To assess the germplasm genetic diversity of Zacco platypus populations in the western Wuling

Mountains and Shennongjia Forestry District, we analyzed the genetic diversity and structure of seven Z. platypus

populations using mitochondrial Cyt b sequences and microsatellite DNA (SSR) markers. Our results revealed

144 variant sites and 33 haplotypes among 185 Cyt b sequences from the seven populations. The haplotype

diversity (H_d) ranged from 0.000 to 0.887, and the nucleotide diversity (π) ranged from 0.000 to 0.275. The

neighbor-joining (N_J) phylogenetic tree and median-joining network of Cyt b haplotypes grouped the seven

populations into three branches: Jiangkou1/Jiangkou2 (JK1/JK2), Shennongjia (SNJ), Xishui/Chishui/Wuchuan/

Nanchuan (XS/CS/WC/NC). Analysis of molecular variance (AMOVA) indicated significant genetic differentiation

among the seven populations and among the three branches. Analyses based on 11 microsatellite DNA loci showed

that the mean number of alleles (N_A) in the seven populations of Z. platypus was 3.66, the mean Shannon’s

information index (I) was 0.689, the mean observed heterozygosity (H_O) was 0.315, the mean expected

heterozygosity (H_E) was 0.354, and the mean polymorphic information content (PIC) was 0.409. The genetic

differentiation index (F_ST) among the populations varied from 0.060 to 0.547, with significant genetic

differentiation among the populations (P<0.05). The UPGMA phylogenetic tree and PCoA showed that the seven Z.

platypus populations clustered into four clades. STRUCTURE clustering analysis revealed that the seven

populations of Z. platypus could be classified into four genetic groups. AMOVA showed significant genetic

differentiation among the seven populations and among the four genetic groups of Z. platypus. Hence, the wild Z.

platypus populations in the western Wuling Mountains and Shennongjia Forestry District exhibit low genetic

diversity and significant genetic differentiation. Strengthening the protection of the riverine ecology and

environment is crucial to ensure the sustainable utilization of wild germplasm resources of Z. platypus.