**2. Materials and Methods**

### *2.1. Study Areas and Materials*

We investigated a total of 30 *C. paliurus* populations across the major distribution areas in 10 provinces of China (Figure 1). These populations range from 290 to 1798 m a.s.l. in altitude, 24.46◦ N–32.42◦ N in latitude and 103.78◦ E–121.79◦ E in longitude. Leaf sampling was carried out in September 2014, because leaf nutrients are relatively stable at this stage [9]. The longitude, latitude and altitude were measured by GPS on the spot. The climate data of the populations was obtained from ClimateAP for the historical period 1991–2014, including mean annual precipitation (MAP), mean annual temperature (MAT), mean temperature in July (MTmax), mean temperature in January (MTmin) and mean annual frost-free period (MAF) (Table 1, http://climateap.net/) [35,36]. The detailed method of sample collection and pre-treatment was described as Liu et al. (2018) [24]. Briefly, each sample consisted of about 400 g mature leaves collected from six to thirty average-sized trees (>20 years) in each population. All samples were dried to a constant weight at 70 ◦C and ground into fine powder in the lab. Soil samples (0–20 cm, *n* = 3) were collected from each location at the same time, sealed in polythene bags and brought back to laboratory.

**Figure 1.** Natural distribution of *C. paliurus* (colored area in line box) and locations of 30 populations sampled (red triangle).



MAP: mean annual precipitation (mm), MAT: mean annual temperature (◦C), MTmax: mean temperature in July (◦C), MTmin: mean temperature in January (◦C), MAF: mean annual frost-free period (d). Sample ID was defined from the abbreviation of the location name. Different letters indicate significant differences (*p* <0.05 by Tukey's test) between populations.
