【作者】 陈仁芳；

【导师】 陈龙清；

【作者基本信息】 华中农业大学 ， 植物学， 2010， 博士

【摘要】 桑属(Morus)为桑科的模式属,由Linne(1753)建立,属内种数、系统发育关系历来存在较大争议。本研究分别从孢粉学、分支系统学、分子系统学、区系地理学等四个方面对桑属的系统学进行研究,以期得到更加科学的桑属分类系统。(1)桑属植物孢粉学研究：选取不同地理分布桑属材料25份,代表桑属10种1变种(Koidzumi分类系统),采用醋酐法,光学显微镜(LM)、扫描电子显微镜(SEM)观察花粉形态、表面纹饰。研究结果显示,本属植物的花粉为椭圆形,极面观为圆形,大小为20.0(13.5-25.0)×13.5(12.3-22.3)μm,P/E=1.11-1.26,属小型花粉。萌发孔1-3,圆形,分属N3P3C4类型,外壁纹饰变化不大,均为颗粒状纹饰,属内花粉差异主要表现在大小和形状(P/E值)以及表面纹饰的细微差异上。根据花粉大小、P/E值、表面纹饰,可将观察的材料10种1变种的亲缘关系由原始到进化排列为：鲁桑M. multicaulis Perrott.→白桑M. alba L.→广东桑M. atropurpurea Roxb→华桑M. cathayana Hemsl.→奶桑M. macroura Miq.→川桑M.notabilis Schneid.→瑞穗桑M. mizuho Hotta.→蒙桑及变种鬼桑M.mongolica Schneid.,var. diabolica Koidz.→山桑M. bombycis Koidz.→鸡桑M. australis Poir.。花粉信息基本支持现行以花柱长短的桑属分类系统,支持短花柱为原始类型,长花柱是进化类型。(2)桑属广义形态分支系统学研究：选取在中国有地理分布的桑种及变种13个,以构树作外类群,通过性状选取、极性化编码,编制出用于分支分析的数据矩阵,用PAUP4.0b10软件(Swofford,2002)在PC机上,最大简约法(MP)、邻接法(Neighbor Joining, NJ)分析,研究结果显示,将桑属分成长花穗类、桑类和山桑类三支。进化顺序由原始到进化依次为：黑桑M. nigra→白桑M. alba→广东桑M. atropurpurea→华桑M. cathayana→川桑M. notabilis→山桑M. bombycis→鸡桑M.australis→细齿桑M. serrata→滇桑M. var. yunnanensis→蒙桑M. mongolica→鬼桑M..mongolica var.diabolica→奶桑M. macroura→长穗桑M. wittiorum.,基本支持小泉源一的分类系统。(3)桑属分子系统学研究：采集不同地理分布材料73份,代表桑属12种1变种,外类群3个。ITS通用引物PCR扩增,直接测序。测序结果用Sequencher4.1.4软件拼接,并从GenBank下载桑科、桑属ITS序列。Clustalxl.83c软件(Thompson et al.,1997)序列比对,根据GenBank已公布的鲁桑(AM042003)18S rRNA基因·3′端、26S rRNA基因5′端、5.8S rDNA基因碱基序列,确定本试验各材料的ITS序列范围,Bioedit软件除去两端非ITS序列部分。Modeltest V3.06(Posada D et al,1998)和PAUP Version4.0b10(Swofford,2002)软件进行碱基替换模型计算为GTR+G,基于模型用PAUPVersion4.0b10和mrbayes软件分析系统位置与进化关系,并用BEASTv1.5.1软件计算桑属的分歧年代。研究结果得到如下结论①基于ITS桑属的系统位置：系统位置排列在非洲硬木树(Milicia excels)(MEU93585)与新西兰鹊肾树(Streblus glaber)(DQ 499105)之间,与桑族、非洲硬木树(MEU93585)亲缘关系最近；MP法分支图以100%靴带(bootstrap)支持率将桑属分为一支,桑属单系得到强烈支持；②基于ITS桑属进化关系：Modeltest V3.06软件计算碱基替换模型为GTR+G。基于此模型Mrbayes分析,分支图首先将新疆黑桑、北美默里桑(FJ605515)分出,靴带支持率77%。其它桑属分为三支,靴带支持率100%。第一支包括：白桑、暹罗桑(AM042005)、瑞穗桑、广东桑、鸡桑、鲁桑(AM041999)蒙桑、鬼桑、印度桑(AM041997)、山桑、赤桑(FJ605516)等,靴带支持率97%。第二支包括：华桑、奶桑、川桑等,靴带支持率72%。第三支利川长穗桑、咸丰长穗桑,靴带支持率100%。其进化顺序依次为：新疆黑桑Xinjiang M. nigra,北美默里桑North America M.murrayana→白桑M.alba,暹罗桑M. rotundiloba (AM042005),瑞穗桑M. mizuho,广东桑M.atropurprea,鸡桑M. australis,鲁桑M.lhou(AM041999),蒙桑M. mongolica,鬼桑M. mongolica var.diabolica,印度桑M.indica(AM041997)、山桑M. bombycis,赤桑M. rubra(FJ605516)→华桑M.cathayana,奶桑M. macroura,川桑M. notabilis,→利川长穗桑Lichuan M.wittorum,咸丰长穗桑Xianfeng M.wittorum。与桑属由北向南地理迁移吻合；③松散分子钟估算本属及内部分支的分歧年代,桑属与桑族非洲硬木树、鹊肾树属新西兰鹊肾树起源年代(height)为53.67mya,新疆黑桑、北美默里桑的起源时间(height)在31.60mya,华桑类的分歧年代(height)在31.16mya,长穗类的分歧年代(height)在20.92mya,而白桑类的白桑、山桑、广东桑、鲁桑、蒙桑、鸡桑、瑞穗桑、滇桑、暹罗桑、赤桑、印度桑,以及华桑类的华桑、奶桑、川桑是近3mya的分化事件。(4)桑属区系地理学研究：通过查阅云南(KUN)、贵州(HGAS)植物标本馆桑属标本,及相关文献资料；汇总确定桑属的地理分布；绘制地理分布图；在此基础上,分析桑属植物的区系特点。研究结果认为,世界有桑属植物26种,分布亚洲、美洲、非洲,欧洲有逸生。东亚有桑属16种,占该属61.5%,中国有桑属植物14种,占该属53.8%,原产我国12种。云南11种,贵州9种,四川8种。中国桑属植物种类丰富,起源古老,区系成份复杂。云、贵、川三省种类最丰富,为现代桑属的遗传多样性中心。桑属按地理成分可划分为：Ⅰ泛北极植物区ⅠE欧亚森林植物亚区：黑桑M. nigra L.;中国—日本森林植物亚区：白桑M.alba L.,蒙桑M. mongolica Schneid.,华桑M. cathayana Hemsl.,山桑M. bombycis Koidz.,鸡桑M. australis Poir.,广东M. atropurpurea Roxb.,滇桑M. yunnanensts Koidz.,川桑M. notabilis Schneid.,裂叶桑M. trilobata (S.S.Chang) Cao.ⅠF.中国-喜马拉雅森林植物区：细齿桑M. serrata Roxb,喜马拉雅桑M.wallichiana Koidz.Ⅱ.古热带植物区ⅡG22滇、缅、泰地区：长穗桑M. wittiorum Hand.-Mazz.,奶桑M. macroura Miq.Ⅲ.新热带植物区：秘鲁桑M. perubjana Planchon.,美洲桑M. insights Bur,非洲桑M. mcsozygio Stapf.根据桑属的区系地理学研究结果,结合分子系统学得出的结论----新疆黑桑、北美默里桑最原始,可以得出如下结论：1.桑属劳亚古陆(Laurasia)高纬度起源(起源中心),；2.随着地球寒、旱化,由北向南迁移；3.桑属起源正是老第三纪始新世(53.67mya),此时整个地球气候还较温暖,白令海峡(Bering Strait)还未关闭,桑属可在北半球美洲、亚洲、欧洲相互迁移。4.Morus起源后,随着古地中海退却,喜马拉雅隆起,气候寒、旱化,逐渐向低纬度迁移;5.到第四纪冰期(2-3mya),北美、欧洲大多数桑种灭绝,而东亚北纬20-40°N山区,受第四纪冰期影响较小,大多数桑种保存下来,成为现代桑属的遗传多样性中心；6.第四纪冰期后桑属继续向南迁移,形成现代美洲、亚洲、欧洲、非洲间断分布格局；7.桑属可由地理分布、抗寒性强弱,间接反映系统演化关系。综合孢粉学、分支分类学、分子系统学和区系地理学的结果,认为桑属中黑桑M. nigra是最原始的种类,长穗桑M.wittorum是最进化的种类。其进化顺序由原始到进化依次为：黑桑M. nigra,默里桑M.murrayana→白桑M.alba,鲁桑M.multicaulis,广东桑M.atropurprea,赤桑M. rubra,暹罗桑M. rotundiloba,印度桑M. indica,瑞穗桑M. mizuho,山桑M. bombycis,蒙桑M. mongolica,鬼桑M. mongolica var.diabolica,鸡桑M. australis,→华桑M.cathayana,川桑M. notabilis,奶桑M. macroura→长穗桑M.wittorum。更多还原

【Abstract】 The Morus L., which is the type genus of Moraceae, was established by Linne in 1753. So far the species number and the phylogeny of Morus have been still controversial. In this study, the genus phylogeny of Morus flora was studied based on the evidences of Palynology, Cladistics, Molecular systematics, floristic plant geography and so on, in order to get more scientific classifier systerm of Morus flora. In order to get a more scientific classifier, the phylogeny of Morus flora was studied based on the evidences of Palynology, Cladistics, Molecular systematics, floristic plant geography and so on.（1） In the study of Palynology of Morus, pollen characteristics of 25 taxa （representing 9 species and 1 varieties） （Koidzumi taxonomy system） of Morus from different distributions regions were described by acetic anhydride method, LM and SEM, to observe pollen mophology and surface decoration. The results showed that the pollens of the genus were small, oval-shaped, round in polar view （P/E=1.11～1.26）. The pollen size was 20.0（13.5-25.0）×13.5（12.3～22.3）μm. Germination aperture was 1-3 in number, round in shape, N3P3C4 in type. The decorations of outer wall were all granular. The differences of pollen characteristics among the species of Morus were the size, shapes （P/E value）, and the surface decoration. According to pollen size, P/E value and the surface ornamentation, the evolutionary order of 9 species and 1 varieties of Morus was as following （from originated to evolved）, M.multicaulis Perrott., M. alba L., M. atropurpurea Roxb., M. cathayana Hemsl.., M. macroura Miq., M.notabilis Schneid., M. mizuho Hotta., M.mongolica Schneid. and M.mongolica var. diabolicaKoidz., M. bombycis Koidz., M. australis Poir.. Pollen information basically supports the current Morus classification system based on the style length. Supporting the short-Style inter lobule is primitive type, while supporting long-Style inter lobule is Evolutionary type.（2） In the study of Cladistics of Morus, collect the evolutionary order of 13 species sampled from different distributions, the evolutionary order of 13 species were collected from different regions （the B. papyrifera L’Herit. Ex Vent is outgroup）. After shape selecting and polarity coding, work out a cladistic analysis of the data matrix.a cladistic analysis of the data matrix was worked out,Use the methods of Maximum Parsimony （MP）, Neighbor Joining （NJ）, by PAUP4.0b10 software （Swofford,2002） on PC to analyse. Then the methods of Maximum Parsimony （MP） and Neighbor Joining （NJ） were used to analyze through software PAUP4.0b10 （Swofford,2002）.The results showed that Morus was separated into three groups including Longispica, Macromorus and Dolichstylae. The evolutionary order of 13 species of Morus was as following （from originated to evolved）, M. nigra L.,M. alba, M. atropurpurea Roxb., M. cathayana Hemsl., M. serrata Roxb., M. notabilis Schneid., M. bombycisKoidz, M. australis.Poir., M. var. yunnanensis Koidz., M. mongolica Schneid., M..mongolica var.diabolica Koidz., M. wittiorum Hand-Mazz, M. macroura Miq. Basic Support Koidzumi classification system. This result is in accordance with Koidzumi classification system.（3） In the study of molecular systematics of Morus, the 73 materials contained 12 species and varieties and 3 out groups collected from different distributions were sequenced directly by amplifying ITS by PCR a total of 73 mulberry genotypes representing 12 species and varieties of the genus Morus and 3 out groups collected from different regions were amplified using ITS primers, and the PCR products were sequenced. Use Sequencher4.1.4 software to splice the sequencing result, Software Sequencher 4.1.4 was used to splice sequencing results. According to GenBank published M.multicaulis （AM042003） 18S rRNA gene 3’end,26S rRNA gene 5’end, 5.8S rDNA gene base sequence, Determine the test range of different materials ITS sequences,use Bioedit software to remove the non-ITS part at the ends. The ranges of ITSs were determined according to the data published on GenBank （AM042003）, and then the non-ITS parts were removed by using Bioedit software. Use Clustalxl.83c software （Thompson et al.,1997） to do sequence alignment. Clustalxl.83c was used to do sequence alignment. Then use Modeltest V3.06（Posada D et al,1998）software and PAUP Version4.0b10（Swofford,2002） software to calculate the model of bases replacement, For the GTR+G.based on which use PAUPVersion4.0b10 and mrbayes software to analyze the systematic position and the evolutionary relationship, and use BEAST v1.5.1 software to calculate the age differences of Morus. Modeltest 3.06 （Posada Detal,1998） and PAUP Version4.0b10 （Swofford,2002） were run for all data sets to select a model of sequence evolution. Based on the model, GTR+G, which was selected the last step, PAUPVersion4.0b10 and MRbayes were used to analyze the systematic position and the evolutionary relationship, and BEAST v1.5.1 was used to calculate the divergence times of Morus. The results showed that the cladogram from joined segments of the three DNA sequences was supported by bootstrap values and compatible to differ the species of Morus. The analysis of DNA sequences indicated that Morus is a monophyletic. Its System Location arranges between African Milicia excels（MEU93585） and New Zealand Streblus glaber（DQ 499105）. It has the closest relationship with the Milicia and the African Milicia excels（MEU93585）. Based on ITS Mrbayes analysis of Morus, genus Morus will be was divided into two broad categories and 4 small categories sub-categories. According to outgroup, Xinjiang M. nigra and North America M.murrayana （FJ605515） are the most primitive. The evolutionary order of Morus was is listed as following （from originated to evolved）, Xinjiang M. nigra, North America M.murrayana→M.alba, M. rotundiloba （AM042005）, M. mizuho, M.atropurprea, M. australis, M.lhou（AM041999）, M. mongolica, M. mongolica var.diabolica, M. indica（AM041997）, M. bombycis,M. rubra（FJ605516）→M.cathayana, M. macroura, M. notabilis→Lichuan M.wittorum, Xianfeng M.wittorum. Estimated differences estimating divergences in age was by loose molecular clock. the origin time The divergence time of Morus, African Milicia excels（MEU93585） and New Zealand Streblus glaber（DQ 499105） （height） is 53.67mya, height₉5%_HPD （highest posterior density）=[43.57.63.46 mya]. The differences in age The divergence time of Xinjiang M. nigra and North America M.murrayana （height） is 31.6mya, height₉5%_HPD= [9.81, 53.64mya]. The differences in age The divergence time of Macromorus and M.cathayana class （height） is 31.16mya,height₉5%_HPD= [10.78,52.87 Ma]. The differences in age The divergence time of Macromorus M.alba class and Longispica （height） is 20.92mya, height₉5%_HPD= [2.74,42.29 Ma]. The differentiation evolution divergence events of M.alba, M. rotundiloba （AM042005）, M. mizuho, M.atropurprea, M. australis, M.lhou（AM041999）, M. mongolica, M. mongolica var.diabolica, M: indica（AM041997）, M. bombycis, M. rubra（FJ605516）, M.cathayana, M. macroura, M. notabilis and M.wittorum begin to have evolutionary radiation nearly 3mya.（4） In the study of geographic flora of Morus, analyze the specimens in the herbarium of KUN and HGAS, and search relative literature data, in order to make sure the geographic distributions of Morus, and work out the geographic distributions graph. Then analyze the floristic characteristics of Morus based on this. we consulted specimens and relative literature in KUN and HGAS, and determined the geographic distribution of Morus. The results show that there are 26 species of Morus distributed in Asia, America, Africa and Europe has Introduction the planting after, natural wild reproduction去掉.In East Asia, has 16 species,Accounted for 61.5% of Morus;16 species accounted for 61.5% of Morus, have been growing there. In China, there are 14 species, Accounted for 53.8% of Morus.including 11 species in Yunnan,9 in Guizhou and 8 in Sichuan. The flora of Chinese mulberries is abundant in the number of species, native and old in origination, complicated in the compositions. For the large species number, the three provinces of—Yunnan, Guizhou and Sichuan are the modern centers of Morus.According to geographical elements, Morus can be devided divided as followingⅠHolarctic regionⅠE Europe and Asia forest region, M. nigra L. Chine-Japan forest plants region, M.alba L., M. mongolica Schneid., M. cathayana Hemsl., M. bombycis Koidz., M. australis Poir., M. atropurpurea Roxb., M. yunnanensts Koidz., M. notabilis Schneid., M. trilobata （S.S.Chang） Cao.ⅠF. China-Himalaya forest region, M. serrata Roxb, M. wallichiana Koidz.ⅡPaleotropical region.ⅡG22 Yunnan, Burma and Thailand region, M. wittiorum Hand.-Mazz., M. macroura Miq..ⅢNeotropical region, M. perubjana Planchon.,M insights Bur,M mcsozygio Stapf.Morus originated in the Northern Hemisphere high latitudes region （center of origin）, migrating from north to south. Geographical distribution and cold resistance can reflect the phylogenetic relationship of Morus indirectly.According to the studies of palynology, branch taxonomy, molecular systematics, and floristic geography, the author conclude that M. nigra L. is the most primitive species, while Xianfeng M.wittorum is the most envolved species. The evolutionary order of Morus was as following （from originated to evolved）, M. nigra, M.murrayana→M.alba, M.multicaulis,M. rotundiloba, M. mizuho, M.atropurprea, M. rubra, M. indica, M. bombycis, M. mongolica, M. mongolica var.diabolica, M. australis,→M.cathayana, M. notabili,M.macroura, M.wittorum. Laurasia high-latitude regions is the origin of the genus Morus, Origin time where the genus Morus originated from 53.67mya ago. As the cold and aridity of the north region, Migration from north to south, Morus migrated from north to south. Now East Asia is the Morus center Center center of Morus genetic diversity. Morus can be geographic distribution, Cold tolerance, Indirectly reflect the phylogenetic relationships. So the geographic distribution and cold tolerance of Morus can reflect the phylogenetic relationship indirectly.更多还原