Stable isotope analysis
To estimate the relative dietary contribution of crops in deer, nitrogen stable isotope analysis of bone collagen was performed. Bone collagen has a relatively slow turnover rate and therefore provides dietary information spanning several years or the lifetime of the individual (Stenhouse & Baxter 1979; Hedges et al., 2007; Koch, 2007). The δ15N values of crops such as vegetables and pasture grasses are much higher than those of wild plants (Hata et al., 2021). Thus, the δ15N values of bone collagen reflect the long-term history of the relative dietary contribution of crops in deer (Hata et al., 2021). Bone collagen was obtained by collagen extraction from bone fragments of the nasal turbinate obtained from the skull specimen. Collagen was extracted following the methods described by Hata et al. (2021). The samples were enclosed in a tin cup and combusted in an elemental analyzer (FlashEA1112; ThermoFisher Scientific, Waltham, MA) interfaced with an isotope ratio mass spectrometer (Thermo Scientific Delta V Advantage, ThermoFisher Scientific), which was used to analyze the nitrogen isotope ratios. Nitrogen isotope ratios are expressed in delta (δ) notation as parts per thousand (‰) relative toR standard as follows:
δ(‰)=[(R sample/R standard)-1] × 1000
where R sample andR standard are15N/14N ratios of the samples and the standard, respectively. The standard is the isotope ratio of atmospheric nitrogen (AIR). The analytical error for the isotope analysis was within 0.1‰ for δ15N.