中国狼不同地理种群遗传多样性及系统进化分析

Genetic diversity and phylogenetic analysis of wolves from different geographical regions of China

为了解中国狼不同地理种群遗传多样性及系统发育情况,从中国境内狼的主要分布区青海、新疆、内蒙古和吉林4个地区采集样品,用分子生物学技术手段成功地获得44个个体线粒体DNA控制区第一高变区(HVRⅠ)序列和40个线粒体Cyt b部分序列。线粒体控制区HVRⅠ共检测到51个变异位点,位点变异率为8.76%;线粒体Cyt b部分序列发现31个变异位点,位点变异率为5.33%,未见插入及缺失现象,变异类型全部为碱基置换。共定义了16个线粒体HVRⅠ单倍型,其中吉林与内蒙种群存在共享单倍型,估计这两地间种群亲缘关系较近。4个地理种群中新疆种群拥有较高的遗传多样性(0.94)。中国狼种群总体平均核苷酸多态性为2.27%,与世界其他国家地区相比,中国狼种群拥有相对较高的遗传多样性。通过线粒体HVRⅠ单倍型构建的系统进化树可以看出,中国狼在进化上分为2大支,其中位于青藏高原的青海种群独立为一支,推测其可能长期作为独立种群进化。基于青海种群与新疆,内蒙种群的线粒体Cyt b遗传距离,推测中国狼2个世系可能在更新世冰川时期青藏高原受地质作用急速隆起后出现分歧,分歧时间大约在1.1 MY前。

Wolves were once widely distributed throughout China. The number of Chinese wolves has drastically declined in recent years as a result of environmental destruction and habitat loss. At present,wolves in China are mainly distributed in sparsely populated western and northern regions of the country. To protect this species,an understanding of genetic diversity and phylogenetic relationships of wolf populations in China is necessary. In this study,we analyzed the partial mitochondrial DNA（ mt DNA） D-loop region and the Cyt b（ Cytochrome b） gene of wolves originating from four distinct geographical populations in Xinjiang,Qinghai,Inner Mongolia,and Jilin. Using standard molecular biology techniques,we generated sequences of the mt DNA control region hypervariable region I（ HVR I） from 44 wolves,and sequences of the Cyt b gene from 40 wolves. The resulting 582-bp HVR I sequence alignment contained 51 variable sites,which corresponded to8. 76% polymorphic loci. Similarly,31 variable sites,all base substitutions,were identified in the aligned Cyt b sequence data set. We detected 16 haplotypes among the 44 HVR I gene sequences. The Inner Mongolian population and the population from Jilin were found to share the same HVR I haplotype. When this data set was combined with previouslygenerated Chinese wolf sequences available in Genbank,a total of 28 haplotypes from approximately 91 Chinese wolves were identified. To explore the genetic diversity of these geographic populations,we calculated nucleotide diversity and haplotype diversity from the HVR I sequences. The population from Xin Jiang was found to have the highest genetic diversity. When we compared these results with previously reported nucleotide diversities of wolves from other countries and regions,we found that Chinese wolves had the highest nucleotide diversity. In the phylogenetic tree generated using the HVR I haplotypes,Chinese wolves are primarily divided into two lineages. The first lineage comprises haplotypes of wolf populations from the Qinghai-Tibet Plateau and surrounding areas. There is no obvious relationship with geographical structure among the remaining wolf haplotype groups. Considering their high level of genetic diversity and restricted geographic distribution,we speculate that the Qinghai-Tibet Plateau populations have evolved in isolation over a long period of time. To investigate the phylogenetic position of Chinese wolves,we downloaded 123 mt DNA partial control region sequences from Genbank,which were obtained from wolves primarily distributed in Eurasia and North America. In the phylogenetic tree generated from these sequences,wolf populations from the Qinghai-Tibet Plateau are clearly separated from wolves from other regions of China. The results of this phylogenetic analysis suggest that wolf populations in the QinghaiTibet Plateau of China comprise an ancient lineage that is still extant in these higher elevation regions. Based on genetic distances calculated using Cty b sequence data,we estimate that the two lineages diverged about 1. 1 million years ago,corresponding to the time period during which the Qinghai-Tibet Plateau was suddenly uplifted by geological processes.Based on these results,we propose that the wolves of the Qinghai-Tibet Plateau and the widespread wolf Canis lupus chanco are not the same subspecies.