DOI: 10.6071/m3tt2j
title: Pronghorn (Antilocapra americana) enamel phosphate δ18O values reflect climate seasonality: implications for paleoclimate reconstruction

description: Stable oxygen isotope compositions from vertebrate tooth enamel are
 commonly used as biogeochemical proxies for paleoclimate reconstructions.
 However, the utility of enamel isotopic values across species varies due
 to differences in rates of enamel deposition and mineralization as well as
 sources of ingested water, body water residence times, and species’
 physiology. We evaluate the use of stable oxygen isotope compositions from
 pronghorn (Antilocapra americana Gray, 1866) enamel for the amplitude
 reconstruction of terrestrial paleoclimate seasonality. We serially
 sampled the third lower molars of pronghorn from Wyoming for oxygen
 isotope composition in phosphate (δ18OPO4) and compared patterns to: (1)
 interpolated and (2) measured yearly variation in central Wyoming
 environmental waters (δ18Ow) as well as to (3) δ18O values from sagebrush
 leaves and stems in the same region. Although we recognize the numerous
 factors influencing the composition of mammal body water, our null
 hypothesis was that δ18OPO4 enamel values reflect δ18Ow with a constant
 offset due to mammalian physiology. We set up our null hypothesis by
 converting δ18Ow values to δ18OPO4 using a published regression based on
 empirical results from mammals. Pronghorn δ18OPO4 values from enamel are
 consistently enriched in 18O relative to the predicted values. We
 hypothesized that pronghorn δ18OPO4 values might also reflect dietary
 water and therefore also converted δ18Oleaf values from plants into
 predicted δ18OPO4 values. We infer that pronghorn obtain at least some of
 their water from 18O-enriched plants because pronghorn enamel δ18OPO4
 values are more similar to predicted δ18OPO4 values from plants than from
 meteoric waters. Modeling of source body water δ18O values show amplitudes
 between 70% and 95% of seasonal variation from Wyoming δ18Ow values.
 Collectively, our findings establish that modern seasonality in source
 water is reliably reflected in modern pronghorn enamel providing the basis
 for exploring changes in the amplitude of seasonality of ancient climates
 using archived tooth collections.
description: Modern and archaeological pronghorn specimens were acquired from
 the University of Wyoming Anthropology museum and Department of
 Archaeology (Appendix I). All specimens were collected from wild
 populations in Wyoming during 1970-1972 and 2010 following deaths that
 were not related to this study. The archaeological specimens date to 1720
 ± 100 years (Frison 1971), thus pre-dating the rapid climate change
 typical of the late 20<sup>th</sup> century (Mann et al. 1998,
 Jones et al. 2001). The lower third molar (m3) is one of the last to
 complete enamel mineralization and erupt in pronghorn (Dow and Wright
 1962), therefore, we included only individuals with erupted third lower
 molars. To recover the most complete isotopic time series, we included
 only individuals showing no or little wear of the m3. We also excluded
 individuals with abscesses or obvious abnormalities of the dentition or
 jaw bone. We extracted lower third molars using a Dremel diamond cutting
 wheel and serially sampled the enamel at ~2 mm intervals using a Dremel
 tool with a diamond taper point bit (part #7144). We collected 2-3 mg of
 powdered enamel for each serial sample. Further, we took bulk samples
 (~4-6 mg) of bone from the mandibular angle just posterior to third lower
 molar for each individual. To analyze the oxygen
 isotope composition of phosphate
 (δ<sup>18</sup>O<sub>PO4</sub>), we weighed
 1.5-2.0 mg of enamel and 3-4 mg of bone from each specimen. Preparation
 procedures for the modern specimens are from a combined approach based on
 Bassett et al. (2007) and Weidemann-Bidlack et al. (2008). We pre-treated
 all samples with 300 μl of 2.5% NaOCl for approximately 20 hours to remove
 organics. Bone samples were usually pre-treated twice to ensure complete
 organic removal (or more if there was continued gas production). Samples
 were then rinsed with deionized (DI) water 5 times and dried overnight at
 50˚C. We then dissolved the remaining powder in 100μl of 0.5M
 HNO<sub>3</sub> overnight. To neutralize the solution and
 precipitate CaF<sub>2</sub>, 75μl of 0.5 M KOH and 200μl of
 0.36 M KF were added. Samples were centrifuged to pellet the
 CaF<sub>2 </sub>and the supernatant was transferred from the
 vials to reaction vessels. We precipitated silver phosphate with 250µl of
 silver amine solution (0.2M AgNO<sub>3</sub>, 0.35M
 NH<sub>4</sub>NO<sub>3</sub>, 0.74M
 NH<sub>4</sub>OH) plus 3-6 drops of 0.1M
 AgNO<sub>3</sub> to initiate the precipitation. Samples were
 placed in a heat block at 50˚C overnight in a fume hood to allow for
 maximum crystal growth. The silver phosphate crystals were rinsed five
 times with ~2 mL of DI water to remove residual silver amine solution.
 After the samples dried overnight at 50˚C, 200 – 300 μg were weighed into
 pressed silver capsules and stored in an oven flushed with
 N<sub>2</sub> until isotopic analysis. Preparation of
 historical specimens used a rapid silver phosphate technique from Mine et
 al. (2018), which demonstrated d<sup>18</sup>O value fidelity
 to the slow precipitation method in Weidemann-Bidlack et al. (2008).
 Briefly, the historical samples were similarly treated for organic
 removal, but hydroxyapatite was dissolved in 50 mL 2M
 HNO<sub>3</sub> while CaF<sub>2</sub> precipitated
 with 30 mL 2.9M HF and neutralized with 50mL of 2M NaOH. Steps to pellet
 and isolate CaF<sub>2</sub> were similar between methods. To
 precipitate silver phosphate, we added 180 mL silver amine solution (0.37M
 AgNO<sub>3</sub> and 1.09M NH<sub>4</sub>OH) and
 adjusted pH to 5.5-6.5 using dilute HNO<sub>3</sub>, which was
 shown to maximize phosphate recovery (Mine et al., 2018). The silver
 phosphate crystals were rinsed and dried similar to the modern samples as
 outlined above. The δ<sup>18</sup>O value
 of silver phosphate [SK1] [DF2] was measured after conversion to CO in a
 Temperature Conversion Elemental Analyzer (TC/EA, Thermo Scientific)
 coupled with a Conflo IV (Thermo Scientific) to a continuous flow isotope
 ratio mass spectrometer (CF-IRMS, Thermo Scientific Delta V). Three
 in-house reference materials (two silver phosphate and one benzoic acid)
 were used to normalize isotopic values and check the effectiveness of
 pyrolysis within and between runs (modern samples at UWSIF: ARCOS [N=4 per
 run], UWSIF21 [N=5 per run], UWSIF33[SK3] [DF4]  [N=6
 per run]; historical samples at SIELO UCM: USGS80 [N=8], USGS 81 [N=8],
 IAEA601 [N=8]). Variation in d<sup>18</sup>O values exhibited
 by these reference materials was &lt; 0.4‰. In addition, we monitored
 the potential for isotopic alteration during sample preparation by
 precipitating silver phosphate from a synthetic hydroxyapatite and NIST
 120c (N=3 and 3 with 1s &lt; 0.3‰). All samples were analyzed in
 triplicate. All d<sup>18</sup>O<sub>PO4</sub>
 values are reported relative to the standard V-SMOW.
 The spacing between enamel carbonate and phosphate
 δ<sup>18</sup>O values is often used as a check for diagenesis
 (Koch et al. 1997) To check the carbonate-phosphate isotopic spacing, we
 also analyzed a subset of enamel samples for carbonate
 d<sup>18</sup>O values as per Koch et al. (1997) and Kohn and
 Cerling (2002). We weighed 1 mg of enamel and 5 mg of bone for each sample
 analysis of δ<sup>18</sup>O<sub>CO3</sub> values.
 To remove organic matter from the bone samples, we used 2-3%
 H<sub>2</sub>O<sub>2</sub> at a ratio of 1 ml per
 25mg of sample, leaving the caps of the microcentrifuge tubes open to
 allow the escape of gas for 24 hours. We did not pre-treat the enamel
 samples to remove organic matter due to the minimal organic content of
 enamel. Only the bone was pre-treated for carbonate analysis. Similar to
 the phosphate preparation, pre-treatment was repeated until gas production
 ceased. We rinsed the bone samples 5 times with DI water to remove
 H<sub>2</sub>O<sub>2</sub> from solution. We then
 added 1M CH<sub>3</sub>COOH with Ca acetate buffer (pH=4.5) to
 remove non-lattice bound carbonates using the same ratio as the preceding
 step. Samples were rinsed five times with DI water, dried for 24 hours in
 a freeze drier, and ~1 mg weighed into exetainer tubes for isotopic
 analysis. Once all samples and reference materials were weighed into
 exetainer tubes, they were dried overnight at 50°C, the headspace flushed
 with He, and 100-200 mL of  &gt;100%
 H<sub>3</sub>PO<sub>4</sub> was added to react for
 24 hours (room temperature). The CO<sub>2</sub> within the
 headspace was sampled for isotopic composition measurement using a gas
 bench (Thermo Scientific) coupled to a CF-IRMS (Thermo Scientific Delta
 Plus) at UWSIF. Three in-house carbonate reference materials were used to
 normalize isotopic values and check for variation within and between runs
 (UWSIF18 [N=4 per run], UWSIF06 [N=4 per run], UWSIF17 [N=4 per
 run]).Variation in d<sup>18</sup>O<sub>CO3</sub>
 values of these reference materials was &lt; 0.2‰. In addition, we
 monitored the potential isotopic alteration during sample preparation with
 one lab bioapatite (MSW0479, ashed manatee bone, N = 1 per run,
 d<sup>18</sup>O<sub>CO3</sub> = 19.0‰) and
 variation in d<sup>18</sup>O<sub>CO3</sub> values
 of a phosporite reference standard (NIST 120c, N = 5,
 d<sup>18</sup>O<sub>CO3</sub> = 28.4 ± 0.3‰).  All
 d<sup>18</sup>O<sub>CO3</sub> values are reported
 relative to the standard V-SMOW. <i>Recovering
 primary isotope time series</i> Modelling is the
 only means by which the primary isotopic time series can be recovered
 (Green et al. 2018), even for mammal taxa with relatively fast rates of
 enamel mineralization and low rates of isotopic overprinting. To recover
 the primary isotopic input signals from our pronghorn tooth isotopic time
 series, we used the mathematical model from Passey and Cerling (2002) and
 Passey <i>et al</i>. (2005) as a transfer function. The Passey
 and Cerling (2002) model takes primary isotope series using measured
 d<sup>18</sup>O values from body water and reconstructs tooth
 enamel d<sup>18</sup>O values, while also accounting for time
 averaging due to amelogenesis and variation in enamel maturation from the
 crown to the root. The Passey <i>et al.</i> (2005) method
 incorporates the Passey and Cerling (2002) time-averaging model and uses
 an inverse linear system to recover the input signals from enamel isotopic
 time series (see Matlab code associated with Passey <i>et
 al</i>., 2005). The model terms for the Passey and Cerling (2002)
 and Passey <i>et al.</i> (2005) models are
 1<sub>a</sub> and 1<sub>m</sub>, which are the
 length of apposition (distance along the tooth from where a new enamel
 layer contacts the enamel-dentine junction and the external layer of the
 tooth; 7 mm) and length of maturation (the length of the tooth that is
 mineralizing at a given time; 13.3 mm assuming an enamel maturation time
 of one month as in small bovids such as sheep; Zazzo et al. 2010),
 respectively. The l<sub>a</sub> parameter was estimated using
 values reported for sheep, which range from 2 mm to 12 mm, depending on
 whether l<sub>a</sub> was measured on the buccal, mesial, or
 lingual side of the tooth and crown height (Zazzo et al. 2012). We used
 the midpoint of these values (7 mm) as a conservative estimate of
 l<sub>a</sub> in pronghorn. We used a constant sample depth
 (l<sub>s</sub>) of 75% of enamel thickness because we drilled
 through approximately 75% of the enamel when serial sampling. The Passey
 and Cerling (2002) and Passey <i>et al.</i> (2005) models were
 run in R version 3.5.1 (Appendix I). To compare
 pronghorn enamel δ<sup>18</sup>O<sub>PO4</sub>
 values to seasonality of environmental waters, we downloaded interpolated
 monthly average isotope values for precipitation from central Wyoming
 (43˚N, 107.5 ˚W at 6,700
 ft of elevation) from waterisotopes.org (Bowen et al. 2005,
 Bowen 2014). These interpolations for the entire United States are based
 in large part on Welker’s USNIP (United States Network for Isotopes in
 Precipitation; Welker 2000, 2012) for the years 1989-1994 and scarce IAEA
 GNIP (Global Network for Isotopes in Precipitation) data from a few years
 in the 1960’s for 6 sites across the United States (Rozanski et al. 1993).
 Environmental water δ<sup>18</sup>O<sub>w</sub>
 values were converted into predicted phosphate values using the following
 equation from Kohn and Cerling (2002), which is based on empirical data
 from mammalian enamel:
 δ<sup>18</sup>O<sub>PO4</sub> = (0.9 x
 δ<sup>18</sup>O<sub>w</sub>) + 23  (2)
 We use equation 2 as a means of setting up our hypothesis, that
 pronghorn δ<sup>18</sup>O<sub>PO4</sub> values
 reflect only δ<sup>18</sup>O<sub>w</sub> and
 physiological offsets. Given that equation 2 was originally derived for
 evaporation insensitive species (i.e., those that rely primarily on
 meteoric waters), underestimation relative to measured
 δ<sup>18</sup>O values can reveal whether a focal species is
 evaporation sensitive. To validate the interpolated
 δ<sup>18</sup>O<sub>w</sub> values we used 1000+
 measured δ<sup>18</sup>O values from precipitation at 9 sites
 in Wyoming (Appendix II) from USNIP for the entire USNIP record 1989-2006
 (Welker 2000, Vachon et al. 2010b, Welker 2012). The high density modern
 precipitation network (USNIP) provides the only site, sub-state, regional
 and continental record of actual meteoric water values that are becoming
 increasing valuable in revealing the range of seasonality in modern
 precipitation (Vachon et al. 2007, Vachon et al. 2010b, a). We made
 comparisons between the interpolated
 δ<sup>18</sup>O<sub>w</sub> values and measured
 values using monthly averages across all sample sites and at the site
 closest to the Laramie and Rawlins region (Albany site), the site closest
 to the area where our modern pronghorn teeth were collected. To evaluate
 the relative contributions of meteoric and river waters, we also
 extrapolated maximum and minimum δ<sup>18</sup>O values from
 river waters in Wyoming (Kendall and Coplen 2001). As with the meteoric
 waters, we converted river water δ<sup>18</sup>O values to
 enamel phosphate values. We also obtained published
 δ<sup>18</sup>O values for water in sagebrush leaves and
 stems, rabbitbrush leaves, and pronghorn incisor enamel from Fenner and
 Frost (2008) for comparison to
 δ<sup>18</sup>O<sub>PO4</sub> values from
 pronghorn molar enamel (this study) as well as
 δ<sup>18</sup>O<sub>w</sub> values (from
 interpolated and measured meteoric precipitation). All plant tissues were
 sampled by Fenner and Frost (2008) during the months of June and July; we
 converted these δ<sup>18</sup>O values into phosphate enamel
 values using equation 2. This conversion of plant leaf water values to
 enamel phosphate values allowed us to set up our alternative hypothesis:
 pronghorn δ<sup>18</sup>O<sub>PO4</sub> values
 from enamel reflect a combination of environmental water, evaporated leaf
 water, and physiological mechanisms. Similarity of pronghorn
 δ<sup>18</sup>O<sub>PO4 </sub>values to
 δ<sup>18</sup>O<sub>w</sub> from precipitation,
 lakes, and rivers or
 δ<sup>18</sup>O<sub>leaf</sub> values should
 provide information on their relative inputs.
description: Appendix I. Raw δ<sup>18</sup>O data from serial
 samples of modern and archaeological pronghorn from Fraser et al. (2021)
 Pronghorn (Antilocapra Americana) enamel
 phosphate δ<sup>18</sup>O values reflect climate seasonality:
 implications for paleoclimate reconstruction. Ecology and Evolution, In
 Press. [Not sure if 2021 will be the correct year or if you want to put
 the entire citation here] Appendix II. R translation of
 the function Emeas1_1 from Passey et al. (2005) from Fraser et al. (2021)
 Pronghorn (Antilocapra Americana) enamel
 phosphate δ<sup>18</sup>O values reflect climate seasonality:
 implications for paleoclimate reconstruction. Ecology and Evolution, In
 Press.. Appendix III. R translation of the function
 mSolv1_1 from Passey et al. (2005) from Fraser et al. (2021) Pronghorn
 (Antilocapra Americana) enamel phosphate δ<sup>18</sup>O
 values reflect climate seasonality: implications for paleoclimate
 reconstruction. Ecology and Evolution, In Press..
 Appendix IV. Running the R translations of Emeas1_1 and mSolv1_1
 with example data from Passey et al. (2005) from Fraser et al. (2021)
 Pronghorn (Antilocapra Americana) enamel
 phosphate δ<sup>18</sup>O values reflect climate seasonality:
 implications for paleoclimate reconstruction. Ecology and Evolution, In
 Press.. Appendix V. Summary data of Wyoming sites for
 which USNIP data were extracted from Fraser et al. (2021) Pronghorn
 (Antilocapra Americana) enamel phosphate δ<sup>18</sup>O
 values reflect climate seasonality: implications for paleoclimate
 reconstruction. Ecology and Evolution, In Press..