2.2.1
Sample selection
Caribou, elk and moose specimens were obtained from the Edmonton area (Edmonton), the Klondike area (Klondike), Herschel Island (Hershel Island) and the Selawik Wildlife Refuge and Surround Areas (Selawik) (Fig. 2.1). Information about sample location and age is summarized in Appendix A. Stable isotopic data for antlers that were serially sampled (sampled several times along the growing length) are listed in Appendix B. The dimensions of the antler are also listed in Appendix B, as well as whether the antler was broken along the length that that it was sampled, or was a complete specimen. All stable isotopic data, including previously reported data for antler and bone for the North Slope (Mann et al., 2013) and Selawik (Druckenmiller, 2008), are also summarized in
Appendix C.
2.2.2
Collagen extraction
For specimens from which only a single sample was taken, this tissue was removed using a Dremel® cutting wheel. Sampling along the length of an antler specimen was performed using a 0.625 cm drill core attached to a drill press. These samples were taken every 10 cm along one side of one beam of the antler, with the base of the antler designated as 0 cm (Fig. 2.2b). Cancellous bone was removed from bone samples to allow for sampling of purely cortical bone. Outer antler tissue was preferentially sampled when the antler was thick. The surfaces of antler and bone were removed using a carbide burr attachment to the Dremel®, and the new surface then washed with deionised water and dried at room temperature.
Lipid extraction was performed on a subset of seven samples using a modified Bligh and Dyer method (Bligh and Dyer, 1959). Prior to collagen extraction, these samples were treated three times each with a 2:1 chloroform:methanol solution (v:v) for 15 minutes.
The samples were then dried at room temperature. Comparison of unextracted versus lipid-extracted fractions showed that the isotopic compositions obtained from the two approaches differ by a maximum of ± 0.1 ‰ (SD) for both 13
C and 15N (Appendix D). For this reason, unextracted and lipid-extracted fractions are considered to be isotopically equivalent in any discussion that follows.
Collagen extraction was performed at room temperature following the modified Longin method (see method in Metcalfe et al., 2010). Samples were dissolved for 24 hours in 0.25 M and subsequently in 0.5 M HCl with the acid changed every 1-3 days until the samples were demineralized. Samples were then rinsed 3 times with deionized water. Humic substances were removed by treatment with 0.1 M NaOH for 20 minutes at room temperature, which was repeated until the liquid remained colourless. The samples were then rinsed seven times with deionized water, and the pH was adjusted to less than 3. They were then placed in a 90°C oven for approximately 16 hours to solubilise the collagen. The solubilised collagen was decanted and dried at 90°C before being weighed for analysis.
2.2.3
Stable isotope measurements
The carbon and nitrogen isotopic compositions of the collagen were measured using a Costech elemental combustion system (ECS 4010) attached to a Thermo-Scientific Delta V stable isotope ratio mass spectrometer (IRMS) operated in continuous-flow mode. The results are presented in Appendices B (serial samples) and C (single samples). The samples were measured over a total of eleven analytical sessions. The carbon isotopic data were calibrated to VPDB using a two-point scale anchored by either NBS-22 (± 0.0 ‰ one standard deviation (SD), n = 24; accepted 13
C = –30.03 ‰; Coplen et al., 2006), and IAEA-CH-6 (± 0.1 ‰ SD, n = 37; accepted 13C = –10.45 ‰; Coplen et al., 2006) or USGS-40 (± 0.1 ‰ SD, n = 44; accepted 13C = –26.39 ‰; Coplen et al., 2006) and USGS-41 (± 0.2 ‰ SD, n = 35; accepted 13C = +37.63 ‰; Coplen et al., 2006). The nitrogen isotopic data were calibrated to AIR using a two-point scale anchored by USGS- 40 (± 0.1 ‰ SD, n = 43; accepted 15N = –4.52 ‰; Qi et al., 2003) and either IAEA-N2 (± 0.2 ‰ SD, n = 35; accepted 15N = +20.39 ‰; Qi et al., 2003) or USGS-41 (± 0.4 ‰
SD, n = 33; accepted 15N = +47.57 ‰; Qi et al., 2003). These standards were also used for calibration of the carbon and nitrogen contents and C/N ratio of each sample. When these standards were not used in the calibration curve, they were used measured as unknowns. IAEA-CH-6 (measured 13C = –10.5 ‰), USGS-40 (measured 13C = –26.4 ‰), USGS-41 (measured δ13
C = +37.8 ‰, measured 15N = +47.0 ‰), IAEA-N1 (measured 15N = +0.5 ‰; accepted 15N = +0.43 ‰; Qi et al., 2003) and IAEA-N2 (measured 15N = +20.4 ‰) all had similar isotopic compositions to their accepted values. Every analytical session also included an internal keratin laboratory standard (MP Biomedicals Inc., Cat. No. 90211, Lot No. 9966H) for which the following average results (SD) were obtained (n = 72): δ13C = –24.1 ± 0.1 ‰, δ15N = +6.4 ± 0.1 ‰, C = 48 ± 2 wt.%, N = 15 ± 1 wt.%, and atomic C/N ratio = 3.7 ± 0.2. These compare well with accepted values of δ13C = –24.0 ‰, δ15N = +6.4 ‰, C = 46.8 wt.%, N = 14.6 wt.% and atomic C/N = 3.7. A subset of samples (n = 25) were analyzed in duplicate or triplicate; reproducibility (SD) ranged from ± 0.0 to ± 0.2 ‰ for δ13C, and from ± 0.0 to ± 0.3 ‰ for δ15N, with an average for both of ± 0.1 ‰.
2.2.4
Radiocarbon dating
Radiocarbon dates were obtained for a subset of samples. Collagen was extracted, combusted, graphitized and dated at the University of Arizona Accelerator Mass
Spectrometry (AMS) Laboratory. Dates are presented as uncalibrated radiocarbon years before present (1950), and are listed in Appendices A, B and C alongside previously published dates (Druckenmiller, 2008; Kristensen and Heffner, 2011; Mann et al., 2013; Meiri, 2010; Zazula, pers. comm., 2015).
2.2.5
Mathematical treatment
Antler-bone pairs were tested to determine if the populations were statistically identical using the Mann-Whitney-Wilcoxon test, as this test does not assume parametric
populations (Bauer, 1972). Carbon and nitrogen isotopic compositions were tested separately, and the data sets were assumed to be independent. A p-value of 0.05 was selected. The test was run in R version 3.1.1 (R Core Team, 2014) using the R Studio interface version 0.98.1083, and the results are summarized in Table 2.1.
The Herschel Island caribou bone and antler samples postdate the Industrial Revolution (post-bomb period; Appendix A). A Suess effect correction was therefore made for the lower carbon isotopic composition of the atmosphere resulting from the burning of fossil fuels following the method of Long et al. (2005). Because the exact date for this
specimen is not known, the mid-point (1987) between the start of the post-bomb time period (1964) and the time of collection (2009) was used to make the Suess effect correction.