本文選題：八角蓮屬 + 鬼臼亞科��； 參考：《浙江大學(xué)》2015年博士論文

【摘要】：八角蓮屬(Dysosma)植物隸屬于小檗科(Berberidaceae)鬼臼亞科(Podophylloideae),其根狀莖含有的生物活性成分鬼臼毒素(Podophylloxotin)等是合成抗癌藥物的前體。該屬植物分布于我國(guó)東南、中南至西南地區(qū),其中,八角蓮(D. versipellis)為廣布種,其余種均為地方特有種。最西部的西藏八角蓮(D. tsayuensis)分布于東喜馬拉雅,最東部的六角蓮(D. pleiantha)間斷分布于我國(guó)東部與臺(tái)灣。該屬有著近200年的分類(lèi)歷史,但由于其形態(tài)性狀的變異性以及采樣不足,使得它與近緣屬的分類(lèi)問(wèn)題和系統(tǒng)發(fā)育問(wèn)題一直沒(méi)有得到很好地解決。以前的系統(tǒng)發(fā)育和親緣地理學(xué)研究?jī)H涉及其中的少數(shù)種,因此有關(guān)屬內(nèi)7個(gè)種的物種形成與分化機(jī)制以及該屬的整合分類(lèi)系統(tǒng)是一個(gè)亟需解決的科學(xué)問(wèn)題�；诒菊n題組對(duì)八角蓮屬近10年的材料積累和廣泛的野外資源調(diào)查取樣,在形態(tài)學(xué)研究的基礎(chǔ)上,利用系統(tǒng)發(fā)育、生物地理以及群體遺傳學(xué)原理與方法,對(duì)鬼臼亞科的系統(tǒng)發(fā)育以及八角蓮屬植物地理分布與物種形成的時(shí)間格局與機(jī)制進(jìn)行了全面深入的研究,獲得以下主要結(jié)果：1)鬼臼亞科的系統(tǒng)發(fā)育及分化時(shí)間：對(duì)鬼臼亞科4屬12個(gè)種進(jìn)行了廣泛的野外取樣,采集了中國(guó)、日本和北美的63個(gè)群體材料,采集群體涵蓋了八角蓮屬的整個(gè)地理分布區(qū)域。以Wang et al.(2007)的小檗科主要類(lèi)群的系統(tǒng)發(fā)育樹(shù)為基礎(chǔ)(matK、rbcL),補(bǔ)充八角蓮屬缺失的6個(gè)種,并對(duì)鬼臼亞科12個(gè)種增加了葉綠體trnL-trnF、atpM-atpl和ITS和ETS的擴(kuò)增,聯(lián)合6個(gè)片段,通過(guò)系統(tǒng)發(fā)育分析構(gòu)建了鬼臼亞科12個(gè)種的分子系統(tǒng)進(jìn)化樹(shù),并結(jié)合兩個(gè)化石校正點(diǎn)(小檗科的crown節(jié)點(diǎn)分化時(shí)間91Ma,Ranzania、Berberis和Mahonia三個(gè)屬的crown節(jié)點(diǎn)時(shí)間45 Ma)和鬼臼亞科二次校正點(diǎn)(8.8士1.4 Ma),利用BEAST進(jìn)行分化時(shí)間估算,揭示了鬼臼亞科屬同及屬內(nèi)種間的系統(tǒng)發(fā)育關(guān)系：鬼臼亞科是一個(gè)單系類(lèi)群(PP=1),山荷葉屬為一個(gè)單系(PP=1)位于鬼臼亞科的基部,八角蓮屬是一個(gè)很好的單系(PP=1),與東亞-北美間斷分布的姐妹類(lèi)群這兒土和足葉草組成一個(gè)支系,但支持率較低(PP=0.67)。八角蓮屬內(nèi)的廣布種八角蓮不是單系,而是與六角蓮形成并系,分子系統(tǒng)樹(shù)支持其它形態(tài)學(xué)種為單系類(lèi)群。分歧時(shí)間估算結(jié)果表明鬼臼亞科的共祖時(shí)問(wèn)為上新世[c.12.67 Ma(10.71-14.56 Ma)]；山荷葉屬的共祖時(shí)間為7.96(5.19-10.61)Ma；八角蓮屬、桃兒七屬和足葉草屬的分化(7.96-9.14 Ma)與中新世的全球氣候變冷有關(guān)。BBM結(jié)果揭示了八角蓮屬起源于東喜馬拉雅,并自上新世由西向東遷移。在八角蓮屬中,位于西部的云南八角蓮、西藏八角蓮和川八角蓮分化時(shí)間較早(7.76-9.14 Ma),其分化時(shí)間與青藏高原早期快速隆升時(shí)間(8-13 Ma)一致,由此可知,早期地理隔離導(dǎo)致了西部類(lèi)群異域成種。2)八角蓮屬種水平親緣地理學(xué)選取八角蓮屬7個(gè)種46個(gè)群體進(jìn)行了親緣地理學(xué)研究,研究結(jié)果揭示了八角蓮屬內(nèi)7個(gè)種的譜系關(guān)系及遺傳多樣性的時(shí)空分布格局。3個(gè)葉綠體片段(trnL-trnF.trnL-ndhJ.trnS-trnfM)序列變異的遺傳多樣性分析揭示了八角蓮屬共有66個(gè)葉綠體單倍型,八角蓮屬水平的遺傳多樣性相對(duì)較高(hT=0.929,πT=3.58×10-3),并具有顯著的譜系地理結(jié)構(gòu)(NST=0.866,GST=0.790,P0.05)。廣布種八角蓮的單倍型多樣性和核苷酸多樣性(hT=0.314；πT= 0.25×10-3)比其它窄域種都高；八角蓮(NsT=0.818,GsT=0.611,P0.05)、小八角蓮(NST=0.865,GsT=0.728,P0.01)和川八角蓮(NST=0.977,GST=0.870,P0.05)的譜系地理結(jié)構(gòu)明顯。TCS單倍型譜系關(guān)系和系統(tǒng)發(fā)育分析結(jié)果均揭示,八角蓮屬共分為6個(gè)與地理分布對(duì)應(yīng)的譜系(Lineasel-Lineage6),它包括分布于中國(guó)西部的云南八角蓮(Lineage 6).西藏八角蓮(Lineage 5)和川八角蓮(Lineage 4)依次分化出來(lái)。分布于中國(guó)中部-東南部的小八角蓮、貴州八角蓮、六角蓮及八角蓮聚成一個(gè)單系[Lineage 1+(Lineage 2+Lincage 3)]；其中,廣布種八角蓮單倍型分為兩個(gè)譜系(西北譜系Lineage 2和東南譜系Lineagel),是一個(gè)并系類(lèi)群；單系類(lèi)群六角蓮(Lineage 3)與八角蓮西北部譜系Lineage 2為姐妹關(guān)系；小八角蓮和貴州八角蓮沒(méi)有獨(dú)立分支,小八角蓮(H1,H52-56)鑲嵌于八角蓮的東南譜系中,并共享單倍型H1；貴州八角蓮的所有個(gè)體和小八角蓮共享一個(gè)單倍型H55�；贗TS序列分析表明,八角蓮屬內(nèi)7個(gè)種沒(méi)有共有單倍型。cpDNA與ITS單倍型的譜系關(guān)系在小八角蓮、貴州八角蓮、八角蓮以及六角蓮之間呈現(xiàn)不一致的現(xiàn)象,表明了這3個(gè)種在同域分布區(qū)可能存在雜交與基因漸滲現(xiàn)象。失配分析表明,八角蓮東南譜系(Lineage 1)和六角蓮譜系(Lineage 3)都經(jīng)歷了顯著的群體擴(kuò)張,但八角蓮西北譜系(Lineage 2)和川八角蓮譜系(Lineage 4)長(zhǎng)期居留原地。八角蓮東南譜系的擴(kuò)張時(shí)間為更新世早期[1.78 Ma(95%CI：1.01-3.03 Ma)],表明氣候變冷導(dǎo)致暖溫帶落葉林下的八角蓮表現(xiàn)出擴(kuò)張信號(hào)。六角蓮的擴(kuò)張時(shí)間為更新世晚期[0.18 Ma (95% CI:0.00072-0.75 Ma)],與倒數(shù)第二個(gè)間冰期時(shí)間一致一致(c.0.25-0.135 Ma),表明氣候變暖使暖溫帶常綠林的六角蓮呈現(xiàn)擴(kuò)張信號(hào)。3)DNA條形碼(DNA barcoding)通過(guò)對(duì)9個(gè)候選的DNA條形碼(ITS、matK、rbcL、trnL-trnF、trnL-nbhJ、 trnS-trnfM、atpH-atpl、rpl32-trnL、rps18-clpp)在鬼臼亞科植物中的鑒定能力進(jìn)行了評(píng)估,比較分析了各條形碼的擴(kuò)增成功率、種內(nèi)和種間變異、barcoding gap,并運(yùn)用BLAST、DISTANCE和構(gòu)建系統(tǒng)發(fā)育樹(shù)方法評(píng)估了不同序列的物種鑒定成功率,結(jié)果表明除rps18-clpp外其余8個(gè)條形碼對(duì)鬼臼亞科12個(gè)種的通用性均為100%,其中通用引物ITS擴(kuò)增的序列種內(nèi)與種間變異的差異、barcodinggap較其他序列具有更明顯的優(yōu)勢(shì),其種鑒定成功率相對(duì)較高(83.3%)。因此,ITS是適合鬼臼亞科植物鑒別最好的DNA條形碼。4)云南八角蓮和八角蓮的比較轉(zhuǎn)錄組通過(guò)Hiseq 2000測(cè)序平臺(tái),對(duì)高海拔的云南八角蓮和低海拔的八角蓮進(jìn)行高通量轉(zhuǎn)錄組測(cè)序,運(yùn)用de novo從頭組裝的方法,分別獲得了53929和44855條de novo基于序列相似性,我們找出兩個(gè)種的直系同源基國(guó)(Orthologs)為4593對(duì),其中3126對(duì)直系同源基因有(？)Ks值,Ka/Ks1的同源基因有202對(duì),0.5 Ka/Ks 1的同源基因有535對(duì),這737對(duì)同源基因只有一對(duì)具有顯著P值(glycosyltransferase gene, Ka/Ks=0.606, P=0.02)。1273對(duì)表現(xiàn)出顯著的凈化選擇(Ka/Ks0.5, P0.05)。另外,4593直系同源基因共含有SSR 461個(gè),其中,去除長(zhǎng)度小于16 bp的、側(cè)翼序列不足夠長(zhǎng)的同源基因,最終成功設(shè)計(jì)了51對(duì)EST-SSR引物。我們檢驗(yàn)了這些引物的通用性,其中26對(duì)(51%)和41對(duì)(80.4%)分別在鬼臼亞科和八角蓮屬具有通用性。最后,我們利用PCR直接擴(kuò)增的方法得到了鬼臼亞科12個(gè)種33樣本的19個(gè)CYP719A(鬼臼毒素合成路徑的關(guān)鍵基因)的基因序列,并分析了它們的分子進(jìn)化式樣,結(jié)果闡明了CYP719A基因在鬼臼亞科經(jīng)歷了強(qiáng)烈的凈化選擇,在八角蓮屬內(nèi)表現(xiàn)為分布海拔較高的物種具有松散的凈化選擇(relaxed purifying selection)或較強(qiáng)的正向選擇(more positive selection)。比較轉(zhuǎn)錄組研究為后續(xù)八角蓮屬物種及其近緣物種的群體遺傳學(xué)與進(jìn)化生物學(xué)研究提供了大量的分子標(biāo)記。綜上所述,鬼臼亞科是一個(gè)單系,山荷葉屬為基部類(lèi)群,北美山荷葉與日本山荷葉成姐妹關(guān)系,八角蓮屬是一個(gè)很好的單系。青藏高原快速隆升導(dǎo)致的地理隔離驅(qū)使了八角蓮屬西部類(lèi)群的異域物種形成；上新世-更新世的氣候動(dòng)蕩變化是八角蓮屬中東部物種分化和遺傳多樣性形成的主要原因,六角蓮是近期分化的物種；ITS是鬼臼亞科鑒定種最好的DNA條形碼。大量的屬水平的EST-SSR引物和單拷貝核基因被開(kāi)發(fā)出來(lái),填補(bǔ)了八角蓮屬遺傳資源缺失的空白,CYP719A在八角蓮屬內(nèi)的分子進(jìn)化模式表明該基因在高海拔的物種中經(jīng)歷了更強(qiáng)的正向選擇或松散的凈化選擇。
[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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：Q949

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