基于cpDNA trnL-F序列的胡黄连保护遗传学研究

Conservative genetics of Neopicrorhiza scrophulariiflora based on cpDNA trnL-F

胡黄连为特产中国-喜马拉雅特有高山植物,作为常用中、藏药材,受到灭绝性采挖,作为濒危和二级保护植物亟待科学的保护。该研究以云南和西藏7个野生居群91个个体为材料,基于cpDNA trnL-F非编码序列测序分析胡黄连的遗传多样性和遗传结构,分析显著进化单元,确立优先保护居群并提出科学的保护策略。结果表明：胡黄连trnL-F序列长度为871～876 bp,根据序列的核苷酸变异共鉴定出5个单倍型,西藏占有2个单倍型,云南占有3个单倍型,西藏和云南2个地区的所有单倍型均不共享。胡黄连具有较低的单倍型多样性（Hd=0.434 19）和核苷酸多样性（Dij=0.004 66）。种群间分化度（Fst=0.864 520）和基因流（Nm=0.04）、居群间的遗传分化水平（G_（ST）=0.916）、AMOVA分析（0.78%的遗传变异发生在居群内,60.97%的遗传变异发生在地区内居群间,38.25%的遗传变异发生在地区间）均表明,胡黄连居群间存在明显遗传分化。多数一致性树将胡黄连划分为3个进化分支（Ⅰ、Ⅱ、Ⅲ）,这3个分支均与地理相关,分支Ⅰ分布于横断山区的4个居群,分支Ⅱ是分布于东喜马拉雅的一个居群,分支Ⅲ是分布于喜马拉雅中段的2个居群。3个分支分属于3个＂进化显著单元（ESU）＂。这3个ESU中白马雪山、茨中、定日、波密、聂拉木五个居群都需要保护,建议现阶段应优先保护的居群是云南白马雪山和西藏波密居群,以就地保护为主。

Neopicrorhiza scrophulariiflora （Scrophulariaceae）, a monotypic genus perennial species, is endemic to the Eastern Himalayas and the Hengduan Mountains region. It only distributes in Yunnan and Tibet in China, ranging from 3 600 m to 4 200 m in elevation. The long and creep rhizomes （Rhizoma Neopicrorhizae） are of high medicinal value and dysentery by traditional Chinese and Tibetan medicine. Mainly because of large-scale acquisitions activity, natural popu-lations of this species have suffered rapid declines and now it is classified as an endangered species under second catego-ry of key protected wild plants in China. In order to protect the decreasing natural genetic resources of N. scrophulariiflo-ra, in this study, the chloroplast DNA （cpDNA） trnL-F noncoding sequence was used to estimate the genetic diversity and genetic structure and the evolutionary significant units （ESU） were proposed. A total of 91 individuals of N. scrophu-lariiflora were collected from seven populations, covering almost all areas of its distribution ranges. Of these seven popu-lations, four were from Yunnan Province and three populations were from Tibet. The statistical results showed that the haplotype sequences length varied from 871 bp to 876 bp. A total of five haplotypes were detected based on trnL-F nucle-otide variation. Yunnan contains three haplotypes and Tibet contains two. However, none of common haplotypes were shared between the populations from Yunnan and Tibet. A normal low level of genetic diversity （Hd = 0.434 19） and nucleotide diversity （Dij = 0.004 66） were identified at the species level. A high level of genetic differentiation （0.96） among populations was revealed. AMOVA results from chloroplast data indicated that 0.78% of the genetic variation was partitioned within population, 60.97% among populations within groups, and 38.25% among groups under the condition that N. scrophulariiflora was divided into two groups including Yunnan and Tibet. The U-statistic test for phylogeographi-cal structure showed that NST was significantly higher than GST（NST 〉GST , P 〈 0.01）, which suggested a distinctly phylo-geographical pattern The gene flow （Nm） was extremely low with only 0.04. The higher NST than GST（P〈0.01） suggested a distinctly phylogeographical pattern. Conjoint Fst （0.864 520）, gene flow, GST and AMOVA results all indicated a sig-nificant high level of genetic differentiation among populations, which could be a consequence of the limited gene flow caused by geographic isolation among populations. Phylogenetic analysis of the haplotypes sequences identified three ten-tative clades （Ⅰ,Ⅱand Ⅲ） according to Majority-rule consensus tree. All of which had distinct geographic range： CladeⅠcomprised four populations （CZ, YZ, SN, BM） which were located at the Hengduan Mountains region; CladeⅡcom-prised one population （BMI）, which was located at the Eastern Himalayas region; and Clade Ⅲ comprised two popula-tions （DR, NLM） located at the Central Himalayas region. Based on the phylogenetic analyses and uniqueness of the populations, three evolutionary significant units （ESU） were identified and conservation strategies were discussed for this endangered species. Baimaxueshan and Cizhong, Bomi, Nielamu and Dingri populations respectively concluded in the three evolutionary significant units and the five populations all contained special haplotypes. Based on these findings, all the populations should be protected. However, in consideration of the actual distribution of every population, Baimax-ueshan population from Yunnan and Bomi population from Tibet should be given priority for conservation and in situ con-servation should be the ideal implement.