Abstract: Recent work on the timing of proboscidean tooth enamel formation has led to advances in the use of stable isotopes to understand diet and behavior.
A MODIFIED TECHNIQUE FOR IN SITU MICRO-SAMPLING OF MAMMUTHUS TOOTH ENAMEL FOR STABLE ISOTOPE ANALYSIS KAYLA KOLIS1, CHRIS WIDGA2, PAUL COUNTRYMAN2, ALAN WANAMAKER3, J. DOUGLAS WALKER4, JOHN FELDMAN4 1 ILLINOIS
COLLEGE, 2 ILLINOIS STATE MUSEUM, 3IOWA STATE UNIVERSITY, 4UNIVERSITY OF KANSAS Illinois State Museum
Abstract: Recent work on the timing of proboscidean tooth enamel formation has led to advances in the use of stable isotopes to understand diet and behavior (Dirks et al 2012, Metcalfe et al 2011). Although these techniques continue to yield excellent insights into the diet and behavior of mastodons and mammoths (e.g., Metcalfe and Longstaffe 2012), sampling techniques typically require the removal of (sometimes large) sections of an enamel ridge-plates. We have modified this technique to accommodate in situ micro-sampling of complete specimens. Precise movement of the specimen in three dimensions is controlled by a Newmark NSCG, 3-axis motion controller. A 4 cm diameter ball joint is mounted to this stage, and used to level the large metal plate (70 cm x 100 cm) that holds the specimen. The specimen is affixed to this plate with orthopedic thermoplastic. Thermoplastic softens above 70° C. Once heated, a cradle can be molded to conform to irregular specimen shapes and mounted to the metal plate using machine screws. A Proxxon 50/E drill equipped with a 500 um diameter burr bit is affixed to a stationary arm above the 3-axis stage. Proxxon 50/E drill
Each specimen is sampled in multiple sets which each correspond to 1 cm of tooth growth. Each 1 cm set consists of ten samples and each sample consists of a series of vertical sub-samples. Each 100 um deep sub-sample is individually collected and processed through the entire thickness of the enamel. All enamel powder is collected with a micro-pipetter in de-ionized water to 1) maximize sample recovery, and 2) lubricate the bit. The sample nearest the enamel-dentin junction is analyzed for δ13C, δ18O or 87Sr/86Sr for paleoecological information. Although this technique is both time- and labor-intensive, it is minimally invasive and is capable of sampling enamel growth structures at a high resolution.
Steel Plate mounted on ball joint
Y-axis control X-axis control
Newmark 3-axis stage
Z-axis control
~5 years of growth
Figure 3. Schematic of a Sample “Set”. Approximately 10 samples/yr of growth. Figure 1. Model of custom-built micromill for micro-sampling large specimens.
Introduction: Stable isotope signatures in incremental growth tissues such as tooth enamel are useful for time-series analyses of diet (δ13C), water budget (δ18O), and geologic origin (87Sr/86Sr). However, the geometry of proboscidean tooth formation requires high precision sampling at a micro-scale to avoid extensive time-averaging of the isotopic signal.
Figure 2. Sampling plan for Mammuthus enamel ridge-plate.
Jones Spring, MO
Jones Spring
Figure 4. Location of Jones Spring, MO. Analyzed 87Sr/86Sr surface samples (grasses) contributing to isoscape of United States Midwest, N=177
Method: 1) The specimen to be sampled is mounted to a steel plate (70 x 100 mm) using orthopedic thermoplastic. Thermoplastic softens when heated >70° C, at which point it conforms to irregular shaped specimens. The stage is leveled by a 4 cm diameter ball joint mounted to a system of computercontrolled linear and vertical positioners (Newmark NSC-G, 3-axis motion controller). This system provides accurate and precise movement of a specimen (within ~20 um) below a stationary drill. A Proxxon 50/E drill equipped with a 500 um diameter burr bit is affixed to a stationary arm above the 3-axis stage.
References: Coplen, T. B., & Kendall, C. 2000. Stable hydrogen and oxygen isotope ratios for selected sites of the US Geological Survey's NASQAN and benchmark surface-water networks (No. USGS-OFR-00-160). United States Geological Survey. Dirks, W., Bromage, D.G., Agenbroad, L.D., 2012. The duration and rate of molar plate formation in Palaeoloxodon cypriotes and Mammuthus columbi from dental histology. Quaternary International 255, 79-85. Metcalfe, J. Z., Longstaffe, F. J., Ballenger, J. A., Haynes, C. V., 2011. Isotopic paleoecology of Clovis mammoths from Arizona. Proceedings of the National Academy of Sciences, 108(44), 17916-17920. Metcalfe, J. Z., Longstaffe, F. J., 2012. Mammoth tooth enamel growth rates inferred from stable isotope analysis and histology. Quaternary Research 77(3), 424-432. Metcalfe, J. Z., Longstaffe, F. J., Hodgins, G. 2013. Proboscideans and paleoenvironments of the Pleistocene Great Lakes: landscape, vegetation, and stable isotopes. Quaternary Science Reviews, 76, 102-113.
Results:
1) δ18O results show periodic negative excursions strongly suggestive of seasonal variability in meteoric precipitation. These data vary by 9-10‰ (SMOW) per oscillation indicating a reliance on un-buffered water sources such as small creeks and streams. Summer δ18Owater values are similar to modern values for streams in the central Ozarks (Coplen and Kendall 2000). However, winter values are very negative, similar to January δ18Oprecipitation in southern Canada. 2) δ13C results are less variable than δ18O, and range from -2 to -4‰ (PDB). This animal had a diet dominated by C4 grasses, although the consumption of minor amounts of C3 browse is also possible. 3)
Cold season
4) Enamel 87Sr/86Sr samples were analyzed at the University of Kansas Isotope Geochemistry lab on a fully-automated VG Sector variable 6collector TIMS system with a 10-sample turret. High-precision Sr and Nd analyses are routine; typical external and internal precisions for these elements are +/- 20ppm. Rb-Sr chemistry follows traditional procedures, with dissolution using HF-HNO3 acid in sealed teflon vessels and elemental separations using HCl elution on cation exchange columns.
Figure 5. Preliminary 87Sr/86Sr isoscape for the Midcontinent showing 1st-order variability. Red contours are values represented in the Jones Spring tooth.
Figure 6. Mandible of Mammuthus jeffersonii, Jones Spring, MO, unit c2, Early Wisconsinan.
Cold season
3) Light isotope samples (δ13C and δ18O) were measured in the Stable Isotope Laboratory at Iowa State University on a Finnigan MAT Delta Plus XL mass spectrometer in continuous flow mode connected to a Gas Bench with a CombiPAL autosampler. Reference standards (NBS-18, NBS-19, LSVEC) were used for isotopic corrections, and to assign the data to the appropriate isotopic scale. At least one reference standard was used for every five samples. The combined uncertainty (analytical uncertainty and average correction factor) for δ13C is ± 0.12‰ (VPDB) and δ18O is ± 0.28‰ (VPDB), respectively. δ18Ocarbonate values were subsequently converted to δ18Owater using the two-step process described in Metcalfe et al 2013.
Un-analyzed samples , N=181
Cold season
2) A single sample consists of multiple 100 um deep sub-samples throughout the thickness of the enamel. The sub-sample (~100 um thick x 5 mm long) nearest the enamel-dentin junction is analyzed for stable isotopes. All samples are collected as suspended powder in DI H2O, centrifuged, decanted and air-dried. Between sub-samples the specimen is flushed with DI H2O and dried with compressed air. Samples did not receive standard acid/base pretreatment due to their small size.
N
(nonlocal, >300 km)
87Sr/86Sr
values of the Jones Spring Mammuthus tooth enamel range from 0.715 to 0.717. These values are significantly different from the local signature, which is 0.710 to 0.712 but similar to grass samples collected from the Precambrian core of the Ozark uplift (0.715-0.720), >300 km away.
Downtooth position, 1 sample/mm
Figure 7. Results of stable isotope analyses. Isolated enamel ridge-plate from Jones Spring, MO, ~35,000 BP.
Conclusions: 1) This technique is an effective way of collecting very small enamel samples from mammoth teeth for stable isotope analyses. Although the technique is both time- and labor-intensive, it is minimally invasive and is capable of sampling enamel growth structures at a high resolution. 2) The Mammuthus specimen from Jones Spring, MO (~35,000 BP) had a diet dominated by C4 plants throughout most of the year. Summer temperatures were broadly similar to modern, but winters were much colder. This animal did NOT migrate seasonally, although between the period of enamel formation and death, this individual moved >300 km.
