药用植物玄参的栽培起源、亲缘地理及东亚玄参系统发育研究
|
摘要
药用植物玄参(Scrophularia ningpoensis Hemsley)是玄参科玄参属多年生草本植物,为我国特产,是著名中药材“浙八味”之一。根药用,有滋阴降火,消肿解毒的功效,在浙江、四川、湖北等地广有栽培。目前,随着中药标准化的推行,中药的种质鉴定和栽培研究也受到了越来越多的关注。本研究通过ISSR (Inter-Simple Sequence Repeats)分子标记研究了栽培玄参的遗传多样性和群体遗传结构,并在此基础上开发了CAR(Sequence Characterized Amplified Region)分子标记,用于鉴定浙江种源的玄参。运用单亲遗传的cpDNA序列变异和双亲遗传的nrDNA的扩增长度多态性(Amplified Fragment Length Polymorphism, AFLP)探讨了玄参的栽培起源。基于ITS和cpDNA序列构建了东亚玄参的系统发育树,阐明了东亚玄参的属内种间关系,并在此基础上对确立的玄参复合种进行了亲缘地理研究。主要研究内容与结果如下：
1.栽培玄参遗传多样性、群体遗传结构及SCAR分子标记的研制
12条ISSR引物对玄参8个栽培群体及2个野生群体的分析结果表明两个野生群体天目山群体TM1W和磐安天网群体TWW的多态位点百分比较高,分别为48.39%和35.48%。栽培群体的多态位点百分比普遍较低,为9.68%-20.97%,其中磐安窈川乡YC,而磐安尚湖PA群体最低。AMOVA分析揭示了遗传多样性所占比率为：种群间76.67%,种群内23.33%,说明各个群体间分化明显。在根据Nei’s遗传距离利用UPGMA法所构建的10个玄参群体的遗传关系聚类图中,大部分栽培群体聚为一大支,其中磐安种源的栽培群体存在较近的亲缘关系,揭示了其道地性的遗传基础。
在ISSR实验的基础上,发现引物UBC874扩增结果中1500bp左右出现的条带是浙江群体所特有的,其他地区的玄参均不存在此条带。通过克隆测序,发现此片段长度为1306bp,针对此片段设计的特异性引物CC874u/CC874d在对所有地区的玄参进行检测后发现,只有浙江的玄参能扩增出单一、清晰的条带。因此这对特异性引物可以作为SCAR分子标记用于浙江种源玄参的鉴定。
2.栽培玄参的起源
进一步运用cpDNA psbA-trbH和trnL-F片段,结合AFLP分子标记对玄参14个栽培群体、14个野生群体进行了研究。结果表明在玄参28个群体379个个体中,两个叶绿体片段共检测到27个多态位点,共得到22种单倍型。其中,栽培群体仅有4种单倍型,每个栽培群体只拥有一种单倍型,没有多态性,单倍型多样性为0.486,核苷酸多样性为0.002；野生群体共有21种单倍型：单倍型多样性为0.919,核苷酸多样性为0.003。揭示仅有限的野生个体参与了玄参的栽培起源,多年的克隆繁殖使栽培玄参的遗传多样性水平已相当低,栽培群体基因流仅0.161,远低于野生群体(0.649)。单倍型MP严格一致树和网状支系图揭示栽培玄参的四个单倍型(E、A、B、P)与多个不同的野生群体共享,表明栽培玄参的多次多地起源,并发现江西冷水野生群体LSW是浙江玄参的最可能起源地。
AFLP中6个引物组合在315个个体中共扩增出289个稳定、清晰、可判读的条带,其中261个条带(90.31%)具有多态性。野生群体的遗传多样性水平普遍高于栽培群体(栽培群体的h=0.0076-0.0875；野生群体的h=0.0791-0.1614)。从扩增的多态条带数目来看,多态性条带的百分比在野生群体中为22.15%-50.87%,在栽培群体中为3.11%-27.68%。并且栽培玄参群体间分化大于野生玄参的：在栽培玄参中76.97%的遗传变异来自于群体间,23.03%的遗传变异来自于群体内(FST=0.7697)；在野生玄参中仅39.60%的遗传变异来自于群体间,60.40%的遗传变异来自于群体内(FST=0.3961)。
AFLP标记的PCoA分析、Neighbor-Joining分析和UPGMA分析结果一致表明栽培玄参遗传一致度较高,并在聚类图中聚为一支,其中与栽培玄参关系最近的是湖南平江HNW和江西冷水LSW两个野生群体,确定了这两个野生群体参与了玄参的栽培起源,而并非来自浙江的野生群体。最终揭示浙江产区的栽培玄参(PAC和XJC)以及引种自浙江的福建光泽栽培群体GZC已与其他地区的栽培玄参之间出现了明显的遗传分化,形成了浙玄参的道地性的基础。研究揭示野生玄参虽然分布较广,但资源同趋减少,认为部分群体拥有丰富的遗传多样性,是选育优质基因和优良品种的重要资源库,可以为玄参的栽培育种提供基础。
3.东亚玄参的系统进化
为进一步搞清药用玄参在东亚玄参属的地位,本研究运用ITS和cpDNA的trnQ-rps16、psbA-trnH和trnL-F三个片段对东亚分布的玄参属(包括中国21个种、同本5种、韩国3种),并以欧美玄参22种为对照(含15种北美玄参的序列来自Genebank)进行了分子系统学分析,结果表明东亚分布的玄参形成了四个谱系：广布种玄参(S. nignpoensis)、丹东玄参(S. kakudensis)、北玄参(Sbuergeriana)和双锯齿玄参(S. yoshimurae)形成的一个非常稳定的复合群；倍体的砾玄参组；分布中国华中-西北-西南的其它玄参类群；以及分布于韩国和日本的特有种(S. takesimensis、S. grayana、S. duplicata-serrata、S. musashiensis)与北美玄参和欧洲的S. nodosa构成的稳定单系。玄参、北玄参、丹东玄参和双锯齿玄参具有相似的化学成分,在分子系统树上表现为一个具很高支持率的单系类群,种与种之间界限不明确,缺乏分辨率,结合地理和形态特征认为东亚的这四个种很明显是一个多倍化过程中的类群,提出将这四个种定为典型的玄参复合种(S. ningpoensis species complex)。此外,首次根据分子系统树推测了东亚玄参的2个来源：一是可能来自北美,即日本-韩国-远东的特有种类,另一支来自欧洲-中亚(以S. canina-S. incisa为基础)。揭示玄参属在东亚形成了2支：包括一支华中-西南至西北分布的玄参类群和另一支华东-华北-东北-韩国-日本分布的药用玄参复合种。结合松散分子钟模型推断东亚玄参的四个谱系形成在52.12mya：华中-西北-西南分布的玄参的早期分化发生在41.25mya的第三纪始新世,喜玛拉雅山脉隆起之前；中亚-西北-华北-东北分布的砾玄参组几乎在同时分化形成(38.81mya)；到中新世(26mya左右),东亚-东北亚的玄参复合种开始出现,而韩国和日本的非复合种玄参应该是在10.7-7.25mya的上新世从北美迁移而来。
4.玄参复合种的亲缘地理研究
对确立的玄参复合种,运用cpDNA psbA-trbH和trnL-F片段,结合AFLP分子标记对其42个群体(玄参28个群体,北玄参7个群体,丹东玄参6个群体,双锯齿玄参1个群体)进行了亲缘地理研究。
cpDNA结果表明538个个体中共有34个多态位点,27种单倍型。其中玄参与北玄参共享单倍型2种；北玄参与丹东玄参共享单倍型1种；双锯齿玄参只拥有一种单倍型,与玄参共享。玄参与北玄参(分布于中国辽宁凌源的群体)共享的单倍型位于网络图的中心位置,根据溯祖理论推测其为祖先单倍型,结合染色体数目的统计(北玄参2n=30,玄参2n=90,丹东玄参2n=36)推测辽宁凌源的的北玄参可能是该复合种现存的祖先类型。江西冷水(LSW)和浙江天目山(TM2W)分别拥有5个和6个cpDNA单倍型,以及特有单倍型S10和S6,推测这2个群体很可能是玄参复合种在末次冰期的避难所。
AFLP分析揭示了玄参复合种的Nei's遗传多样性(h)：玄参、北玄参、丹东玄参和双锯齿玄参依次为：0.2202,0.1543,0.1512,0.0685。将所有群体按照玄参、北玄参、丹东玄参和双锯齿玄参分为四个组后进行分组分层AMOVA分析,仅检测到32.62%的遗传变异发生于组间,表明种间分化不显著。PCoA分析、Neighbor-Joining分析和STRUCTURE分析揭示了这四个种的关系：认为从核基因水平,除了双锯齿玄参外,玄参、北玄参和丹东玄参之间的分化是存在的,虽然这种分化程度有限(仅32.62%),但还是可以进行物种的区分；而相对保守、单亲遗传的cpDNA序列反应的群体历史信息表明,这四个种可能来自于同一个祖先,并且分化时间不长。综合cpDNA单倍型,核DNA序列和AFLP分析,推测玄参复合种的分化和迁移路线：玄参复合种是玄参属随着东亚古气候从干旱生境(现存二倍体砾玄参组植物)向湿润生境转变的结果。二倍体的北玄参是该复合种的根基,现存河北辽宁交界的凌源野生北玄参LYW群体是它的典型代表。认为该复合种的祖先在中新世早期(20mya左右),当时气候开始变温暖潮湿,北玄参向东迁移分化形成丹东玄参,向南迁移形成玄参和双锯齿玄参。
cpDNA单倍型和AFLP分析结果都表明台湾的双锯齿玄参与玄参的关系非常密切,来自同一个基因池,首次提出了对双锯齿玄参的分类地位进行修订,建议降为亚种置于玄参(S. ningpoensis Hemsley)种下。
Scrophularia ningpoensis Hemsley, known as one of the famous Traditional Chinese Medicine (TCM)--"Zhebawei", used in the Chinese Materia Medica (CMM) belonging to the family Scrophulariaceae, has a long history of widespread use in China. Root of this medicinal herb is used to treat inflammation, laryngitis, tonsillitis, abscesses of carbuncles, constipation and this species is widely cultivated in Zhejiang, Sichuan, Hubei Provinces and so on. Nowadays, with the standardization of TCM, domestication of medicinal herbs and molecular authentication become more and more important. In this study, firstly, we used the ISSR (Inter-Simple Sequence Repeats) molecular markers to detect the genetic diversity and population genetic structure in cultivated S. ningpoensis. Further more, one pair of SCAR (Sequence Characterized Amplified Region) primers was developed to identify S. ningpoensis originated from Zhejiang Province. Secondly, cpDNA sequences and AFLP (Amplified Fragment Length Polymorphism) markers were applied to study the domestication of S. ningpoensis. Thirdly, phylogenetic trees of East-Asian Scrophularia were constructed based on ITS and cpDNA sequences. Finally, the Phylogeography of S. ningpoensis species complex was studied by cpDNA sequences and AFLP molecular markers. There are4main conclusions of our research:
1. Genetic diversity and population genetic structure of cultivated S. ningpoensis and the development of SCAR markers
Twelve ISSR universal primers were applied in8cultivated population and2wild populations of S. ningpoensis, which revealed that two wild populations (TM1W, TWW) harbored higher polymorphic percentage (48.39%,35.48%) than cultivated populations (9.68%-20.97%). In cultivated populations, the polymorphic percentage of population YC and PA were the lowest. AMOVA analysis suggested that genetic variance occurred among populations is76.67%and that occurred within population is23.33%, which resulted from differentiation in populations. UPGMA dendrogram for ten populations of S. ningpoensis based on Nei's genetic distance revealed that cultivated populations grouped in one clade, within which populations from Pan'an County were closer than others.
Based on ISSR analysis, primer UBC874provided an approximately1500bp band unique to populations originated from Zhejiang Province. After gel purified, cloned and sequenced, this DNA fragment turned out to be1306bp. A pair of22bp SCAR primers (CC874u and CC874d) was designed for the amplification of this DNA fragment. All samples from different regions were amplified by SCAR primers CC874u and CC874d and PCR products show that a single band about1000bp was only in accessions originated from Zhejiang Province, which proved that CC874u and CC874d are useful for identifying S. ningpoensis originated from Zhejiang Province.
2. Domestication of S. ningpoensis
cpDNA (psbA-trbH and trnL-F) and AFLP molecular markers were used in14cultivated populations and14wild populations of S. ningpoensis.27polymorphic sites classified into22haplotypes were detected by cpDNA. There were only4haplotypes in cultivated popoulations and every population only harbored single haplotype:the haplotype diversity is0.486and nucleotide diversity is0.002; whereas21haplotypes were in wild populations:haplotype diversity is0.919, nucleotide diversity is0.003. Results of haplotype network showed that four haplotypes in cultivated populations shared with many wild populations were located in different clades, which suggested that S. ningpoensis might experience multiple origin events and LSW wild population in Jiangxi Province probably was involved in the origin of cultivated S. ningpoensis in Zhejiang Province.
Two hundreds eighty nine bands were amplified in315individuals by6pairs of AFLP markers and261bands were polymorphic (90.31%). Genetic diversity in wild populations were higher than cultivated populations (h in cultivated populations were from0.0076to0.0875; in wild populations were from0.0791to0.1614). As to the percentage of polymorphic bands, in wild populations were from22.15%to50.87%, in the cultivated were from3.11%to27.68%. Moreover, the differentiation in cultivated populations was larger than which in the wild:in cultivated populations, genetic variance occurred among populations were76.97%, genetic variance occurred in populations were23.03%; in the wild, those two statistical numbers were39.60%and60.40%respectively.
Results of PCoA、Neighbor-Joining and UPGMA analysis were consistent, which showed that all cultivated populations grouped together with two wild populations: HNW and LSW, located in Hunan and Jiangxi Provinces respectively. This clarified that the origin of cultivated S. ningpoensis were Hunan and Jiangxi Province, precisely, the HNW and LSW wild populations. That point contrary to current belief that Zhejiang Province was involved in the origin of cultivated S. ningpoensis. Moreover, cultivated populations from Zhejiang (PAC, XJC, GZC—introduced from Zhejiang) occurred in same cluster represented genetic identity, which can be the genetic evidence for geo-authentic S. ningpoensis from Zhejiang. Due to habitat deterioration and over exploitation, the wild genetic resources of S. ningpoensis have suffered rapid declines. But some native populations (LSW, JHW etc.) have high genetic diversity may contain special genes that are very important for the plant's growth and use.
3. Phylogeny of East Asian Scrophularia
Samples including21species of Scrophularia in China mainland,5species in Japan,3species in South Korea and7species in America-Europe were all surveyed by ITS and cpDNA fragments (trnQ-rps16, psbA-trnH, trnL-F), combined with sequences of15North American species which were download from Genebank. The molecular phylogenetic tree revealed that Scrophularia in East Asian were divided into four lineages:S. ningpoensis species complex including S. ningpoensis, S. buergeriana, S. kakudensis and S. yoshimurae; diploid clade with Sect. Tomiphyllum; the rest Scrophularia species distributed in China; South Korea-Japan-North America clade. S. ningpoensis, S. buergeriana, S. kakudensis and S. yoshimurae form a monophyletic clade with high bootstrap support and shared with very similar morphological characters can be treated as S. ningpoensis species complex. Our study also threw light on the origin of East Asian Scrophularia:Species in South Korea and Japan might be derived from North America; Species in China might be from Europe-Central Asian and then evolved into two lineages. Based on relaxed clock model in BEAST analysis, time dating was estimated:four lineages in East Asian formed at52.12mya; S. ningpoensis species complex rose at26mya in the Miocene epoch; other Scrophularia species in China were evolved at41.25mya in the Eocene epoch, the same time as diploid clade; South Korea and Japan groups were migrated from North America at10.7-7.25mya in the Pliocene epoch.
4. Phylogeography of S. ningpoensis species complex
Samples of S. ningpoensis species complex comprises28populations of S. ningpoensis,7populations of S. buergeriana,6populations of S. kakudensis and one population of S. yoshimurae. cpDNA fragments (psbA-trbH and trn"L-F) combined AFLP molecular markers were used for all the42populations.
Thirty four polymorphic sites and27haplotypes were detected in538individuals by cpDNA sequences. Two haplotypes were share by S. ningpoensis and S. buergeriana; one haplotype was share by S. buergeriana and S. kakudensis; S. yoshimurae only contained one haplotype which was share with S. ningpoensis. The haplotype shared by S. ningpoensis and S. buergeriana (population in Lingyuan, Liaoning) located in the center of the haplotype network. By Coalescence Theory, the haplotype in the center of the network can be the candidate of ancestors. Considered with chromosome numbers in Scrophularia (S. buergeriana:2n=30; S. ningpoensis:2n=90; S. kakudensis:2n=36), we indicated that LYW population of S. buergeriana might be the ancestor of S. ningpoensis species complex. Two wild populations LSW (Jiangxi Province) and TM2W (Mt. Tianmu) harbored five and six cpDNA haplotypes respectively, and contain S10and S6rare haplotypes. So we indicated the glacial refugia for S. ningpoensis were Jiangxi and Mt. Tianmu.
Results of AFLP analysis showed the Nei's genetic diversity in S. ningpoensis species complex were:S. ningpoensis was0.2202, S. buergeriana was0.1543, S, kakudensis was0.1512and S. yoshimurae was0.0685. Hierarchical analysis of molecular variance revealed that genetic variance occurred among species were32.62%; genetic variances occurred among populations in species were38.10%and that within populations were29.28%. Results of PCoA, Neighbor-Joining and STRUCTURE analysis all clarified the differentiation among three species:S. ningpoensis, S. buergeriana and kakudensis. So we confirmed the taxonomic status of these three species. But results from cpDNA sequences suggested that these three species must from the same ancestor. S. ningpoensis species complex might rise in early Miocene epoch when Weather changed from dry to warm and humid. At that time,s. buergeriana (wild population in Liaoning, China--LYW) considered as diploid migrated towards east to form S. kakudensis, and migrated towards south to form S. ningpoensis and S. yoshimurae.
S. yoshimurae and S. ningpoensis grouped together and came from the same gene pool. So these two species might be synonym and we suggested that S. yoshimurae can be treated as a subspecies of S. ningpoensis Hemsley.
1.栽培玄参遗传多样性、群体遗传结构及SCAR分子标记的研制
12条ISSR引物对玄参8个栽培群体及2个野生群体的分析结果表明两个野生群体天目山群体TM1W和磐安天网群体TWW的多态位点百分比较高,分别为48.39%和35.48%。栽培群体的多态位点百分比普遍较低,为9.68%-20.97%,其中磐安窈川乡YC,而磐安尚湖PA群体最低。AMOVA分析揭示了遗传多样性所占比率为：种群间76.67%,种群内23.33%,说明各个群体间分化明显。在根据Nei’s遗传距离利用UPGMA法所构建的10个玄参群体的遗传关系聚类图中,大部分栽培群体聚为一大支,其中磐安种源的栽培群体存在较近的亲缘关系,揭示了其道地性的遗传基础。
在ISSR实验的基础上,发现引物UBC874扩增结果中1500bp左右出现的条带是浙江群体所特有的,其他地区的玄参均不存在此条带。通过克隆测序,发现此片段长度为1306bp,针对此片段设计的特异性引物CC874u/CC874d在对所有地区的玄参进行检测后发现,只有浙江的玄参能扩增出单一、清晰的条带。因此这对特异性引物可以作为SCAR分子标记用于浙江种源玄参的鉴定。
2.栽培玄参的起源
进一步运用cpDNA psbA-trbH和trnL-F片段,结合AFLP分子标记对玄参14个栽培群体、14个野生群体进行了研究。结果表明在玄参28个群体379个个体中,两个叶绿体片段共检测到27个多态位点,共得到22种单倍型。其中,栽培群体仅有4种单倍型,每个栽培群体只拥有一种单倍型,没有多态性,单倍型多样性为0.486,核苷酸多样性为0.002；野生群体共有21种单倍型：单倍型多样性为0.919,核苷酸多样性为0.003。揭示仅有限的野生个体参与了玄参的栽培起源,多年的克隆繁殖使栽培玄参的遗传多样性水平已相当低,栽培群体基因流仅0.161,远低于野生群体(0.649)。单倍型MP严格一致树和网状支系图揭示栽培玄参的四个单倍型(E、A、B、P)与多个不同的野生群体共享,表明栽培玄参的多次多地起源,并发现江西冷水野生群体LSW是浙江玄参的最可能起源地。
AFLP中6个引物组合在315个个体中共扩增出289个稳定、清晰、可判读的条带,其中261个条带(90.31%)具有多态性。野生群体的遗传多样性水平普遍高于栽培群体(栽培群体的h=0.0076-0.0875；野生群体的h=0.0791-0.1614)。从扩增的多态条带数目来看,多态性条带的百分比在野生群体中为22.15%-50.87%,在栽培群体中为3.11%-27.68%。并且栽培玄参群体间分化大于野生玄参的：在栽培玄参中76.97%的遗传变异来自于群体间,23.03%的遗传变异来自于群体内(FST=0.7697)；在野生玄参中仅39.60%的遗传变异来自于群体间,60.40%的遗传变异来自于群体内(FST=0.3961)。
AFLP标记的PCoA分析、Neighbor-Joining分析和UPGMA分析结果一致表明栽培玄参遗传一致度较高,并在聚类图中聚为一支,其中与栽培玄参关系最近的是湖南平江HNW和江西冷水LSW两个野生群体,确定了这两个野生群体参与了玄参的栽培起源,而并非来自浙江的野生群体。最终揭示浙江产区的栽培玄参(PAC和XJC)以及引种自浙江的福建光泽栽培群体GZC已与其他地区的栽培玄参之间出现了明显的遗传分化,形成了浙玄参的道地性的基础。研究揭示野生玄参虽然分布较广,但资源同趋减少,认为部分群体拥有丰富的遗传多样性,是选育优质基因和优良品种的重要资源库,可以为玄参的栽培育种提供基础。
3.东亚玄参的系统进化
为进一步搞清药用玄参在东亚玄参属的地位,本研究运用ITS和cpDNA的trnQ-rps16、psbA-trnH和trnL-F三个片段对东亚分布的玄参属(包括中国21个种、同本5种、韩国3种),并以欧美玄参22种为对照(含15种北美玄参的序列来自Genebank)进行了分子系统学分析,结果表明东亚分布的玄参形成了四个谱系：广布种玄参(S. nignpoensis)、丹东玄参(S. kakudensis)、北玄参(Sbuergeriana)和双锯齿玄参(S. yoshimurae)形成的一个非常稳定的复合群；倍体的砾玄参组；分布中国华中-西北-西南的其它玄参类群；以及分布于韩国和日本的特有种(S. takesimensis、S. grayana、S. duplicata-serrata、S. musashiensis)与北美玄参和欧洲的S. nodosa构成的稳定单系。玄参、北玄参、丹东玄参和双锯齿玄参具有相似的化学成分,在分子系统树上表现为一个具很高支持率的单系类群,种与种之间界限不明确,缺乏分辨率,结合地理和形态特征认为东亚的这四个种很明显是一个多倍化过程中的类群,提出将这四个种定为典型的玄参复合种(S. ningpoensis species complex)。此外,首次根据分子系统树推测了东亚玄参的2个来源：一是可能来自北美,即日本-韩国-远东的特有种类,另一支来自欧洲-中亚(以S. canina-S. incisa为基础)。揭示玄参属在东亚形成了2支：包括一支华中-西南至西北分布的玄参类群和另一支华东-华北-东北-韩国-日本分布的药用玄参复合种。结合松散分子钟模型推断东亚玄参的四个谱系形成在52.12mya：华中-西北-西南分布的玄参的早期分化发生在41.25mya的第三纪始新世,喜玛拉雅山脉隆起之前；中亚-西北-华北-东北分布的砾玄参组几乎在同时分化形成(38.81mya)；到中新世(26mya左右),东亚-东北亚的玄参复合种开始出现,而韩国和日本的非复合种玄参应该是在10.7-7.25mya的上新世从北美迁移而来。
4.玄参复合种的亲缘地理研究
对确立的玄参复合种,运用cpDNA psbA-trbH和trnL-F片段,结合AFLP分子标记对其42个群体(玄参28个群体,北玄参7个群体,丹东玄参6个群体,双锯齿玄参1个群体)进行了亲缘地理研究。
cpDNA结果表明538个个体中共有34个多态位点,27种单倍型。其中玄参与北玄参共享单倍型2种；北玄参与丹东玄参共享单倍型1种；双锯齿玄参只拥有一种单倍型,与玄参共享。玄参与北玄参(分布于中国辽宁凌源的群体)共享的单倍型位于网络图的中心位置,根据溯祖理论推测其为祖先单倍型,结合染色体数目的统计(北玄参2n=30,玄参2n=90,丹东玄参2n=36)推测辽宁凌源的的北玄参可能是该复合种现存的祖先类型。江西冷水(LSW)和浙江天目山(TM2W)分别拥有5个和6个cpDNA单倍型,以及特有单倍型S10和S6,推测这2个群体很可能是玄参复合种在末次冰期的避难所。
AFLP分析揭示了玄参复合种的Nei's遗传多样性(h)：玄参、北玄参、丹东玄参和双锯齿玄参依次为：0.2202,0.1543,0.1512,0.0685。将所有群体按照玄参、北玄参、丹东玄参和双锯齿玄参分为四个组后进行分组分层AMOVA分析,仅检测到32.62%的遗传变异发生于组间,表明种间分化不显著。PCoA分析、Neighbor-Joining分析和STRUCTURE分析揭示了这四个种的关系：认为从核基因水平,除了双锯齿玄参外,玄参、北玄参和丹东玄参之间的分化是存在的,虽然这种分化程度有限(仅32.62%),但还是可以进行物种的区分；而相对保守、单亲遗传的cpDNA序列反应的群体历史信息表明,这四个种可能来自于同一个祖先,并且分化时间不长。综合cpDNA单倍型,核DNA序列和AFLP分析,推测玄参复合种的分化和迁移路线：玄参复合种是玄参属随着东亚古气候从干旱生境(现存二倍体砾玄参组植物)向湿润生境转变的结果。二倍体的北玄参是该复合种的根基,现存河北辽宁交界的凌源野生北玄参LYW群体是它的典型代表。认为该复合种的祖先在中新世早期(20mya左右),当时气候开始变温暖潮湿,北玄参向东迁移分化形成丹东玄参,向南迁移形成玄参和双锯齿玄参。
cpDNA单倍型和AFLP分析结果都表明台湾的双锯齿玄参与玄参的关系非常密切,来自同一个基因池,首次提出了对双锯齿玄参的分类地位进行修订,建议降为亚种置于玄参(S. ningpoensis Hemsley)种下。
Scrophularia ningpoensis Hemsley, known as one of the famous Traditional Chinese Medicine (TCM)--"Zhebawei", used in the Chinese Materia Medica (CMM) belonging to the family Scrophulariaceae, has a long history of widespread use in China. Root of this medicinal herb is used to treat inflammation, laryngitis, tonsillitis, abscesses of carbuncles, constipation and this species is widely cultivated in Zhejiang, Sichuan, Hubei Provinces and so on. Nowadays, with the standardization of TCM, domestication of medicinal herbs and molecular authentication become more and more important. In this study, firstly, we used the ISSR (Inter-Simple Sequence Repeats) molecular markers to detect the genetic diversity and population genetic structure in cultivated S. ningpoensis. Further more, one pair of SCAR (Sequence Characterized Amplified Region) primers was developed to identify S. ningpoensis originated from Zhejiang Province. Secondly, cpDNA sequences and AFLP (Amplified Fragment Length Polymorphism) markers were applied to study the domestication of S. ningpoensis. Thirdly, phylogenetic trees of East-Asian Scrophularia were constructed based on ITS and cpDNA sequences. Finally, the Phylogeography of S. ningpoensis species complex was studied by cpDNA sequences and AFLP molecular markers. There are4main conclusions of our research:
1. Genetic diversity and population genetic structure of cultivated S. ningpoensis and the development of SCAR markers
Twelve ISSR universal primers were applied in8cultivated population and2wild populations of S. ningpoensis, which revealed that two wild populations (TM1W, TWW) harbored higher polymorphic percentage (48.39%,35.48%) than cultivated populations (9.68%-20.97%). In cultivated populations, the polymorphic percentage of population YC and PA were the lowest. AMOVA analysis suggested that genetic variance occurred among populations is76.67%and that occurred within population is23.33%, which resulted from differentiation in populations. UPGMA dendrogram for ten populations of S. ningpoensis based on Nei's genetic distance revealed that cultivated populations grouped in one clade, within which populations from Pan'an County were closer than others.
Based on ISSR analysis, primer UBC874provided an approximately1500bp band unique to populations originated from Zhejiang Province. After gel purified, cloned and sequenced, this DNA fragment turned out to be1306bp. A pair of22bp SCAR primers (CC874u and CC874d) was designed for the amplification of this DNA fragment. All samples from different regions were amplified by SCAR primers CC874u and CC874d and PCR products show that a single band about1000bp was only in accessions originated from Zhejiang Province, which proved that CC874u and CC874d are useful for identifying S. ningpoensis originated from Zhejiang Province.
2. Domestication of S. ningpoensis
cpDNA (psbA-trbH and trnL-F) and AFLP molecular markers were used in14cultivated populations and14wild populations of S. ningpoensis.27polymorphic sites classified into22haplotypes were detected by cpDNA. There were only4haplotypes in cultivated popoulations and every population only harbored single haplotype:the haplotype diversity is0.486and nucleotide diversity is0.002; whereas21haplotypes were in wild populations:haplotype diversity is0.919, nucleotide diversity is0.003. Results of haplotype network showed that four haplotypes in cultivated populations shared with many wild populations were located in different clades, which suggested that S. ningpoensis might experience multiple origin events and LSW wild population in Jiangxi Province probably was involved in the origin of cultivated S. ningpoensis in Zhejiang Province.
Two hundreds eighty nine bands were amplified in315individuals by6pairs of AFLP markers and261bands were polymorphic (90.31%). Genetic diversity in wild populations were higher than cultivated populations (h in cultivated populations were from0.0076to0.0875; in wild populations were from0.0791to0.1614). As to the percentage of polymorphic bands, in wild populations were from22.15%to50.87%, in the cultivated were from3.11%to27.68%. Moreover, the differentiation in cultivated populations was larger than which in the wild:in cultivated populations, genetic variance occurred among populations were76.97%, genetic variance occurred in populations were23.03%; in the wild, those two statistical numbers were39.60%and60.40%respectively.
Results of PCoA、Neighbor-Joining and UPGMA analysis were consistent, which showed that all cultivated populations grouped together with two wild populations: HNW and LSW, located in Hunan and Jiangxi Provinces respectively. This clarified that the origin of cultivated S. ningpoensis were Hunan and Jiangxi Province, precisely, the HNW and LSW wild populations. That point contrary to current belief that Zhejiang Province was involved in the origin of cultivated S. ningpoensis. Moreover, cultivated populations from Zhejiang (PAC, XJC, GZC—introduced from Zhejiang) occurred in same cluster represented genetic identity, which can be the genetic evidence for geo-authentic S. ningpoensis from Zhejiang. Due to habitat deterioration and over exploitation, the wild genetic resources of S. ningpoensis have suffered rapid declines. But some native populations (LSW, JHW etc.) have high genetic diversity may contain special genes that are very important for the plant's growth and use.
3. Phylogeny of East Asian Scrophularia
Samples including21species of Scrophularia in China mainland,5species in Japan,3species in South Korea and7species in America-Europe were all surveyed by ITS and cpDNA fragments (trnQ-rps16, psbA-trnH, trnL-F), combined with sequences of15North American species which were download from Genebank. The molecular phylogenetic tree revealed that Scrophularia in East Asian were divided into four lineages:S. ningpoensis species complex including S. ningpoensis, S. buergeriana, S. kakudensis and S. yoshimurae; diploid clade with Sect. Tomiphyllum; the rest Scrophularia species distributed in China; South Korea-Japan-North America clade. S. ningpoensis, S. buergeriana, S. kakudensis and S. yoshimurae form a monophyletic clade with high bootstrap support and shared with very similar morphological characters can be treated as S. ningpoensis species complex. Our study also threw light on the origin of East Asian Scrophularia:Species in South Korea and Japan might be derived from North America; Species in China might be from Europe-Central Asian and then evolved into two lineages. Based on relaxed clock model in BEAST analysis, time dating was estimated:four lineages in East Asian formed at52.12mya; S. ningpoensis species complex rose at26mya in the Miocene epoch; other Scrophularia species in China were evolved at41.25mya in the Eocene epoch, the same time as diploid clade; South Korea and Japan groups were migrated from North America at10.7-7.25mya in the Pliocene epoch.
4. Phylogeography of S. ningpoensis species complex
Samples of S. ningpoensis species complex comprises28populations of S. ningpoensis,7populations of S. buergeriana,6populations of S. kakudensis and one population of S. yoshimurae. cpDNA fragments (psbA-trbH and trn"L-F) combined AFLP molecular markers were used for all the42populations.
Thirty four polymorphic sites and27haplotypes were detected in538individuals by cpDNA sequences. Two haplotypes were share by S. ningpoensis and S. buergeriana; one haplotype was share by S. buergeriana and S. kakudensis; S. yoshimurae only contained one haplotype which was share with S. ningpoensis. The haplotype shared by S. ningpoensis and S. buergeriana (population in Lingyuan, Liaoning) located in the center of the haplotype network. By Coalescence Theory, the haplotype in the center of the network can be the candidate of ancestors. Considered with chromosome numbers in Scrophularia (S. buergeriana:2n=30; S. ningpoensis:2n=90; S. kakudensis:2n=36), we indicated that LYW population of S. buergeriana might be the ancestor of S. ningpoensis species complex. Two wild populations LSW (Jiangxi Province) and TM2W (Mt. Tianmu) harbored five and six cpDNA haplotypes respectively, and contain S10and S6rare haplotypes. So we indicated the glacial refugia for S. ningpoensis were Jiangxi and Mt. Tianmu.
Results of AFLP analysis showed the Nei's genetic diversity in S. ningpoensis species complex were:S. ningpoensis was0.2202, S. buergeriana was0.1543, S, kakudensis was0.1512and S. yoshimurae was0.0685. Hierarchical analysis of molecular variance revealed that genetic variance occurred among species were32.62%; genetic variances occurred among populations in species were38.10%and that within populations were29.28%. Results of PCoA, Neighbor-Joining and STRUCTURE analysis all clarified the differentiation among three species:S. ningpoensis, S. buergeriana and kakudensis. So we confirmed the taxonomic status of these three species. But results from cpDNA sequences suggested that these three species must from the same ancestor. S. ningpoensis species complex might rise in early Miocene epoch when Weather changed from dry to warm and humid. At that time,s. buergeriana (wild population in Liaoning, China--LYW) considered as diploid migrated towards east to form S. kakudensis, and migrated towards south to form S. ningpoensis and S. yoshimurae.
S. yoshimurae and S. ningpoensis grouped together and came from the same gene pool. So these two species might be synonym and we suggested that S. yoshimurae can be treated as a subspecies of S. ningpoensis Hemsley.
引文
Adams KL, Palmer JD (2003) Evolution of mitochondrial gene content:gene loss and transfer to the nucleus. Molecular Phylogenetics and Evolution 29,380-395.
Ajibade SR, Weeden NF, Chite SM (2000) Inter simple sequence repeat analysis of genetic relationships in the genus Vigna. Euphytica 111,47-55.
Allaby RG, Fuller DQ, Brown TA (2008) The genetic expectations of a protracted model for the origins of domesticated crops. Proceedings of the National Academy of Sciences 105, 13982-13986.
Althoff DM, Gitzendanner MA, Segraves KA (2007) The utility of amplified fragment length polymorphisms in Phylogenetics:a comparison of homology within and between genomes. Systematic Biology 56,477-484.
Anthony, Combes, Astorga, et al. (2002) The origin of cultivated Coffea arabica L. varieties revealed by AFLP and SSR markers. Theoretical and Applied Genetics 104,894-900.
Arens P, Coops H, Jansen J, Vosman B (1998) Molecular genetic analysis of black poplar (Populus nigra L.) along Dutch rivers. Molecular Ecology 7,11-18.
Arroyo GR, Ruiz-Garci L, Bolling L, et al. (2006) Multiple origins of cultivated grapevine (Vitis vinifera L. ssp. sativa) based on chloroplast DNA polymorphisms. Molecular Ecology 15, 3707-3714.
Avise JC (1978) Variances and frequency distributions of genetic distance in evolutionary phylads. Heredity 40,225-237.
Avise JC, Giblin-Davidson C, Laerm J, Patton JC, Lansman RA (1979) Mitochondrial DNA clones and matriarchal phylogeny within and among geographic populations of the pocket gopher, Geomys pinetis. Proceedings of the National Academy of Sciences 76,6694.
Avise JC, Walker DE (1998) Pleistocene phylogeographic effects on avian populations and the speciation process. Proceedings of the Royal Society of London. Series B:Biological Sciences 265,457.
Badr A, Sch R, et al. (2000) On the Origin and Domestication History of Barley(Hordeum vulgare). Molecular Biology and Evolution 17,499-510.
Barringer K (1993) Five new tribes in the Scrophulariaceae. Novon 3,15-17.
Bensch S, Akesson M (2005) Ten years of AFLP in ecology and evolution:why so few animals? Molecular Ecology 14,2899-2914.
Beutler E, Gelbart T, Han JH, Koziol JA, Beutler B (1989) Evolution of the genome and the genetic code:selection at the dinucleotide level by methylation and polyribonucleotide cleavage. Proceedings of the National Academy of Sciences 86,192-196.
Bigazzi M (1993) A survey on the intraclear inclusions in the Schrophulariaceae and their systematic significance. Nordic Journal of Botany 13,19-31.
Blattner FR, Badani-Mendez AG (2001) RAPD data do not support a second centre of barley domestication in Morocco. Genetic Resources and Crop Evolution 48,13-19.
Bremer B, Bremer K, Chase M, et al. (2009) An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants:APG III. Botanical Journal of the Linnean Society 161(2),105-121.
Bremer K, Friis E, Bremer B (2004) Molecular phylogenetic dating of asterid flowering plants shows early Cretaceous diversification. Systematic Biology:53,496-505.
Brown TA, Jones MK, Powell W, Allaby RG (2009) The complex origins of domesticated crops in the Fertile Crescent. Trends in Ecology & Evolution 24,103-109.
Buckler I, Thornsberry JM, Kresovich S (2001) Molecular diversity, structure, and domestication of grasses. Genetic Resources and Crop Evolution 77,213-218.
Burger JC, Chapman MA, Burke JM (2008) Molecular insights into the evolution of crop plants. American Journal of Botany 95,113-122.
Burke JM, Tang S, Knapp SJ, Rieseberg LH (2002) Genetic Analysis of Sunflower Domestication. Genetics 161,1257-1267.
Caicedo AL, Schaal BA (2004) Population structure and phylogeography of Solanum pimpinellifolium inferred from a nuclear gene. Molecular Ecology 13,1871-1882.
Camacho FJ, Liston A (2001) Population structure and genetic diversity of Botrychium pumicola (Ophioglossaceae) based on inter-simple sequence repeats (ISSR). American Journal of Botany 88,1065-1070.
Carlbom C (1969) Evolution relationships in the genus Scrophularia L. Hereditas 61,287-301. Chandna R, Gupta S, Ahmad A, Iqbal M, Prasad M (2010) Variability in Indian bread wheat (Triticum aestivum L.) varieties differing in nitrogen efficiency as assessed by microsatellite markers. Protoplasma 242,55-67.
Clegg MT, Gaut BS, Learn GH, Morton BR (1994) Rates and patterns of chloroplast DNA evolution. Proceedings of the National Academy of Sciences 91,6795-6801.
Clement M, Posada D, Crandall KA (2000) TCS:a computer program to estimate gene genealogies. Molecular Ecology 9,1657-1659.
Corte-Real HBSM, Holland PWH, Dixon DR (1994) Inheritance of a nuclear DNA polymorphism assayed in single bivalve larvae. Marine Biology 120,415-420.
Cottrell JE, Munro RC, Tabbener HE, et al. (2002) Distribution of chloroplast DNA variation in British oaks (Quercus robur and Q. petraea):the influence of postglacial colonisation and human management. Forest Ecology and Management 156,181-195.
Coulibaly S, Pasquet RS, Papa R, Gepts P (2002) AFLP analysis of the phenetic organization and genetic diversity of Vigna unguiculata L. Walp. reveals extensive gene flow between wild and domesticated types. Theoretical and Applied Genetics 104,358-366.
Cronquist A (1981) An integrated system of classification of flowering plants. Columbia Univ Press, New York.
Davierwala AP, Chowdari KV, Kumar S, et al. (2000) Use of Three Different Marker Systems to Estimate Genetic Diversity of Indian Elite Rice Varieties. Genetica 108,269-284.
de Santos Galindez J, Diaz Lanza AMA, Fernandez Matellano L (2002) Biologically Active Substances from the Genus Scrophularia. Pharmaceutical Biology 40,45-59.
Demesure B, Sodzi N, Petit RJ (1995) A set of universal primers for amplification of polymorphic non-coding regions of mitochondrial and chloroplast DNA in plants. Molecular Ecology4,129-134.
Diaz AM, Abad MJ, Fernandez L, et al. (2004) Phenylpropanoid glycosides from Scrophularia scorodonia:In vitro anti-inflammatory activity. Life Sciences 74,2515-2526.
Doebley J (1989a) Molecular evidence for a missing wild relative of maize and the introgression of its chloroplast fenome into Zea Perennis. Evolution 43,1555-1559.
Doebley J (1989b) Isozymic evidence and the evolution of crop plants, In Isozymes in Plant Biology, D. Soltis and P. Soltis, eds., pp.165-191, Portland, Oregon:Dioscorides Press.
Doebley J, Lukens L (1998) Transcriptional Regulators and the Evolution of Plant Form. The Plant Cell Online 10,1075-1082.
Doebley J, Stec A (1993) Inheritance of the Morphological Differences Between Maize and Teosinte:Comparison of Results for Two F(2) Populations. Genetics 134,559-570.
Doebley JF, Gaut S, Smith D (2006) The Molecular Genetics of Crop Domestication. Cell 127, 1309-1321.
Doyle J (1991) DNA protocols for plants-CTAB total DNA isolation. Molecular techniques in taxonomy,283-293.
Drummond AJ, Rambaut A (2007) BEAST:Bayesian evolutionary analysis by sampling trees. Bmc Evolutionary Biology 7,214.
Dumolin-Lapegue S, Pemonge MH, Petit RJ (1997) An enlarged set of consensus primers for the study of organelle DNA in plants. Molecular Ecology 6,393-397.
Edgar RC (2004) MUSCLE:a multiple sequence alignment method with reduced time and space complexity. Bmc Bioinformatics 5,1-19.
Edwards SV, Fertil B, Giron A, Deschavanne PJ (2002) Genomic schism in birds revealed by phylogenetic analysis of DNA strings. Systematic Biology 51,599-613.
Ehrich D (2006) AFLPDAT:a collection of r functions for convenient handling of AFLP data. Molecular Ecology Notes 6,603-604.
Elias M, Panaud O, Robert T (2000) Assessment of genetic variability in a traditional cassava (Manihot esculenta Crantz) farming system, using AFLP markers. Heredity 85,219-230.
Excoffier L (1995) AMOVA, Analysis of molecular variance, vers.1.55. Univ. of Geneva, Geneva, Switzerland.
Eyre-Walker A, Gaut RL, Hilton H, Feldman DL, Gaut BS (1998) Investigation of the bottleneck leading to the domestication of maize. Proceedings of the National Academy of Sciences 95,4441-4446.
Felsenstein J (1985) Confidence limits on phylogenies:An approach using the bootstrap. Evolution 39,783-791.
Felsenstein J (1993) PHYLIP:phylogenetic inference package, version 3.5c. Department of Genetics, University of Washington.
Forbes FB, Hemsley (1890) Scrophularia ningpoensis Hemsl. Botanical Journal of Linner Society 26,178.
Franch J (1900) S. microdonta. Bull.Soc.Bot.France 47,1900.
Fu YX, Li WH (1993) Statistical tests of neutrality of mutations. Genetics 133,693-709.
Fuller DQ (2007) Contrasting Patterns in Crop Domestication and Domestication Rates:Recent Archaeobotanical Insights from the Old World. Annals of Botany 100,903-924.
Fuller DQ, Qin L, Zheng Y, et al. (2009) The domestication process and domestication rate in rice:spikelet bases from the lower Yangtze. Science 323,1607-1610.
Garcia-Mas J, Oliver M, Gomez-Paniagua H, de Vicente MC (2000) Comparing AFLP, RAPD and RFLP markers for measuring genetic diversity in melon. Theoretical and Applied Genetics 101,860-864.
Ge XJ, Liu MH, Wang WK, Schaal BA, Chiang TY (2005) Population structure of wild bananas, Musa balbisiana, in China determined by SSR fingerprinting and cpDNA PCR-RFLP. Molecular Ecology 14,933-944.
Gepts P, Osborn T, Rashka K, Bliss F (1986) Phaseolin-protein variability in wild forms and landraces of the common bean(Phaseolus vulgaris):evidence for multiple centers of domestication. Economic Botany 40,451-468.
Golding GB (1987) The detectiong of deleterious selection using ancestors inferred from a phylogenetic history. Genetic Resources and Crop Evolution 49,71-82.
Gong W, Chen C, Dobes C, Fu CX, Koch MA (2008) Phylogeography of a living fossil: Pleistocene glaciations forced Ginkgo biloba L. (Ginkgoaceae) into two refuge areas in China with limited subsequent postglacial expansion. Molecular Phylogenetics and Evolution 48, 1094-1105.
Gong W, Fu C, Luo Y, Qiu Y (2006) Molecular Identification of Sinopodophyllum hexandrum and Dysosma Species using cpDNA Sequences and PCR-RFLP Markers. Planta Medica 72, 650-652.
Grivet D, Heinze B, Vendramin GG, Petit RJ (2001) Genome walking with consensus primers: application to the large single copy region of chloroplast DNA. Molecular Ecology Notes 1, 345-349.
Gross BL, Olsen KM (2010) Genetic perspectives on crop domestication. Trends in Plant Science 15,529-537.
Grusz AL, Windham MD, Pryer KM (2009) Deciphering the origins of apomictic polyploids in the Cheilanthes yavapensis complex (Pteridaceae). American Journal of Botany 96, 1636-1645.
Gugerli F, Senn J, Anzidei M, et al. (2001) Chloroplast microsatellites and mitochondrial nadl intron 2 sequences indicate congruent phylogenetic relationships among Swiss stone pine (Pinus cembra), Siberian stone pine (Pinus sibirica), and Siberian dwarf pine (Pinus pumila). Molecular Ecology 10,1489-1497.
Hamilton MB (1999) Four primer pairs for the amplification of chloroplast intergenic regions with intraspecific variation. Molecular Ecology Notes 8,513-525.
Hammer K (1984) Das Domestikationssydrom. Genetic Resources and Crop Evolution 32,11-34.
Han TH, van Eck HJ, De Jeu MJ, Jacobsen E (1999) Optimization of AFLP fingerprinting of organisms with a large-sized genome:a study on Alstroemeria spp. Theoretical and Applied Genetics 98,465-471.
Hare MP (2001) Prospects for nuclear gene phylogeography. Trends in Ecology and Evolution 16, 700-706.
Harris H (1966) Enzyme polymorphism in man. Proc. R. Soc. Lond.164,298-310.
Hartl DL, Clark AG (1989) Principles of Population Genetics., Sunderland, Mass:Sinauer Assoc. Inc. Pub.
He J, Chen L, Si Y, et al. (2009) Population structure and genetic diversity distribution in wild and cultivated populations of the traditional Chinese medicinal plant Magnolia officinalis subsp. biloba (Magnoliaceae). Genetica 135,233-243.
Hernandez P, Martin A, Dorado G (1999) Development of SCARs by direct sequencing of RAPD products:a practical tool for the introgression and marker-assisted selection of wheat. Molecular Breeding 5,245-253.
Hilliard OM (1994) The Manuleae-a tribe of Scrophulariaceae Edinburgh University Press, Edinburgh.
Hong DY (1983) The distribution of Scrophulariaceae in the Holarctic with special reference to the floristic relationships between eastern Asia and eastern North America. Annals of the Missouri Botanical Garden.70,701-712.
Hong DY, Yang HB, Jin C, et al. (1998) Scrophularia. In:wu Z-Y (edit) Flora of China. Vol.18 Missouri Botanocal Garden's Scientific Publications, St. Louis.
Hu P, Luo GA, Zhao ZZ, Jiang ZH (2005) Multi-component HPLC fingerprinting of Radix Salviae Miltiorrhizae and its LC-MS-MS identification. Chemical and Pharmaceutical Bulletin 3,677-683.
Hua R, Sun S, Zhou Q, Noda I, Wang B (2003) Discrimination of Fritillary according to geographical origin with Fourier transform infrared spectroscopy and two-dimensional correlation IR spectroscopy. Journal of Pharmaceutical and Biomedical Analysis 33, 199-209.
Hubby JL, Leqontin RC (1966) A molecular approach to the study of genic heterozygosity in natural populations. I. the number of alleles at different loci in Drosophila pseudoobscura. Genetics 54,577-594.
Hwang SY, Lin TP, Ma CS, Lin CL (2003) Postglacial population growth of Cunninghamia konishii (Cupressaceae) inferred from phylogeographical and mismatch analysis of chloroplast DNA variation. Molecular Ecology 12,2689-2695.
Iwatsuki (1993) Flora of Japan:http://foj.c.u-tokyo.ac.jp/gbif/.
Jaenicke-Despres V, Buckler ES, Smith BD, et al. (2003) Early Allelic Selection in Maize as Revealed by Ancient DNA. Science 302,1206-1208.
Jansen R, Greenbaum D, Gerstein M (2002) Relating Whole-Genome Expression Data with Protein-Protein Interactions. Genome Research 12,37-46.
Kajimoto T, Hidaka M, Shoyama K, Nohara T (1989) Iridoids from Scrophularia ningpoensis. Phytochemistry 28,2701-2704.
Kim SR, Kim YC (2000) Neuroprotective phenylpropanoid esters of rhamnose isolated from roots of Scrophularia buergeriana. Phytochemistry 54,503-509.
Kimura M (1983) The Neutral Theory of Molecular Evolution Cambridge University Press., New York.
King AR, Ferris C (1998) Chloroplast DNA phylogeography of Alnus glutinosa (L.) Gaertn. Molecular Ecology 7,1151-1161.
Kong HH, Wang AL, Lee J, Fu CX (2007) Studies of Systematic evolutioni and karyotypic variation in Smilax and Heterosmilax (smilacaceae). Acta Phytotaxon Sin 45 (3),257-273.
Kovach WL (1999) MVSP-A multivariate statistical Package for Windows, ver.3.1 Kovach Computing Services, Pentraeth, Wales, UK.
Kress WJ, Prince LM, Williams KJ (2002) The phylogeny and a new classification of the gingers (Zingiberaceae):evidence from molecular data. American Journal of Botany 89, 1682-1696.
Kuroda Y, Kaga A, Tomooka N, Vaughan DA (2006) Population genetic structure of Japanese wild soybean (Glycine soja) based on microsatellite variation. Molecular Ecology 15, 959-974.
Lee G, Doh E, Lee M, Ko B, Oh S (2010) Discrimination of three Scrophularia plants utilizing 'Scrophularia Radix'by DNA markers based on internal transcribed spacer (ITS) sequences. Genomics 32,181-189.
Lee TB (2003) Coloured Flora of Korea Academy Book Co., Seoul.
Lee YN (1967) Chromosome numbers of flowering plants in Korea. Journal of Korean Research Institute Ewha Women's University 11,455-478.
Leliaert F, Verbruggen H, Wysor B, Clerck OD (2009) DNA taxonomy in morphologically plastic taxa:Algorithmic species delimitation in the Boodlea complex (Chlorophyta: Cladophorales). Molecular Phylogenetics and Evolution 53,122-133.
Lersten NR, Curtis JD (1997) Anatomy and distribution of foliar idioblasts in Scrophularia and Verbascum (Scrophulariaceae). American Journal of Botany 84,1638.
Lewontin RC (1972) Testing the Theory of Natural Selection. Nature 236,181-182.
Li C, Zhou A, Sang T (2006) Rice Domestication by Reducing Shattering. Science 311, 1936-1939.
Li J, Kelley S, Tredici P, Donoghue MJ (2001) Phylogenetic relationships of Torreya (Taxaceae) inferred from sequences of nuclear ribosomal DNA ITS region, harv. Pap. Bot.6,275-281.
Li J, Zhang D, Donoghue MJ (2003) Phylogeny and biogeography of Chamaecyparis (Cupressaceae) inferred from DNA sequences of the nuclear ribosomal ITS region. Rhodora 105,106-117.
Li Y, Jiang S, Gao W, Zhu D (2000) Phenylpropanoid glycosides from Scrophularia ningpoensis. Phytochemistry 54,923-925.
Liu, Chen, Wu, Xia (1998) Flora of Taiwan:http://www.efloras.org/flora_page.aspx?flora_id= 10
Londo JP, Chiang YC, Hung KH, Chiang TY, Schaal BA (2006) Phylogeography of Asian wild rice. Oryza rufipogon, reveals multiple independent domestications of cultivated rice. Oryza sativa. Proc. Natl. Acad. Sci. USA 103,9578-9583.
Lu G, Chan K, Liang Y, et al. (2005) Development of high-performance liquid chromatographic fingerprints for distinguishing Chinese Angelica from related umbelliferae herbs. Journal of Chromatography A 1073,383-392.
Lum MR, Potter E, Dang T, et al. (2005) Identification of Botanicals and Potential Contaminants through RFLP and Sequencing. Planta Medica 71,841,846.
Lumaret R, Ouazzani N (2001) Plant genetics:Ancient wild olives in Mediterranean forests. Nature 413,700.
Lynch M, Milligan BG (1994) Analysis of population genetic structure with RAPD markers, Molecular Ecology 3,91-99.
Ma X (1984) Chromosome Observations of Some Medical Plants in Xinjian. Acta Phytotaxonomica Sinica 22,243-249.
Matsuoka Y, Vigouroux Y, Goodman MM, et al. (2002) A single domestication for maize shown by multilocus microsatellite genotyping. Proceedings of the National Academy of Sciences 99,6080-6084.
Maude FP (1940) Chromosome Numbers in Some British Plants. New Phytologist 39,17-32.
McKey D, Elias M, Pujol B, Duputie A (2010) The evolutionary ecology of clonally propagated domesticated plants. New Phytologist 186,318-332.
Mehra PN, Vasudevan KN (1972) Scrophularia dentata 4257. In:Love A (edit) IOPB chromosome number reports XXXVI. Taxon 21,343.
Micharel DP, Dorian QF (2009) The nature of selection during plant domestication. Nature 457, 843-848.
Miller A, Schaal B (2005) Domestication of a Mesoamerican cultivated fruit tree, Spondias purpurea. Proceedings of the National Academy of Sciences 102,12801.
Mitton JB, Kreiser BR, Latta RG (2000) Glacial refugia of limber pine (Pinus flexilis James) inferred from the population structure of mitochondrial DNA. Molecular Ecology 9,91-97.
Miyazawa M, Okuno Y (2003) Volatile components from the roots of Scrophularia ningpoensis Hemsl. Flavour and Fragrance Journal 18,398-400.
Moraes RM, Momm HG, Silva B, et al. (2005) Geographic information system method for assessing chemo-diversity in Medicinal plants. Planta Medica 71,1157-1164.
Morrell PL, Clegg MT (2007) Genetic evidence for a second domestication of barley (Hordeum vulgare) east of the Fertile Crescent. Proceedings of the National Academy of Sciences 104, 3289.
Mueller UG, Wolfenbarger LL (1999) AFLP genotyping and figerprinting. Trends in Ecology and Evolution 14,389-394.
Muller MH, Prosperi JM, Santoni S, Ronfort J (2003) Inferences from mitochondrial DNA patterns on the domestication history of alfalfa (Medicago sativa). Molecular Ecology 12, 2187-2199.
Nei M (1987) Molecular evolutionary genetics Oxford University Press, New York, USA.
Nei M, Tajima F (1983) Maximum likelihood estimation of the number of nucleotide substitutions from restriction sites data. Genetics 105,205-217.
Nesbitt TC, Tanksley SD (2002) Comparative Sequencing in the Genus Lycopersicon: Implications for the Evolution of Fruit Size in the Domestication of Cultivated Tomatoes. Genetics 162,365-379.
Nguyen A, Fontaine J, Malonne H, et al. (2005) A sugar ester and an iridoid glycoside from Scrophularia ningpoensis. Phytochemistry 66,1186-1191.
Nishikawa (1985) Chromosome counts of flowering plants of Hokkaido (Japan). Journal of Hokkaido 36,25-40.
Obeso JR (2002) The costs of reproduction in plants. New Phytologist 155,321-348.
Olmstead RG (1995) Evidence for the Polyphyly of the Scrophularia Based on Chloroplast rbcL and ndhF Sequences. Annals of the Missouri Botanical Garden 82,176-193.
Olmstead RG (2001) Disintegration of the Scrophulatiaceae. American Journal of Botany 88, 348-361.
Olsen KM, Schaal BA (1999) Evidence on the origin of cassava:phylogeography of Manihot esculenta. Proceedings of the National Academy of Sciences 96,5586-5591.
Olsen KM, SchaalL BA (2007) Insights on the evolution of a vegetatively propagated crop species. Molecular ecology 16,2838-2840.
Otero-arnaiz A, Casas A, Hamrick JL, Cruse-sanders J (2005) Genetic variation and evolution of Polaskia chichipe (Cactaceae) under domestication in the Tehuacan Valley, central Mexico. Molecular Ecology 14,1603-1611.
Ozkan H, Brandolini A, Schafer-Pregl R, Salamini F (2002) AFLP Analysis of a Collection of Tetraploid Wheats Indicates the Origin of Emmer and Hard Wheat Domestication in Southeast Turkey. Molecular Biology and Evolution 19,1797-1801.
Palme AE (2002) Chloroplast DNA variation, postglacial recolonization and hybridization in hazel, Corylus avellana. Molecular Ecology 11,1769-1779.
Palmer JD (1992) Mitochondrial DNA in systematics:applications and limitations. In:Soltis P S, Soltis D E, Doyle J J, Ed. Molecular Systematics of Plants Champman and Hall, New York.
Palumbi SR, Baker CS (1994) Contrasting population structure from nuclear intron sequences and mtDNA of humpback whales. Molecular Biology and Evolution 11,426-435.
Panwar P, Nath M, Yadav V, Kumar A (2010) Comparative evaluation of genetic diversity using RAPD. SSR and cytochrome P450 gene based markers with respect to calcium content in finger millet (Eleusine coracana L. Gaertn.). Journal of Genetics 89,121-133.
Paran I, Michelmore RW (1993) Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce. Theoretical and Applied Genetics 85,985-993.
Passinho-Soares H, Felix D, Kaplan MA, Margis-Pinheiro M, Margis R (2006) Authentication of Medicinal Plant Botanical Identity by Amplified Fragmented Length Polymorphism Dominant DNA Marker:Inferences from the Plectranthus Genus. Planta Medica 72,929, 931.
Pereira RJ, Wake DB (2009) Genetic leakage after adaptive and nonadaptive divergence in the ensatina eschscholtzii ring species. Evolution 63,2288-2301.
Petit E, Balloux F, Goudet J (2001) Set-biased dispersal in a migraatory bat:a characterization using sex-specific demographic parameters. Evolution 55,635-640.
Petit RJ, Csaikl MU, Bords S, et al. (2002) Chloroplast DNA variation in European white oaks: Phylogeography and patterns of diversity based on data from over 2600 populations. Forest Ecology and Management 156,5-26.
Pickersgill B (1997) Genetic resources and breeding of Capsicum spp. Euphytica 96,129-133.
Piperno DR, Flannery KV (2001) The earliest archaeological maize (Zea mays L.) from highland Mexico:New accelerator mass spectrometry dates and their implications. Proceedings of the National Academy of Sciences 98,2101-2103.
Posada D (2008) jModelTest:Phylogenetic Model Averaging. Molecular Biology and Evolution 25,1253-1256.
Pritchard JK, Stephens M, Donnelly P (2000) Inference of Population Structure Using Multilocus Genotype Data. Genetics 155,945-959.
Provan J, Powell W, Hollingsworth PM (2001) Chloroplast microsatellites:new tools for studies in plant ecology and evolution. Trends in Ecology and Evolution 16,142-147.
Provan J, Soranzo N, Wilson NJ, Goldstein DB, Powell W (1999) A Low Mutation Rate For Chloroplast Microsatellites. Genetics 153,943-947.
Qian J, Hunkler D, Rimpler H (1992) Iridoid-related aglycone and its glycosides from Scrophularia ningpoensis. Phytochemistry 31,905-911.
Qiu Y, Fu C, Comes HP (2011) Plant molecular phylogeography in China and adjacent regions: Tracing the genetic imprints of Quaternary climate and environmental change in the world's most diverse temperate flora. Molecular Phylogenetics and Evolution 59,225-244.
Qiu YX, Sun Y, Zhang XP, et al. (2009) Molecular phylogeography of East Asian Kirengeshoma (Hydrangeaceae) in relation to Quaternary climate change and landbridge configurations. New Phytologist 183,480-495.
Qiu YX, Zong M, Yao Y, et al. (2009) Genetic variation in wild and cultivated Rhizoma corydalis revealed by ISSRs markers. Planta medica 75,94-98.
Quicke DLJ, Jones OR, Epstein DR (2007) Correcting the Problem of false incongruence due to noise imbalance in the Incongruence Length Difference (ILD) Test. Systematic Biology 56, 496-503.
Ren N, Timko MP (2001) AFLP analysis of genetic polymorphism and evolutionary relationships among cultivated and wild Nicotiana species. Genome 44,559-571.
Richardson IBK (1993) Scrophulariaceae. In V. H. Heywood (edit) Flowering plants of the world Oxford University Press, New York.
Rogers AR, Harpending H (1992) Population growth makes waves in the distribution of pairwise genetic differences. Molecular Biology and Evolution 9,552-569.
Rozas J, Sanchez-DelBarrio JC, Messeguer X, Rozas R (2003) DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 19,2496-2497.
Sagare AP, Kuo CL, Chueh FS, Tsay HS (2001) De novo regeneration of Scrophularia yoshimurae Yamazaki (Scrophulariaceae) and quantitative analysis of harpagoside, an iridoid glucoside, formed in aerial and underground parts of in vitro propagated and wild plants by HPLC. Biological& pharmaceutical bulletin 24,1311-1315.
Sang T, Crawford DJ, Stuessy TF (1995) Documentation of reticulate evolution in peonies (Paeonia) using internal transcribed spacer sequences of nuclear ribosomal DNA: implications for biogeography and concerted evolution. Proceedings of the National Academy of Sciences 92,6813-6817.
Sang T, Crawford DJ, Stuessy TF (1997) Chloroplast DNA phylogeny, reticulate evolution, and biogeography of Paeonia (Paeoniaceae). American Journal of Botany 84,1120.
Schaal AB, Hayworth AD, Olsen MK, Rauscher TJ, Smith TW (1998) Phylogeographic studies in plants:problems and prospects. Molecular Ecology 7,465-474.
Scherer H (1939) Chromosomenzahlen aus der schleswig-holsteinischen. Flora, I. Planta 29, 636-642.
Scheunert A, Heubl G (2011) Phylogenetic relationships among New World Scrophularia L. (Scrophulariaceae):new insights inferred from DNA sequence data. Plant Systematics and Evolution 291,69-89.
Schneider S (2000) ARLEQUIN, version 2.000:a software for population genetics data analysis (ed. Roessli D). Genetics and Biometry Laboratory, University of Geneva, Geneva, Switzerland.
Shi W, Yang CF, Chen JM, Guo YH (2008) Genetic variation among wild and cultivated populations of the Chinese medicinal plant Coptis chinensis (Ranunculaceae). Plant Biology 10,485-491.
Slade RW, Moritz C, Heideman A (1994) Multiple nuclear-gene phylogenies:application to pinnipeds and comparison with a mitochondrial DNA gene phylogeny. Molecular Biology and Evolution 11,341-356.
Slade RW, Moritz C, Heideman A, Hale PT (1993) Rapid assessment of single-copy nuclear DNA variation in diverse species. Molecular Ecology 2,359-373.
Slatkin M (1987) Gene flow and the geographic structure of natural populations. Science 236,787-792.
Slatkin M (1993) Isolation by distance in equilibrium and non-equilibrium populations. Evolution 47,264-279.
Slatkin M, Hudson RR (1991) Pairwise Comparisons of Mitochondrial DNA Sequences in Stable and Exponentially Growing Populations. Genetics 129,555-562.
Smith BD (1989) Origins of Agriculture in Eastern North America. Science 246,1566-1571.
Smith BD (2001) Documenting plant domestication:The consilience of biological and archaeological approaches. Proceedings of the National Academy of Sciences 98,1324-1326.
Sonnante G, Stockton T, Nodari RO, Becerra Velasquez VL, Gepts P (1994) Evolution of genetic diversity during the domestication of common-bean (Phaseolus vulgaris L.). Theoretical and Applied Genetics 89,629-635.
Stamatakis A, Hoover P, Rougemont J (2008) A Rapid Bootstrap Algorithm for the RAxML Web Servers. Systematic Biology 57,758-771.
Stamatakis A, Ott M, Ludwig T (2005) RAxML-OMP:An Efficient Program for Phylogenetic I nference on SMPs. Computer Science 3606,288-302.
Stiefelhagen H (1910) Systematische und pflanzengeographische studien zur Kenntnis der Gattung Scrophularia. Botanical Jahrb Sysematicst.44,406-496.
Strand AE, Leebens-Mack J, Milligan BG (1997) Nuclear DNA-based markers for plant evolutionary biology. Molecular Ecology 6,113-118.
Stuessy TF (1990) Plant taxonomy. Columbia univ. press, New York.
Swofford DL (2002) PAUP*:phylogenetic analysis using parsimony (* and other methods), version 4.0b10, Sunderland, MA, USA:Sinauer Associates.
Sytsma KJ, Schaal BA (1985) Genetic Variation, Differentiation, and Evolution in a Species Complex of Tropical Shrubs Based on Isozymic Data. Evolution 39,582-593.
Taberlet P, Gielly L, Pautou G, Bouvet J (1991) Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Molecular Biology 17,1105-1109.
Tajima F (1989) Statistical-method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123,585-595.
Techen N, Khan IA, Pan Z, Scheffler BE (2006) The Use of Polymerase Chain Reaction (PCR) for the Identification of Ephedra DNA in Dietary Supplements. Planta Medica 72,241,247.
Templeton AR (1998) The detectiong of deleterious selection using ancestors inferred from a phylogenetic histor. Molecular Ecology 7.
Templeton AR (2006) Population genetics and microevolutionary theory John Wiley & Sons, Inc., Hoboken, New Jersey, USA.
Tenaillon MI, U'Ren J, Tenaillon O, Gaut BS (2004) Selection Versus Demography:A Multilocus Investigation of the Domestication Process in Maize. Molecular Biology and Evolution 21,1214-1225.
The Angiosperm Phylogeny Group (1998) An ordinal classification for the families of flowering plants. Annals of the Missouri Botanical Garden 85,531-553.
The Angiosperm Phylogeny Group (2003) An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants:APG II. Botanical Journal of the Linnean Society 141,399-436.
Thieret JW (1967) Supraspecific classification in the Scrophulariaceae:a review. Sida 3,87-106.
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL X Windows Interface:Flexible Strategies for Multiple Sequence Alignment Aided by Quality Analysis Tools. Nucleic Acids Research 25,4876-4882.
Tian F, Stevens NM, Buckler ES (2009) Tracking footprints of maize domestication and evidence for a massive selective sweep on chromosome 10. Proceedings of the National Academy of Sciences 106,9979-9986.
Tzedakis PC, Lawson IT, Frogley MR, Hewitt GM, Preece RC (2002) Buffered tree population changes in a Quaternary refugium:evolutionary implications. Science 297,2044.
Vaarama A, Hiirsalmi H (1967) Chromosome studies on some Old World species on the genus Scrophularia. Hereditas 58,333-358.
Vos P, Hogers R, Bleeker M, et al. (1995) AFLP:a new technique for DNA fingerprinting. Nucleic Acids Research 23,4407-4414.
Weismann A (1892) Essays upon heredity and kindred biological Problems. Vol.2. Edited by Poulton, E. B. and Shipley, A. E. the Clarendon Press., Oxford.
Wen J, Shi S (1999) A phylogenetic and biogeographic study of Hamamelis (Hamamelidaceae), an eastern Asian and eastern North American disjunct genus. Biochemical Systematics and Ecology 27,55-66.
White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR protocols a guide to methods and applications Academic Press, San Diego.
Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research 18, 6531-6535.
Wills DM, Burke JM (2006) Chloroplast DNA Variation Confirms a Single Origin of Domesticated Sunflower (Helianthus annuus L.). Journal of Heredity 97,403-408.
Wolfe KH, Li WH, Sharp PM (1987) Rates of nucleotide substitution vary greatly among plant mitochondrial, chloroplast, and nuclear DNAs. Proceedings of the National Academy of Sciences 84,9054-9058.
Woo Y, Kim H, Cho J, Chung H (1999) Discrimination of herbal medicines according to geographical origin with near infrared reflectance spectroscopy and pattern recognition techniques. Journal of Pharmaceutical and Biomedical Analysis 21,407-413.
Wright S (1931) Statistical methods in biology. Journal of the American Statistical Association 26,155-161.
Wright S (1978) Evolution and the genetics of populations. University of Chicago Press, Chicago, IL.
Wright SI, Bi IV, Schroeder SG, et al. (2005) The Effects of Artificial Selection on the Maize Genome. Science 308,1310-1314.
Xiang Q, Soltis DE, Soltis PS, Manchester SR, Crawford DJ (2000) Timing the Eastern Asian-Eastern North American Floristic Disjunction:Molecular Clock Corroborates Paleontological Estimates. Molecular Phylogenetics and Evolution 15,462-472.
Xiang QY, Crawford DJ, Wolfe AD (1998) Origin and biogeography of Aesculus L.(Hippocastanaceae):a molecular phylogenetic perspective. Evolution 52,988-997.
Yamane K, Lu N, Ohnishi O (2009) Multiple origins and high genetic diversity of cultivated radish inferred from polymorphism in chloroplast simple sequence repeats. Breeding Science 59,55-65.
Yang ST, Chen C, Zhao YP, et al. (2011) Association between Chemical and Genetic Variation of Wild and Cultivated Populations of Scrophularia ningpoensis Hemsl. Planta Medica 77, 865-871.
Ye Q, Qiu Y, Quo Y, et al. (2006) Species-specific SCAR markers for authentication of Sinocalycanthus chinensis. Journal of Zhejiang University-Science B 7,868-872.
Yeh F, Yang RC, Boyle T, Ye ZH (1997) POPGENE, the User-Friendly Shareware for Population Genetic Analysis. Molecular Biology and Biotechnology Centre, University of Alberta, Edmonton.
Yuan QJ, Zhang ZY, Hu J, et al. (2010) Impacts of recent cultivation on genetic diversity pattern of a medicinal plant, Scutellaria baicalensis (Lamiaceae). Bmc Genetics 11,29.
Zeder MA (2006) Central questions in the domestication of plants and animals. Evolutionary Anthropology:Issues, News, and Reviews 15,105-117.
Zeder MA, Emshwiller E, Smith BD, Bradley DG (2006) Documenting domestication:the intersection of genetics and archaeology. Trends in Genetics 22,139-155.
Zhang D, Hewitt GM (2003) Nuclear DNA analyses in genetic studies of populations:practice, problems and prospects. Molecular Ecology 12,563-584.
Zhang D, Sang T (1999) Physical mapping of ribosomal RNA genes in peonies (Paeonia, Paeoniaceae) by fluorescent in situ hybridization:implications for phylogeny and concerted evolution. American Journal of Botany 86,735-740.
Zhang DQ, Gao LM, Yang YP (2010) Genetic diversity and structure of a traditional Chinese medicinal plant species, Fritillaria cirrhosa (Liliaceae) in southwest China and implications for its conservation. Biochemical Systematics and Ecology 38,236-242.
Zhao YP, Qiu YX, Gong W, Li JH, Fu CX (2007) Authentication of Actinidia macrosperma Using PCR-RFLP Based on trnK Sequences. Botaniy Study 48(3),46-49.
Zhao ZZ, Hu Y, Liang ZT, et al. (2006) Authentication is fundamental for standardization of Chinese Medicines. Planta Medica 72,865-874.
Zietkiewicz E, Rafalski A, Labuda D (1994) Degome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification. Genomics 20(2),176-183.
陈大霞,李隆云,彭锐,吴叶宽,蔡应繁(2009)玄参3种栽培类型遗传关系和遗传多样性的SRAP研究。中国中药杂志34,138-142。
戴金秋,邢振荣(1994)浙江产玄参的化学成分。现代应用药学11,16-1。
龚维(2007) 孑遗植物银杏的分子亲缘地理学研究,浙江大学。
管毕财(2008)特有濒危植物八角莲保护遗传学和分子亲缘地理学,浙江大学。
胡世林(1989)《中国道地药材》黑龙江科学记数出版社,哈尔滨。
胡世林(1995)现代道地论概要.中国医药信息杂志 2,7。
国家药典委员会(2005)《中华人民共和国药典》2005年版。化学工业出版社,北京。
黄璐琦,陈美兰,肖培根(2004)中药材道地性研究的现代生物学基础及模式假说。中国中药杂志29(6),494-496。
黄璐琦,张瑞贤(1997)“道地药材”的生物学探讨。中国药学杂志9(32),563。
李密密 (2011)山茶科紫茎属和折柄茶属系统进化研究,浙江大学。
李时珍(1590)《本草纲目》中国医药出版社,北京。
刘承伟,毕志明,祝艳斐,李萍(2007)玄参中4种主要活性成分的HPLC定量分析。中国药学杂志 42,3。
马兴华,覃若林,邢文斌(1984)新疆某些药用植物的染色体观察。植物分类学报 22,243-249。
田欣,李德铢(2002)DNA序列在植物系统学研究中的应用。云南植物研究24,170-184。
万德光(2008)《中药品质研究--理论、方法与实践》。上海科学技术出版社,上海.
王家葵,王佳黎(2007)《中药材品种沿革及道地性》。中国医药科技出版社,北京。
吴云(2006)玄参主要病虫害及防治技术。植物医生 19,30-02。
谢丽华(2000)中药玄参薄层色谱鉴别法的建立。中国中药杂志 25,654-656。
杨舒婷(2011)玄参化学多样性及其与遗传变异和环境因子之间的关系研究,浙江大学。
俞静静,王琴,吕圭源(2006)玄有效部位的药理研究进展。中华实用中西医杂志 19, 1866-1867。
章国镇(2006)《神农本草经(精选图文本)》。福建科学技术出版社,福建。
赵志新,梁宗锁,姜在民,薛媛菲,杨永宙(2007)玄参ISSR-PCR反应体系的建立与优化。药物生物技术14,318-323。
中国科学院中国植物志编委会(1979)《中国植物志》。科学出版社,北京。
周浓(2008)玄参的质量评价及其指纹图谱研究,西南大学。
祝艳斐,毕志明,刘承伟(2008)内皮细胞提取和高效液相色谱一电喷雾匕行时间质谱联用预测玄参中的活性成分。中国药科大学学报3,228-231。
邹节明(2005)苦玄参HPLC(?)指纹图谱研究。中国药学杂志 40,13-15。
Ajibade SR, Weeden NF, Chite SM (2000) Inter simple sequence repeat analysis of genetic relationships in the genus Vigna. Euphytica 111,47-55.
Allaby RG, Fuller DQ, Brown TA (2008) The genetic expectations of a protracted model for the origins of domesticated crops. Proceedings of the National Academy of Sciences 105, 13982-13986.
Althoff DM, Gitzendanner MA, Segraves KA (2007) The utility of amplified fragment length polymorphisms in Phylogenetics:a comparison of homology within and between genomes. Systematic Biology 56,477-484.
Anthony, Combes, Astorga, et al. (2002) The origin of cultivated Coffea arabica L. varieties revealed by AFLP and SSR markers. Theoretical and Applied Genetics 104,894-900.
Arens P, Coops H, Jansen J, Vosman B (1998) Molecular genetic analysis of black poplar (Populus nigra L.) along Dutch rivers. Molecular Ecology 7,11-18.
Arroyo GR, Ruiz-Garci L, Bolling L, et al. (2006) Multiple origins of cultivated grapevine (Vitis vinifera L. ssp. sativa) based on chloroplast DNA polymorphisms. Molecular Ecology 15, 3707-3714.
Avise JC (1978) Variances and frequency distributions of genetic distance in evolutionary phylads. Heredity 40,225-237.
Avise JC, Giblin-Davidson C, Laerm J, Patton JC, Lansman RA (1979) Mitochondrial DNA clones and matriarchal phylogeny within and among geographic populations of the pocket gopher, Geomys pinetis. Proceedings of the National Academy of Sciences 76,6694.
Avise JC, Walker DE (1998) Pleistocene phylogeographic effects on avian populations and the speciation process. Proceedings of the Royal Society of London. Series B:Biological Sciences 265,457.
Badr A, Sch R, et al. (2000) On the Origin and Domestication History of Barley(Hordeum vulgare). Molecular Biology and Evolution 17,499-510.
Barringer K (1993) Five new tribes in the Scrophulariaceae. Novon 3,15-17.
Bensch S, Akesson M (2005) Ten years of AFLP in ecology and evolution:why so few animals? Molecular Ecology 14,2899-2914.
Beutler E, Gelbart T, Han JH, Koziol JA, Beutler B (1989) Evolution of the genome and the genetic code:selection at the dinucleotide level by methylation and polyribonucleotide cleavage. Proceedings of the National Academy of Sciences 86,192-196.
Bigazzi M (1993) A survey on the intraclear inclusions in the Schrophulariaceae and their systematic significance. Nordic Journal of Botany 13,19-31.
Blattner FR, Badani-Mendez AG (2001) RAPD data do not support a second centre of barley domestication in Morocco. Genetic Resources and Crop Evolution 48,13-19.
Bremer B, Bremer K, Chase M, et al. (2009) An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants:APG III. Botanical Journal of the Linnean Society 161(2),105-121.
Bremer K, Friis E, Bremer B (2004) Molecular phylogenetic dating of asterid flowering plants shows early Cretaceous diversification. Systematic Biology:53,496-505.
Brown TA, Jones MK, Powell W, Allaby RG (2009) The complex origins of domesticated crops in the Fertile Crescent. Trends in Ecology & Evolution 24,103-109.
Buckler I, Thornsberry JM, Kresovich S (2001) Molecular diversity, structure, and domestication of grasses. Genetic Resources and Crop Evolution 77,213-218.
Burger JC, Chapman MA, Burke JM (2008) Molecular insights into the evolution of crop plants. American Journal of Botany 95,113-122.
Burke JM, Tang S, Knapp SJ, Rieseberg LH (2002) Genetic Analysis of Sunflower Domestication. Genetics 161,1257-1267.
Caicedo AL, Schaal BA (2004) Population structure and phylogeography of Solanum pimpinellifolium inferred from a nuclear gene. Molecular Ecology 13,1871-1882.
Camacho FJ, Liston A (2001) Population structure and genetic diversity of Botrychium pumicola (Ophioglossaceae) based on inter-simple sequence repeats (ISSR). American Journal of Botany 88,1065-1070.
Carlbom C (1969) Evolution relationships in the genus Scrophularia L. Hereditas 61,287-301. Chandna R, Gupta S, Ahmad A, Iqbal M, Prasad M (2010) Variability in Indian bread wheat (Triticum aestivum L.) varieties differing in nitrogen efficiency as assessed by microsatellite markers. Protoplasma 242,55-67.
Clegg MT, Gaut BS, Learn GH, Morton BR (1994) Rates and patterns of chloroplast DNA evolution. Proceedings of the National Academy of Sciences 91,6795-6801.
Clement M, Posada D, Crandall KA (2000) TCS:a computer program to estimate gene genealogies. Molecular Ecology 9,1657-1659.
Corte-Real HBSM, Holland PWH, Dixon DR (1994) Inheritance of a nuclear DNA polymorphism assayed in single bivalve larvae. Marine Biology 120,415-420.
Cottrell JE, Munro RC, Tabbener HE, et al. (2002) Distribution of chloroplast DNA variation in British oaks (Quercus robur and Q. petraea):the influence of postglacial colonisation and human management. Forest Ecology and Management 156,181-195.
Coulibaly S, Pasquet RS, Papa R, Gepts P (2002) AFLP analysis of the phenetic organization and genetic diversity of Vigna unguiculata L. Walp. reveals extensive gene flow between wild and domesticated types. Theoretical and Applied Genetics 104,358-366.
Cronquist A (1981) An integrated system of classification of flowering plants. Columbia Univ Press, New York.
Davierwala AP, Chowdari KV, Kumar S, et al. (2000) Use of Three Different Marker Systems to Estimate Genetic Diversity of Indian Elite Rice Varieties. Genetica 108,269-284.
de Santos Galindez J, Diaz Lanza AMA, Fernandez Matellano L (2002) Biologically Active Substances from the Genus Scrophularia. Pharmaceutical Biology 40,45-59.
Demesure B, Sodzi N, Petit RJ (1995) A set of universal primers for amplification of polymorphic non-coding regions of mitochondrial and chloroplast DNA in plants. Molecular Ecology4,129-134.
Diaz AM, Abad MJ, Fernandez L, et al. (2004) Phenylpropanoid glycosides from Scrophularia scorodonia:In vitro anti-inflammatory activity. Life Sciences 74,2515-2526.
Doebley J (1989a) Molecular evidence for a missing wild relative of maize and the introgression of its chloroplast fenome into Zea Perennis. Evolution 43,1555-1559.
Doebley J (1989b) Isozymic evidence and the evolution of crop plants, In Isozymes in Plant Biology, D. Soltis and P. Soltis, eds., pp.165-191, Portland, Oregon:Dioscorides Press.
Doebley J, Lukens L (1998) Transcriptional Regulators and the Evolution of Plant Form. The Plant Cell Online 10,1075-1082.
Doebley J, Stec A (1993) Inheritance of the Morphological Differences Between Maize and Teosinte:Comparison of Results for Two F(2) Populations. Genetics 134,559-570.
Doebley JF, Gaut S, Smith D (2006) The Molecular Genetics of Crop Domestication. Cell 127, 1309-1321.
Doyle J (1991) DNA protocols for plants-CTAB total DNA isolation. Molecular techniques in taxonomy,283-293.
Drummond AJ, Rambaut A (2007) BEAST:Bayesian evolutionary analysis by sampling trees. Bmc Evolutionary Biology 7,214.
Dumolin-Lapegue S, Pemonge MH, Petit RJ (1997) An enlarged set of consensus primers for the study of organelle DNA in plants. Molecular Ecology 6,393-397.
Edgar RC (2004) MUSCLE:a multiple sequence alignment method with reduced time and space complexity. Bmc Bioinformatics 5,1-19.
Edwards SV, Fertil B, Giron A, Deschavanne PJ (2002) Genomic schism in birds revealed by phylogenetic analysis of DNA strings. Systematic Biology 51,599-613.
Ehrich D (2006) AFLPDAT:a collection of r functions for convenient handling of AFLP data. Molecular Ecology Notes 6,603-604.
Elias M, Panaud O, Robert T (2000) Assessment of genetic variability in a traditional cassava (Manihot esculenta Crantz) farming system, using AFLP markers. Heredity 85,219-230.
Excoffier L (1995) AMOVA, Analysis of molecular variance, vers.1.55. Univ. of Geneva, Geneva, Switzerland.
Eyre-Walker A, Gaut RL, Hilton H, Feldman DL, Gaut BS (1998) Investigation of the bottleneck leading to the domestication of maize. Proceedings of the National Academy of Sciences 95,4441-4446.
Felsenstein J (1985) Confidence limits on phylogenies:An approach using the bootstrap. Evolution 39,783-791.
Felsenstein J (1993) PHYLIP:phylogenetic inference package, version 3.5c. Department of Genetics, University of Washington.
Forbes FB, Hemsley (1890) Scrophularia ningpoensis Hemsl. Botanical Journal of Linner Society 26,178.
Franch J (1900) S. microdonta. Bull.Soc.Bot.France 47,1900.
Fu YX, Li WH (1993) Statistical tests of neutrality of mutations. Genetics 133,693-709.
Fuller DQ (2007) Contrasting Patterns in Crop Domestication and Domestication Rates:Recent Archaeobotanical Insights from the Old World. Annals of Botany 100,903-924.
Fuller DQ, Qin L, Zheng Y, et al. (2009) The domestication process and domestication rate in rice:spikelet bases from the lower Yangtze. Science 323,1607-1610.
Garcia-Mas J, Oliver M, Gomez-Paniagua H, de Vicente MC (2000) Comparing AFLP, RAPD and RFLP markers for measuring genetic diversity in melon. Theoretical and Applied Genetics 101,860-864.
Ge XJ, Liu MH, Wang WK, Schaal BA, Chiang TY (2005) Population structure of wild bananas, Musa balbisiana, in China determined by SSR fingerprinting and cpDNA PCR-RFLP. Molecular Ecology 14,933-944.
Gepts P, Osborn T, Rashka K, Bliss F (1986) Phaseolin-protein variability in wild forms and landraces of the common bean(Phaseolus vulgaris):evidence for multiple centers of domestication. Economic Botany 40,451-468.
Golding GB (1987) The detectiong of deleterious selection using ancestors inferred from a phylogenetic history. Genetic Resources and Crop Evolution 49,71-82.
Gong W, Chen C, Dobes C, Fu CX, Koch MA (2008) Phylogeography of a living fossil: Pleistocene glaciations forced Ginkgo biloba L. (Ginkgoaceae) into two refuge areas in China with limited subsequent postglacial expansion. Molecular Phylogenetics and Evolution 48, 1094-1105.
Gong W, Fu C, Luo Y, Qiu Y (2006) Molecular Identification of Sinopodophyllum hexandrum and Dysosma Species using cpDNA Sequences and PCR-RFLP Markers. Planta Medica 72, 650-652.
Grivet D, Heinze B, Vendramin GG, Petit RJ (2001) Genome walking with consensus primers: application to the large single copy region of chloroplast DNA. Molecular Ecology Notes 1, 345-349.
Gross BL, Olsen KM (2010) Genetic perspectives on crop domestication. Trends in Plant Science 15,529-537.
Grusz AL, Windham MD, Pryer KM (2009) Deciphering the origins of apomictic polyploids in the Cheilanthes yavapensis complex (Pteridaceae). American Journal of Botany 96, 1636-1645.
Gugerli F, Senn J, Anzidei M, et al. (2001) Chloroplast microsatellites and mitochondrial nadl intron 2 sequences indicate congruent phylogenetic relationships among Swiss stone pine (Pinus cembra), Siberian stone pine (Pinus sibirica), and Siberian dwarf pine (Pinus pumila). Molecular Ecology 10,1489-1497.
Hamilton MB (1999) Four primer pairs for the amplification of chloroplast intergenic regions with intraspecific variation. Molecular Ecology Notes 8,513-525.
Hammer K (1984) Das Domestikationssydrom. Genetic Resources and Crop Evolution 32,11-34.
Han TH, van Eck HJ, De Jeu MJ, Jacobsen E (1999) Optimization of AFLP fingerprinting of organisms with a large-sized genome:a study on Alstroemeria spp. Theoretical and Applied Genetics 98,465-471.
Hare MP (2001) Prospects for nuclear gene phylogeography. Trends in Ecology and Evolution 16, 700-706.
Harris H (1966) Enzyme polymorphism in man. Proc. R. Soc. Lond.164,298-310.
Hartl DL, Clark AG (1989) Principles of Population Genetics., Sunderland, Mass:Sinauer Assoc. Inc. Pub.
He J, Chen L, Si Y, et al. (2009) Population structure and genetic diversity distribution in wild and cultivated populations of the traditional Chinese medicinal plant Magnolia officinalis subsp. biloba (Magnoliaceae). Genetica 135,233-243.
Hernandez P, Martin A, Dorado G (1999) Development of SCARs by direct sequencing of RAPD products:a practical tool for the introgression and marker-assisted selection of wheat. Molecular Breeding 5,245-253.
Hilliard OM (1994) The Manuleae-a tribe of Scrophulariaceae Edinburgh University Press, Edinburgh.
Hong DY (1983) The distribution of Scrophulariaceae in the Holarctic with special reference to the floristic relationships between eastern Asia and eastern North America. Annals of the Missouri Botanical Garden.70,701-712.
Hong DY, Yang HB, Jin C, et al. (1998) Scrophularia. In:wu Z-Y (edit) Flora of China. Vol.18 Missouri Botanocal Garden's Scientific Publications, St. Louis.
Hu P, Luo GA, Zhao ZZ, Jiang ZH (2005) Multi-component HPLC fingerprinting of Radix Salviae Miltiorrhizae and its LC-MS-MS identification. Chemical and Pharmaceutical Bulletin 3,677-683.
Hua R, Sun S, Zhou Q, Noda I, Wang B (2003) Discrimination of Fritillary according to geographical origin with Fourier transform infrared spectroscopy and two-dimensional correlation IR spectroscopy. Journal of Pharmaceutical and Biomedical Analysis 33, 199-209.
Hubby JL, Leqontin RC (1966) A molecular approach to the study of genic heterozygosity in natural populations. I. the number of alleles at different loci in Drosophila pseudoobscura. Genetics 54,577-594.
Hwang SY, Lin TP, Ma CS, Lin CL (2003) Postglacial population growth of Cunninghamia konishii (Cupressaceae) inferred from phylogeographical and mismatch analysis of chloroplast DNA variation. Molecular Ecology 12,2689-2695.
Iwatsuki (1993) Flora of Japan:http://foj.c.u-tokyo.ac.jp/gbif/.
Jaenicke-Despres V, Buckler ES, Smith BD, et al. (2003) Early Allelic Selection in Maize as Revealed by Ancient DNA. Science 302,1206-1208.
Jansen R, Greenbaum D, Gerstein M (2002) Relating Whole-Genome Expression Data with Protein-Protein Interactions. Genome Research 12,37-46.
Kajimoto T, Hidaka M, Shoyama K, Nohara T (1989) Iridoids from Scrophularia ningpoensis. Phytochemistry 28,2701-2704.
Kim SR, Kim YC (2000) Neuroprotective phenylpropanoid esters of rhamnose isolated from roots of Scrophularia buergeriana. Phytochemistry 54,503-509.
Kimura M (1983) The Neutral Theory of Molecular Evolution Cambridge University Press., New York.
King AR, Ferris C (1998) Chloroplast DNA phylogeography of Alnus glutinosa (L.) Gaertn. Molecular Ecology 7,1151-1161.
Kong HH, Wang AL, Lee J, Fu CX (2007) Studies of Systematic evolutioni and karyotypic variation in Smilax and Heterosmilax (smilacaceae). Acta Phytotaxon Sin 45 (3),257-273.
Kovach WL (1999) MVSP-A multivariate statistical Package for Windows, ver.3.1 Kovach Computing Services, Pentraeth, Wales, UK.
Kress WJ, Prince LM, Williams KJ (2002) The phylogeny and a new classification of the gingers (Zingiberaceae):evidence from molecular data. American Journal of Botany 89, 1682-1696.
Kuroda Y, Kaga A, Tomooka N, Vaughan DA (2006) Population genetic structure of Japanese wild soybean (Glycine soja) based on microsatellite variation. Molecular Ecology 15, 959-974.
Lee G, Doh E, Lee M, Ko B, Oh S (2010) Discrimination of three Scrophularia plants utilizing 'Scrophularia Radix'by DNA markers based on internal transcribed spacer (ITS) sequences. Genomics 32,181-189.
Lee TB (2003) Coloured Flora of Korea Academy Book Co., Seoul.
Lee YN (1967) Chromosome numbers of flowering plants in Korea. Journal of Korean Research Institute Ewha Women's University 11,455-478.
Leliaert F, Verbruggen H, Wysor B, Clerck OD (2009) DNA taxonomy in morphologically plastic taxa:Algorithmic species delimitation in the Boodlea complex (Chlorophyta: Cladophorales). Molecular Phylogenetics and Evolution 53,122-133.
Lersten NR, Curtis JD (1997) Anatomy and distribution of foliar idioblasts in Scrophularia and Verbascum (Scrophulariaceae). American Journal of Botany 84,1638.
Lewontin RC (1972) Testing the Theory of Natural Selection. Nature 236,181-182.
Li C, Zhou A, Sang T (2006) Rice Domestication by Reducing Shattering. Science 311, 1936-1939.
Li J, Kelley S, Tredici P, Donoghue MJ (2001) Phylogenetic relationships of Torreya (Taxaceae) inferred from sequences of nuclear ribosomal DNA ITS region, harv. Pap. Bot.6,275-281.
Li J, Zhang D, Donoghue MJ (2003) Phylogeny and biogeography of Chamaecyparis (Cupressaceae) inferred from DNA sequences of the nuclear ribosomal ITS region. Rhodora 105,106-117.
Li Y, Jiang S, Gao W, Zhu D (2000) Phenylpropanoid glycosides from Scrophularia ningpoensis. Phytochemistry 54,923-925.
Liu, Chen, Wu, Xia (1998) Flora of Taiwan:http://www.efloras.org/flora_page.aspx?flora_id= 10
Londo JP, Chiang YC, Hung KH, Chiang TY, Schaal BA (2006) Phylogeography of Asian wild rice. Oryza rufipogon, reveals multiple independent domestications of cultivated rice. Oryza sativa. Proc. Natl. Acad. Sci. USA 103,9578-9583.
Lu G, Chan K, Liang Y, et al. (2005) Development of high-performance liquid chromatographic fingerprints for distinguishing Chinese Angelica from related umbelliferae herbs. Journal of Chromatography A 1073,383-392.
Lum MR, Potter E, Dang T, et al. (2005) Identification of Botanicals and Potential Contaminants through RFLP and Sequencing. Planta Medica 71,841,846.
Lumaret R, Ouazzani N (2001) Plant genetics:Ancient wild olives in Mediterranean forests. Nature 413,700.
Lynch M, Milligan BG (1994) Analysis of population genetic structure with RAPD markers, Molecular Ecology 3,91-99.
Ma X (1984) Chromosome Observations of Some Medical Plants in Xinjian. Acta Phytotaxonomica Sinica 22,243-249.
Matsuoka Y, Vigouroux Y, Goodman MM, et al. (2002) A single domestication for maize shown by multilocus microsatellite genotyping. Proceedings of the National Academy of Sciences 99,6080-6084.
Maude FP (1940) Chromosome Numbers in Some British Plants. New Phytologist 39,17-32.
McKey D, Elias M, Pujol B, Duputie A (2010) The evolutionary ecology of clonally propagated domesticated plants. New Phytologist 186,318-332.
Mehra PN, Vasudevan KN (1972) Scrophularia dentata 4257. In:Love A (edit) IOPB chromosome number reports XXXVI. Taxon 21,343.
Micharel DP, Dorian QF (2009) The nature of selection during plant domestication. Nature 457, 843-848.
Miller A, Schaal B (2005) Domestication of a Mesoamerican cultivated fruit tree, Spondias purpurea. Proceedings of the National Academy of Sciences 102,12801.
Mitton JB, Kreiser BR, Latta RG (2000) Glacial refugia of limber pine (Pinus flexilis James) inferred from the population structure of mitochondrial DNA. Molecular Ecology 9,91-97.
Miyazawa M, Okuno Y (2003) Volatile components from the roots of Scrophularia ningpoensis Hemsl. Flavour and Fragrance Journal 18,398-400.
Moraes RM, Momm HG, Silva B, et al. (2005) Geographic information system method for assessing chemo-diversity in Medicinal plants. Planta Medica 71,1157-1164.
Morrell PL, Clegg MT (2007) Genetic evidence for a second domestication of barley (Hordeum vulgare) east of the Fertile Crescent. Proceedings of the National Academy of Sciences 104, 3289.
Mueller UG, Wolfenbarger LL (1999) AFLP genotyping and figerprinting. Trends in Ecology and Evolution 14,389-394.
Muller MH, Prosperi JM, Santoni S, Ronfort J (2003) Inferences from mitochondrial DNA patterns on the domestication history of alfalfa (Medicago sativa). Molecular Ecology 12, 2187-2199.
Nei M (1987) Molecular evolutionary genetics Oxford University Press, New York, USA.
Nei M, Tajima F (1983) Maximum likelihood estimation of the number of nucleotide substitutions from restriction sites data. Genetics 105,205-217.
Nesbitt TC, Tanksley SD (2002) Comparative Sequencing in the Genus Lycopersicon: Implications for the Evolution of Fruit Size in the Domestication of Cultivated Tomatoes. Genetics 162,365-379.
Nguyen A, Fontaine J, Malonne H, et al. (2005) A sugar ester and an iridoid glycoside from Scrophularia ningpoensis. Phytochemistry 66,1186-1191.
Nishikawa (1985) Chromosome counts of flowering plants of Hokkaido (Japan). Journal of Hokkaido 36,25-40.
Obeso JR (2002) The costs of reproduction in plants. New Phytologist 155,321-348.
Olmstead RG (1995) Evidence for the Polyphyly of the Scrophularia Based on Chloroplast rbcL and ndhF Sequences. Annals of the Missouri Botanical Garden 82,176-193.
Olmstead RG (2001) Disintegration of the Scrophulatiaceae. American Journal of Botany 88, 348-361.
Olsen KM, Schaal BA (1999) Evidence on the origin of cassava:phylogeography of Manihot esculenta. Proceedings of the National Academy of Sciences 96,5586-5591.
Olsen KM, SchaalL BA (2007) Insights on the evolution of a vegetatively propagated crop species. Molecular ecology 16,2838-2840.
Otero-arnaiz A, Casas A, Hamrick JL, Cruse-sanders J (2005) Genetic variation and evolution of Polaskia chichipe (Cactaceae) under domestication in the Tehuacan Valley, central Mexico. Molecular Ecology 14,1603-1611.
Ozkan H, Brandolini A, Schafer-Pregl R, Salamini F (2002) AFLP Analysis of a Collection of Tetraploid Wheats Indicates the Origin of Emmer and Hard Wheat Domestication in Southeast Turkey. Molecular Biology and Evolution 19,1797-1801.
Palme AE (2002) Chloroplast DNA variation, postglacial recolonization and hybridization in hazel, Corylus avellana. Molecular Ecology 11,1769-1779.
Palmer JD (1992) Mitochondrial DNA in systematics:applications and limitations. In:Soltis P S, Soltis D E, Doyle J J, Ed. Molecular Systematics of Plants Champman and Hall, New York.
Palumbi SR, Baker CS (1994) Contrasting population structure from nuclear intron sequences and mtDNA of humpback whales. Molecular Biology and Evolution 11,426-435.
Panwar P, Nath M, Yadav V, Kumar A (2010) Comparative evaluation of genetic diversity using RAPD. SSR and cytochrome P450 gene based markers with respect to calcium content in finger millet (Eleusine coracana L. Gaertn.). Journal of Genetics 89,121-133.
Paran I, Michelmore RW (1993) Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce. Theoretical and Applied Genetics 85,985-993.
Passinho-Soares H, Felix D, Kaplan MA, Margis-Pinheiro M, Margis R (2006) Authentication of Medicinal Plant Botanical Identity by Amplified Fragmented Length Polymorphism Dominant DNA Marker:Inferences from the Plectranthus Genus. Planta Medica 72,929, 931.
Pereira RJ, Wake DB (2009) Genetic leakage after adaptive and nonadaptive divergence in the ensatina eschscholtzii ring species. Evolution 63,2288-2301.
Petit E, Balloux F, Goudet J (2001) Set-biased dispersal in a migraatory bat:a characterization using sex-specific demographic parameters. Evolution 55,635-640.
Petit RJ, Csaikl MU, Bords S, et al. (2002) Chloroplast DNA variation in European white oaks: Phylogeography and patterns of diversity based on data from over 2600 populations. Forest Ecology and Management 156,5-26.
Pickersgill B (1997) Genetic resources and breeding of Capsicum spp. Euphytica 96,129-133.
Piperno DR, Flannery KV (2001) The earliest archaeological maize (Zea mays L.) from highland Mexico:New accelerator mass spectrometry dates and their implications. Proceedings of the National Academy of Sciences 98,2101-2103.
Posada D (2008) jModelTest:Phylogenetic Model Averaging. Molecular Biology and Evolution 25,1253-1256.
Pritchard JK, Stephens M, Donnelly P (2000) Inference of Population Structure Using Multilocus Genotype Data. Genetics 155,945-959.
Provan J, Powell W, Hollingsworth PM (2001) Chloroplast microsatellites:new tools for studies in plant ecology and evolution. Trends in Ecology and Evolution 16,142-147.
Provan J, Soranzo N, Wilson NJ, Goldstein DB, Powell W (1999) A Low Mutation Rate For Chloroplast Microsatellites. Genetics 153,943-947.
Qian J, Hunkler D, Rimpler H (1992) Iridoid-related aglycone and its glycosides from Scrophularia ningpoensis. Phytochemistry 31,905-911.
Qiu Y, Fu C, Comes HP (2011) Plant molecular phylogeography in China and adjacent regions: Tracing the genetic imprints of Quaternary climate and environmental change in the world's most diverse temperate flora. Molecular Phylogenetics and Evolution 59,225-244.
Qiu YX, Sun Y, Zhang XP, et al. (2009) Molecular phylogeography of East Asian Kirengeshoma (Hydrangeaceae) in relation to Quaternary climate change and landbridge configurations. New Phytologist 183,480-495.
Qiu YX, Zong M, Yao Y, et al. (2009) Genetic variation in wild and cultivated Rhizoma corydalis revealed by ISSRs markers. Planta medica 75,94-98.
Quicke DLJ, Jones OR, Epstein DR (2007) Correcting the Problem of false incongruence due to noise imbalance in the Incongruence Length Difference (ILD) Test. Systematic Biology 56, 496-503.
Ren N, Timko MP (2001) AFLP analysis of genetic polymorphism and evolutionary relationships among cultivated and wild Nicotiana species. Genome 44,559-571.
Richardson IBK (1993) Scrophulariaceae. In V. H. Heywood (edit) Flowering plants of the world Oxford University Press, New York.
Rogers AR, Harpending H (1992) Population growth makes waves in the distribution of pairwise genetic differences. Molecular Biology and Evolution 9,552-569.
Rozas J, Sanchez-DelBarrio JC, Messeguer X, Rozas R (2003) DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 19,2496-2497.
Sagare AP, Kuo CL, Chueh FS, Tsay HS (2001) De novo regeneration of Scrophularia yoshimurae Yamazaki (Scrophulariaceae) and quantitative analysis of harpagoside, an iridoid glucoside, formed in aerial and underground parts of in vitro propagated and wild plants by HPLC. Biological& pharmaceutical bulletin 24,1311-1315.
Sang T, Crawford DJ, Stuessy TF (1995) Documentation of reticulate evolution in peonies (Paeonia) using internal transcribed spacer sequences of nuclear ribosomal DNA: implications for biogeography and concerted evolution. Proceedings of the National Academy of Sciences 92,6813-6817.
Sang T, Crawford DJ, Stuessy TF (1997) Chloroplast DNA phylogeny, reticulate evolution, and biogeography of Paeonia (Paeoniaceae). American Journal of Botany 84,1120.
Schaal AB, Hayworth AD, Olsen MK, Rauscher TJ, Smith TW (1998) Phylogeographic studies in plants:problems and prospects. Molecular Ecology 7,465-474.
Scherer H (1939) Chromosomenzahlen aus der schleswig-holsteinischen. Flora, I. Planta 29, 636-642.
Scheunert A, Heubl G (2011) Phylogenetic relationships among New World Scrophularia L. (Scrophulariaceae):new insights inferred from DNA sequence data. Plant Systematics and Evolution 291,69-89.
Schneider S (2000) ARLEQUIN, version 2.000:a software for population genetics data analysis (ed. Roessli D). Genetics and Biometry Laboratory, University of Geneva, Geneva, Switzerland.
Shi W, Yang CF, Chen JM, Guo YH (2008) Genetic variation among wild and cultivated populations of the Chinese medicinal plant Coptis chinensis (Ranunculaceae). Plant Biology 10,485-491.
Slade RW, Moritz C, Heideman A (1994) Multiple nuclear-gene phylogenies:application to pinnipeds and comparison with a mitochondrial DNA gene phylogeny. Molecular Biology and Evolution 11,341-356.
Slade RW, Moritz C, Heideman A, Hale PT (1993) Rapid assessment of single-copy nuclear DNA variation in diverse species. Molecular Ecology 2,359-373.
Slatkin M (1987) Gene flow and the geographic structure of natural populations. Science 236,787-792.
Slatkin M (1993) Isolation by distance in equilibrium and non-equilibrium populations. Evolution 47,264-279.
Slatkin M, Hudson RR (1991) Pairwise Comparisons of Mitochondrial DNA Sequences in Stable and Exponentially Growing Populations. Genetics 129,555-562.
Smith BD (1989) Origins of Agriculture in Eastern North America. Science 246,1566-1571.
Smith BD (2001) Documenting plant domestication:The consilience of biological and archaeological approaches. Proceedings of the National Academy of Sciences 98,1324-1326.
Sonnante G, Stockton T, Nodari RO, Becerra Velasquez VL, Gepts P (1994) Evolution of genetic diversity during the domestication of common-bean (Phaseolus vulgaris L.). Theoretical and Applied Genetics 89,629-635.
Stamatakis A, Hoover P, Rougemont J (2008) A Rapid Bootstrap Algorithm for the RAxML Web Servers. Systematic Biology 57,758-771.
Stamatakis A, Ott M, Ludwig T (2005) RAxML-OMP:An Efficient Program for Phylogenetic I nference on SMPs. Computer Science 3606,288-302.
Stiefelhagen H (1910) Systematische und pflanzengeographische studien zur Kenntnis der Gattung Scrophularia. Botanical Jahrb Sysematicst.44,406-496.
Strand AE, Leebens-Mack J, Milligan BG (1997) Nuclear DNA-based markers for plant evolutionary biology. Molecular Ecology 6,113-118.
Stuessy TF (1990) Plant taxonomy. Columbia univ. press, New York.
Swofford DL (2002) PAUP*:phylogenetic analysis using parsimony (* and other methods), version 4.0b10, Sunderland, MA, USA:Sinauer Associates.
Sytsma KJ, Schaal BA (1985) Genetic Variation, Differentiation, and Evolution in a Species Complex of Tropical Shrubs Based on Isozymic Data. Evolution 39,582-593.
Taberlet P, Gielly L, Pautou G, Bouvet J (1991) Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Molecular Biology 17,1105-1109.
Tajima F (1989) Statistical-method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123,585-595.
Techen N, Khan IA, Pan Z, Scheffler BE (2006) The Use of Polymerase Chain Reaction (PCR) for the Identification of Ephedra DNA in Dietary Supplements. Planta Medica 72,241,247.
Templeton AR (1998) The detectiong of deleterious selection using ancestors inferred from a phylogenetic histor. Molecular Ecology 7.
Templeton AR (2006) Population genetics and microevolutionary theory John Wiley & Sons, Inc., Hoboken, New Jersey, USA.
Tenaillon MI, U'Ren J, Tenaillon O, Gaut BS (2004) Selection Versus Demography:A Multilocus Investigation of the Domestication Process in Maize. Molecular Biology and Evolution 21,1214-1225.
The Angiosperm Phylogeny Group (1998) An ordinal classification for the families of flowering plants. Annals of the Missouri Botanical Garden 85,531-553.
The Angiosperm Phylogeny Group (2003) An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants:APG II. Botanical Journal of the Linnean Society 141,399-436.
Thieret JW (1967) Supraspecific classification in the Scrophulariaceae:a review. Sida 3,87-106.
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL X Windows Interface:Flexible Strategies for Multiple Sequence Alignment Aided by Quality Analysis Tools. Nucleic Acids Research 25,4876-4882.
Tian F, Stevens NM, Buckler ES (2009) Tracking footprints of maize domestication and evidence for a massive selective sweep on chromosome 10. Proceedings of the National Academy of Sciences 106,9979-9986.
Tzedakis PC, Lawson IT, Frogley MR, Hewitt GM, Preece RC (2002) Buffered tree population changes in a Quaternary refugium:evolutionary implications. Science 297,2044.
Vaarama A, Hiirsalmi H (1967) Chromosome studies on some Old World species on the genus Scrophularia. Hereditas 58,333-358.
Vos P, Hogers R, Bleeker M, et al. (1995) AFLP:a new technique for DNA fingerprinting. Nucleic Acids Research 23,4407-4414.
Weismann A (1892) Essays upon heredity and kindred biological Problems. Vol.2. Edited by Poulton, E. B. and Shipley, A. E. the Clarendon Press., Oxford.
Wen J, Shi S (1999) A phylogenetic and biogeographic study of Hamamelis (Hamamelidaceae), an eastern Asian and eastern North American disjunct genus. Biochemical Systematics and Ecology 27,55-66.
White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR protocols a guide to methods and applications Academic Press, San Diego.
Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research 18, 6531-6535.
Wills DM, Burke JM (2006) Chloroplast DNA Variation Confirms a Single Origin of Domesticated Sunflower (Helianthus annuus L.). Journal of Heredity 97,403-408.
Wolfe KH, Li WH, Sharp PM (1987) Rates of nucleotide substitution vary greatly among plant mitochondrial, chloroplast, and nuclear DNAs. Proceedings of the National Academy of Sciences 84,9054-9058.
Woo Y, Kim H, Cho J, Chung H (1999) Discrimination of herbal medicines according to geographical origin with near infrared reflectance spectroscopy and pattern recognition techniques. Journal of Pharmaceutical and Biomedical Analysis 21,407-413.
Wright S (1931) Statistical methods in biology. Journal of the American Statistical Association 26,155-161.
Wright S (1978) Evolution and the genetics of populations. University of Chicago Press, Chicago, IL.
Wright SI, Bi IV, Schroeder SG, et al. (2005) The Effects of Artificial Selection on the Maize Genome. Science 308,1310-1314.
Xiang Q, Soltis DE, Soltis PS, Manchester SR, Crawford DJ (2000) Timing the Eastern Asian-Eastern North American Floristic Disjunction:Molecular Clock Corroborates Paleontological Estimates. Molecular Phylogenetics and Evolution 15,462-472.
Xiang QY, Crawford DJ, Wolfe AD (1998) Origin and biogeography of Aesculus L.(Hippocastanaceae):a molecular phylogenetic perspective. Evolution 52,988-997.
Yamane K, Lu N, Ohnishi O (2009) Multiple origins and high genetic diversity of cultivated radish inferred from polymorphism in chloroplast simple sequence repeats. Breeding Science 59,55-65.
Yang ST, Chen C, Zhao YP, et al. (2011) Association between Chemical and Genetic Variation of Wild and Cultivated Populations of Scrophularia ningpoensis Hemsl. Planta Medica 77, 865-871.
Ye Q, Qiu Y, Quo Y, et al. (2006) Species-specific SCAR markers for authentication of Sinocalycanthus chinensis. Journal of Zhejiang University-Science B 7,868-872.
Yeh F, Yang RC, Boyle T, Ye ZH (1997) POPGENE, the User-Friendly Shareware for Population Genetic Analysis. Molecular Biology and Biotechnology Centre, University of Alberta, Edmonton.
Yuan QJ, Zhang ZY, Hu J, et al. (2010) Impacts of recent cultivation on genetic diversity pattern of a medicinal plant, Scutellaria baicalensis (Lamiaceae). Bmc Genetics 11,29.
Zeder MA (2006) Central questions in the domestication of plants and animals. Evolutionary Anthropology:Issues, News, and Reviews 15,105-117.
Zeder MA, Emshwiller E, Smith BD, Bradley DG (2006) Documenting domestication:the intersection of genetics and archaeology. Trends in Genetics 22,139-155.
Zhang D, Hewitt GM (2003) Nuclear DNA analyses in genetic studies of populations:practice, problems and prospects. Molecular Ecology 12,563-584.
Zhang D, Sang T (1999) Physical mapping of ribosomal RNA genes in peonies (Paeonia, Paeoniaceae) by fluorescent in situ hybridization:implications for phylogeny and concerted evolution. American Journal of Botany 86,735-740.
Zhang DQ, Gao LM, Yang YP (2010) Genetic diversity and structure of a traditional Chinese medicinal plant species, Fritillaria cirrhosa (Liliaceae) in southwest China and implications for its conservation. Biochemical Systematics and Ecology 38,236-242.
Zhao YP, Qiu YX, Gong W, Li JH, Fu CX (2007) Authentication of Actinidia macrosperma Using PCR-RFLP Based on trnK Sequences. Botaniy Study 48(3),46-49.
Zhao ZZ, Hu Y, Liang ZT, et al. (2006) Authentication is fundamental for standardization of Chinese Medicines. Planta Medica 72,865-874.
Zietkiewicz E, Rafalski A, Labuda D (1994) Degome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification. Genomics 20(2),176-183.
陈大霞,李隆云,彭锐,吴叶宽,蔡应繁(2009)玄参3种栽培类型遗传关系和遗传多样性的SRAP研究。中国中药杂志34,138-142。
戴金秋,邢振荣(1994)浙江产玄参的化学成分。现代应用药学11,16-1。
龚维(2007) 孑遗植物银杏的分子亲缘地理学研究,浙江大学。
管毕财(2008)特有濒危植物八角莲保护遗传学和分子亲缘地理学,浙江大学。
胡世林(1989)《中国道地药材》黑龙江科学记数出版社,哈尔滨。
胡世林(1995)现代道地论概要.中国医药信息杂志 2,7。
国家药典委员会(2005)《中华人民共和国药典》2005年版。化学工业出版社,北京。
黄璐琦,陈美兰,肖培根(2004)中药材道地性研究的现代生物学基础及模式假说。中国中药杂志29(6),494-496。
黄璐琦,张瑞贤(1997)“道地药材”的生物学探讨。中国药学杂志9(32),563。
李密密 (2011)山茶科紫茎属和折柄茶属系统进化研究,浙江大学。
李时珍(1590)《本草纲目》中国医药出版社,北京。
刘承伟,毕志明,祝艳斐,李萍(2007)玄参中4种主要活性成分的HPLC定量分析。中国药学杂志 42,3。
马兴华,覃若林,邢文斌(1984)新疆某些药用植物的染色体观察。植物分类学报 22,243-249。
田欣,李德铢(2002)DNA序列在植物系统学研究中的应用。云南植物研究24,170-184。
万德光(2008)《中药品质研究--理论、方法与实践》。上海科学技术出版社,上海.
王家葵,王佳黎(2007)《中药材品种沿革及道地性》。中国医药科技出版社,北京。
吴云(2006)玄参主要病虫害及防治技术。植物医生 19,30-02。
谢丽华(2000)中药玄参薄层色谱鉴别法的建立。中国中药杂志 25,654-656。
杨舒婷(2011)玄参化学多样性及其与遗传变异和环境因子之间的关系研究,浙江大学。
俞静静,王琴,吕圭源(2006)玄有效部位的药理研究进展。中华实用中西医杂志 19, 1866-1867。
章国镇(2006)《神农本草经(精选图文本)》。福建科学技术出版社,福建。
赵志新,梁宗锁,姜在民,薛媛菲,杨永宙(2007)玄参ISSR-PCR反应体系的建立与优化。药物生物技术14,318-323。
中国科学院中国植物志编委会(1979)《中国植物志》。科学出版社,北京。
周浓(2008)玄参的质量评价及其指纹图谱研究,西南大学。
祝艳斐,毕志明,刘承伟(2008)内皮细胞提取和高效液相色谱一电喷雾匕行时间质谱联用预测玄参中的活性成分。中国药科大学学报3,228-231。
邹节明(2005)苦玄参HPLC(?)指纹图谱研究。中国药学杂志 40,13-15。