Genetic Diversity and Population Structure of Gansu Zokors(Myospalax cansus) in Eastern Qinghai Inferred from Mitochondrial D-loop Sequences

Gansu zokors(Myospalax cansus) are small, solitary, subterranean rodents that inhabit the Loess Plateau in China. The genetic diversity and population genetic structure of M. cansus were determined by analyzing the sequence variation of a 530 bp fragment of the mitochondrial D-loop region in 158 natural individuals from eight locations in eastern Qinghai(Fig. 1, Table 1). Total DNA was extracted following the Joe and David method from 0.3 g of ethanol-fixed tissue. The D-loop sequence was amplified using primers FR(5′-TACCATCCTCCGTGAAACCA-3′) and RV(5′-CTAATAATAAGGCCAGGACC-3′), and PCR was performed in a 50 μl reaction volume. PCR products were purified and directly sequenced in both strands of the DNA using forward and reverse primers for amplification on a Mega BACE 1000 DNA Analysis System. Sequences were recorded on both strands with an overlap of 70%. The sequences were checked and aligned using Clustal Ⅹ with default settings and refined manually. Genetic diversity was estimated with Arlequin 3.10, using two different diversity indices: Haplotype diversity(h) and Nucleotide diversity(π). To estimate the extent of genetic differentiation among populations, pairwise genetic distances(FST) were calculated using Arlequin 3.10, and their significance was estimated by performing 10 000 permutations among individuals and populations. The same program was also used to calculate the gene flow(Nm), which is based on FST estimates, equivalent to the effective number of migrants between populations per generation. Analysis of molecular variance(AMOVA) was carried out to examine the significance of population structure. The phylogeographical pattern was examined by constructing haplotype networks using the median-joining network approach performed in Network 4.6.1.1. The hypothesis of neutral evolution was tested by Tajima′s D function test and Fu′s Fs-test with 10 000 permutations using Arlequin 3.10. Mismatch distributions of pairwise substitutional differences among haplotypes were also examined using Arlequin 3.10. The IBD 1.52 algorithm was used to test the correlation between genetic distance and geographical distance. The genetic distances were expressed by FST among populations, excluding the PA1 population which showed high genetic departure. The geographical distances(km) were estimated as straight-line distances between any pair of locations. The correlation between Nm and geographical distance was also estimated. Overall, 26 polymorphic sites were identified and 39 haplotypes were defined(Table 1 and 2). The number of haplotypes(Nh) and polymorphic sites(Np) were also shown in Table 1. Haplotype diversity varied from 0.257 1 to 0.874 5, while nucleotide diversity varied from 0.000 82 to 0.005 20(Table 1). Genetic diversity estimates revealed extensive haplotype diversity(0.953 2) and limited nucleotide diversities(0.006 36) in all populations(Table 1). Interestingly, the mismatch distribution analysis showed a unimodal pattern, reflecting a sudden population expansion(Fig. 3). A significantly large negative value of Fu′s FS was found when the total haplotypes were analyzed(FS =﹣22.10, P 0.01), which indicated a recent population expansion, as suggested by the mismatch distribution analysis. The estimated time of population expansion was 0.19﹣0.077 Mya, mostly corresponding to the interglacial period(0.17﹣0.021 Mya) before the last glacial maximum(LGM). The median-joining network was star-like throughout the studied range of M. cansus, showing that most individuals from different populations were highly interconnected with each other and they did not exhibit reciprocal monophyly(Fig. 2). All populations' pairwise FST values were statistically significant(P 0.01), ranging from 0.249 12(HZ1-LD2) to 0.775 70(DT1-PA1)(Table 3), indicating that all populations were significantly differentiated from one another. The values of Nm based on FST estimates in Table 4 showed that the levels of gene flow were relatively low. Among 28 values, only three were greater than 1 and the smallest was only 0.144 58. It was consistent with the significant population differentiation. The differentiation was confirmed by the percentage of variation among populations and within populations in AMOVA analysis, which were 51.18% and 48.82%, respectively(Table 5). The Mantel tests, excluding the PA1 population, revealed a significant negative correlation between genetic flow(Nm) and geographical distance(r =﹣0.598, P = 0.001, Fig. 3a), and a significant relationship between the genetic distance(FST) and geographical distance(r = 0.608, P 0.05, Fig. 3b) among populations, suggesting that distance isolation played a remarkable role in genetic differentiation. The data suggest that the weak dispersal ability of subterranean animals has shaped the peculiar population genetic diversity and genetic structure of Gansu zokors.