鬼臼亚科系统发育与八角莲属的亲缘地理学研究

发布时间：2018-04-30 10:54

本文选题：八角莲属 + 鬼臼亚科　；参考：《浙江大学》2015年博士论文

【摘要】：八角莲属(Dysosma)植物隶属于小檗科(Berberidaceae)鬼臼亚科(Podophylloideae),其根状茎含有的生物活性成分鬼臼毒素(Podophylloxotin)等是合成抗癌药物的前体。该属植物分布于我国东南、中南至西南地区,其中,八角莲(D. versipellis)为广布种,其余种均为地方特有种。最西部的西藏八角莲(D. tsayuensis)分布于东喜马拉雅,最东部的六角莲(D. pleiantha)间断分布于我国东部与台湾。该属有着近200年的分类历史,但由于其形态性状的变异性以及采样不足,使得它与近缘属的分类问题和系统发育问题一直没有得到很好地解决。以前的系统发育和亲缘地理学研究仅涉及其中的少数种,因此有关属内7个种的物种形成与分化机制以及该属的整合分类系统是一个亟需解决的科学问题。基于本课题组对八角莲属近10年的材料积累和广泛的野外资源调查取样,在形态学研究的基础上,利用系统发育、生物地理以及群体遗传学原理与方法,对鬼臼亚科的系统发育以及八角莲属植物地理分布与物种形成的时间格局与机制进行了全面深入的研究,获得以下主要结果：1)鬼臼亚科的系统发育及分化时间：对鬼臼亚科4属12个种进行了广泛的野外取样,采集了中国、日本和北美的63个群体材料,采集群体涵盖了八角莲属的整个地理分布区域。以Wang et al.(2007)的小檗科主要类群的系统发育树为基础(matK、rbcL),补充八角莲属缺失的6个种,并对鬼臼亚科12个种增加了叶绿体trnL-trnF、atpM-atpl和ITS和ETS的扩增,联合6个片段,通过系统发育分析构建了鬼臼亚科12个种的分子系统进化树,并结合两个化石校正点(小檗科的crown节点分化时间91Ma,Ranzania、Berberis和Mahonia三个属的crown节点时间45 Ma)和鬼臼亚科二次校正点(8.8士1.4 Ma),利用BEAST进行分化时间估算,揭示了鬼臼亚科属同及属内种间的系统发育关系：鬼臼亚科是一个单系类群(PP=1),山荷叶属为一个单系(PP=1)位于鬼臼亚科的基部,八角莲属是一个很好的单系(PP=1),与东亚-北美间断分布的姐妹类群这儿土和足叶草组成一个支系,但支持率较低(PP=0.67)。八角莲属内的广布种八角莲不是单系,而是与六角莲形成并系,分子系统树支持其它形态学种为单系类群。分歧时间估算结果表明鬼臼亚科的共祖时问为上新世[c.12.67 Ma(10.71-14.56 Ma)]；山荷叶属的共祖时间为7.96(5.19-10.61)Ma；八角莲属、桃儿七属和足叶草属的分化(7.96-9.14 Ma)与中新世的全球气候变冷有关。BBM结果揭示了八角莲属起源于东喜马拉雅,并自上新世由西向东迁移。在八角莲属中,位于西部的云南八角莲、西藏八角莲和川八角莲分化时间较早(7.76-9.14 Ma),其分化时间与青藏高原早期快速隆升时间(8-13 Ma)一致,由此可知,早期地理隔离导致了西部类群异域成种。2)八角莲属种水平亲缘地理学选取八角莲属7个种46个群体进行了亲缘地理学研究,研究结果揭示了八角莲属内7个种的谱系关系及遗传多样性的时空分布格局。3个叶绿体片段(trnL-trnF.trnL-ndhJ.trnS-trnfM)序列变异的遗传多样性分析揭示了八角莲属共有66个叶绿体单倍型,八角莲属水平的遗传多样性相对较高(hT=0.929,πT=3.58×10-3),并具有显著的谱系地理结构(NST=0.866,GST=0.790,P0.05)。广布种八角莲的单倍型多样性和核苷酸多样性(hT=0.314；πT= 0.25×10-3)比其它窄域种都高；八角莲(NsT=0.818,GsT=0.611,P0.05)、小八角莲(NST=0.865,GsT=0.728,P0.01)和川八角莲(NST=0.977,GST=0.870,P0.05)的谱系地理结构明显。TCS单倍型谱系关系和系统发育分析结果均揭示,八角莲属共分为6个与地理分布对应的谱系(Lineasel-Lineage6),它包括分布于中国西部的云南八角莲(Lineage 6).西藏八角莲(Lineage 5)和川八角莲(Lineage 4)依次分化出来。分布于中国中部-东南部的小八角莲、贵州八角莲、六角莲及八角莲聚成一个单系[Lineage 1+(Lineage 2+Lincage 3)]；其中,广布种八角莲单倍型分为两个谱系(西北谱系Lineage 2和东南谱系Lineagel),是一个并系类群；单系类群六角莲(Lineage 3)与八角莲西北部谱系Lineage 2为姐妹关系；小八角莲和贵州八角莲没有独立分支,小八角莲(H1,H52-56)镶嵌于八角莲的东南谱系中,并共享单倍型H1；贵州八角莲的所有个体和小八角莲共享一个单倍型H55。基于ITS序列分析表明,八角莲属内7个种没有共有单倍型。cpDNA与ITS单倍型的谱系关系在小八角莲、贵州八角莲、八角莲以及六角莲之间呈现不一致的现象,表明了这3个种在同域分布区可能存在杂交与基因渐渗现象。失配分析表明,八角莲东南谱系(Lineage 1)和六角莲谱系(Lineage 3)都经历了显著的群体扩张,但八角莲西北谱系(Lineage 2)和川八角莲谱系(Lineage 4)长期居留原地。八角莲东南谱系的扩张时间为更新世早期[1.78 Ma(95%CI：1.01-3.03 Ma)],表明气候变冷导致暖温带落叶林下的八角莲表现出扩张信号。六角莲的扩张时间为更新世晚期[0.18 Ma (95% CI:0.00072-0.75 Ma)],与倒数第二个间冰期时间一致一致(c.0.25-0.135 Ma),表明气候变暖使暖温带常绿林的六角莲呈现扩张信号。3)DNA条形码(DNA barcoding)通过对9个候选的DNA条形码(ITS、matK、rbcL、trnL-trnF、trnL-nbhJ、 trnS-trnfM、atpH-atpl、rpl32-trnL、rps18-clpp)在鬼臼亚科植物中的鉴定能力进行了评估,比较分析了各条形码的扩增成功率、种内和种间变异、barcoding gap,并运用BLAST、DISTANCE和构建系统发育树方法评估了不同序列的物种鉴定成功率,结果表明除rps18-clpp外其余8个条形码对鬼臼亚科12个种的通用性均为100%,其中通用引物ITS扩增的序列种内与种间变异的差异、barcodinggap较其他序列具有更明显的优势,其种鉴定成功率相对较高(83.3%)。因此,ITS是适合鬼臼亚科植物鉴别最好的DNA条形码。4)云南八角莲和八角莲的比较转录组通过Hiseq 2000测序平台,对高海拔的云南八角莲和低海拔的八角莲进行高通量转录组测序,运用de novo从头组装的方法,分别获得了53929和44855条de novo基于序列相似性,我们找出两个种的直系同源基国(Orthologs)为4593对,其中3126对直系同源基因有(？)Ks值,Ka/Ks1的同源基因有202对,0.5 Ka/Ks 1的同源基因有535对,这737对同源基因只有一对具有显著P值(glycosyltransferase gene, Ka/Ks=0.606, P=0.02)。1273对表现出显著的净化选择(Ka/Ks0.5, P0.05)。另外,4593直系同源基因共含有SSR 461个,其中,去除长度小于16 bp的、侧翼序列不足够长的同源基因,最终成功设计了51对EST-SSR引物。我们检验了这些引物的通用性,其中26对(51%)和41对(80.4%)分别在鬼臼亚科和八角莲属具有通用性。最后,我们利用PCR直接扩增的方法得到了鬼臼亚科12个种33样本的19个CYP719A(鬼臼毒素合成路径的关键基因)的基因序列,并分析了它们的分子进化式样,结果阐明了CYP719A基因在鬼臼亚科经历了强烈的净化选择,在八角莲属内表现为分布海拔较高的物种具有松散的净化选择(relaxed purifying selection)或较强的正向选择(more positive selection)。比较转录组研究为后续八角莲属物种及其近缘物种的群体遗传学与进化生物学研究提供了大量的分子标记。综上所述,鬼臼亚科是一个单系,山荷叶属为基部类群,北美山荷叶与日本山荷叶成姐妹关系,八角莲属是一个很好的单系。青藏高原快速隆升导致的地理隔离驱使了八角莲属西部类群的异域物种形成；上新世-更新世的气候动荡变化是八角莲属中东部物种分化和遗传多样性形成的主要原因,六角莲是近期分化的物种；ITS是鬼臼亚科鉴定种最好的DNA条形码。大量的属水平的EST-SSR引物和单拷贝核基因被开发出来,填补了八角莲属遗传资源缺失的空白,CYP719A在八角莲属内的分子进化模式表明该基因在高海拔的物种中经历了更强的正向选择或松散的净化选择。
[Abstract]:Dysosma plants belong to the family Berberidaceae (Podophylloideae) of the family berberisfamily (Berberidaceae). The rhizome containing the bioactive component of the podophyllotoxin (Podophylloxotin) is a precursor to the synthesis of anticancer drugs. The genus is distributed in Southeast China, from middle to south to southwest, among which, the D. versipellis is widely distributed, and the rest of the genus (D.). The species are endemic species. The most Western Tibet octagonal (D. tsayuensis) is distributed in East Himalaya, and the most Eastern six hornies (D. pleiantha) are discontinuous in eastern China and Taiwan. This genus has nearly 200 years of classification history, but because of its variation in morphological characters and lack of sampling, it has made it with the classification problems and lines of the genera. The problem of phylogenetic development has not been well solved. Previous phylogenetic and genetic geographic studies only involve a few of them. Therefore, the mechanism of species formation and differentiation of the 7 species of the genus and the integrated classification system of this genus are an urgent problem to be solved. Based on this group, the materials of the genus aniseed for nearly 10 years On the basis of morphological studies, the systematic development of podophylloid and the temporal pattern and mechanism of the geographical distribution and species formation of the genus octagonal plants were thoroughly studied on the basis of the morphological studies, and on the basis of morphological studies. Fruit: 1) phylogeny and differentiation time of podophyllosubfamily: 12 species of 4 genera and 12 species of podophylloid were widely sampled, and 63 population materials from China, Japan and North America were collected. The collection group covered the whole geographical distribution area of the genus octagon. The phylogenetic tree of the main groups of berberid in Wang et al. (2007) was based on the phylogenetic tree of the main group of berberid (mat K, rbcL), supplemented with 6 species of anisetin deletion, and 12 species of podophylloid, adding chloroplast trnL-trnF, atpM-atpl and ITS and ETS amplification, combined 6 fragments, constructed 12 species of podophylloid molecular phylogenetic tree by phylogenetic analysis, and combined two fossil correction points (crown node differentiation time 91Ma, Ranza). The crown node time of three genera of NIA, Berberis and Mahonia (45 Ma) and two calibration points of podophylloid (8.8. 1.4 Ma), using BEAST to estimate the differentiation time, revealing the phylogenetic relationship between the genus and interspecies of the genus podophyllosubfamily: podophyllosubfamily is a monophyletic group (PP= 1), and the genus monophylla is a monophylla (PP=1) located in the podophylloid family. The base part of the genus octagonal is a very good monophyle (PP=1), which is a branch of the sister groups of the East Asian and North American discontinuous distribution, but the support rate is lower (PP=0.67). The wide species of octagonal lotus in the genus octagon is not a single line, but is formed with six horns. The molecular system tree supports the other morphological species as monophyletic groups. The common ancestor of podophylloid is [c.12.67 Ma (10.71-14.56 Ma) of the Pliocene; the total progenitor time of the genus Hyde is 7.96 (5.19-10.61) Ma, octagonal lotus, the differentiation of peach seven and Poda (7.96-9.14 Ma) and the global climate change in the middle Miocene revealed that the genus octagon originated from the eastern joy horse. Laya, and migrated from west to East in the Pliocene. In the genus octagonal lotus, Yunnan octagonal lotus, Tibet octagonal lotus and Sichuan aniseed (7.76-9.14 Ma), the differentiation time is consistent with the early rapid uplift time (8-13 Ma) of the Qinghai Tibet Plateau, thus it can be seen that early geographical isolation led to the western group of exotic species.2) octagonal. The genetic diversity of the 7 species of the genus aniseus from 46 groups of 7 species of the genus aniseus was studied. The results revealed the genealogical relationship of 7 species in the genus aniseus and the spatial and temporal distribution pattern of genetic diversity. The genetic diversity of the sequence variation of trnL-trnF.trnL-ndhJ.trnS-trnfM (chloroplast fragments) was revealed by the analysis of the genetic diversity. There are 66 haplotypes of chloroplasts in the genus lotus. The genetic diversity of the level of aniseed is relatively high (hT=0.929, PI T=3.58 x 10-3), and has a distinct genealogical structure (NST=0.866, GST=0.790, P0.05). The haplotype diversity and nucleotide diversity (hT=0.314; PI T= 0.25 * 10-3) of the species of aniseed are higher than those of other narrow species; NsT=0.818, GsT=0.611, P0.05), the genealogical geographical structure of NST=0.865, GsT=0.728, P0.01 and NST=0.977, GST=0.870, P0.05 (NST=0.977, GST=0.870, P0.05) and the phylogenetic relationship of.TCS haplotype and phylogenetic analysis revealed that the genus octagon was divided into 6 pedigree (Lineasel-Lineage6) corresponding to the earth's distribution, including the west of China. The Yunnan octagonal Lotus (Lineage 6). Tibet octagonal Lotus (Lineage 5) and Sichuan alba (Lineage 4) are divided in turn. They are distributed in the middle and southeastern part of China, small octagonal lotus, Guizhou latitum, six latitum and octagonal lotus, and a single line [Lineage 1+ (Lineage 2+Lincage 3)]. The northern pedigree Lineage 2 and the southeastern pedigree Lineagel) are a group of groups; the monophyletic group of six Lotus (Lineage 3) and the pedigree Lineage 2 of the northwestern aniseed are sister; the small octagonal and Guizhou octagonal lotus have no independent branch, and the small octagonal (H1, H52-56) is inlaid in the southeastern pedigree of the eight hornblero and shares the haplotype H1; Guizhou octagonal. The sharing of a haplotype H55. based on ITS sequence analysis of a haplotype H55. showed that the genealogical relationship between the 7 species in the genus octagon and ITS haplotype in the 7 species of the genus octagonal was not consistent between the eight aniseed, the octagonal lotus, the latitum and the six horns, indicating that the 3 species may exist in the same domain. In the hybridization and gene introgression, the mismatch analysis showed that the southeast pedigree pedigree (Lineage 1) and the six nicia lotus pedigree (Lineage 3) experienced significant population expansion, but the north-west pedigree (Lineage 2) and the Sichuan anisome pedigree (Lineage 4) stayed in place for a long time. The expansion time of the southeast eight pedigree pedigree was [1.78 Ma (95%CI) in the early Pleistocene. 1.01-3.03 Ma): the results showed that the cold climate caused the expansion of the aniseed under the warm temperate deciduous forest. The expansion time of six lotus was [0.18 Ma (95% CI:0.00072-0.75 Ma) in the late Pleistocene, consistent with the second interglacial times (c.0.25-0.135 Ma), indicating that the warming made the six lotus of the warm temperate evergreen forest expand. .3 DNA barcode (DNA barcoding) is used to evaluate the identification ability of 9 candidate DNA barcodes (ITS, matK, rbcL, trnL-trnF, trnL-nbhJ, trnS-trnfM, atpH-atpl, etc.) in the subfamily podophylloid. LAST, DISTANCE and phylogenetic tree method were constructed to evaluate the success rate of species identification in different sequences. The results showed that the generality of the 12 species of podophylloid in the other 8 barcodes, except rps18-clpp, was 100%, of which the difference in the intraspecific and interspecific mutation of the universal primer ITS amplification was more obvious than that of the other sequences. Potential, the success rate of identification is relatively high (83.3%). Therefore, ITS is the best DNA barcode for identification of podophylloid plants.) the comparative transcriptional group of Yunnan octagonal and octagonal lotus through Hiseq 2000 sequencing platform, the high altitude of octagonal lotus and low altitudes of the high flux transcriptional group were sequenced, and de novo was assembled from the head. Method, we obtained 53929 and 44855 de novo based on sequence similarity, we found that two species of direct homologous base states (Orthologs) were 4593 pairs, of which 3126 were (?) Ks values, 202 pairs of Ka/Ks1 homologous genes, 0.5 Ka/Ks 1 homologous genes, and 737 pairs of homologous genes with a significant P value (glycosyl) Transferase gene, Ka/Ks=0.606, P=0.02).1273 showed significant purification options (Ka/Ks0.5, P0.05). In addition, 4593 direct homologous genes contained 461 SSR, of which the removal length was less than 16 BP, and the flanking sequences were not long enough homologous genes. Finally, 51 pairs of EST-SSR primers were successfully set up. We tested the generality of these primers. 26 of them (51%) and 41 pairs (80.4%) were general-purpose in podophylloid and octagonal. Finally, we obtained the gene sequence of 19 CYP719A (key genes of podophyllotoxin synthesis pathway) in 12 species of podophylloid 33 from 12 species of podophylloid subfamily, and analyzed their molecular evolution patterns. The results clarified CYP719A The gene in the subfamily podophylloid has experienced a strong purification selection. In the genus Aneta, the species with higher altitudes have loose purification selection (relaxed purifying selection) or strong positive selection (more positive selection). The comparative transcriptional group studies the population genetics of the subsequent eight species of lotus and its related species. The study of evolutionary biology provides a large number of molecular markers. To sum up, the podophylloid family is a single lineage, the genus Mt. lotus leaf is the base group, the North American lotus leaf and the Japanese mountain lotus leaf are sister, and the octagonal lotus is a very good monophyletic. The geographical isolation caused by the rapid uplift of the Qinghai Tibet Plateau drives the foreign regions of the western anisetin group. The main cause of species differentiation and genetic diversity in the middle and eastern part of the genus octagonal is the major cause of species differentiation and genetic diversity in the middle and eastern part of the Pliocene and the Pleistocene. The six horns are the most recently differentiated species; ITS is the best DNA barcode for the identification species of podophylloid. A large number of EST-SSR primers and single copy nuclear genes have been developed to fill the octagonal. In the absence of genetic resources in the genus lotus, the molecular evolutionary pattern of CYP719A in the genus aniseus indicates that the gene has experienced stronger positive selection or loose selection in high altitude species.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：Q949

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