DOI: 10.5061/dryad.sqv9s4n7c
title: Diet composition and selection of Père David's deer in Hubei Shishou Milu National Nature Reserve, China

description: Hubei Shishou Milu National Nature Reserve is an ideal place to restore
 the wild population of Père David's deer (Elaphurus davidianus).
 Understanding foraging ecology and diet composition is essential for
 assessing population development or establishing long-term effective
 conservation measures for endangered species. However, little is known
 about the diet composition of Père David's deer and its diet
 selection mechanism. In this study, we used stable isotope technology to
 investigate the diet composition of Père David's deer according to
 various tissues (i.e., fur, muscle, liver, heart, and feces) and seasons,
 and evaluated the correlation between the nutrient composition of plants
 and diet composition. Bayesian isotope analysis showed that the autumn and
 winter diet estimated by fur and fecal samples indicated a diet dominated
 by C3 grasses (42.7 to 57.2%, mean), while the summer diet estimated by
 muscle and liver samples was dominated by C3 forbs (30.9 to 41.6%, mean).
 The Pearson correlation test indicated that the contribution of winter
 diet composition reflected by fur and fecal samples was associated with
 correlations with crude protein (r = 0.666, P &lt; 0.01) and soluble
 sugars (r = 0.695, P &lt; 0.01). The results indicated that crude
 protein and soluble sugars were important factors influencing the winter
 diet selection of Père David's deer. In the context of the current
 reintroduction facing many challenges, such as habitat fragmentation,
 wetland degradation, and human disturbance, comprehensively evaluating the
 diet selection mechanism of Père David’s deer under different resource
 specificities and temporal changes should be considered in the future.
description: Crude fat determination was
 determined according to the direct extraction method. Freeze-dried plant
 samples (500 mg) were dissolved in 10 ml of HCl for 50 min. Then, 10 ml of
 ethanol was added, and the fat was extracted with 25 ml of petroleum
 ether, mixed and shaken and the supernatant was collected. The supernatant
 was dried (105 °C for 2 h)
 and weighed to calculate the crude fat content.
 The C/N and N contents were
 determined using an elemental analyzer (Flash EA 1112HT, Thermo Fisher
 Scientific, USA) in the laboratory of the Food Inspection and Quarantine
 Center, Shenzhen Custom, China. The soluble sugar content was determined
 according to the anthrone -
 H<sub>2</sub>SO<sub>4</sub> method.
 A standard curve was established with
 glucose standards. Freeze-dried plant samples (250 mg) were extracted by
 adding 10 ml of distilled water in boiling water for 30 min, and the
 supernatant was mixed with ethyl anthranilate-concentrated
 H<sub>2</sub>SO<sub>4</sub>, shaken, and held in a
 boiling water bath for 1 min. The absorbance was measured at 630 nm by UV
 spectrophotometry. The crude protein content was determined by the
 Coomassie brilliant blue method. A standard curve was established with
 bovine serum protein standards. Freeze-dried plant samples (100 mg) were
 weighed and extracted with distilled water for 2 h at room temperature,
 the supernatant was mixed with Kaumas Brilliant Blue G250, and the
 absorbance was measured at 595 nm with UV
 spectrophotometry. Condensed tannin (proanthocyanidins) was
 determined by the acid-butanol method. Freeze-dried plant samples (50 mg)
 were dissolved in 1 ml of methanol and mixed with 6 mL of 95% butan-1-ol
 and 5% concentrated HCl in
 10 mL test tubes. The tubes were sealed and placed in a water bath at 95
 °C for 1 h. The color was
 observed after cooling to room temperature. The alcoholysis under acidic
 conditions converts the extended units of condensed tannins into colored
 anthocyanins. The darker the color is, the higher the tannin
 concentration. After the pretreatments, the carbon and nitrogen
 stable isotope ratios of the samples were analyzed using an elemental
 analyzer (Flash EA 1112HT, Thermo Fisher Scientific, USA) coupled with an
 isotope ratio mass spectrometer (Delta V Advantage, Thermo Fisher
 Scientific, USA) in the laboratory of the Food Inspection and Quarantine
 Center, Shenzhen Custom, China. Approximately 0.200 mg (± 0.001 mg) of sample plant and
 tissues was weighed and inserted into 4 mm × 6 mm tin cups. The carbon and
 nitrogen stable isotope ratios of the sample were expressed in delta
 notation (difference between sample and standard) as parts per thousand
 (‰): δ
 = [(R<sub>sample</sub>/R<sub>standard</sub>)
 -1]×1000 where R is the abundance ratio of heavy isotopes
 to light isotopes in the sample.
 <sup>13</sup>C/<sup>12</sup>C and
 <sup>15</sup>N/<sup>14</sup>N. R<sub>sample
 </sub>is the measured isotope ratio;
 R<sub>standard</sub> is the isotope ratio of reference
 materials. All results are reported relative to atmospheric nitrogen as
 the standard for δ<sup>15</sup>N and to Cretaceous belemnite
 (Belemnitella Americana) from the Peedee Formation of South Carolina for
 δ<sup>13</sup>C. Laboratory standards (glycine and urea) were
 run every 12 samples to correct any instances of instrument drift. The
 analytical precision was ± 0.1‰ for <sup>13</sup>C and ± 0.2‰
 for <sup>15</sup>N.
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