基于线粒体控制区的序列变异分析青海东部甘肃鼢鼠遗传多样性

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

甘肃鼢鼠(Myospalax cansus)是一种终年营地下独居生活的小型掘土类动物。本文通过测定mt DNA的控制区部分序列(530 bp)变异,分析青海东部地区8个甘肃鼢鼠地理种群遗传多样性与遗传结构。158个样本共发现26个变异位点,定义了39种单倍型,整体的平均单倍型多样性高(h=0.953 2)、核苷酸多样性低(π=0.006 36)。歧点分布和中性检验均说明青海东部甘肃鼢鼠种群在历史上存在着快速扩张的事件。基于邻接法构建的网络关系图中,单倍型呈星状分布,没有按地理位置形成对应类群。基因流(Nm)数据显示多数地理种群间基因交流贫乏,AMOVA结果显示种群内与种群间遗传变异分别为48.82%和51.18%,遗传分化明显。IBD分析表明,甘肃鼢鼠的遗传分化与地理距离呈正相关,说明距离隔离对甘肃鼢鼠种群分化具有重要作用。甘肃鼢鼠的这种遗传多样性与种群遗传结构特点,可能是地下生活方式靠挖掘迁移带来的较小扩散能力的结果。

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.