Reconstructing diagenetic conditions of bone at the Gray Fossil Site, Tennessee, USA

Accepted Manuscript Reconstructing diagenetic conditions of bone at the Gray Fossil Site, Tennessee, USA

Sarah W. Keenan, Annette Summers Engel PII: DOI: Reference:

S0031-0182(17)30090-1 doi: 10.1016/j.palaeo.2017.01.037 PALAEO 8176

To appear in:

Palaeogeography, Palaeoclimatology, Palaeoecology

Received date: Revised date: Accepted date:

27 April 2016 11 January 2017 25 January 2017

Please cite this article as: Sarah W. Keenan, Annette Summers Engel , Reconstructing diagenetic conditions of bone at the Gray Fossil Site, Tennessee, USA. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Palaeo(2017), doi: 10.1016/j.palaeo.2017.01.037

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ACCEPTED MANUSCRIPT Reconstructing diagenetic conditions of bone at the Gray Fossil Site, Tennessee, USA Sarah W. Keenan a,b*, Annette Summers Engel a a

Department of Earth & Planetary Sciences, University of Tennessee, 1412 Circle Drive,

Current address: Department of Biosystems Engineering & Soil Sciences, University of

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b

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Knoxville, TN 37996, United States

Corresponding Author. Email address: [email protected] (S.W. Keenan)

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*

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Tennessee, 2506 E.J. Chapman Drive, Knoxville, TN 37996, United States

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Abstract

The preservation of vertebrate fossils requires the transformation of the original bioapatite

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mineral matrix, predominantly hydroxylapatite, into a thermodynamically more stable phase

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during diagenesis. Fossil bone geochemistry and structure provides a wealth of information on the conditions present during diagenesis, including site redox and pH, and about sedimentation

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rates during burial. The Gray Fossil Site preserves Late Neogene lacustrine deposits that serve as

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one of a few records of North American paleoclimate during a critical period of the Cenozoic. Despite having more than 40 vertebrate species and diverse plant assemblages, none of the prior

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studies used bone composition to understand what paleoenvironmental and diagenetic conditions must have been like to facilitate bone preservation and fossilization. Alligator sp. phalanges were examined using multiple geochemical tools, including Fourier-transform infrared spectroscopy, X-ray diffraction, and electron microprobe analyses. Evidence of bioerosion within thin cortical and isolated trabecular regions of bone suggests biotic utilization of organics (collagen) and/or the mineral(s) following deposition. Chemically zoned and laminated cortical Alligator bone is interpreted as preserved lines of arrested growth that developed during life, and supports 1

ACCEPTED MANUSCRIPT previous interpretations of a regional seasonal climate. Lastly, the Alligator bones are mineralogically heterogeneous as fluorinated and iron-containing apatite phases. Compared to modern bone, iron concentrations were high, at up to 3 weight percent. The uptake of Fe by the Alligator bones examined in this study suggests that fluids present during diagenesis that led to

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bone preservation within the lacustrine sediment at the Gray Fossil Site were likely acidic,

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anoxic, and reducing.

Keywords

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Diagenesis; fossilization; paleoenvironment; Neogene; fossil bone; geochemistry

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Highlights

Gray Fossil Site Alligator sp. bones are used to infer paleosinkhole geochemistry.

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Gray Fossil Site bones are Fe-enriched compared to modern bone.

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Bone diagenesis involved acidic, anoxic, and reducing fluids.

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Preserved lines of arrested growth in GFS alligator bone indicate seasonality.

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1.1 Introduction

Fossil bones have been recovered from sediments associated with rivers (e.g.,

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Behrensmeyer, 1988), lakes (e.g., Wallace and Wang, 2004), floodplains or wetlands (e.g., Keenan and Scannella, 2014), sinkholes (e.g., Laury, 1980), and even from speleothems in caves (Auler et al., 2006). The preservation of bones as fossils requires a suite of physical and geochemical conditions to facilitate the transformation of inorganic and organic phases in bone (i.e., bioapatite) into a mineralized form that is more thermodynamically stable in geochemically dynamic depositional conditions (Lee-Thorp, 2002; Goodwin et al., 2007; Kohn, 2008; Koenig et al., 2009; Kohn and Moses, 2012; Keenan, 2016). As such, the transformation of unaltered 2

ACCEPTED MANUSCRIPT bioapatite into fossil apatite mineral phases must be enhanced by solution pH and redox state, as well as by the ionic strength of fluids, sediment mineralogy, sedimentation rate, and mineral saturation states (Berna et al., 2004; Trueman, 2013). Reconstructing diagenetic conditions that affected bone in a past depositional setting is difficult, although some insight can be gained from

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studying the mineralogy and geochemical composition of the fossils themselves (Hubert et al.,

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1996; Goodwin et al., 2007; Kohn, 2008).

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The Gray Fossil Site (GFS) near Gray, Tennessee (USA), was accidentally discovered in 2000 during highway construction (Fig. 1) (Clark et al., 2005). To date, over 40 vertebrate

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species have been identified from disarticulated fossil bones and articulated skeletons preserved

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from the lacustrine deposits that accumulated in a large sinkhole at the GFS (Mead et al., 2012; Kohl, 2014). Diverse assemblages of plants (e.g., grape vines; Gong et al., 2010) and animals

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like the Red Panda (Wallace and Wang, 2004; Wang and Wallace, 2004), as well as the largest

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accumulation of articulated tapirs (Tapirus polkensis) yet discovered, are preserved at the site. The GFS records unrivaled biodiversity and environmental information about eastern North

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America during the Late Neogene (Late Miocene to Pliocene, ca. 7.0 – 4.5 Ma), as one of only

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two localities east of the Mississippi River known to preserve terrestrial flora and fauna from this time period. The second site, the Pipe Creek Sinkhole, is located in Grant County, Indiana

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(Farlow and Argast, 2006). The Neogene is characterized by the global transition from warmer to cooler temperatures, which could have led to seasonality in mid-latitudes, the spread and dominance of C4 vegetation across much of North America, and eventually extensive glaciations (Cerling et al., 1993; Cerling et al., 1997; Strömberg, 2005). The diagenetic conditions that facilitated preservation of the fossil bones at GFS have largely been unknown, mostly because the existing sedimentological and stratigraphic evidence

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ACCEPTED MANUSCRIPT collected to date (Shunk et al., 2006; Shunk et al., 2009a), as well as isotopic evidence (DeSantis et al., 2008), provides limited information about what geochemical conditions facilitated the transformation of hydroxylapatite into a more thermodynamically stable mineral. Based on our previous work (Keenan et al., 2015; Keenan and Engel, 2017), it is now possible to gain insight

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into early paleoenvironmental and diagenetic conditions from the fossil bone, such as the redox

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state and pH of the paleosinkhole bottom waters. We used Fourier transform infrared

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spectroscopy (FTIR), X-ray diffraction (XRD), and electron microprobe analysis (EMP) to evaluate Alligator sp. bone major element geochemistry and structural crystallinity. The GFS

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results were compared to fossil bone geochemistry from the Pipe Creek Sinkhole, a fossiliferous

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paleosinkhole deposit that spans the same geologic interval, in Grant County, Indiana (USA). Collectively, our results provide a new understanding about the diagenetic conditions that led to

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bone preservation at the GFS.

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1.2 GFS geology and formational history

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Karst features, including high densities of caves and sinkholes, dominate the geomorphology of the Valley and Ridge province of East Tennessee, both in the modern (Moore,

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2006) and geologic past (Zobaa et al., 2011). The region is underlain by Ordovician-aged Knox

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Group limestone and dolostone units (Clark et al., 2005) that follow northeast-to-southwest trending structural valleys flanked by ridges underlain by siliciclastics (Whitelaw et al., 2008). Relict weathering and paleokarst development is evident in some areas, including the GFS (e.g., Anthony and Granger, 2004). The single, large sinkhole at the GFS formed by the progressive collapse of underlying subsurface voids (i.e., caves) and at least 11 smaller sinkholes (Whitelaw et al., 2008). Based on stratigraphy and sedimentology evidence (Shunk et al., 2006; Shunk et al., 2009a) and high-resolution gravity surveys (Whitelaw et al., 2008), the GFS is approximately

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ACCEPTED MANUSCRIPT 150 m in diameter, with depths ranging from 11 to 42 m (Whitelaw et al., 2008). Angular limestone boulders that disrupt the sediment bedding likely represent collapse features, similar to those interpreted at the Pipe Creek Sinkhole, another paleokarst site (Shunk et al., 2009b). Freshwater lake or pond formation at GFS, due to limited drainage and a shallow groundwater

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table, led to differential sediment infilling but deposition of contemporaneous terrestrial, semi-

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aquatic, and aquatic biotic assemblages (Shunk et al., 2006; their Fig. 2). Water depth was likely

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shallow, and flow was slow or stagnant, which may have periodically led to dried up water bodies in some of the smaller sinkholes (Worobiec et al., 2013). Unlike the Mammoth Site in

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Hot Springs, South Dakota (USA), there is no evidence of animals being mired in the GFS

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paleosinkhole (Laury, 1980).

Gaps remain in our understanding of GFS infilling history, paleolake geochemistry, and

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the conditions that favored bone fossilization. Initial sedimentation interpretations by Shunk et

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al. (2006) were based on analyses of predominantly gray to brown clay at ca. 29 m depth that transitions up-section to laminated rhythmite facies consisting of organic-rich, and silty, dark

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gray clay (Clark et al., 2005; Shunk et al., 2009a; Zobaa et al., 2011). Clays are interbedded with

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quartz, chert, feldspar, dolomite, and reworked paleosol fragments, as well as minor occurrences of siderite, pyrite, gypsum, Fe-and Mn-oxides (Shunk et al., 2006). Isolated sediment layers

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preserve evidence of bioturbation and opaques associated with phytoclasts indicate periodic oxygenation of the sinkhole bottom (Zobaa et al., 2011). But, seasonally-driven, graded and laminated sedimentation with pyrite, preserved organic matter, and an absence of bioturbation in some layers suggest poorly oxygenated or even anoxic bottom waters (Shunk et al., 2006). The oxygenation state of the paleolake was likely spatially and temporally variable. Consequently, we hypothesized that the sediment record alone provides insufficient information about the

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ACCEPTED MANUSCRIPT paleoenvironmental conditions that led to bone preservation and fossilization, but the fossil bones should provide information about the diagenetic conditions that led to preservation and fossilization. We present the first analysis of GFS fossil bone mineralogy and geochemistry. 2.1 Materials and Methods

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Alligator sp. bones, including phalanges, were collected from the GFS on 30 September

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and 22 October, 2000, shortly after the site was discovered and during road construction (Clark

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et al., 2005). Permission to use only phalange fragments (n = 5; 1.1 to 1.5 g) for destructive analyses was granted by the McClung Museum of Natural History and Culture, University of

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Tennessee, Knoxville, where the bones are catalogued. Due to the fragmentary nature of the

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samples, all of the bones were assigned a single catalogue number (40WG122). For this study, samples were assigned letters to designate the individual fragments (e.g., A, B). It is unknown if

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the bones were derived from a single individual or multiple individuals, and no sediment was

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retained from where the bones were collected. However, as all bone samples were collected at the same time from a single location (i.e., sediment depth) at the GFS, the bones likely

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experienced similar early depositional and diagenetic conditions.

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Although no sediment was visible on the GFS bone surfaces, phalange fragments were cleaned using antistatic, anti-abrasive, cellulose fiber KimwipesTM (Kimberly-ClarkTM

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Corporation, Irving, Texas, USA) dampened with autoclaved deionized, 18 MΩ (Milli-Q) water to remove any adhering clay-sized particles or dust that potentially accumulated during storage. Bone fragments were gently cleaned with 70% ethanol (with deionized water) and allowed to air dry. Two phalange fragments were selected for EMP analysis and three were subsequently powdered in an agate mortar and pestle for FTIR. To prevent contamination, all cleaning and processing was performed wearing nitrile gloves and the mortar and pestle was cleaned using

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ACCEPTED MANUSCRIPT autoclaved deionized Milli-Q water followed by 100% reagent grade acetone prior to and after use. Powdered bone aliquots were stored in sterile 1.5 μL microcentrifuge tubes until analysis with FTIR and XRD. Powdered aliquots are available upon request from the McClung Museum (catalogue number 40WG122).

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Bone from a modern salvaged, juvenile alligator from the Rockefeller Wildlife Refuge,

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Grand Chenier, Louisiana (under the Louisiana Natural Heritage Program permit LNHP-10-009),

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was included in this study to compare the extent of geochemical and structural change of the GFS alligator bones with modern, unaltered alligator bone. Because the phalanges recovered

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from this salvaged alligator were too small to subsample for EMP and FTIR, a vertebrae was

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used after removal of soft tissues by dermestid beetles and manual removal of any adhering organic detritus. The bone was rinsed in Milli-Q water, placed into a sonicator (Branson 2510)

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filled with Milli-Q water for 60 minutes, dried through a series of ethanol washes from 70% to

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100% (Hamilton, 1999), and left to dry at 37°C for 24 hours. The bone was cut in half using a Dremel handsaw. One half was powdered for FTIR and XRD using the same protocol as

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described for the GFS samples, and the other half was prepared for EMP analysis, as outlined for

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the GFS samples (below). We used the modern bone to show: 1) mineralogical change in fossil bone (crystallite-scale); and 2) geochemical change as a consequence of diagenesis. Although

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there could be structural and chemical differences between a phalange and vertebra due to physiology, the differences were expected to be less than the relative differences observed in the structure and chemistry between a modern and fossil bone. This is because crystallite properties (e.g., mineral chemistry and crystallinity) do not vary significantly between bone types in a single skeleton (Donnelly et al., 2012). Moreover, there appears to be no significant difference in bone density, Ca content, organic content, and mineral content as a function of bone type (e.g.,

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ACCEPTED MANUSCRIPT Roschger et al., 2003; Donnelly et al., 2012), although some earlier studies identified differences within trabecular bone from a single skeleton (e.g., Aerssens et al., 1997). Consequently, the type of modern bone examined in this study (i.e. phalange or vertebra) would not change the results and implications of the comparison to fossil bone.

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Structural and compositional information was obtained using FTIR and XRD of

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powdered samples at the University of Tennessee, Department of Earth & Planetary Sciences.

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FTIR provides a powerful tool to assess the structure and composition of bones, particularly information about conditions present during diagenesis from the amount and location of

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carbonate incorporation in the lattice, and changes to crystallinity and crystal ordering. FTIR

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analyses were run on an Agilent Cary 660 FTIR using a diffuse reflectance accessory. Powdered bone samples were mixed with KBr (1-5% sample, 99-95% KBr) and transferred to a stainless

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steel sample holder. Each sample was measured at least five times with fresh powders mixed for

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each analysis. The resulting dataset for each sample was averaged using ResolutionsPro software, and the averaged spectra were used in subsequent data analyses. Spectra were

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measured with a resolution of 4 cm-1 and 32 scans per analysis. Following previously published

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methods, crystallinity (measured as infrared splitting factor, IRSF) (Weiner and Bar-Yosef, 1990), amide content (Am/P), organic weight % (Trueman et al., 2008), carbonate/phosphate

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ratio (Pucéat et al., 2004), and carbonate content (ratio of type B carbonate to the primary phosphate peak [BPI], type A carbonate to the primary phosphate peak [API], and the ratio of type B carbonate to type A carbonate [BAI]) (Sponheimer and Lee-Thorp, 1999) were calculated. Powdered bone samples (20 to 100 mg) examined with FTIR were also subjected to XRD analyses on a Rigaku instrument under 40 kV and 30 mA operating conditions with a continuous

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ACCEPTED MANUSCRIPT scan speed of 2° per minute. Samples were scanned from 4° to 70°. Aliquots of the powdered samples examined with FTIR were placed onto a glass sample holder and inserted into the instrument. Diffractograms were input into the Rigaku software package PDXL for peak identification, characterization of mineralogy, and calculation of mineral content (%).

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Crystallinity indices (CI) were calculated using the diffractograms, following the methods of

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Person et al. (1995) and Munro et al. (2007). XRD results were previously reported in Keenan et

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al. (2015) and are briefly summarized here.

Studies of modern and fossil bone with EMP provide a quantitative tool to assess major

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(e.g., P, Ca), minor (e.g., Na, F), and, if above the detection limit, trace element composition

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(e.g., rare earth elements, REE) and compositional zoning of the mineral phase(s) (Hubert et al., 1996; Tütken et al., 2004; Keenan et al., 2015). In modern bone, approximately 30% of the bulk

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composition consists of organic carbon, lipids, and water, which results in totals of ~70% from

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EMP analyses for inorganic phases. In fossilized bone, consistently low totals that are less than 100% reflect the incorporation of carbonate into the mineral lattice, either structurally or as

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secondary carbonate phases within pore spaces. Despite potentially low totals, EMP analysis can

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quantify bulk element chemistry (Hubert et al., 1996; Tütken et al., 2004) and provide an easily accessible and relatively rapid tool to visualize elemental distributions within mineral phases.

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For this study, spots with totals less than 80% were excluded from subsequent analyses (e.g., two from the cortical bone of sample D). For EMP analysis, two cleaned phalange fragments were embedded in resin (EpoThin Epoxy Resin—Buehler) under vacuum in a glass desiccation chamber to ensure complete diffusion of resin into pore spaces and to remove any air bubbles. Once set, sections were polished with progressively finer grit size to achieve a final uniform surface of 1 μm using

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ACCEPTED MANUSCRIPT MetaDi (monocrystalline diamond suspension, Buehler). The sections were photographed under reflected light at 50X resolution, and used to construct composite maps to guide EMP analyses. Sections were carbon-coated prior to EMP analysis. Quantitative analyses of major (i.e., P, Ca, Na), minor (i.e., Mn, Mg, Fe, S, Si, Al), and trace (i.e., La, Ce, Cl) element chemistry and

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element mapping were completed at the University of Tennessee, Department of Earth &

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Planetary Sciences, on a Cameca SX-100. Instrument calibration and accuracy were checked by

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measuring known standards and re-calibrating when an element was less than 98% of the anticipated weight percentage. Spot analyses were done under standard operating conditions

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(Pyle et al., 2002), with an electron beam of 10 nA, an accelerating potential of 15 kV, and a 10

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μm spot size. Elements were analyzed in a fixed order under the same spectrometer conditions as previously reported (Table 1 in Keenan et al., 2015). Analytical precision is presented as

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standard deviations for each element, most of which are less than 20 ppm. Data were internally

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corrected using PAP, an approach similar to ZAF that accounts for the effects of atomic number, absorption, and fluorescence, with the Cameca software PeakSight.

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Element maps complemented spot analyses by visualizing potential sub-micron

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variations in apatite chemistry and chemical heterogeneity. Several areas of cortical and trabecular bone were mapped to evaluate the variation of selected elements (e.g., Fe, P, Na, F,

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Ca) after examining spot analysis data and noting that certain compounds (e.g., FeO, P2O5) had larger standard deviations than expected (> 20 ppm). Mapping was done for Na as NaO, a highly mobile ion in an aqueous environment and in living bone tissue (Bergstrom and Wallace, 1954), and F, which can increase in bioapatite during diagenesis (e.g., Kohn, 2008). Quantifying F by EMP analyses can cause ion volatilization if beam current, count time, or time under vacuum are high (e.g., Pyle et al., 2002), so care was taken to diminish analytical bias for F measurements.

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ACCEPTED MANUSCRIPT After mapping, maps were false colored using PeakSight to visually assess the changes in concentration, presented as changes to the number of counts measured by the detector with brighter colors reflecting higher counts or greater concentrations. False color element mixing provides a visualization of variations in multiple elements together (e.g., P and Fe).

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Comparisons to the Pipe Creek Sinkhole dataset obtained with EDX (Farlow and Argast,

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2006) required converting data obtained for the GFS bones to reflect weight percent (wt. %) of

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each element. The total wt. % of each element was calculated by determining the percentage contribution of an element (e.g., P) using the gram formula weight of the oxidized compound.

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For example, EMP analyses present P as P2O5. Based on the gram formula weight, P represents

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43.6% of the P2O5 compound. Using the molar contribution, the total amount of each element can then be calculated using the weight percentage values obtained by EMP analysis. To make

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the data comparable, the GFS data were then normalized to 100% on a C- and H-free basis, as

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implemented by Farlow and Argast (2006) in their dataset. Minor or trace elements (e.g., Ba, Sr, Ce, La, Cl) measured in this study by EMP analysis but not examined by Farlow and Argast

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(2006) were excluded from GFS dataset normalization. These minor and trace elements

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contributed 0.42 wt. % to the GFS bone geochemistry, and excluding them to make the two datasets comparable was within the standard deviation (2.7 wt. %) measured for the total

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elemental concentrations of the GFS bones. 3.1 Results

3.1.1 GFS bone structure, mineralogy, and geochemical variability In general, the GFS alligator phalange bones, as well as other bones examined at the McClung Museum and at the GFS site in Gray, Tennessee, are dark brown in color, and are light-weight and fragile, but well-preserved, as they retain macrostructural features (e.g., cortical

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ACCEPTED MANUSCRIPT and trabecular bone). From the alligator bones examined in this study, there were no secondary phases infilling porespaces at the macro-and micro-scale. Numerous small (< 1 μm to 20 μm) circular holes or pits and tube-like structures restricted to thin regions of cortical bone underlain by trabeculae were evident from BSE

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imaging prior to chemical analysis with the EMP (Fig. 2A, B, C). Feature density and

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distribution patterns were non-uniform along the external bone margin. Surface modifications

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were largely restricted to the outermost surfaces, likely in direct contact with the sediment, and largely absent from inner trabecular margins that line vascular canals, as well as ~ 550 μm thick

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cortical bone (Fig. 2D). The size and shape of these features ranged from sub-micron to ~ 20 μm

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diameter holes. Tube structures extended several microns up to 100 μm from the bone surface inwards.

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The structure and elemental composition of the GFS alligator fossil bone differed when

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compared to modern bone (Table 1). Based on FTIR analyses, the GFS samples had lower organic weight % compared to modern bone, and lower Am/P ratio, which also supported a

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reduction in organics. The IRSF values, a measure of crystallite or structural ordering, were

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higher in the GFS fossils than in unaltered bone (Table 1). However, based on XRD analysis, each of the three fossil phalanges had similar calculated CI values, between 0.22 and 0.27, which

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suggested a poorly recrystallized phase (Person et al., 1995; Munro et al., 2007). The GFS samples had reduced carbonate/phosphate ratios compared to modern bone, indicating either loss of carbonate relative to phosphate or phosphate enrichment. Both the BPI and API ratios from FTIR measurements were lower in the fossils than the modern, which would be caused by the incorporation of carbonate at both sites in the apatite lattice. In all three GFS samples examined for this and in a previous study (Keenan et al., 2015), BAI ratios were elevated due to greater

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ACCEPTED MANUSCRIPT incorporation of carbonate at the apatite lattice B site. But, based on XRD analyses (Keenan et al., 2015), the phalanges consisted predominantly of a fluorinated rather than carbonated apatite mineral phase. A calcium-iron-phosphate phase was also detected in all three fossils. Because of the absence of minerals infilling pore spaces, the Fe-bearing phases are likely incorporated

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structurally within the apatite lattice and formed mixed Ca-Fe-PO4 phases.

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The GFS phalanges consisted predominantly of a fluorinated apatite. and the cortical region appeared to be uniform in elemental composition based on XRD and EMP analyses (Fig.

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3A). F content in the GFS samples was ~2.00 wt. % compared to 0.07 wt. % in modern bone. But, compared to modern alligator bone, the GFS samples also contained higher Fe2+ and lower

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Na concentrations (Table 2). Spot EMP analyses suggested a fairly uniform composition (e.g.,

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standard deviations less than 20 ppm) for all major and minor elements examined with no distinct zonation apparent using a spot size of 10 μm. Spot EMP analyses showed that the

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modern bone had lower overall totals (~67-74 wt. %) compared to the GFS fossils, with totals

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~89 wt. % that were likely due to organic loss, as supported by FTIR analyses. Moreover, the standard deviations calculated from EMP analyses for several elements (e.g., FeO, P2O5) were

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high but did not correlate with low totals, which might be explained by analysis of a poorly

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polished area of the section or inclusion of a canal in the spot area. The element map for P concentration (as P2O5) from the GFS cortical fossil bone suggested that P was relatively uniform compositionally (Fig. 3B). But, after EMP mapping of selected elements, it was apparent that the spot analyses did not reveal the full chemical heterogeneity within the bone from mapping Fe and Na independently and after creating mixed P, Fe, and Na false color element maps (Figs. 3C, D, E). The cortical bone contained visible, distinctive zonation on scales less than 10 μm, and elemental concentrations were non-uniformly

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ACCEPTED MANUSCRIPT distributed throughout the cortex (Fig. 3E). In particular, there were areas of the cortex with an enrichment of Fe developed within the apatite mineral as Fe-phosphate phases. Enrichment of Fe in the cortex was also present in areas where Na (as well as Ca) was relatively depleted. Moreover, the GFS fossils had distinct zones of alternating Fe enrichment and comparatively

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depleted areas in the Fe-phosphate phases. The Fe concentrations were significantly different

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based on pixel intensity or count number after converting the Fe map to grayscale and assessing

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average pixel intensities from a total of five depleted and five comparatively enriched zones (ttest, P = 0.001). Additional element maps constructed around a heavily bioeroded section of the

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cortex further highlighted bone chemical heterogeneity. In regions immediately surrounding pits

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and tunnels, P and Fe concentrations were lower, but Na concentrations were unaffected (Fig. 5A, B, C).

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The GFS trabecular bone composition was also heterogeneous. Spot analyses with high

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standard deviations suggested variable chemical compositions, but a 10 μm spot size masked the fine, micron-scale variations. Element maps constructed from several areas of the trabeculae

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(Figs. 4, 5) revealed chemical variability similar to what was observed from the cortical region.

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However, in contrast to the cortex, chemical variations in the trabecular bone did not appear to be zonal. Mixed element maps of Haversian canal within trabecular bone (Fig. 4B, C) showed

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chemical variations preserved within the GFS bone, and Fe enrichment around some Haversian canals highlighted the underlying bone microstructure, as observed in other fossils by Goodwin et al. (2007). However, other areas of the trabeculae lacked these features (Fig. 4). 3.1.2 Comparison of GFS fossil geochemistry to Pipe Creek Sinkhole fossil geochemistry To evaluate the potential for similar diagenetic processes at two sites formed in contemporaneous depositional environments and geologic settings, EMP-based bone

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ACCEPTED MANUSCRIPT geochemistry data from the GFS bones were compared to bone chemistry from the Pipe Creek Sinkhole (PCS) (Farlow and Argast, 2006). The PCS preserves a comparable Late Neogene record of terrestrial fauna and flora that were also deposited within a paleosinkhole and lacustrine system (Shunk et al., 2009b). Bones preserved at both sites contained the same order

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of magnitude concentrations of Ca, P, trace to below detection Si, and elevated concentrations of

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F (Table 3). But, GFS alligator phalanges were 2- to 3-fold enriched in Fe, with an order of

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magnitude less Mn compared to PCS material (average of 0.03 wt.% compared to 0.39 wt. %) (Table 3). Bones from both sites also had physical evidence of bioerosion. However, the

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paleosinkhole chemistries were likely different because siderite (FeCO3) infilled pore spaces in

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PCS bone.

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4.1 Discussion

Deciphering the early diagenesis of bones and interpreting the types of geochemical

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processes that may lead to fossilization are formidable challenges to paleobiologists. However,

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recrystallization of the original hydroxylapatite in bone to a more thermodynamically favorable apatite phase is a direct reflection of ambient geochemical conditions during early diagenesis

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(e.g., Goodwin et al., 2007; Hinz and Kohn, 2010; Suarez et al., 2010; Kohn and Moses, 2012;

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Keenan and Engel, 2017). As such, the fossil bones themselves serve as an archive of paleoenvironmental conditions during diagenesis. Distinct chemical zonation in the GFS Alligator sp. cortical bone suggests that the apatite must have had some underlying heterogeneity prior to burial and diagenesis. We interpret the zonation to be lines of arrested growth (LAGs) that developed during the Neogene-aged alligator’s life during periods of seasonal feeding or fasting (Rasch et al., 2000; Woodward et al., 2011). Alligators and other reptiles experience seasonal growth, driven largely by physiological

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ACCEPTED MANUSCRIPT responses to ambient temperature changes (e.g., Lang, 1979). During periods of active movement, high metabolism, and feeding, bone actively forms; during cooler months, however, bone deposition rates dramatically decrease. This results in the formation of LAGs, which have also been identified in fossil reptilian taxa, including dinosaurs (Horner et al., 1999; Kohler et al.,

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2012). Diagenesis may enhance chemical differences between the LAGs present in life (e.g.,

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Rasch et al., 2000). For the GFS bones in particular, preservation of LAGs in an ectothermic

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reptile corroborates some palynological and sedimentological evidence that climate of East Tennessee during the Late Neogene was more humid and warmer than at present, and that there

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were seasons with annual periods of warmer and cooler conditions (Wallace and Wang, 2004;

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Shunk et al., 2006; Shunk et al., 2009a; Zobaa et al., 2011).

The circular pits and tunnels abundant within GFS cortical bone and isolated trabecular

M

regions are interpreted as bioerosion caused by microbial and/or invertebrate boring activity

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(e.g., Jans et al. 2004; Jans, 2008; Turner-Walker, 2008). Evidence of bioerosion from the PCS, in the form of numerous pits and tunnels, has also been observed along the cortical margin of

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bone, as well as some regions in the trabeculae (Farlow and Argast, 2006). Although the exact

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timing (i.e., either syn-diagenetic or post-recrystallization) of bioerosion cannot be unequivocally determined, the lack of borings within the thicker cortical surfaces and inner trabeculae suggests

AC

that bone colonization by microorganisms and/or invertebrates occurred during burial and early diagenesis when organics (e.g., lipids and proteins) were still present on and within the bone. Limited bioerosion was noted within the inner trabecular region, which suggests that these GFS bones were buried early during diagenesis. Burial would have prevented extensive erosive activity within the trabeculae that is rich with labile carbon and nitrogen. The preservation of bioeroded bone provides insight into the timing or rate of burial and

16

ACCEPTED MANUSCRIPT diagenesis. Whale falls in marine deposits are an extreme example of clear biotic degradation of bone (e.g., Goffredi et al., 2005). But, in general, bones preserving extensive bioerosion in the terrestrial vertebrate record are rare (Trueman and Martill, 2002; Farlow and Argast, 2006; Turner-Walker, 2012). Therefore, for bioerosion to be preserved, transformation of the original

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hydroxylapatite into a more stable mineral phase(s), in the case for the GFS bones being a Ca-

IP

Fe-phosphate phase, must have occurred either synchronously with, or relatively quickly after,

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bioerosion. This is because increased surface area caused by bioerosion should either enhance dissolution of a mineral phase that was thermodynamically unfavorable in the paleolake waters

US

or enhance ion movement and substitution to a more thermodynamically stable mineral phase.

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Recent experiments of early diagenesis reveal the presence of putative bioerosion in bone after as little as one week, as well as in bones buried for one to three years (Keenan and Engel, 2017).

M

Consequently, the development of bioerosion of the GFS bones (and possibly the PCS bones)

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could have occurred as rapidly as one week following the exposure of bones to the environment. The IRSF values were higher in the GFS fossils than in unaltered, modern bone, indicating more

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structural ordering and supporting that recrystallization has occurred (Table 1) (Trueman et al.,

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2008). However, based on XRD analysis, each of the three fossil phalanges had similar calculated CI values, between 0.22 and 0.27, which indicate early postmortem recrystallization

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of the bioapatite that usually results in a poorly recrystallized phase (Person et al., 1995; Munro et al., 2007). The preservation of poorly recrystallized bioapatite would support rapid diagenetic alteration, preserving the bioeroded bone. Previous sedimentology research of the lacustrine deposits suggests that sediments accumulated seasonally or in response to significant rainfall events (e.g., Shunk et al., 2006). These events would have likely buried bones rapidly and removed them from oxic to suboxic conditions at the sediment-water interface within days to

17

ACCEPTED MANUSCRIPT weeks or months of deposition. Apatite minerals are among the most diverse mineral assemblages because they can accommodate numerous and varied substitutions at every site within the lattice (Pan and Fleet, 2002; Kohn, 2008). Divalent or trivalent metals, as well as REE, can replace Ca at either of the

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two sites, Ca(I) and Ca(II) (e.g., Pan and Fleet, 2002), although the replacement lattice site

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depends on several variables, such as charge balance within the lattice, local electrostatic

CR

repulsion, the substituting ion radius, and other ions already incorporated into the lattice (e.g., Cl, F) (Fleet and Pan, 1995; Jiang et al., 2002). As a consequence of recrystallization of the original

US

bioapatite and enrichment in F and Fe, the GFS bones are now mineralogically a fluorinated and

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Fe-enriched apatite phase. Fe incorporation was partially afforded by the loss of Na (and Ca) in the original apatite mineral, based on observed depletions of Na and Ca. A similar coupled

M

exchange between Fe and Ca has been observed in other fossils, including fossil crocodilians

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(Goodwin et al., 2007). For redox-sensitive elements like Fe, we expect that the majority of Fe in the GFS bones is as Fe2+, although we also recognize that the apatite lattice can accommodate

PT

both valences at both Ca sites (Jiang et al., 2002). Moreover, determining the valency of

CE

incorporated Fe is not straight-forward. Nevertheless, Ca sites are the most likely positions in the bioapatite lattice for Fe incorporation (Pan and Fleet, 2002), and once Fe was incorporated into

AC

the lattice, limited valency change would have occurred. It is possible that Fe2+ substitution into the lattice occurred from interactions with reducing solutions because in vitro experiments of Fe valency in tooth enamel (bone was not assessed) show that Fe valency is the same as the solution (Bauminger et al., 1985). At GFS, pyrite and siderite clasts in isolated sediment layers at the GFS provide evidence that bottom water conditions were reducing and anoxic (Shunk et al., 2006). These conditions

18

ACCEPTED MANUSCRIPT must have persisted during bone recrystallization to preserve Fe2+ and to prevent oxidation to Fe3+ either in solution or as an Fe-oxide solid associated with the bone surface or within the apatite lattice (Jiang et al., 2002). Like the evidence from preserved bioerosion features, Fe uptake could have been relatively rapid based on recent experiments of modern alligators buried

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under anaerobic conditions in a wetland where Fe2+ uptake in cortical bone occurred after one

IP

year of burial (Keenan and Engel, 2017).

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The formation of an Fe-enriched apatite mineral phase implies that bottom-water and sediment conditions at GFS were acidic, as Fe-phosphates are more thermodynamically stable

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under acidic conditions (Moore and Reddy, 1994). Because the overall paleolake water was

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likely buffered to circumneutral pH due to being in thermodynamic equilibrium with carbonate bedrock, preservation of Fe-phosphate mineralization was likely due to localized acidity within

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the sediments and fluids immediately surrounding the bones. Localized acidity could have been

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generated from organic matter decomposition, derived from the abundant vegetation deposited into the lake, as well as decaying animals and the alligator(s) itself (e.g., Schweitzer et al., 2014).

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For instance, soft tissues (e.g., muscle, adipose tissue, skin) likely degraded rapidly following

trabeculae.

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5.1 Conclusions

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burial, which would have left behind labile organics, including organic acids, held within

In this study, our goal was to evaluate GFS bone major element geochemistry and structural crystallinity by using analytical techniques, including FTIR, XRD, and EMP. Although examining a single suite of bones recovered from one horizon within the bone-rich lacustrine deposits at GFS may provide only a snap-shot of the diagenetic conditions that led to bone preservation, there is still great potential at the GFS to evaluate diagenetic processes,

19

ACCEPTED MANUSCRIPT geochemical conditions, and the evolution of the paleolake geochemistry through time from the stratigraphic succession of reptile and mammal bones. For the first time, the geochemical and mineralogical evaluation of Alligator sp. bones preserved at the GFS provides a window into the diagenetic conditions of the burial environment as suboxic, acidic, and reducing. During or

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shortly after exposure to aggressive environmental microbial or invertebrate communities that

IP

caused bone bioerosion at the sediment-water interface prior to burial, and once removed from

CR

the suboxic sediment surface after as little as one week time, the reducing and acidic conditions below the sediment-water interface. Free Fe2+ ions in solution would have favored the

US

incorporation of Fe within the apatite lattice at the expense of Ca2+. This resulted in a

AN

comparatively more thermodynamically stable, mixed Fe-Ca-phosphate apatite phase. As a way to further explore the bone diagenetic history of the GFS, comparisons between site sediment

M

geochemistry and bone geochemistry may provide additional insight into the source or sources of

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substituting elements into the apatite lattice (e.g., changes in redox conditions or organic degradation mobilizing Fe). Lastly, comparisons to modern sinkhole deposits in karst areas

PT

having a similar seasonal climate as that inferred for the Late Neogene of East Tennessee could

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offer information on the rates, timing, and processes leading to faunal accumulation within

AC

sinkholes, as well as the extent of bone diagenetic alteration over time. Acknowledgements

We thank the McClung Museum of Natural History and Culture at the University of Tennessee for access and permission to destructively analyze the Gray Fossil Site bones used in this study. Larry Taylor is graciously acknowledged for access to the electron microprobe and for enlightening discussions. Allan Patchen assisted with EMP analyses. We also would like thank Chris Fedo for microscope access and reading a previous version of this manuscript. Linda Kah

20

ACCEPTED MANUSCRIPT and Mark Radosevich also provided helpful feedback. One anonymous reviewer and Steven Driese provided critical feedback that greatly enhanced the quality of this work. This work was funded by the Jones Endowment and the Department of Earth & Planetary Sciences at the University of Tennessee.

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References

CR

Aerssens, J., Boonen, S., Joly, J., Dequeker, J., 1997. Variations in trabecular bone composition with anatomical site and age: potential implications for bone quality assessment. J.

US

Endocrinol., 155, 411-421.

Anthony, D.M., Granger, D.E., 2004. A Late Tertiary origin for multilevel caves along the

AN

western escarpment of the Cumberland Plateau, Tennessee and Kentucky, established by

M

cosmogenic 26Al and 10Be. J. Cave Karst Stud. 66, 46-55. Auler, A.S., Pilo, L.B., Smart, P.L., Wang, X.F., Hoffmann, D., Richards, D.A., Edwards, R.L.,

ED

Neves, W.A., Cheng, H., 2006. U-series dating and taphonomy of Quaternary vertebrates

PT

from Brazilian caves. Palaeogeogr. Palaeoclimatol. Palaeoecol. 240, 508-522. Bauminger, E., Ofer, S., Gedalia, I., Horowitz, G., Mayer, I., 1985. Iron uptake by teeth and

CE

bones: a Mössbauer-effect study. Calcif. Tissue Int. 37, 386-389.

AC

Behrensmeyer, A.K., 1988. Vertebrate preservation in fluvial channels. Palaeogeogr. Palaeoclimatol. Palaeoecol. 63, 183-199. Bergstrom, W.H., Wallace, W.M., 1954. Bone as a sodium and potassium reservoir. J. Clinic. Invest. 33, 867-873. Berna, F., Matthews, A., Weiner, S., 2004. Solubilities of bone mineral from archaeological sites: the recrystallization window. J. Archaeol. Sci. 31, 867-882. Cerling, T.E., Harris, J.M., MacFadden, B.J., Leakey, M.G., Quade, J., Eisenmann, V.,

21

ACCEPTED MANUSCRIPT Ehleringer, J.R., 1997. Global vegetation change through the Miocene/Pliocene boundary. Nature 389, 153-158. Cerling, T.E., Wang, Y., Quade, J., 1993. Expansion of C4 ecosystems as an indicator of global ecological change in the Late Miocene. Nature 361, 344-345.

T

Clark, G.M., Kohl, M., Moore, H.L., Sasowsky, I.D., 2005. The Gray Fossil Site: a spectacular

IP

example in Tennessee of ancient regolith occurrences in carbonate terranes, Valley and Ridge

CR

subprovince, southern Appalachians U.S.A., in: Beck, B.F. (Ed.), Tenth Multidisciplinary Conference on Sinkholes and the Engineering and Environmental Impacts of Karst. American

US

Society of Civil Engineers, San Antonio, Texas, pp. 82-90.

AN

DeSantis, L.R.G., Wallace, S.C., 2008. Neogene forests from the Appalachians of Tennessee, USA: geochemical evidence from fossil mammal teeth. Palaeogeogr. Palaeoclimatol.

M

Palaeoecol. 266, 59-68.

ED

Donnelly, E., Meredith, D.S., Nguyen, J.T., Boskey, A.L., 2012. Bone tissue composition varies across anatomic sites in the proximal femur and iliac crest. J. of Orthop. Res. 30, 700-706.

PT

Farlow, J.O., Argast, A., 2006. Preservation of fossil bone from the Pipe Creek Sinkhole (Late

CE

Neogene, Grant County, Indiana, U.S.A.). J. Paleontol. Soc. of Korea 22, 51-75. Fleet, M.E., Pan, Y.M., 1995. Site preference of rare-earth elements in fluorapatite. Am. Mineral.

AC

80, 329-335.

Goffredi, S.K., Orphan, V.J., Rouse, G.W., Jahnke, L., Embaye, T., Turk, K., Lee, R., Vrijenhoek, R.C., 2005. Evolutionary innovation: a bone-eating marine symbiosis. Environ. Microbiol. 7, 1369-1378. Gong, F.D., Karsai, I., Liu, Y.S., 2010. Vitis seeds (Vitaceae) from the late Neogene Gray Fossil Site, northeastern Tennessee, USA. Rev. Palaeobot. Palyno. 162, 71-83.

22

ACCEPTED MANUSCRIPT Goodwin, M.B., Grant, P.G., Bench, G., Holroyd, P.A., 2007. Elemental composition and diagenetic alteration of dinosaur bone: Distinguishing micron-scale spatial and compositional heterogeneity using PIXE. Palaeogeogr. Palaeoclimatol. Palaeoecol. 253, 458-476. Hamilton, D.L., 1999. Methods for conserving archaeological material from underwater sites.

T

Texas A&M University, College Station, Texas.

CR

uptake in fossils. Geochim. Cosmochim. Acta 74, 3213-3231.

IP

Hinz, E.A., Kohn, M.J., 2010. The effect of tissue structure and soil chemistry on trace element

Horner, J.R., de Ricqles, A., Padian, K., 1999. Variation in dinosaur skeletochronology

US

indicators: implications for age assessment and physiology. Paleobiology 25, 295-304.

AN

Hubert, J.F., Panish, P.T., Chure, D.J., Prostak, K.S., 1996. Chemistry, microstructure, petrology, and diagenetic model of Jurassic dinosaur bones, Dinosaur National Monument, Utah. J.

M

Sediment. Res. 66, 531-547.

ED

Jans, M.M.E., 2008. Microbial bioerosion of bone—a review, in: Wisshak, M., Tapanila, L. (Eds.), Current developments in bioerosion. Springer-Verlag, Berlin, pp. 397-413.

PT

Jans, M.M.E., Nielsen-Marsh, C.M., Smith, C.I., Collins, M.J., Kars, H., 2004. Characterisation

CE

of microbial attack on archaeological bone. J. Archaeol. Sci. 31, 87-95. Jiang, M., Terra, J., Rossi, A.M., Morales, M.A., Saitovitch, E.M.B., Ellis, D.E., 2002. Fe2+/Fe3+

AC

substitution in hydroxyapatite: theory and experiment. Phys. Rev. B 66, 224107.1-224107.15. Keenan, S.W., 2016. From bone to fossil: a review of the diagenesis of bioapatite. Am. Mineral. 101, 1943-1951. Keenan, S.W., Engel, A.S., 2017. Early diagenesis and recrystallization of bone. Geochim. Cosmochim. Acta 196, 209-223. Keenan, S.W., Scannella, J.B., 2014. Paleobiological implications of a Triceratops bonebed from

23

ACCEPTED MANUSCRIPT the Hell Creek Formation, Garfield County, northeastern Montana, in: Hartman, J., Wilson, G., Horner, J.R., Clemens, W. (Eds.), Through the end of the Cretaceous in the type locality of the Hell Creek Formation in Montana and adjacent areas. Geol. Society Am. S. 503, 349364.

T

Keenan, S.W., Engel, A.S., Roy, A., Bovenkamp-Langlois, G.L., 2015. Evaluating the

IP

consequences of diagenesis and fossilization on bioapatite lattice structure and composition.

CR

Chem. Geol. 413, 18-27.

Koenig, A.E., Rogers, R.R., Trueman, C.N., 2009. Visualizing fossilization using laser ablation-

US

inductively coupled plasma-mass spectrometry maps of trace elements in Late Cretaceous

AN

bones. Geology 37, 511-514.

Kohl, M., 2014. Gray Fossil Site current list of fossils (2014).

M

https://www.tn.gov/environment/article/geo-gray-current-list-of-fossils (accessed 17 June

ED

2016).

Kohler, M., Marin-Moratalla, N., Jordana, X., Aanes, R., 2012. Seasonal bone growth and

PT

physiology in endotherms shed light on dinosaur physiology. Nature 487, 358-361.

CE

Kohn, M.J., 2008. Models of diffusion-limited uptake of trace elements in fossils and rates of fossilization. Geochim. Cosmochim. Acta 72, 3758-3770.

AC

Kohn, M.J., Moses, R.J., 2012. Trace element diffusivities in bone rule out simple diffusive uptake during fossilization but explain in vivo uptake and release. Proc. Natl. Acad. Sci. USA 110, 419-424. Lang, J.W., 1979. Thermophilic response of the American alligator and the American crocodile to feeding. Copeia, 48-59. Laury, R.L., 1980. Paleoenvironment of a Late Quaternary mammoth-bearing sinkhole deposit,

24

ACCEPTED MANUSCRIPT Hot Springs, South Dakota. Geol. Soc. Am. Bull. 91, 465-475. Lee-Thorp, J., 2002. Two decades of progress towards understanding fossilization processes and isotopic signals in calcified tissue minerals. Archaeometry 44, 435-446. Lundelius, E.L.J., 2006. Cave site contributions to vertebrate history. Alcheringa 30.S1, 195-210.

T

Mead, J.I., Schubert, B.W., Wallace, S.C., Swift, S.L., 2012. Helodermatid lizard from the Mio-

IP

Pliocene oak-hickory forest of Tennessee, eastern USA, and a review of monstersaurian

CR

osteoderms. Acta Palaeontol. Pol. 57, 111-121.

Moore, H.L., 2006. A proactive approach to planning and designing highways in east Tennessee

US

karst. Environ. Eng. Geosci. 12, 147-160.

AN

Moore, P.A., Jr., Reddy, K.R., 1994. Role of Eh and pH on phosphorus geochemistry in sediments of Lake Okeechobee, Florida. J. Environ. Qual. 23, 955-964.

M

Munro, L.E., Longstaffe, F.J., White, C.D., 2007. Burning and boiling of modern deer bone:

ED

effects on crystallinity and oxygen isotope composition of bioapatite phosphate. Palaeogeogr. Palaeoclimatol. Palaeoecol. 249, 90-102.

PT

Pan, Y.M., Fleet, M.E., 2002. Compositions of the apatite-group minerals: Substitution

CE

mechanisms and controlling factors. Rev. Mineral Geochem. 48, 13-49. Person, A., Bocherens, H., Saliege, J.F., Paris, F., Zeitoun, V., Gerard, M., 1995. Early

AC

diagenetic evolution of bone phosphate: an X-ray diffractometry analysis. J. Archaeol. Sci. 22, 211-221.

Pucéat, E., Reynard, B., Lécuyer, C., 2004. Can crystallinity be used to determine the degree of chemical alteration of biogenic apatites? Chem. Geol. 205, 83-97. Pyle, J.M., Spear, F.S., Wark, D.A., 2002. Electron microprobe analysis of REE in apatite, monazite and xenotime: protocols and pitfalls. Rev. Mineral Geochem. 48, 337-362.

25

ACCEPTED MANUSCRIPT Rasch, R., Tucker, A.D., Daddow, L., Wentrup-Byrne, E., 2000. Electron microprobe investigations of growth marks in crocodile osteoderms, in: Grigg, G.C., Seebacher, F., Franklin, C.E. (Eds.), Crocodilian Biology and Evolution. Surrey Beatty & Sons, Chipping Norton, Australia, pp. 144-155.

T

Roschger, P., Gupta, H.S., Berzlanovich, A., Ittner, G., Dempster, D.W., Fratzl, P., Cosman, F.,

CR

distribution in cancellous human bone. Bone, 32, 316-323.

IP

Parisien, M., Lindsay, R., Nieves, J.W., Klaushofer, K., 2003. Constant mineralization density

Schweitzer, M.H., Zheng, W., Cleland, T.P., Goodwin, M.B., Boatman, E., Theil, E., Marcus,

US

M.A., Fakra, S.C., 2014. A role for iron and oxygen chemistry in preserving soft tissues, cells

AN

and molecules from deep time. Proc. R. Soc. B 281, 20132741.Shunk, A.J., Driese, S.G., Clark, G.M., 2006. Late Miocene to earliest Pliocene sedimentation and climate record

M

derived from paleosinkhole fill deposits, Gray Fossil Site, northeastern Tennessee, U.S.A.

ED

Palaeogeogr. Palaeoclimatol. Palaeoecol. 231, 265-278. Shunk, A.J., Driese, S.G., Dunbar, J.A., 2009a. Late Tertiary paleoclimatic interpretation from

PT

lacustrine rhythmites in the Gray Fossil Site, northeastern Tennessee, USA. J. Paleolimnol.

CE

42, 11-24.

Shunk, A.J., Driese, S.G., Farlow, J.O., Zavada, M.S., Zobaa, M.K., 2009b. Late Neogene

AC

paleoclimate and paleoenvironment reconstructions from the Pipe Creek Sinkhole, Indiana, USA. Palaeogeogr. Palaeoclimatol. Palaeoecol. 274, 173-184. Soriano, M.A., Luzon, A., Yuste, A., Pocovi, A., Perez, A., Simon, J.L., Gil, H., 2012. Quaternary alluvial sinkholes: record of environmental conditions of karst development, examples from the Ebro Basin, Spain. J. Cave Karst Stud. 74, 173-185. Sponheimer, M., Lee-Thorp, J.A., 1999. Alteration of enamel carbonate environments during

26

ACCEPTED MANUSCRIPT fossilization. J. Archaeol. Sci. 26, 143-150. Strömberg, C.A.E., 2005. Decoupled taxonomic radiation and ecological expansion of openhabitat grasses in the Cenozoic of North America. Proc. Natl. Acad. Sci. USA 102, 1198011984.

T

Suarez, C.A., Macpherson, G.L., González, L.A., Grandstaff, D.E., 2010. Heterogeneous rare

IP

earth element (REE) patterns and concentrations in a fossil bone: implications for the use of

CR

REE in vertebrate taphonomy and fossilization history. Geochim. Cosmochim. Acta 74, 2970–2988.

US

Trueman, C.N., 2013. Chemical taphonomy of biomineralized tissues. Palaeontology 56, 475-

AN

486.

Trueman, C.N., Martill, D.M., 2002. The long-term survival of bone: the role of bioerosion.

M

Archaeometry 44, 371-382.

ED

Trueman, C.N., Privat, K., Field, J., 2008. Why do crystallinity values fail to predict the extent of diagenetic alteration of bone mineral? Palaeogeogr. Palaeoclimatol. Palaeoecol. 266, 160-167.

PT

Trueman, C.N.G., Behrensmeyer, A.K., Tuross, N., Weiner, S., 2004. Mineralogical and

CE

compositional changes in bones exposed on soil surfaces in Amboseli National Park, Kenya: diagenetic mechanisms and the role of sediment pore fluids. J. of Archaeol. Sci. 31, 721-739.

AC

Turner-Walker, G., 2008. The chemical and microbial degradation of bones and teeth, in: Pinhasi, R., Mays, S. (Eds.), Advances in Human Palaeopathology. John Wiley & Sons, Ltd., pp. 3-29. Turner-Walker, G., 2012. Early bioerosion in skeletal tissues: persistence through deep time. Neues. Jahrb. Geol. P-A. 265, 165-183. Tütken, T., Pfretzschner, H.U., Vennemann, T.W., Sun, G., Wang, Y.D., 2004. Paleobiology and

27

ACCEPTED MANUSCRIPT skeletochronology of Jurassic dinosaurs: implications from the histology and oxygen isotope compositions of bones. Palaeogeogr. Palaeoclimatol. Palaeoecol. 206, 217-238. Wallace, S.C., Wang, X.M., 2004. Two new carnivores from an unusual late Tertiary forest biota in eastern North America. Nature 431, 556-559.

T

Wang, X.M., Wallace, S., 2004. Two new immigrants from the Old World: the earliest and most

IP

primitive Red Panda (Parailurus) and a New Eurasian badger (Arctomeles) from Late

CR

Miocene/Early Pliocene Gray Fossil Site, eastern Tennessee. J. Vertebr. Paleontol. 24, 126A. Weiner, S., Bar-Yosef, O., 1990. States of preservation of bones from prehistoric sites in the

US

near-east: a survey. J. of Archaeol. Sci. 17, 187-196.

AN

Whitelaw, J.L., Mickus, K., Whitelaw, M.J., Nave, J., 2008. High-resolution gravity study of the Gray Fossil Site. Geophysics 73, B25-B32.

M

Woodward, H.N., Horner, J.R., Farlow, J.O., 2011. Osteohistological evidence for determinate

ED

growth in the American alligator. J. Herpetol. 45, 339-342. Worobiec, E., Szulc, J., 2010. A Middle Miocene palynoflora from sinkhole deposits from Upper

PT

Silesia, Poland and its palaeoenvironmental context. Rev. Palaeobot. Palyno. 163, 1-10.

CE

Worobiec, E., Liu, Y.S., Zavada, M.S., 2013. Palaeoenvironment of late Neogene lacustrine sediments at the Gray Fossil Site, Tennessee, USA. Annales Societatis Geologorum Poloniae

AC

83, 51-63.

Zobaa, M.K., Zavada, M.S., Whitelaw, M.J., Shunk, A.J., Oboh-Lkuenobe, F.E., 2011. Palynology and palynofacies analyses of the Gray Fossil Site, eastern Tennessee: their role in understanding the basin-fill history. Palaeogeogr. Palaeoclimatol. Palaeoecol. 308, 433-444.

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ACCEPTED MANUSCRIPT Table 1 Crystallinity (IRSF) 2.813 ± 0.843

Age Modern Alligator Bone

Modern

Am/P

Weight % organic matter

1.600 ± 0.144

37.638 ± 0.977

Carbonate/ Phosphate 2.378 ± 0.573

BPI 2.130 ± 0.614

API 2.522 ± 0.873

BAI 0.845 ± 0.030

4.5-7.0 Mya

2.895 ± 0.070

0.253 ± 0.018

17.221 ± 0.828

0.933 ± 0.053

0.952 ± 0.059

0.904 ± 0.061

1.053 ± 0.016

Gray Fossil Site (B)

4.5-7.0 Mya

2.929 ± 0.161

0.261 ± 0.012

17.555 ± 0.489

0.902 ± 0.055

0.915 ± 0.060

0.890 ± 0.063

1.027 ± 0.015

Gray Fossil Site (C)

4.5-7.0 Mya

3.130 ± 0.249

0.235 ± 0.249

16.380 ± 0.846

0.911 ± 0.042

0.908 ± 0.043

0.862 ± 0.046

1.053 ± 0.017

AC

CE

PT

ED

M

AN

US

CR

IP

T

Gray Fossil Site (A)

29

ACCEPTED MANUSCRIPT Table 2

Cortical

n=7

28. 8 (13 )

0.0 2 (2)

0.4 3 (8)

B.D .

B.D.

B.D.

Trabecu lar

n=5

28. 9 (9)

B. D

0.5 5 (8)

B.D .

B.D.

B.D.

All regions

n = 12

28. 8 (11 )

0.0 2 (1)

0.4 8 (10 )

B.D .

B.D.

B.D.

Cortical

n = 32

35. 2 (9)

0.0 2 (2)

0.0 7 (6)

0.32 (10)

0.10 (10)

0.11 (11)

Cortical (near bioerosi on)

n=7

34. 6 (8)

B. D.

0.0 4 (3)

0.13 (15)

0.10 (14)

0.11 (15)

Trabecu lar

n = 11

35. 7 (2)

B. D.

B. D.

N. M.

B.D.

All regions

n = 50

35. 2 (9)

B. D.

0.0 5 (6)

0.22 (16)

Cortical

n=6

35. 1 (2)

0.0 2 (1)

B. D.

Trabecu lar

n=4

34. 9 (2)

0.0 2 (1)

All regions

n = 10

0.0 2 (1)

B. D.

Ca O

38. 1 (17 ) 38. 4 (15 ) 38. 2 (15 )

Mn O

Fe O

Sr O

Ba O

H2 O

Na2 O

F

Cl

Tot al

B.D .

B. D

B. D.

B. D.

1.1 1 (8)

0.7 9 (21)

0.0 8 (5)

0.3 4 (13 )

69. 7 (32)

B.D .

B. D.

B. D.

B.D

B. D.

48. 1 (9)

0.0 7 (2)

47. 4 (8)

0.0 4 (2)

AN

Ce2 O3

N. M.

0.09 (11)

48. 0 (8)

0.0 5 (2)

ED

35. 0 (2)

La2 O3

48. 2 (4)

B.D.

B.D.

48. 5 (23 )

B.D .

B. D.

B.D .

B.D.

B.D.

48. 1 (5)

B.D .

B.D .

B.D.

B.D.

48. 4 (4)

B.D .

0.02 (1)

PT

Gray Fossil Site (E)

CE

B.D.

M

Gray Fossil Site (D)

Al2 O3

0.09 (10)

B. D.

1.0 6 (5)

0.7 3 (12)

0.0 5 (3)

0.6 1 (9)

70. 4 (24)

B. D.

B. D.

1.0 9 (7)

0.7 6 (17)

0.0 7 (4)

0.4 6 (18 )

70. 0 (28)

2.3 0 (38 ) 2.6 4 (63 ) 2.7 0 (25 ) 2.4 3 (43 ) 2.8 0 (18 ) 3.1 4 (15 ) 2.9 3 (24 )

0.0 5 (2)

0.1 8 (6)

0.5 5 (6)

0.2 5 (5)

B. D.

89. 0 (18)

0.0 4 (1)

0.1 7 (4)

0.5 8 (19 )

0.1 6 (2)

B. D.

87. 9 (17)

0.0 6 (3)

0.1 2 (3)

0.5 5 (6)

0.2 2 (3)

B. D.

89. 6 (6)

0.0 5 (2)

0.1 6 (5)

0.5 5 (9)

0.2 3 (5)

B. D.

89. 0 (17)

0.0 4 (2)

0.1 1 (2)

0.5 0 (7)

0.1 9 (3)

B. D.

89. 5 (4)

0.0 5 (1)

0.1 3 (2)

0.5 9 (6)

0.1 9 (4)

B. D.

89. 2 (6)

0.0 4 (2)

0.1 2 (2)

0.5 3 (8)

0.1 9 (3)

B. D.

89. 4 (5)

2.0 1 (1 1) 1.9 1 (3 7) 2.0 3 (1 1) 2.0 0 (1 7) 2.1 3 (1 4) 1.9 2 (1 4) 2.0 5 (1 7)

AC

Moder n Alliga tor

2

T

SO

IP

Si O2

CR

P2 O5

US

Numb er of Analys es Per Region or Speci men

Spot Analysi s Locatio n

30

ACCEPTED MANUSCRIPT Table 3 O

F

Na

Al

Si

S

Mn

Fe

37.36

15.99

41.69

3.34

0

B.D.

0

0

0.47

1.04

Pond turtle shell (n = 10)

37.13

16.23

41.92

3.49

0

0

0

0

0.51

0.72

Hesperotestudo sp. (tortoise) shell (n = 15)

37.84

16.19

40.91

3.81

B.D.

0

0

0.32

0.19

0.70

Large mammal rib? (n = 42)

38.02

16.17

41.21

3.41

0

0

0

0

0.39

0.80

Alligator sp. phalange (D) (n = 50)

38.95

17.46

38.77

2.26

0.20

0.13

0.01

0.03

0.05

2.15

Alligator sp. phalange (E) (n = 10)

39.01

17.26

38.71

2.25

0.16

0.01

0.01

0.01

0.02

2.57

PT

ED

M

AN

US

CR

IP

T

P

CE

GFS Bones

Ca Rana pipiens complex (frog) limb bone (n = 7)

AC

PCS Bones

31

AC

CE

PT

ED

M

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

32

AC

CE

PT

ED

M

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

33

AC

CE

PT

ED

M

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

34

AC

CE

PT

ED

M

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

35

AC

CE

PT

ED

M

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

36

ACCEPTED MANUSCRIPT Highlight Gray Fossil Site Alligator sp. bones are used to infer paleosinkhole geochemistry.

-

Gray Fossil Site bones are Fe-enriched compared to modern bone.

-

Bone diagenesis involved acidic, anoxic, and reducing fluids.

-

Preserved lines of arrested growth in GFS alligator bone indicate seasonality.

AC

CE

PT

ED

M

AN

US

CR

IP

T

-

37