have been developed in the past for laboratory rats , wild murids ,  and wild arvicolids , . 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 –, , Neogene , ,  and Pleistocene – climate change. Carbon isotope records of fossil small rodents also were used to reconstruct vegetational change and palaeodiets in the Cenozoic , , –.
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  has investigated intra-population and intra-jaw variations (δ18OPO4) 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.  concluded that a quantity of>5 different post-weaning teeth should be used for isotope palaeo-thermometry. Further intra-population δ18OPO4 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  and Longinelli et al. , but no data for distinct sympatric species from the same locality are available so far.
Materials and Methods
The samples originate from fresh barn owl (Tyto alba) pellets accumulated over a maximum of four years, collected in April 1995 at “Hof Gülker”, 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 Bülow . Long term mean annual local temperatures in this region (IAEA-GNIP station Emmerich) average 10°C, and the mean annual precipitation is 744 mm. The long term weighted mean of the monthly δ18O record of local precipitation between 1980 and 2005 is −7.3‰. 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 δ18O value is −7.7. This is very close to the long term record . 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 , . 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.
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., –). Avian predator populations, responsible for pellet accumulations, are positively correlated to populations of their small mammal prey (e.g., –), 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 δ18OPO4/CO3-δ18OLW calibration equations and their application for palaeoclimate reconstructions .
No specific permits were required for the described field studies.
Oxygen and carbon isotope analyses of the carbonate in the bioapatite (δ18OCO3, δ13C).
Only teeth from taxonomically 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 δ18OCO3 and δ13C 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. . Approximately 10 mg of sample powder were soaked with 30% H2O2 (0.1 ml mg−1) 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 mg−1) and rinsed again five times with millipore water, followed by drying overnight at 50°C. Typically, 1 to 1.3 mg of pretreated sample powder were reacted for 15 min. with 100% H3PO4 at 70°C 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 Göttingen. 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 δ-notation in per mil (‰), relative to the international isotope reference standards Vienna Standard Mean Ocean Water (VSMOW) for δ18O and Vienna Pee Dee Belemnite (VPDB) for δ13C .
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 ±0.1‰ (1σ) in δ18O and ±0.04‰ (1σ) in δ13C (n = 232, analysed between November 2010 and February 2012). For the NBS 120c Florida phosphate rock standard (pretreated as described above), we obtained a δ18O value of 30.0±0.2‰ (1σ) and a δ13C value of −6.4±0.04‰ (1σ) (n = 10). Our internal bioapatite standard AG-Lox (African elephant enamel) had a δ18O value of 30.0±0.08‰ (1σ) and a δ13C value of -12.0±0.03‰ (1σ) (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 (δ18OPO4).
Analyses of the phosphate moiety of the bioapatite (δ18OPO4) 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.  and described in detail by Tütken et al. . 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 50°C.
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. .
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 comparisons. 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 δ18O by 95% degree of confidence within ±1σ were determined by a standard bootstrapping approach according to the method presented by Fox-Dobbs et al. . 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 ±1σ 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 ±1σ 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.
Oxygen and Carbon Isotope Data of the Carbonate in the Bioapatite (δ18OCO3, δ13C)
The data, which are summarised in Table S1, include 135 individual δ18OCO3 and δ13C analyses of bulk incisor and molar teeth, as well as δ18OCO3 and δ13C values of bone material from seven modern arvicolid and murid species from a single locality in NW Germany (see Materials and Methods section).
Oxygen Isotope Composition of the Incisors
Intra-population variations in δ18OCO3 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 δ18OCO3 values of the arvicolid incisors are 26.8±1.4‰ (A. terrestris), 27.2±1.8‰ (M. glareolus), 27.3±1.1‰ (M. agrestis) and 27.3±1.2‰ (M. arvalis). The murid incisors have mean δ18OCO3 values of 27.9±1.0‰ (A. sylvaticus), 28.3±1.0‰ (M. musculus) and 29.0±0.7‰ (R. norvegicus) (Fig. 3, Table 1). Between the mean δ18OCO3 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 δ18OCO3 were detected (one-way ANOVA, F = 0.304, P = 0.82). Within murids, mean δ18OCO3 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).
1: A. terrestris, 2: M. glareolus, 3: M. agrestis, 4: M. arvalis, 5: A. sylvaticus, 6: M. musculus, 7: R. norvegicus.
Carbon Isotope Composition of the Incisors
The δ13C 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 −17.7±0.6‰, −16.2±0.7‰ and −19.7±0.6‰, respectively. The intra-population δ13C variations of M. arvalis and the murid A. sylvaticus are much larger, with ranges of 4.0‰ and 5.4‰ and mean values of −18.8±1.4‰ and −15.9±1.7‰, respectively. Very large variations were observed in the two murids, M. musculus and R. norvegicus, that have an intra-population δ13C range of 12.7‰ and 10.2‰ with mean values of -12.1±5.3‰ and -9.6±3.4‰, respectively (Fig. 3, Table 1). Mean δ13C values differed significantly between the analysed species (one-way ANOVA, F = 20.632, P<0.01). By pair-wise comparison, mean δ13C values of two murids (M. musculus and R. norvegicus) differed significantly from those of all arvicolids as well as from A. sylvaticus. The mean δ13C 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 δ13C 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 δ18OCO3 values (n = 5 of each species) deviate from the incisor values of of the same individuals by −4.2 to+1.5‰. 85% (30 out of 35 specimens) have higher δ18OCO3 values in their incisors than in their bones (Fig. 4, Table S1). Mean values are 25.6±1.2‰ (A. terrestris), 25.7±1.4‰ (M. glareolus), 24.6±1.3‰ (M. agrestis) and 26.6±1.3‰ (M. arvalis). The murid bones have mean δ18OCO3 values of 25.8±0.8‰ (A. sylvaticus), 28.2±1.6‰ (M. musculus) and 25.8±1.0‰ (R. norvegicus) (Table 2).
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).
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 δ13C of bone material deviates by+1.9 to −1.6‰ from that of the incisors, with the exceptions of one A. sylvaticus (−2.9‰) and one M. musculus (+4.3‰). Mean values are −17.8±0.9‰ (A. terrestris), −17.7±0.4‰ (M. glareolus), −19.5±0.6‰ (M. agrestis) and −18.4±0.8‰ (M. arvalis). The murid bones have higher mean δ13C values of −16.4±1.3‰ (A. sylvaticus), −11.6±5.5‰ (M. musculus) and −7.9±2.2‰ (R. norvegicus). No systematic differences between incisor and bone material can be observed, as is the case for δ18OCO3 (Fig. 5, Table 2).
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).
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 δ18OCO3 and 0.1 to 0.6‰ in δ13C. The overall intra-jaw range (I1, M1–M3) varies from 0.3 to 1.9‰ in δ18OCO3, while in most cases the incisors have higher values than the molars (Fig. 6, Table S1). In δ13C, 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 26.1±1.6‰, 26.1±1.5‰ and 26.2±1.3‰ in δ18OCO3 and −16.9±0.6‰, −16.8±0.6‰ and −16.7±0.5‰, respectively (n = 10 of each tooth type).
Filled triangles = incisors, open diamonds = M1, open squares = M2, open down triangles = M3.
Filled triangles = incisors, open diamonds = M1, open squares = M2, open down triangles = M3.
Oxygen Isotope Analyses of the Phosphate in the Bioapatite (δ18OPO4) of A. terrestris
The mean δ18OPO4 of skeletal apatite from A. terrestris are 15.4±1.1‰ (bulk incisors, n = 5), 14.9±1.3‰ (bulk M1, n = 5) and 14.9±0.8‰ (bone, n = 5). The corresponding δ18OCO3 means are 26.5±1.2‰, 25.8±1.1‰ and 25.6±1.2‰, leading to an Δ18OCO3-PO4 of 11.2±1.0‰ (incisors), 10.9±0.4‰ (M1) and 10.7±0.9‰ (bone), respectively. The overall average Δ18OCO3-PO4 is 10.9±0.8‰ (Fig. 8, Table 3).
Triangles represent the incisors, diamonds the first molars and circles the bones. The average value for all samples is 10.9±0.8‰ (solid and dashed lines).
Minimum Sample Size Calculations
Minimum sample sizes, calculated with the bootstrapping approach used by Fox-Dobbs et al.  are in the same range or smaller than previously published data from large mammals (e.g., –, ; Table 4). From all randomly chosen subsamples with nsub ≥4, 95% or more range within 1σ 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 1σ of the mean of the whole dataset.
Inter- and Intra-specific Variations in δ18OCO3
Mean δ18OCO3 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 δ18OCO3 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.
Due to the lack of published small mammal δ18OCO3 data, the intra-population variability of the analysed species can only be compared to published δ18OPO4 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 δ18OPO4 of 0.7 to 0.8‰ for fat dormice (Glis glis). However, only three specimens were investigated by Lindars et al. . Bones of Pitymus sp., M. arvalis and A. terrestris had δ18OPO4 intra-population variations of 0.8 to 2.2‰, 0.4 ‰ and 1.3 to 2.3‰, respectively . These ranges are lower than or reach the lower limit of the rodent δ18OCO3 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 δ18OCO3 values for North American bison (Bison bison) with 2.4 to 3.0‰ , Baird’s tapir (Tapirus bairdii) with 1.4 to 2.6‰ , feral and domestic horses (Equus caballus) with 2.0 to 6.5‰ , , , and bobcats (Lynx rufus) with 3.1‰ intra-population variability at a single locality . 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‰ , 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 .
Inter- and Intra-specific Variations in δ13C
Mean δ13C 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 δ13C have a wide range from 1.8 to 12.7‰, reflecting variations in the δ13C 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‰ , , , .
The average carbon isotope fractionation between diet and bioapatite in rodents (determined on bone material) is+9.9‰ , , . This suggests average diet δ13C values between −29.6 and −26.1‰ for the arvicolids. For the murids, the average diet δ13C values are higher: −25.8‰ in A. sylvaticus, −22.0‰ in M. musculus and −19.5‰ 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 , . An exclusively herbivorous diet is also known for M. glareolus in various regions . The diet of M. arvalis and A. sylvaticus consists of a considerable but varying amount of invertebrates ,  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 δ13C 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 opportunistic diet that commonly includes human food supplies and food waste –. Thus, the high variability in δ13C 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 δ18OCO3 and δ13C between Incisor and Bone Material
The δ18OCO3 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 , , whereas no pronounced remodeling of bone tissue occurs in rodents. Thus, bones archive a nearly life-long record . This may also explain the phenomenon of why nearly 90% of the analysed individuals have higher δ18OCO3 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 δ18OCO3 of a large number of individuals are likely biased towards the 18O-enriched precipitation 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 δ18OCO3 values.
The δ13C values of the bulk incisor teeth, with two exceptions (Table S1), deviate less than 2‰ from the δ13C results of the respective bone material, and no general trend to a skewed variation, as is the case for δ18OCO3, 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 δ18OCO3 and δ13C of A. terrestris
The δ18OCO3 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 , whereas first and second molars of Microtus underly a complete renewal in a time interval of eight to twelve weeks . Considering this, as well as the fact that the molars mostly have lower δ18OCO3 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 δ13C between molar teeth and incisors of all analysed individuals is less than 2‰, comparable to the bone-incisor variations and reflecting varying carbon isotope compositions of the ingested food (Table S1). Given that the owl pellets represent a seasonally biased taphocoenosis, the lower δ13C 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., , ).
Variations among M1 to M3 of the same animal are generally lower, both in δ18OCO3 (0.2 to 1‰) as well as in δ13C (0.1 to 0.6‰), indicating a more or less contemporaneous growth and mineralisation of these molars.
The Δ18OCO3-PO4 in A. terrestris
Comparing incisors, molars and bone, the differences between δ18OCO3 and δ18OPO4 of each element overlap within error (Table 3). The overall Δ18OCO3-PO4 of 10.9±0.8‰ is high and at the upper limit of previously reported values for large and medium-sized mammals (7.5 to 10.6‰; –, , , ) that include perissodactyls, artiodactyls and carnivorans. The only reported data for rodents (laboratory rats, Rattus norvegicus) from two different settings show a highly variable Δ18OCO3-PO4 of 8.5 and 11.4‰, respectively . Further studies are needed to evaluate a possible species or body temperature dependence as suggested by Martin et al.  or a connection to seasonal variations in the δ18O of drinking water and/or a food source of phosphate as supposed by Kirsanow and Tuross .
Minimum Sample Size Calculations
The bootstrapping results of our δ18OCO3 data from rodents (more than 95% of the subsamples are within ±1σ 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 , ,  as well as for the horses from the study of Wang et al. . For goats and yaks, a considerably higher minimum sample size for reliable estimates of the population mean δ18O within a 95% confidence level has been determined , 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 δ18O Reconstruction of Local Water
The δ18OCO3 variability of different sympatric rodent taxa from the studied locality is low. This suggests the reliability of a δ18OCO3/PO4-δ18OLW 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 δ18O 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 δ18OPO4-δ18OLW calibration equations for different rodent taxa (Table 5). The δ18OPO4 values were calculated from the measured δ18OCO3 values, applying the Δ18OCO3-PO4 of 10.9±0.8‰ determined for Arvicola terrestris. Thus, combining the data of all seven analysed species, the calculated mean δ18OPO4 values are 16.8±0.8‰ (n = 70) for incisor teeth and 15.1±1.1‰ (n = 35) for bones. These values were plotted versus the long-term local mean δ18OLW of −7.3‰ in relation to existing δ18OPO4-δ18OLW regression lines for rodents , ,  (Fig. 9).
Dashed lines show the existing δ18OPO4-δ18OLW regression lines for rodents developed mainly on bone material by Luz & Kolodny (1985) and Longinelli et al. (2003) , . The solid line shows the δ18OPO4-δ18OLW calibration equation from Navarro et al. (2004) , which is mainly based on tooth material.
The regression line of Luz and Kolodny  is based on bone material from laboratory rats, and that of Longinelli et al.  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.  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  covers only a δ18OLW range outside the δ18O 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 δ18O 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.  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.  (Fig. 9).
This indicates that the choice of the skeletal element used for the calibration between the δ18O of biogenic apatite and δ18OLW 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 , 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., ). 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 , . Therefore, future δ18OPO4-δ18OLW calibration 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.
The observed inter-specific variations in δ18OCO3 within arvicolid and murid incisors are low. Intra-specific variations in the δ18O 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 within 1σ of the mean of the sample population. The observed minor differences in the intra-specific δ18O 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 δ18O within a single individual, caused by seasonal biased δ18OLW ingested by individuals. This underscores the importance of using analogous tissue material when δ18O data from fossil rodents are evaluated with δ18OPO4-δ18OLW calibration equations based on modern taxa. Comparisons of the δ18O data from the analysed arvicolids and murids with the existing calibration equations relative to the δ18O 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 Δ18OCO3-PO4 of 10.9±0.8‰ 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 δ18O data from rodent bioapatite compared to large- and medium-sized mammals were observed. Nevertheless, it has to be taken into account that fossil rodent taphocoenoses in temperate regions mostly underlie seasonal and spatial predator-prey population dynamics.
The δ13C data mirror the different nutritional strategies of the analysed species (i.e., dietary specialisation vs. dietary opportunism). 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.
Detailed summary of analytical results
The authors are grateful to B. von Bülow, 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.
Conceived and designed the experiments: AG AP TT. Performed the experiments: AG TT. Analyzed the data: AG. Contributed reagents/materials/analysis tools: AP TT. Wrote the paper: AG TT AP.
- 1. DeNiro MJ, Epstein S (1978) Influence of diet on the distribution of carbon isotopes in animals. Geochimica et Cosmochimica Acta 42: 495–506.
- 2. Longinelli A (1984) Oxygen isotopes in mammal bone phosphate: a new tool for paleohydrological and paleoclimatological research? Geochimica et Cosmochimica Acta 48: 385–390.
- 3. Luz B, Kolodny Y, Horowitz M (1984) Fractionation of oxygen isotopes between mammalian bone phosphate and environmental drinking water. Geochimica et Cosmochimica Acta 48: 1689–1693.
- 4. Luz B, Kolodny Y (1985) Oxygen isotope variations in phosphate of biogenic apatites, IV. Mammal teeth and bones. Earth and Planetary Science Letters 75: 29–36.
- 5. Land LS, Lundelius EL, Valastro S (1980) Isotopic ecology of deer bones. Palaeogeography, Palaeoclimatology, Palaeoecology 32: 143–151.
- 6. Kohn MJ, Cerling TE (2002) Stable isotope compositions of biological apatite. Reviews in Mineralogy and Geochemistry 48: 455–488.
- 7. Levin NE, Cerling TE, Passey BH, Harris JM, Ehleringer JR (2006) A stable isotope aridity index for terrestrial environments. Proceedings of the National Academy of Sciences 103: 11201–11205.
- 8. Tütken T, Vennemann TW, Janz H, Heizmann EPJ (2006) Palaeoenvironment and palaeoclimate of the Middle Miocene lake in the Steinheim Basin, SW Germany: A reconstruction from C, O, and Sr isotopes of fossil remains. Palaeogeography, Palaeoclimatology, Palaeoecology 241: 457–491.
- 9. Tütken T, Vennemann T (2009) Stable isotope ecology of Miocene large mammals from Sandelzhausen, southern Germany. Paläontologische Zeitschrift 83: 207–226.
- 10. Balasse M, Ambrose SH, Smith AB, Price TD (2002) The seasonal mobility model for prehistoric herders in the south-western cape of South Africa assessed by isotopic analysis of sheep tooth enamel. Journal of Archaeological Science 29: 917–932.
- 11. Balasse M, Smith AB, Ambrose SH, Leigh SR (2003) Determining sheep birth seasonality by analysis of tooth enamel oxygen isotope ratios: the Late Stone Age site of Kasteelberg (South Africa). Journal of Archaeological Science 30: 205–215.
- 12. Cerling TE, Hart JA, Hart TB (2004) Stable isotope ecology in the Ituri Forest. Oecologica 138: 5–12.
- 13. MacFadden BJ, Higgins P (2004) Ancient ecology of 15-million-year-old browsing mammals within C3 plant communities from Panama. Oecologica 140: 169–182.
- 14. Drucker DG, Bridault A, Hobson KA, Szuma E, Bocherens H (2008) Can carbon-13 in large herbivores track canopy effect in temperate and boreal ecosystems? Evidence from modern and ancient ungulates. Palaeogeography, Palaeoclimatology, Palaeoecology 266: 69–82.
- 15. Feranec RS, MacFadden BJ (2006) Isotopic discrimination of resource partitioning among ungulates in C3-dominated communities from the Miocene of Florida and California. Paleobiology 32: 191–205.
- 16. Navarro N, Lécuyer C, Montuire S, Langlois C, Martineau F (2004) Oxygen isotope compositions of phosphate from arvicoline teeth and Quaternary climatic changes, Gigny, French Jura. Quaternary Research 62: 172–182.
- 17. Ruddy M (2008) Water vole incisors as records of palaeoclimate. Quaternary Newsletter 116: 51–54.
- 18. Rogers KL, Wang Y (2002) Stable isotopes in pocket gopher teeth as evidence of a Late Matuyama climate shift in the southern Rocky Mountains. Quaternary Research 57: 200–207.
- 19. Yeakel JD, Bennett NC, Koch PL, Dominy NJ (2007) The isotopic ecology of African mole rats informs hypotheses on the evolution of human diet. Proceedings of the Royal Society (B) 274: 1723–1730.
- 20. Grimes ST, Mattey DP, Hooker JJ, Collinson ME (2003) Eocene-Oligocene palaeoclimate reconstruction using oxygen isotopes: problems and solutions from the use of multiple palaeoproxies. Geochimica et Cosmochimica Acta 67: 4033–4047.
- 21. Grimes ST, Mattey DP, Collinson ME, Hooker JJ (2004) Using mammal tooth phosphate with freshwater carbonate and phosphate palaeoproxies to obtain mean palaeotemperatures. Quaternary Science Reviews 23: 967–976.
- 22. Grimes ST, Hooker JJ, Collinson ME, Mattey DP (2005) Summer temperatures of late Eocene to early Oligocene freshwaters. Geology 33: 189–192.
- 23. Grimes ST, Collinson ME, Hooker JJ, Mattey DP, Grassineau NV, et al. (2004) Distinguishing the diets of coexisting fossil theridomyid and glirid rodents using carbon isotopes. Palaeogeography, Palaeoclimatology, Palaeoecology 208: 103–119.
- 24. Hopley PJ, Latham AG, Marshall JD (2006) Palaeoenvironments and palaeodiets of mid-Pliocene micromammals from Makapansgat Limeworks, South Africa: A stable isotope and dental microwear approach. Palaeogeography, Palaeoclimatology, Palaeoecology 233: 235–251.
- 25. Hynek SA, Passey BH, Prado JL, Brown FH, Cerling TE, et al. (2012) Small mammal carbon isotope ecology across the Miocene-Pliocene boundary, northwestern Argentina. Earth and Planetary Science Letters 321–322: 177–188.
- 26. Tóth E, Görög Á, Lécuyer C, Moissette P, Balter V, et al. (2010) Palaeoenvironmental reconstruction of the Sarmatian (Middle Miocene) Central Paratethys based on palaeontological and geochemical analyses of foraminifera, ostracods, gastropods and rodents. Geological Magazine 147: 299–314.
- 27. Héran MA, Lécuyer C, Legendre S (2010) Cenozoic long-term terrestrial climatic evolution in Germany tracked by δ18O of rodent tooth phosphate. Palaeogeography, Palaeoclimatology, Palaeoecology 285: 331–342.
- 28. Hoppe KA, Stuska S, Amundson R (2005) Implications for palaeodietary and palaeoclimatic reconstructions of intrapopulation variability in the oxygen and carbon isotopes of teeth from modern feral horses. Quaternary Research 64: 138–146.
- 29. Hoppe KA (2006) Correlation between the oxygen isotope ratio of North American bison teeth and local waters: Implication for palaeoclimatic reconstructions. Earth and Planetary Science Letters 244: 408–417.
- 30. Wang Y, Kromhout E, Zhang C, Xu Y, Parker W, et al. (2008) Stable isotopic variations in modern herbivore tooth enamel, plants and water on the Tibetan Plateau: Implications for paleoclimate and paleoelevation reconstructions. Palaeogeography, Palaeoclimatology, Palaeoecology 260: 359–374.
- 31. Fricke HC, O’Neil JR (1996) Inter- and intra-tooth variation in the oxygen isotope composition of mammalian tooth enamel phosphate: implications for palaeoclimatological and palaeobiological research. Palaeogeography, Palaeoclimatology, Palaeoecology 126: 91–99.
- 32. Clementz MT, Koch PL (2001) Differentiating aquatic mammal habitat and foraging ecology with stable isotopes in tooth enamel. Oecologica 129: 461–472.
- 33. Kohn MJ, Schoeninger MJ, Valley JW (1996) Herbivore tooth oxygen isotope compositions: Effects of diet and physiology. Geochimica et Cosmochimica Acta 60: 3889–3896.
- 34. Bocherens H, Koch PL, Mariotti A, Geraards D, Jaeger J-J (1996) Isotopic biogeochemistry (13C, 18O) of mammalian enamel from African Pleistocene Hominid Sites. Palaios 11: 306–318.
- 35. Hoppe KA, Amundson R, Vavra M, McClaran MP, Anderson D (2004) Isotopic analysis of tooth enamel carbonate from modern North American feral horses: implications for palaeoenvironmental reconstructions. Palaeogeography, Palaeoclimatology, Palaeoecology 203: 299–311.
- 36. Longinelli A, Iacumin P, Davanzo S, Nikolaev V (2003) Modern reindeer and mice: revised phosphate-water isotope equations. Earth and Planetary Science Letters 214: 491–498.
- 37. Bryant JD, Froelich PN (1995) A model of oxygen isotope fractionation in body water of large mammals. Geochimica et Cosmochimica Acta 59: 4523–4537.
- 38. D’Angela D, Longinelli A (1990) Oxygen isotopes in living mammal’s bone phosphate: further results. Chemical Geology 86: 75–82.
- 39. Rozanski K, Johnsen SJ, Schotterer U, Thompson LG (1997) Reconstruction of past climates from stable isotope records of palaeo-precipitation preserved in continental archives. Hydrological Sciences 42: 725–745.
- 40. Dansgaard W (1964) Stable isotopes in precipitation. Tellus 16: 436–468.
- 41. Rozanski K, Araguás-Araguás L, Gonfiantini R (1993) Isotopic patterns in modern global precipitation. In: Swart PK, Lohmann KC, McKenzie J, Savin S, editors. Climate change in continental isotopic records ( = Geophysical Monograph 78). Washington D. C.: American Geophysical Union. 1–36.
- 42. Fricke HC, O’Neil JR (1999) The correlation between 18O/16O ratios of meteoric water and surface temperature: its use in investigating terrestrial climate change over geologic time. Earth and Planetary Science Letters 170: 181–196.
- 43. Iacumin P, Bocherens H, Mariotti A, Longinelli A (1996) Oxygen isotope analyses of co-existing carbonate and phosphate in biogenic apatite: a way to monitor diagenetic alteration of bone phosphate? Earth and Planetary Science Letters 142: 1–6.
- 44. Bryant JD, Koch PL, Froelich PN, Showers WJ, Genna BJ (1996) Oxygen isotope partitioning between phosphate and carbonate in mammalian apatite. Geochimica et Cosmochimica Acta 60: 5145–5148.
- 45. Martin C, Bentaleb I, Kaandorp R, Iacumin P, Chatri K (2008) Intra-tooth study of modern rhinoceros enamel δ18O: Is the difference between phosphate and carbonate δ18O a sound diagenetic test? Palaeogeography, Palaeoclimatology, Palaeoecology 266: 183–187.
- 46. Zazzo A (2001) Validation méthodologique de l’utilisation des compositions isotopiques (13C, 18O) des bioapatites fossiles pour les reconstitutions des paléoenvironnements continentaux. Paris: université Pierre et Marie Curie. 504 p.
- 47. Shahack-Gross R, Tchernov E, Luz B (1999) Oxygen isotopic composition of mammalian skeletal phosphate from the Natufian Period, Hayonim Cave, Israel: Diagenesis and Paleoclimate. Geoarchaeology 14: 1–13.
- 48. Kirsanow K, Tuross N (2011) Oxygen and hydrogen isotopes in rodent tissues: Impact of diet, water and ontogeny. Palaeogeography, Palaeoclimatology, Palaeoecology 310: 9–16.
- 49. Zazzo A, Lécuyer C, Sheppard SMF, Grandjean P, Mariotti A (2004) Diagenesis and the reconstruction of palaeoenvironments: A method to restore original δ18O values of carbonate and phosphate from fossil tooth enamel. Geochimica et Cosmochimica Acta 68: 2245–2258.
- 50. Jones AM, Iacumin P, Young ED (1999) High-resolution δ18O analysis of tooth enamel phosphate by isotope ratio monitoring gas chromatography mass spectrometry and ultraviolet laser fluorination. Chemical Geology 153: 241–248.
- 51. Farquhar GD, Ehleringer JR, Hubick KT (1989) Carbon isotope discrimination and photosynthesis. Annual Review of Plant Physiology and Plant Molecular Biology 40: 503–537.
- 52. O’Leary MH (1988) Carbon isotopes in photosynthesis. BioScience 38: 328–336.
- 53. Bender MM (1971) Variations in the 13C/12C ratios of plants in relation to the pathway of photosynthetic carbon dioxide fixation. Phytochemistry 10: 1239–1244.
- 54. Bowes G (1993) Facing the inevitable: plants and increasing atmospheric CO2. Annual Review of Plant Physiology and Plant Molecular Biology 44: 309–332.
- 55. Deines P (1980) The isotopic composition of reduced organic carbon. In: Fritz P, Fontes C, editors. Handbook of environmental geochemistry, Vol 1. New York: Elsevier. 239–406.
- 56. Mariotti A (1991) Le carbone 13 en abondance naturelle, traceur de la dynamique de la matière organique des sols et de l’évolution des paléoenvironnements continentaux. Cahiers Orstom, série Pédologie 26: 299–313.
- 57. Bender MM, Rouhani I, Vines HM, Black Jr CC (1973) 13C/12C ratio changes in crassulacean acid metabolism plants. Plant Physiology 52: 427–430.
- 58. Ting IP (1985) Crassulacean acid metabolism. Annual Review of Plant Physiology 36: 595–622.
- 59. Sayed OH (2001) Crassulacean acid metabolism 1975–2000, a check list. Photosynthetica 39: 339–352.
- 60. MacFadden BJ, Solounias N, Cerling TE (1999) Ancient diets, ecology, and extinction of 5-million-year-old horses from Florida. Science 283: 824–827.
- 61. Quade J, Cerling TE, Barry JC, Morgan ME, Pilbeam DR, et al. (1992) A 16-Ma record of paleodiet using carbon and oxygen isotopes in fossil teeth from Pakistan. Chemical Geology (Isotope Geoscience Section) 94: 183–192.
- 62. MacFadden BJ, Cerling TE (1996) Mammalian herbivore communities, ancient feeding ecology and carbon isotopes: a 10-million-year sequence from the Neogene of Florida. Journal of Vertebrate Paleontology 16: 103–115.
- 63. MacFadden BJ, Wang Y, Cerling TE, Anaya F (1994) South American fossil mammals and carbon isotopes: a 25 million-year sequence from the Bolivian Andes. Palaeogeography, Palaeoclimatology, Palaeoecology 107: 257–268.
- 64. Cerling TE, Harris JM, Ambrose SH, Leakey MG, Solounias N (1997) Dietary and environmental reconstruction with stable isotope analysis of herbivore tooth enamel from the Miocene locality of Fort Ternan, Kenya. Journal of Human Evolution 33: 635–650.
- 65. Cerling TE, Harris JM, Leakey MG (1999) Browsing and grazing in elephants: the isotope record of modern and fossil proboscideans. Oecologica 120: 364–374.
- 66. Cerling TE, Wang Y, Quade J (1993) Expansion of C4 ecosystems as an indicator of global ecological change in the late Miocene. Nature 361: 344–345.
- 67. Heaton THE (1999) Spatial, species and temporal Variations in the 13C/12C ratios of C3 plants: implications for palaeodiet studies. Journal of Archaeological Science 26: 637–649.
- 68. Vogel JC (1978) Recycling of carbon in a forest environment. Oecologica Plantarum 13: 89–94.
- 69. Van Der Merwe NJ, Medina E (1989) Photosynthesis and 13C/12C ratios in Amazonian rain forests. Geochimica et Cosmochimica Acta 53: 1091–1094.
- 70. Van Der Merwe NJ, Medina E (1991) The canopy effect, carbon isotope ratios and foodwebs in Amazonia. Journal of Archaeological Science 18: 249–259.
- 71. Medina E, Minchin P (1980) Stratification of δ13C values of leaves in Amazonian rain forests. Oecologica 45: 377–378.
- 72. Ehleringer JR, Cooper TA (1988) Correlation between carbon isotope ratio and microhabitat in desert plants. Oecologica 76: 562–566.
- 73. Drucker D, Bocherens H, Bridault A, Billiou D (2003) Carbon and nitrogen isotopic composition of red deer (Cervus elaphus) collagen as a tool for tracking palaeoenvironmental change during the Late-Glacial and Early Holocene in the northern Jura (France). Palaeogeography, Palaeoclimatology, Palaeoecology 195: 375–388.
- 74. Quade J, Cerling TE, Andrews P, Alpagut B (1995) Paleodietary reconstruction of Miocene faunas from Pasalar, Turkey using stable carbon and oxygen isotopes of fossil tooth enamel. Journal of Human Evolution 28: 373–384.
- 75. Harris JM, Cerling TE (2002) Dietary adaptions of extant and Neogene African suids. Journal of the Zoological Society of London 256: 45–54.
- 76. Feranec RS (2007) Stable carbon isotope values reveal evidence of resource partitioning among ungulates from modern C3-dominated ecosystems in North America. Palaeogeography, Palaeoclimatology, Palaeoecology 252: 575–585.
- 77. Nelson SV (2007) Isotopic reconstructions of habitat change surrounding the extinction of Sivapithecus, a Miocene hominoid, in the Siwalik Group of Pakistan. Palaeogeography, Palaeoclimatology, Palaeoecology 243: 204–222.
- 78. Zanazzi A, Kohn MJ (2008) Ecology and physiology of White River mammals based on stable isotope ratios of teeth. Palaeogeography, Palaeoclimatology, Palaeoecology 257: 22–37.
- 79. Still C, Berry JA, Collatz GJ, DeFries R (2003) Global distribution of C3 and C4 vegetation: Carbon cycle implications. Global Biogeochemical Cycles 17: 6.1–6.14.
- 80. Passey BH, Robinson TF, Ayliffe LK, Cerling TE, Sponheimer M, et al. (2005) Carbon isotope fractionation between diet, breath CO2, and bioapatite in different mammals. Journal of Archaeological Science 32: 1459–1470.
- 81. Kemp TS (2007) The origin and evolution of mammals. New York: Oxford University Press. 331 p.
- 82. Grimes ST, Collinson ME, Hooker JJ, Mattey DP (2008) Is small beautiful? A review of the advantages and limitations of using small mammal teeth and the direct laser fluorination analysis technique. Palaeogeography, Palaeoclimatology, Palaeoecology 266: 39–50.
- 83. Lindars ES, Grimes ST, Mattey DP, Collinson ME, Hooker JJ, et al. (2001) Phosphate δ18O determination of modern rodent teeth by direct laser fluorination: An appraisal of methodology and potential application to palaeoclimate reconstruction. Geochimica et Cosmochimica Acta 65: 2535–2548.
- 84. Bülow Bv (1997) Kleinsäuger im NSG Rhader Wiesen in Dorsten. Natur und Heimat 57: 37–40.
- 85. IAEA/WISER (2011) Water Isotope System for Data Analysis, Visualization and Electronic Retrieval, IAEA Water Resources Programme. Available at http://nds121.iaea.org/wiser/accessed 21. May 2011.
- 86. Koenigswald Wv, Golenishev FN (1979) A method for determining growth rates in continuously growing molars. Journal of Mammalogy 60: 397–400.
- 87. Klevezal GA, Pucek M, Sukhovskaya LI (1990) Incisor growth in voles. Acta Theriologica 35: 331–344.
- 88. Klevezal GA (1996) Recording structures of mammals. Determination of age and reconstruction of life history. Mina MV, Oreshkin AV, translator. Rotterdam, Brookfield: Balkema. 274 p.
- 89. Stubbe M (1982) Dynamik der Kleinnagergesellschaft (Rodentia: Arvicolidae, Muridae) im Hakel. Hercynia N F 19: 110–120.
- 90. Ylönen H, Altner HJ, Stubbe M (1991) Seasonal dynamics of small mammals in an isolated woodlot and its agricultural surroundings. Annales Zoologici Fennici 28: 7–14.
- 91. Bäumler W (1986) Populationsdynamik von Mäusen in verschiedenen Waldgebieten Bayerns. Anzeiger für Schädlingskunde, Pflanzenschutz, Umweltschutz 59: 112–117.
- 92. Norrdahl K, Korpimäki E (2002) Seasonal changes in the numerical responses of predators to cyclic vole populations. Ecography 25: 428–438.
- 93. Schönfeld M, Girbig G, Sturm H (1977) Beiträge zur Populationsdynamik der Schleiereule, Tyto alba. Hercynia N F 14: 303–351.
- 94. Schönfeld M, Girbig G (1975) Beiträge zur Brutbiologie der Schleiereule, Tyto alba, unter besonderer Berücksichtigung der Abhängigkeit von der Feldmausdichte. Hercynia N F 12: 257–319.
- 95. Salamolard M, Butet A, Leroux A, Bretagnolle V (2000) Responses of an avian predator to variations in prey density at a temperate latitude. Ecology 81: 2428–2441.
- 96. Koch PL, Tuross N, Fogel ML (1997) The effects of sample treatment and diagenesis on the isotopic integrity of carbonate in biogenic hydroxylapatite. Journal of Archaeological Science 24: 417–429.
- 97. Coplen TB (1994) Reporting of stable hydrogen, carbon, and oxygen abundances. Pure and Applied Chemistry 66: 273–276.
- 98. Dettmann DL, Kohn MJ, Quade J, Ryerson FJ, Ojha TP, et al. (2001) Seasonal stable isotope evidence for a stron Asian monsoon throughout the past 10.7 m.y. Geology 29: 31–34.
- 99. Vennemann TW, Fricke HC, Blake RE, O’Neil JR, Colman A (2002) Oxygen isotope analyses of phosphates: a comparison of techniques for analysis of Ag3PO4. Chemical Geology 185: 321–336.
- 100. Fox-Dobbs K, Bump JK, Peterson RO, Fox DL, Koch PL (2007) Carnivore-specific stable isotope variables and variation in the foraging ecology of modern and ancient wolf populations: case studies from Isle Royale, Minnesota, and La Brea. Canadian Journal of Zoology 85: 458–471.
- 101. DeSantis LG (2011) Stable isotope ecology of extant tapirs from the Americas. Biotropica 43: 746–754.
- 102. Ambrose SH, Norr L (1993) Experimental evidence for the relationship of the carbon isotope ratios of whole diet and dietary protein to those of bone collagen and carbonate. In: Lambert J, Grupe G, editors. Prehistoric human bone, archaeology at the molecular level. Berlin: Springer. 1–37.
- 103. Krapp F, Niethammer J (1982) Microtus agrestis (Linnaeus, 1761) - Erdmaus. In: Niethammer J, Krapp F, editors. Handbuch der Säugetiere Europas. Wiesbaden: Akademische Verlagsgesellschaft. 349–373.
- 104. Reichstein H (1982) Arvicola terrestris (Linnaeus, 1758) - Schermaus. In: Niethammer J, Krapp F, editors. Handbuch der Säugetiere Europas. Wiesbaden: Akademische Verlagsgesellschaft. 217–252.
- 105. Viro P, Niethammer J (1982) Clethrionomys glareolus (Schreber, 1780) - Rötelmaus. In: Niethammer J, Krapp F, editors. Handbuch der Säugetiere Europas. Wiesbaden: Akademische Verlagsgesellschaft. 109–146.
- 106. Niethammer J (1978) Apodemus sylvaticus (Linnaeus, 1758) - Waldmaus. In: Niethammer J, Krapp F, editors. Handbuch der Säugetiere Europas. Wiesbaden: Akademische Verlagsgesellschaft. 337–358.
- 107. Niethammer J, Krapp F (1982) Microtus arvalis (Pallas, 1779) - Feldmaus. In: Niethammer J, Krapp F, editors. Handbuch der Säugetiere Europas. Wiesbaden: Akademische Verlagsgesellschaft. 284–318.
- 108. Freye HA, Freye H (1960) Die Hausmaus. Wittenberg: A. Ziemsen. 104 p.
- 109. Becker K (1978) Rattus norvegicus (Berkenhout, 1769) - Wanderratte. In: Niethammer J, Krapp F, editors. Handbuch der Säugetiere Europas. Wiesbaden: Akademische Verlagsgesellschaft. 401–420.
- 110. Reichstein H (1978) Mus musculus Linnaeus, 1758 - Hausmaus. In: Niethammer J, Krapp F, editors. Handbuch der Säugetiere Europas. Wiesbaden: Akademische Verlagsgesellschaft. 421–451.
- 111. Lowdon JA, Dyck W (1974) Seasonal variations in the isotope ratios of carbon in maple leaves and other plants. Canadian Journal of Earth Sciences 11: 79–88.
- 112. Pellegrini M, Lee-Thorp JA, Donahue RE (2011) Exploring the variation of the δ18Op and δ18Oc relationship in enamel increments. Palaeogeography, Palaeoclimatology, Palaeoecology 310: 71–83.
- 113. Gehler A, Tütken T, Pack A (2011) Triple oxygen isotope analysis of bioapatite as tracer for diagenetic alteration of bones and teeth. Palaeogeography, Palaeoclimatology, Palaeoecology 310: 84–91.