Palaeogeography, Palaeoclimatology, Palaeoecology

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1 Palaeogeography, Palaeoclimatology, Palaeoecology (2012) Contents lists available at SciVerse ScienceDirect Palaeogeography, Palaeoclimatology, Palaeoecology journal homepage: Paleoecology of late Pleistocene Holocene faunas of eastern and central Wyoming, USA, with implications for LGM climate models Matthew J. Kohn a,, Moriah P. McKay b a Department of Geosciences, Boise State University, Boise, ID 83725, United States b Department of Geological Sciences, University of South Carolina, Columbia, SC 29208, United States article info abstract Article history: Received 7 September 2011 Received in revised form 23 January 2012 Accepted 30 January 2012 Available online 11 February 2012 Keywords: Pleistocene Carbon isotopes Oxygen isotopes Tooth enamel Paleoecology Oxygen and carbon isotope compositions of teeth were measured for a variety of fossil herbivores, omnivores and carnivores from Natural Trap Cave (NTC) and Little Box Elder Cave (LBEC), central eastern Wyoming, USA. These sites host some of the best preserved and most abundant late Pleistocene to early Holocene mammal fossils, and provide key information about ecological and climatic change attending the Pleistocene Holocene transition. Average Pleistocene compositions are consistent with C3-dominated diets, but C4 grass consumption increased in Bison from the late Pleistocene to today. A single Sangamonian Equus tooth also has elevated 13 C, suggesting that C4 biomass increased during interglacials in general. Tooth δ 18 O values imply local water δ 18 O values at NTC of 15 to 16 during the Last Glacial Maximum (LGM) and 14 today, consistent with modern regional studies and general circulation models (GCM's) for the LGM. Water δ 18 O values at LBEC were ~ 12. Although herbivore compositions are consistent with theoretical expectations, carnivores exhibit lower δ 13 C and higher δ 18 O values relative to herbivores than anticipated. These discrepancies could reflect poorly understood carnivore physiologies or diets, and emphasize the need for further investigation of isotope systematics in carnivores. Isotope zoning in several herbivore teeth shows negative correlations between carbon and oxygen isotopes: high and low δ 18 O values representing summer and winter seasons occur with low and high δ 13 C values respectively. This trend could indicate seasonally changing diets, e.g. consumption of drier foods or conifers during the winter, or burning of stored fat during the winter. Increasing δ 18 O values from the Pleistocene to Holocene in water-dependent taxa are consistent with an increasing proportion of summer moisture derived from the Gulf of Mexico, as predicted by isotopeenabled GCM's. Mean annual precipitation during the late Pleistocene is estimated at 350 mm/yr, similar to modern day (c. 200 mm/yr), indicating that dry conditions have prevailed for the last 25 ka in Wyoming. A revised analytical expression for C4 plant abundance in the western US is proposed that accounts for climate variables and p CO2. Combination of this expression with the climate predictions of the PMIP2 ensemble average GCM explains LGM C4 plant abundances at NTC, southern Texas, and Florida. Changes to C4 plant abundances may provide sensitive tests of GCM accuracies Elsevier B.V. All rights reserved. 1. Introduction Characterizing past ecologies and climates in comparison to modern day conditions helps us understand processes of climate change and critically evaluate climate models. Late Pleistocene fossils at Natural Trap Cave (NTC) and Little Box Elder Cave (LBEC), Wyoming (Fig. 1), provide an unusual opportunity to compare Last Glacial Maximum (LGM) and modern conditions, and to explore the Pleistocene Holocene transition. This transition is important for several reasons. Documented isotopic and climatic changes are used to validate the accuracy of general circulation models that incorporate stable isotopes (e.g., Joussaume and Jouzel, 1993; Hoffmann et al., 2000; Jouzel et al., 2000; Risi et al., Corresponding author. address: mattkohn@boisestate.edu (M.J. Kohn). 2010) or that are used to predict future climate change (e.g., Joussaume and Taylor, 1995; Braconnot et al., 2007; Meehl et al., 2007). The isotope compositions of diverse faunas prior to the late Pleistocene megafaunal extinction also allow investigation of niche partitioning and diet selectivity in anthropogenically undisturbed ecosystems, as well as floral changes attending glacial interglacial cycles (e.g., Koch et al., 1998; Coltrain et al., 2004; Kohn et al., 2005; Palmqvist et al., 2008). Unlike many faunal sites, an abundance of canid ( dog ), felid ( cat ), and ursid ( bear ) fossils, in addition to numerous ungulates, further allows comparisons among carnivores, omnivores, and herbivores for understanding predator prey relations, effects of trophic level on isotope compositions, and physiological or dietary causes of isotopic outliers. In this study, we report stable carbon and oxygen isotope compositions of teeth from a diverse suite of animals recovered from these two caves to explore a series of interrelated ecological, physiological /$ see front matter 2012 Elsevier B.V. All rights reserved. doi: /j.palaeo

2 M.J. Kohn, M.P. McKay / Palaeogeography, Palaeoclimatology, Palaeoecology (2012) W 100W 90W 80W TR 40N NTC H LBEC 30N K04 HG HM H06 K/F Fig. 1. Outline of conterminous United States, Wyoming, and areas studied by Koch et al. (1998) and Feranec (2004b; K/F ), and Koch et al. (2004; K04 ); dot shows study area of Huang et al. (2006; H06 ). Sample localities: NTC=Natural Trap Cave and LBEC=Little Box Elder Cave. Topographically high areas in Wyoming are outlined. Holocene sites are: TR (Tongue River; Melton and Davis, 1999), H (Hawken; Lovvorn et al., 2001), HM (Hudson-Meng; Gadbury et al., 2000; Larson et al., 2001), and HG (Hell Gap; CARD, 2011). and climatic questions. These questions include: (1) How did late Pleistocene environments differ from the present, particularly with respect to C4 plant abundances and water compositions? (2) Relative to herbivores, are theoretical models of carnivore compositions accurate, and are carnivore herbivore isotopic offsets consistent with other empirical measurements? (3) What possibilities explain isotopically anomalous taxa relative to better-studied and more typical species? (4) What seasonal changes to diet or climate are inferable from intra-tooth isotope zoning? (5) What changes to mean annual precipitation (MAP) and δ 18 O values occurred across the Pleistocene Holocene transition, and how do they compare with GCM predictions? (6) How do estimates of C4 abundance trends, as derived from δ 13 C values of herbivore teeth, compare with predictions linked to GCM's? 2. Stable isotopes of teeth Teeth consist mineralogically of hydroxyapatite with major substitution of CO 3 for PO 4 and OH groups. In this study we analyzed the CO 3 component for δ 13 C and δ 18 O values. For reviews of stable isotopes in teeth, see Koch (1998, 2007), MacFadden (2000), Kohn and Cerling (2002) and Kohn and Dettman (2007). Oxygen isotopes in herbivore tooth enamel correlate with local water compositions (Kohn, 1996), but drought-tolerant species exhibit additional negative correlations with relative humidity (Ayliffe and Chivas, 1990; Luz et al., 1990). Oxygen isotopes in carnivores are assumed to correlate with local water as well (Kohn, 1996), but the modern data set is too small to verify this assumption generally, and isotope data from felid hair suggests poor correlations (Pietsch et al., 2011). Carbon isotopes correlate with diet. In mixed C3 C4 ecosystems, grazers exhibit elevated δ 13 C values compared to browsers in proportion to the ratio of C4 to C3 consumed. δ 13 C values of C3 plants and the animals that consume them increase with decreasing MAP (e.g., Farquhar et al., 1989), and in principle the δ 13 C value of tooth enamel in C3 ecosystems can be used to infer past MAP (Kohn, 2010). Teeth mineralize progressively from the tip or occlusal surface toward the root. Because local water compositions vary seasonally (lower in winter and higher in summer), oxygen isotope zoning both provides a measure of climate seasonality and indicates when different portions of a tooth mineralized. When paired with carbon isotope analysis, changes in diet can be identified and correlated with season. In principle, an ontogenetic shift from nursing to an adult diet can change isotope compositions both within teeth that mineralize over a significant period as well as among teeth that mineralize at different times. Although this effect is well documented for human oxygen and carbon isotope compositions (Wright and Schwarcz, 1998), results for other animals are scattered and inconclusive. Some data for mammoths suggest that neonate tooth enamel has lower δ 13 C and higher δ 18 O values than adults (Metcalfe et al., 2010). Other data from 11 modern mammal species, however, show no δ 13 C differences in the plasma of nursing juveniles and their mothers (Jenkins et al., 2001). Oxygen isotope compositions of teeth that formed during and after nursing are not predicted to be different (Kohn et al., 1998), and studies of large wild herbivores have failed to identify a clear oxygen isotope effect (Kohn et al., 1998; Gadbury et al., 2000; Murphy et al., 2007; Forbes et al., 2010). Although the timing of tooth formation and mineralization has been discussed extensively for herbivores, carnivores have received less attention. We have found little direct information on rates of tooth enamel mineralization in modern carnivores, but diverse sources document the timing of weaning and of the initial appearance or eruption of different teeth in the jaw (Saunders, 1964; Slaughter et al., 1974; Smuts et al., 1978; Mazak, 1981; Currier, 1983; Gittleman, 1986; Pusey and Packer, 1994; Biknevicius, 1996; Hillson, 1996; Miles and Grigson, 2003; Strömquist et al., 2009; see Supplemental file). In general, relative to birth, the first permanent cheek teeth in felids and canids erupt 2 times later than the timing of weaning. For example, weaning in Vulpes occurs on average at 1.8 months, whereas eruption of most cheek teeth occurs at 4 6 months.in Panthera pardus, weaning occurs on average at 4.6 months, whereas canines, molars, and premolars do not erupt until 8 10 months. Canine growth in Panthera leo is well studied. Whereas weaning occurs at 7 months, mineralization of the canine does not initiate until 9 11 months, initial eruption occurs soon after (11 15 months), and final mineralization concludes by months. Stable isotope zoning in Smilodon canines implies similarly lengthy durations of mineralization (Feranec, 2004a). Considering that meat consumption commences well prior to weaning, the isotopic effects of milk consumption on cheek teeth and canines in canids and felids should be negligible. Consequently, we emphasized their analysis in this study. In contrast, weaning in Ursus arctos at ~2 years postdates initial eruption of canines (8 months) and cheek teeth (12 24 months), although canine growth can continue past 50 months. Thus, in principle milk consumption could affect ursid isotope compositions. 3. Specimens and methods 3.1. Specimens and research sites Specimens were provided by the University of Kansas Museum of Natural History and the University of Colorado at Boulder. In our discussion and interpretations, we emphasize NTC more than LBEC because of the larger number of specimens and generally better chronology and stratigraphy. Note that county-wide grass surveys in Wyoming indicate that the C4 photosynthetic pathway comprises c. one-quarter of generic and one-sixth of species diversity in Big Horn County (NTC) and c. one-third of generic and one-quarter of species diversity in Converse County (LBEC) (USDA and NRCS,

3 44 M.J. Kohn, M.P. McKay / Palaeogeography, Palaeoclimatology, Palaeoecology (2012) ). C4 biomass has not been measured directly in either area, but regional correlations and data compilations (Paruelo and Lauenroth, 1996) suggest at most 20% C4 biomass at both NTC and LBEC. Natural Trap Cave (Fig. 1) is a karst feature within the Mississippian Madison Formation on the western slope of the Big Horn Mountains, near Lovell, Wyoming. At ~1400 m elevation, local topography obscures the sinkhole cave's opening to a ~25 m drop. Excavations of NTC from 1974 to 1985 recovered over 40,000 specimens, or ~5% of the deposit (Wang and Martin, 1993). Taxa analyzed included numerous herbivores (Bootherium bombifrons, Bison sp., Antilocapra americana, Ovis canadensis catclawensis, Camelops sp., Equus sp., Mammuthus sp., Bos taurus, Odocoileus sp., and two unidentified artiodactyls), several carnivores (Canis lupus, Gulo gulo, Miracinonyx trumani, and Panthera atrox), and several omnivores (Vulpes vulpes, Canis latrans, Arctodus simus, andu. arctos). Ages for NTC are based on 14 C measurements on bone collagen (Chomko and Gilbert, 1987; Walker, 1987; CARD, 2011) and converted to YBP using IntCal09 (Reimer et al., 2009). Most ages range from c. 26 ka to 11 ka. Several teeth from the Sangamon interglacial, dated at ~110 ka (Chorn et al., 1988), were also analyzed. Pleistocene data were augmented with isotope measurements of a historical B. taurus tooth, several published Holocene bone collagen analyses (Bison bison; Haynes, 1968; Melton and Davis, 1999; Gadbury et al., 2000; Larson et al., 2001; Lovvorn et al., 2001), and modern measurements of A. americana teeth (Fenner and Frost, 2009). Many specimens are correlated stratigraphically, permitting direct comparisons among taxa, although in some cases their ages are only bracketed with 14 C dates, leading to relatively large absolute uncertainties. Little Box Elder Cave (Fig. 1) is located ~30 km west of Douglas, Wyoming, at ~1675 m elevation; it formed either by solution of the Mississippian Amsden Formation or by lateral cutting of a nearby creek. Excavations between 1949 and 1963 recovered over 15,000 specimens, or approximately two-thirds of the deposit (Anderson, 1968). Taxa analyzed include herbivores (Equus sp., B. bison, Cervus elephus, Odocoileus hemionus, O. canadensis, and Oreamnos sp.), carnivores (Lynx rufus, Puma concolor, C. lupus, and Taxidea taxus) and omnivores (V. vulpes, A. simus, and U. arctos). Little Box Elder Cave is commonly assumed to span the Pleistocene Holocene transition (Kurten and Anderson, 1980). 14 C ages range from latest Pleistocene to early Holocene (Walker, 1987) Analytical methods We used the method of Kohn et al. (2005) for sampling and analysis of enamel. This involves cutting a strip of enamel lengthwise along each tooth, subsampling every 1 2 mm, cleaning subsamples of adhering dentine, and preparing and analyzing the resulting purified enamel using the method of Koch et al. (1997). This latter method involves grinding the enamel to a fine powder, pretreating in H 2 O 2 and an acetic acid Ca-acetate buffer, and analyzing in an automated carbonate extraction device, on-line with a mass spectrometer. For this study, analyses were collected with a VG Optima mass spectrometer at the University of South Carolina. Three to five aliquots of NIST120c were prepared and analyzed with each batch of unknowns. Six to seven analyses of NBS-19 calcite were also collected to verify mass spectrometer operation and reference gas calibrations. Data are reported in permil relative to V-SMOW for oxygen, and V- PDB for carbon (Tables 1 2; Supplemental files). Means for NIST- 120c were δ 18 O=28.8±0.8 (2σ) and δ 13 C= 6.4±0.4 (2σ). Errors include intra-run and day-to-day variations in sample preparation and analysis. Comparisons of mean values were assessed statistically using Mann Whitney non-parametric tests; t-tests yield similar Table 1 Average compositions of taxa at Natural Trap Cave and Little Box Elder Cave, Wyoming, USA. Taxon n δ 13 C Natural Trap Cave Bootherium 7/ Bison 7/ Antilocapra 6/ Camelops 3/ Ovis 8/ Equus 21/ Mammuthus 5/ Cervid 1/ Bos 1/ Odocoileus 1/ C. lupus 8/ Gulo 3/ Miracinonyx 5/ Panthera 2/ Vulpes 3/ Arctodus 3/ C. latrans 1/ Ursus 1/ Little Box Elder Cave Equus 6/ Bison 4/ Odocoileus 2/ Ovis 5/ Oreamnos 2/ Cervus 2/ Lynx 3/ Taxidea 2/ C. lupus 3/ Felis 1/ Ursus 3/ Arctodus 1/ Vulpes 3/ Note: n=number of teeth sampled/total number of analyses. 2σ δ 18 O 2σ

4 M.J. Kohn, M.P. McKay / Palaeogeography, Palaeoclimatology, Palaeoecology (2012) Table 2 Secular changes to tooth enamel compositions for select taxa at Natural Trap Cave, Wyoming, USA. Age (a) 2σ (a) n δ 13 C Antilocapra 25, , , , a Bison/Bos 23, , , , , b a a a a a Equus 110,000 10, , , , , , , , , , , Ovis 110,000 10, , , , , b a Published data from Melton and Davis (1999), Gadbury et al. (2000), Larson et al. (2001), Lovvorn et al. (2001), and CARD (2011). b Carbon isotope data from LBEC (δ 18 O not included because of systematic geographic differences). 2s.e. δ 18 O 2s.e. results. Select results are presented here; comprehensive interspecies and time-slice comparisons are provided in McKay (2008). Mean values across taxa (e.g., all NTC LGM herbivores, all large LBEC carnivores, etc.) were calculated by weighting according to measured variation of individual taxa; corresponding errors were estimated from the weighted standard deviation of observations about the mean (cf. Kohn and Spear, 1991a) Modeling Oxygen isotope modeling used the approach of Kohn (1996), modifying key oxygen fluxes and input/output compositions to explore compositional sensitivities (Table 3). Mean annual precipitation (MAP) was estimated based on C3 plant δ 13 C using the approach of Kohn (2010). Because plant compositions have not been measured, we instead use the isotope compositions of tooth enamel from herbivores that consumed C3 plants only, correcting for the known fractionation between diet and tooth enamel (Passey et al., 2005) and changes to δ 13 C atm. We adopt an average δ 13 C atm value of 7.0 during the late Pleistocene, 6.5 during the pre-industrial Holocene, and 8.0 for today (Smith et al., 1999). Relative to modern δ 13 C atm = 8.0, average δ 13 C values of deciduous C3 angiosperms are lower than 24.3 (Kohn, 2010). Correcting for fractionations between diet and tooth enamel (Passey et al., 2005: 14.5 for artiodactyls and 14 for other herbivores), we adopt an isotopic cutoff for C3 consumption by artiodactyls (all herbivores at LBEC and NTC except Equus) of 9.8 today, 8.8 during the late Pleistocene, and 8.3 during the pre-industrial Holocene. Higher values imply consumption of other 13 C-enriched foods, including CAM and C4 plants. Isotopic cutoffs for Equus are 0.5 lower. Note that changes to p CO2 alone are not thought to influence terrestrial C3 plant δ 13 C(Arens et al., 2000). Estimating MAP from δ 13 C values requires rewriting the relationship between modern plant δ 13 C (referenced to δ 13 C atm = 8.0 ) and MAP to solve for MAP as a function of δ 13 C, elevation (in meters) and latitude (absolute value in ): " # MAP ¼ 10ˆ δ13 C þ 10:29 0: elev þ 0:0124 AbsðlatÞ 300: 5:61 In addition, we evaluate the uncertainty in calculated MAP according to the standard error propagation equation (cf. Kohn and Spear, 1991b):! σ 2 MAP MAP MAP ¼ σ j i X i X i σ j ρ ij j ð1þ ð2þ

5 46 M.J. Kohn, M.P. McKay / Palaeogeography, Palaeoclimatology, Palaeoecology (2012) Table 3 Models of herbivore and carnivore δ 18 O. Model Drinking H 2 O (%, δ 18 O) Food H 2 O (%, δ 18 O) Water econ. index Dry food δ 18 O H 2 O vapor, liquid output (%) Model δ 18 O Standard herbivore 23, , , Standard carnivore 57, , , Low WEI 2, , , High δ 18 OH 2 O 57, , , High δ 18 O protein 57, , , High vapor output 46, , , Maximum carnivore 2, , , Note: δ 18 O values are in relative to V-SMOW; water economy index is the ratio of daily water turnover in ml to energy expenditure in KJ; the % vapor and liquid H 2 O output is relative to total oxygen (which includes CO 2 ). High vapor output model has reduced fecal water content (50%, rather than 60%) and urine output (15% rather than 25%). where MAP/ X i and MAP/ X j are the partial derivatives of Eq. (1) with respect to the variables X i and X j, σ i and σ j are the uncertainties in X i and X j, and ρ ij is the correlation coefficient between X i and X j. Correlation coefficients are known from the original linear regression, and errors can be assumed from reproducibilities or data scatter. Partial derivatives, however, are most easily estimated numerically (Roddick, 1987). In this approach, we perturb each parameter in Eq. (1) by a small amount (Δ i, typically ~1%), and determine a new value for MAP, here denoted MAP*. The partial derivative of Eq. (1) with respect to the parameter X i ( MAP/ X i ) is then simply (MAP* MAP)/Δ i. We then successively multiply partial derivatives, errors and correlation coefficients, sum over all parameters and errors, and take the square root. In general, the magnitude of each error increases with increasing MAP, increasing elevation, and decreasing latitude, but the 2σ uncertainty is approximately 50% of MAP above ~500 mm/yr, and never falls below ~±120 mm/yr (Fig. 2). 4. Results 4.1. Mean isotope compositions Average δ 18 O values at NTC (Fig. 3) are systematically ~3 lower than at LBEC (Fig. 4; pb0.001), but δ 13 C values generally overlap, and relative isotopic differences among groups are consistent at the two sites. For example, relative to Pleistocene herbivores, Pleistocene carnivores consistently show elevated δ 18 O, c. 3 4, both at NTC (Fig. 3; pb0.001) and LBEC (Fig. 4; pb0.001). Some studies have shown similar results (Bösl et al., 2006; Garcia Garcia et al., 2009; Feranec et al., 2010), MAP (mm/yr) ±2σ Elevation = 1500m Latitude = 45 Equivalent modern C3 Composition δ 13 C (, V-PDB) Fig. 2. MAP vs. δ 13 C at an elevation of 1500 m and latitude of 45, with ±2σ error envelopes from regression. Error is approximately 50% of MAP, except at low MAP (c. ±120 mm/yr). but others show overlapping carnivore herbivore compositions (Kohn et al., 2005; Palmqvist et al, 2008). Pleistocene carnivore δ 13 C values are consistently lower than herbivores at NTC by ~2 (pb0.001; 2.3 for all carnivores, and 2.2 considering large carnivores only). This difference is slightly larger than anticipated from careful carnivore herbivore comparisons of bone (Clementz et al., 2009). Large carnivores at LBEC (C. lupus and Felis)havec.3 higher δ 13 C values than small carnivores (Lynx and Taxidea; pb0.001), with a near-zero offset between large carnivores and herbivores (p=0.96). Carnivore herbivore comparisons at LBEC may be compromised by uncertainty in whether individual carnivore specimens reflect late Pleistocene or Holocene ecosystems. Collagen isotopic compositions at NTC (McNulty et al., 2002) and measured isotopic offsets between bioapatite and collagen (Clementz et al., 2009) imply bioapatite δ 13 C values of c. 10 to 11.5 for herbivores and 13 to 15 for carnivores, 1 2 lower than reported here. The small number of collagen analyses and wide spread of ages does not allow close comparison of these different datasets, however, except to note that relative isotopic spacings among taxa are preserved regardless of tissue. Tooth enamel analyses from 6 large herbivore teeth (Higgins and MacFadden, 2009) overlap ours. Bears exhibit δ 18 O values intermediate between carnivores and herbivores (Figs. 3 4). Arctodus shows distinctly lower δ 13 C values, as does δ 18 O (, V-SMOW) Natural Trap Cave Odocoileus (Hol.) Arctodus (pre-hol) Vulpes (pre-hol) Panthera Gulo Carnivore Wtd Mean Canis lupus Miracinonyx Antilocapra Cervid Equus Bootherium Mammuthus Herbivore Wtd Mean δ 13 C (, V-PDB) Ovis Canis latrans (Hol.) Ursus (Hol.) Bos (Hol.) Bison Camelops Fig. 3. Summary of data from Natural Trap Cave (see Tables 1 2 and Supplemental file for data). Data show resource partitioning among taxa, and systematic and large isotopic offset between weighted mean compositions of carnivores vs. herbivores. Carnivore mean omits bears and small canids (Arctodus, C. latrans, and Vulpes); herbivore mean omits Holocene taxa (Bos and Odocoileus). The size of the shaded ellipses reflects the 2σ uncertainty in the weighted mean value. A difference in mean δ 13 C values between carnivores and herbivores is anticipated from predator prey isotopic fractionations (Clementz et al., 2009); oxygen isotope differences are less easily interpreted. Hol. and pre-hol. labels indicate whether outliers are Holocene or pre-holocene.

6 M.J. Kohn, M.P. McKay / Palaeogeography, Palaeoclimatology, Palaeoecology (2012) δ 18 O (, V-SMOW) Little Box Elder Cave Ursus Arctodus Lynx Taxidea Equus Vulpes Canis Lupus Cervus Carnivore Wtd Mean Odocoileus Felis Ovis Herbivore Wtd Mean Oreamnos Bison δ 13 C (, V-PDB) Fig. 4. Summary of data from Little Box Elder Cave (see Tables 1 2 and Supplemental file for data). Data show resource partitioning among taxa, and systematic and large offset between weighted mean compositions of carnivores vs. herbivores. Carnivore mean omits bears and small canid (Arctodus, Vulpes, Ursus); herbivore mean includes all taxa. The size of the shaded ellipses reflects the 2σ uncertainty in the weighted mean value. A difference in mean δ 13 C values between carnivores and herbivores is anticipated from predator prey isotopic fractionations (Clementz et al., 2009); oxygen isotope differences are less easily interpreted. Ursus at LBEC, but Ursus at NTC has high δ 13 C values. Besides bears, compositional extremes at NTC (Fig. 3) includec. latrans (high δ 18 Oand δ 13 C values), Vulpes (high δ 18 O values), and Odocoileus (high δ 18 Oand δ 13 C values; p for all comparisons). At LBEC, compositional extremes include Vulpes (high δ 18 O values; p=0.04) and Bison (low δ 18 O, high δ 13 Cvalues;Fig. 4; pb0.001) Isotope zoning Compositional zoning along large herbivore teeth shows quasisinusoidal variations (Fig. 5), both in δ 18 O and δ 13 C. C- and O- isotopes, however, are negatively correlated, i.e. 18 O-enriched portions of a tooth (summer compositions) generally have low δ 13 Cvalues, and 18 O-depleted portions (winter compositions) have high δ 13 C values. Negative correlations are common among herbivores (Fig. 6), although for most p 0.1, and not obviously dependent on age (cf. Holocene Bos,post-LGM Bootherium and Antilocapra, and LGM Mammuthus). Carnivore teeth are generally too small or insufficiently zoned to show this directly, but one also has a possible negative correlation (KU26133 Miracinonyx; p=0.318) Secular isotope changes Carbon isotopes (Fig. 7) show steadily increasing δ 13 C values for Bison and Bos from the LGM to the present (pb0.001). Equus data from the LGM to Pleistocene Holocene transition do not statistically resolve a trend (p=0.95), but show distinctly higher δ 13 C values for A -7.0 Ovis δ 13 C (, V-PDB) B δ 13 C (, V-PDB) Occlusal Surface δ 13 C Bison δ 13 C Time δ 18 O Cervical Margin δ 18 C (, V-SMOW) δ 18 C (, V-SMOW) Occlusal Surface Time Cervical Margin Distance (mm) Fig. 5. Zoning profiles measured along (A) Bison and (B) Ovis teeth, showing strong and weak zoning in δ 18 O and δ 13 C respectively. Note general correspondence between maximum δ 18 O and minimum δ 13 C values. 2σ error bars represent within-run variations on standards.

7 48 M.J. Kohn, M.P. McKay / Palaeogeography, Palaeoclimatology, Palaeoecology (2012) δ 18 O (, V-SMOW) Antilocapra Miracinonyx Cervid Bootherium Bootherium Bos Cervid Antilocapra Miracinonyx (+ 2 ) δ 13 C (, V-PDB) Fig. 6. δ 18 O vs. δ 13 C from individual zoned teeth, showing common negative correlations (cf. Fig. 5); p-values are (Bootherium), (cervid), (Antilocapra), (Miracinonyx) and (Bos). Lines for Bos, Miracinonyx, cervid and Bootherium illustrate general consistency among different taxa; line for Antilocapra illustrates different slope. 2σ error bars represent within-run variations on standards. δ 13 C values for Miracinonyx have been increased by 2 to account for systematic offsets between carnivores and herbivores. the Sangamon interglacial (pb0.001) relative to other Pleistocene compositions, comparable to Holocene Bison and Bos. Secular changes for Ovis, C. lupus, and Antilocapra show no consistent major shifts from the LGM to present (p>0.05). Statistically, a carbon isotope difference occurs between Sangamon vs. LGM and post-lgm Ovis (pb0.02), but because the (essentially homogeneous) Sangamon tooth composition falls within the range of other Pleistocene teeth, we do not view the difference as significant. Secular changes in δ 18 O values have been discussed previously (Kohn and McKay, 2010), and for water dependent taxa (Equus, Bootherium, Mammuthus, Bison/Bos) show an average 1.8±1.6 (2 s.e.) increase from the Bos LGM to the Holocene (Fig. 8). Ovis and Antilocapra compositions do not independently confirm this trend (p 0.10) but are consistent with it (Fig. 8). C. lupus shows no secular change to δ 18 O values (p>0.8), although data are restricted to the Pleistocene. 5. Interpretations 5.1. Herbivore isotope compositions: water compositions and vegetation Applying modern empirical calibrations for Equus, Bovinae, and elephants to data for Equus, Bison, Bootherium, and Mammuthus, Kohn and McKay (2010) inferred water δ 18 O values at NTC of ~ 16 during the LGM and ~ 14 today; the latter value is consistent with regional compilations of precipitation and local water δ 18 O values (Coplen and Kendall, 2000; Friedman, 2000; Dutton et al., 2005). Using the empirically-derived compositions for NTC, generalized theoretical models for herbivores (Kohn, 1996; Table 3) are grossly accurate, predicting LGM herbivore tooth enamel δ 18 O values of c. 17 vs for average herbivore compositions there (Fig. 3). Using either empirical calibrations or inverting theoretical models, water compositions at LBEC are estimated to have been ~ 12 during the post-lgm Pleistocene. We do not know which specimens from LBEC are Holocene, so we cannot independently estimate water δ 18 Ovaluesatthattime. During the LGM, all δ 13 C values fall below the cutoff for C3 consumption (Fig. 7), so in principle no CAM, C4, or isotopically unusual C3 plants (e.g. conifers) are required to explain compositions. High δ 13 C values do imply relatively arid conditions, which we discuss further in Section 5.5. Browsers (Odocoileus, Bootherium, and possibly Antilocapra), exhibit lower δ 13 C values than grazers and mixed feeders (Bison, Camelops, Mammuthus and possibly Ovis; pb0.001), as expected because more open habitats tend towards 13 C-enrichment (e.g. see Koch et al., 1998; Kohn et al., 2005). Although low p CO2 during the LGM could stabilize or maintain C4 biomass relative to C3 (Collatz et al., 1998; Koch et al., 2004), lower temperatures offset the C4 advantage (e.g. Ward et al., 2008). Also, C4 grass growth depends on availability of summer moisture. All general circulation models for the LGM predict lower temperatures at NTC, and many predict drier summers (e.g., Hoffmann et al., 2000; Shin et al., 2003; Braconnot et al., 2007), essentially destabilizing C4 grasses at that time; implications for GCM accuracies and C4 biomass predictions are discussed further in Section C (, V-PDB) Equus Bison, Bos Ovis Canis lupus Antilocapra Sangamon Interglacial May include non-c3 plants (artiodactyl) Bison trajectory TR Non-bison trajectory LGM post-lgm Holocene Age (ka) Fig. 7. δ 13 C vs. time for Natural Trap Cave. δ 13 C values of hypergrazer (Bison) increases in warmer climates, probably reflecting increase in percentage of C4 grass in local ecosystem. Equus show an analogous decrease from 110 ka to LGM. Browser (Antilocapra), mixed feeder (Ovis), and carnivore (C. lupus) show no significant change. Error bars represent 2σ age uncertainties, and 2σ variation in compositions. Dashed line shows upper limit of δ 13 C values for C3 plants (Kohn, 2010), as adjusted for the δ 13 C value of atmospheric CO 2 (Smith et al., 1999). HG H Bos HM δ 18 O (, V-SMOW) Equus Bison, Bso Ovis Canis lupus Antilocapra Sangamon Interglacial ? Water-dependent herbivore trajectory LGM post-lgm Holocene Age (ka) Fig. 8. δ 18 O vs. time for Natural Trap Cave. δ 18 O values for water-dependent herbivores increase from Last Glacial Maximum to Holocene (see also Kohn and McKay, 2010); other herbivore compositions are consistent with this trend, but carnivore data are not. The δ 18 O value from Sangamonian Equus is anomalously low. HM Bos

8 M.J. Kohn, M.P. McKay / Palaeogeography, Palaeoclimatology, Palaeoecology (2012) During the latest Pleistocene, Bison and Ovis δ 13 C values exceed the C3 cutoff (slightly), as does Bison during the Holocene, and Equus during the Sangamonian. In fact, δ 13 C values for Bison increase systematically by 2 to 7 from the LGM to the Holocene. Considering that Bison is a hypergrazer, this increase likely reflects an increasing proportion of C4 grass in diets and the overall environment. That is, C4 abundances could well have been 0% during the LGM, and increased in the Holocene. Insofar as we can determine, this is the first documentation of increasing C4 abundances across the Pleistocene Holocene transition from carbon isotopes in tooth enamel from North America. The compositions of all herbivores except Bison and Equus, however, are consistent with an exclusively C3 diet, especially when considering seasonal shifts and isotope biases discussed in Section Carnivore isotope compositions: evaluation of isotopic models Theoretical models for carnivores (Kohn, 1996; Table 3) predict similar or slightly lower δ 18 O values relative to prey. Thus, carnivore values of ~22 at NTC, and oxygen isotopic differences between carnivores and herbivores (Δ 18 O carn herb ) of 4 to 5 are much greater than anticipated. Some parameters in the carnivore models are either adjustable for a specific physiology, such as daily water intake and urine output, or constrained by few data, such as the oxygen isotope fractionation between protein and water. Consequently we explore the range of carnivore compositions that such models can explain, and their implications for carnivore diet and physiology. Most intake oxygen is derived from liquid water, both in food and as drinking water (Luz and Kolodny, 1985). Large herbivore tooth compositions imply body water compositions of c. 8 (slightly higher than predicted local leaf water compositions), and drinking water compositions of 15 to 16 (Kohn and McKay, 2010). Major changes to carnivore drinking water intake (low WEI model), food δ 18 O values (high δ 18 O H 2 O and protein models), and high vapor output all increase Δ 18 O carn herb by only 0.3 to 1.4 (Table 3). Thus, error in a single variable cannot explain the model-data discrepancy. Taking extremes in all parameters (maximum carnivore model) yields Δ 18 O carn herb ~2.5. While this result is still below observed values, it does illustrate that average carnivores may have physiologies and diets far different than assumed by Kohn (1996). Most importantly, carnivores in Wyoming may minimize liquid water output and consume prey items much more 18 O enriched than currently modeled. Clearly, more research on carnivore oxygen isotope compositions, possibly in controlled experiments, is needed. The magnitude of Δ 13 C carn herb is slightly larger than anticipated. Most work on bone suggests a value of 1 to 1.5 (Clementz et al., 2009), whereas we observe tooth enamel differences of 2 to 2.5 at NTC. Previous studies might have underestimated Δ 13 C carn herb, but several other explanations are possible including: (1) Large herbivores may not be representative of carnivore diets (Clementz et al., 2009), and instead more common prey may have had lower δ 13 C values. This view receives some support from the isotopic difference between small and large carnivores at LBEC, although large canids and felids typically prey on large herbivores. (2) Carnivores preferentially consume fat-rich portions of prey, and fats have lower δ 13 C values than other components (e.g., DeNiro and Epstein, 1977, 1978; Hilderbrand et al., 1996). (3) Carnivore teeth mineralize rapidly post-weaning, so may be seasonally biased towards summer and autumn compositions when herbivore δ 13 C values are lowest. Again, experimental studies of carnivores may help address these discrepancies between expected and observed isotopic fractionations. The δ 13 C values of C. lupus appear to remain invariant through time, but we have no modern or Sangamonian data from the region with which to compare glacial and interglacial compositions. Arguably, the higher δ 13 C values of C. lupus and Felis at LBEC could indicate an increase in C4 biomass into the Holocene, but the absence of direct dating of carnivores precludes definitive interpretation Extreme isotopic compositions Whereas generalized models can explain many compositions, we offer two main explanations for the extreme compositions observed for several taxa omnivory and climate-induced changes to diet. First, ursids and small canids are omnivorous, so the diets and compositions of Vulpes, C. latrans, Ursus and Arctodus may have experienced greater seasonal change than in other animals. If so, each tooth might reflect only one small window within the total isotopic range sampled by these opportunistic animals. Thus, the >7 carbon isotope difference in the single Ursus tooth at NTC vs. LBEC, and the nearly 5 difference between Ursus and Arctodus compositions at NTC might simply reflect the range of δ 13 C values that these animals sampled seasonally. This view is supported by minimal isotopic zoning (b1 ) recorded in many teeth. Alternatively, different isotopic values might reflect individual selectivity, so that some animals systematically record different compositions than other individuals of the same species, even in the same area and time. This view is supported by the NTC Ursus tooth, which exhibits nearly 4 isotopic variation in δ 18 O values, but only 0.5 variation in δ 13 C values. A second explanation for isotopic extremes notes that many of these compositions at NTC occur for Holocene specimens. Possibly Pleistocene Holocene climate change caused divergence in isotope compositions of different taxa, either because the environment diversified isotopically, so the same diet had a different composition, or individual taxa preferentially shifted their diets to isotopically extreme foods. For example, Odocoileus carbon isotope compositions in the southeastern US shifted downwards from the Pleistocene to Holocene, probably reflecting Holocene development of denser forests (Koch et al., 1998; Kohn et al., 2005). These possibilities might be distinguished with a larger modern dataset that includes a range of herbivores and carnivores similar to those we analyzed from late Pleistocene NTC. We also note that few isotopic data exist for some ecosystem components, such as arthropods, which are consumed by omnivores. It appears that the oxygen isotope composition of insect chitin and cellulose are broadly similar (Motz, 2000), but the fractionation between water and chitin is only ~20 (Wang et al., 2009; Nielson and Bowen, 2010), whereas the water-cellulose fractionation is ~27 (Sternberg, 1989). Thus insect body water may have a much higher δ 18 O value than other water sources, including plants. Data from one study suggest that bumblebees are ~10 enriched in 18 O over local water in southern England (Wolf et al., 1996; Darling and Talbot, 2003; Darling et al., 2003), whereas leaf water in the same environment is probably only 5 7 enriched (e.g. Table 3). An experimental study of vapor uptake in cockroaches (Ellwood et al., 2011) indicates they can be over 10 enriched in 18 O relative to water in dry environments. Although requiring further study, possibly insectivory leads to 18 O enriched compositions, and can help explain unusually high δ 18 O values for Vulpes and C. latrans, either directly or by consumption of insectivorous rodents Intratooth zoning The 2 3 range in δ 18 O values along the length of herbivore teeth (Fig. 5) likely reflects seasonal changes to local water and plant compositions, with high and low values in the summer and winter respectively (Bryant et al., 1996; Fricke and O'Neil, 1996; Kohn, 1996; Kohn et al., 1998). The isotopic seasonality of modern precipitation in the western United States is commonly ~10 (Henderson and Shuman, 2009), so the measured range in teeth requires some degree of damping, either within soils, streams or lakes in the environment (Coplen and Kendall, 2000; Henderson and Shuman, 2009), within the animal

9 50 M.J. Kohn, M.P. McKay / Palaeogeography, Palaeoclimatology, Palaeoecology (2012) (Kohn et al., 2002), or during the formation and maturation of tooth enamel (Passey and Cerling, 2002). Carbon isotope variations in herbivore teeth are considerably smaller, with a common range b1 (Figs. 5 6). Because the δ 18 O maxima and minima recorded in teeth suggest that both summer and winter compositions are recorded, the small variation in δ 13 C values suggests that large herbivores did not substantially vary their diets seasonally, possibly in contrast to omnivores. Some published data also illustrate seasonal inverse correlations between 18 O and 13 C in large mammals (e.g., Bocherens et al., 2001; Wang et al., 2008; Feranec et al., 2009; Biasatti et al., 2010, 2012; Zin-Maung- Maung-Thein et al., 2011). Inverse correlations might result from a combination of factors related to the seasonality of plant growth and availability of different foods. Possible explanations for inverse 18 O 13 C correlations include the following: (1) Monsoonal circulation. The standard interpretation of inverse 18 O 13 C correlations in southeast Asia is that intense, 18 O-depleted, summer monsoonal precipitation stabilizes 13 C- enriched C4 grasses (Wang et al., 2008; Biasatti et al., 2010, 2012). Although this process explains Asian data well, there is no evidence for C4 grasses in Wyoming during the LGM, when similar negative correlations also occur in our data, and summer precipitation in Wyoming is 18 O-enriched, not depleted. This explanation does not seem viable for our study area. (2) Moisture. Plant δ 13 C values decrease with increasing moisture availability (Farquhar et al., 1989; Kohn, 2010). If the summer growing season were relatively wet, then high δ 18 O summer precipitation values would correspond with low δ 13 C plant values relative to other seasons. (3) Seasonal dietary selection of same plants. New plant growth does not generally occur during the winter, so herbivores must rely on other food sources. Different tissues of the same plant have different compositions, and leaf litter, bark, and twigs are enriched in 13 C relative to fresh leaves (Dawson et al., 2002). Reliance on different tissues of the same plant during the winter could increase δ 13 C values of herbivores. (4) Seasonal dietary shift to conifers. Conifers have δ 13 C values ~2 higher than deciduous angiosperms (Diefendorf et al., 2010), so a winter dietary switch to conifers could increase herbivore δ 13 C values. (5) Winter fat burning. Herbivores commonly accumulate fat reserves in the summer and autumn to guard against scarce winter food supplies. Lipids are 2 4 depleted in 13 C relative to coexisting proteinaceous tissues (e.g., DeNiro and Epstein, 1978; Hilderbrand et al., 1996). Because proteins are 5 enriched relative to total diet (e.g. Ambrose and DeNiro, 1986; Passey et al., 2005; Clementz et al., 2009), however, herbivore lipid reserves should be at least 2 enriched relative to summer and autumn plant sources. Thus, preferential use of fat reserves in the winter could increase δ 13 C values. There are too few data from carnivores to draw firm conclusions regarding seasonal isotopic variations. An inverse correlation between δ 18 O and δ 13 C values, as suggested by one Miracinonyx tooth, could simply indicate that carnivore compositions track herbivores. That is, as herbivore δ 13 C values increase in the winter and decrease in the summer, so too do carnivore values. Alternatively and analogously to herbivores, if lipid δ 13 C values in carnivores are higher than in original prey, then winter use of fat reserves could cause an increase in δ 13 C and an inverse δ 18 O δ 13 C correlation Critical evaluation of GCM's, 1. MAP and precipitation δ 18 O If we can identify exclusive C3 consumers, then their isotope compositions can be used to estimate MAP (Kohn, 2010). In the case of NTC, we can consider either LGM compositions only, when no taxon exceeds the C3 cutoff, or taxa other than Bison, Sangamonian Equus, and possibly Ovis. Either approach yields average δ 13 Cvalues of ~ 10.0±0.5, which is equivalent to modern C3 plant values of ~ 25.5 after accounting for isotopic fractionations and Pleistocene vs modern δ 13 C atm. At an elevation of 1500 m and latitude of 45 N, this value implies MAP~150±200 mm/yr (Fig. 2); approximately threequarters of the error results from the MAP calibration, which in turn reflects scatter in modern data. The remainder of the error corresponds to the assumed uncertainty in mean δ 13 C of ±0.5. Average MAP today near NTC is ~200 mm/yr (Kohn and McKay, 2010), so within uncertainty we can resolve no change to MAP between the late Pleistocene and today. Even considering uncertainties, our data are consistent with a relatively dry late Pleistocene, with MAP 350 mm/yr. This result is consistent with models of vegetation structure over North America during the LGM (e.g., Cowling, 1999), which suggest much more open habitats at NTC and LBEC than occurring today. Oxygen isotope compositions for the more water-dependent taxa (Bison/Bos and Equus, Fig. 8) systematically increase from the LGM to the present. The same trend is observed for Bootherium and Mammuthus (Kohn and McKay, 2010), and possibly also less waterdependent Ovis and Antilocapra (Fig. 8). As discussed previously (Kohn and McKay, 2010), this trend is interpreted to reflect changes to atmospheric circulation and the δ 18 O value of local precipitation. In general, precipitation that is 18 O-depleted vs. -enriched derives from the Pacific during the winter vs. the Gulf of Mexico during the summer. Lower δ 18 O values during the LGM are consistent with GCM's that predict a decrease in summer precipitation and in annual precipitation δ 18 O overall (e.g., Hoffmann et al., 2000; Shin et al., 2003; Braconnot et al., 2007). A decrease in summer precipitation, however, would argue against wetter summers as the cause of inverse seasonal correlations between δ 18 O and δ 13 C in zoned teeth (see above). Sangamonian Equus compositions are surprisingly low, however, even below LGM values. Possibly, the single Equus specimen has non-representative δ 18 O values, although its δ 13 C value is consistent with systematic carbon isotopic trends. Alternatively, Sangamonian atmospheric circulation may have been different from Holocene circulation, with a greater proportion of Pacific moisture Critical evaluation of GCM's, 2. Shifts to C4 plant abundance Following Connin et al. (1998) and Koch et al. (2004), we further explore using modern correlations of C4 abundance with climate variables (Paruelo and Lauenroth, 1996) to evaluate the consistency of GCM's with observed increases and decreases in C4 biomass. Climate variables required for predictions include mean annual temperature (MAT) and precipitation (MAP) as well as the ratio of June July August (summer) precipitation to MAP (JJA/MAP). Unlike previous studies, we quantitatively consider theoretical models of C3 vs. C4 competition (Collatz et al., 1998) to correct for changes in p CO2. Essentially, we assume that the decrease in C3 C4 cross-over temperature associated with decreased p CO2 offsets the GCM-predicted decrease in mean annual temperature: X C4 ¼ 0:9837 þ 0:000594ðMAP; mmþþ1:3528ðjja=mapþ þ 0:2710 lnðmat ΔT X Þ: ð3þ where X C4 is the fraction C4 biomass and ΔT X is the change in cross-over temperature associated with a change to p CO2 (T X is negative for decreased p CO2 ). X C4 is calculated both for modern conditions and the LGM; because absolute C4 biomass is difficult to evaluate from proxy data, changes to X C4 are evaluated qualitatively. Using NTC as an example, modern conditions of MAT=7.1 C, MAP=211 mm/yr, and JJA/MAP=0.31 predict 9% C4 biomass, consistent with regional trends (Paruelo and Lauenroth, 1996). The PMIP2 ensemble averages predict lower MAP (c. 100 mm/yr), no change to JJA/MAP, and a C decrease in MAT. However, relative to a modern reference p CO2