*2.4. Genetic Diversity and Population Structure*

The genetic diversity parameters of MJ and ZB were calculated by Stacks-2.55 [15], including observed heterozygosity (Ho), expected heterozygosity (He), genetic differentiation (Fst), the inbreeding coefficient (Fis), and nucleotide diversity (π).

Genetic variation was determined by population structure analysis and genetic differentiation assessment. The R package poppr [17] was used to calculate a distance matrix and draw a heatmap [18]. The R packages igraph and poppr were used to cluster multi-locus genotypes (MLGs) to generate minimum spanning networks (MSNs) [19]. A phylogenetic tree was constructed by PHYLIP (version 3.695, Phylip Team, Washington, DC, USA) software [20] to analyze the relationships between samples. Compared with Structure, Admixture software can be used to estimate the ancestral components of individuals faster [21]. Therefore, ADMIXTURE (version 1.3.0, David H. Alexander et al., Los Angeles, CA, USA) software [22] was used to consider various numbers of putative clusters (ranging from 1 to 10) and compute the ten-fold cross-validation error to select the most suitable number of clusters [23]. The PCA module of GCTA (version 1.93.2, Jian Yang et al., Hangzhou, Zhejiang, China) software [24] was used for principal component analysis (PCA).

#### **3. Results**

#### *3.1. Genomic Data Statistics and SNP Discovery*

Raw reads were obtained by RAD-seq of 30 individuals of *L. kasmira*. After trimming barcodes and filtering, a total of 286,264,230 high-quality clean reads were obtained from 30 individuals. The number of clean reads per individual ranged from 6,672,063 to 21,514,652, with an average of 9,542,141. In all the libraries, the GC content was stable between 39% and 40%, and the Phred score was 20 ≥ 93.64% (Table 2). A total of 9822 high-quality SNPs were screened out for further analysis after the selection of SNPs with an MAF < 0.05 and in HWE.


**Table 2.** Statistics for the genomic data obtained from 30 *L. kasmira* individuals.
