植物DNA条形码技术

DNA条形码技术是利用标准的、具有足够变异的、易扩增且相对较短的DNA片段在物种内的特异性和种间的多样性而创建的一种新的生物身份识别系统,从而实现对物种的快速自动鉴定。尽管这一技术在理论上和具体应用上仍存在很多争论。但DNA条形码概念自2003年由加拿大分类学家Paul Hebert首次提出后就在世界范围内受到了广泛关注。在植物类群中条形码的研究和应用尚处于探索阶段,稍落后于对动物类群的研究,这主要表现在：（1）DNA条形码的选择及其评价仍没有统一的标准：（2）对类群较全面的形态分类学修订和植物DNA条形码研究的结合十分缺乏：（3）以往研究在取样上尺度较大,而对具体类群的研究较少,一个科或一个属只用有限的种类作为代表,同一种内的取样个体数量也不足,这样虽然表面上看来利用选定的DNA条形码可以较容易地把代表物种区分开,但实际上目前建议的植物DNA条形码（例如由生命条形码咨询委员会植物工作组最近提出的rbcL和matK）由于其分子进化速率较慢,在种级水平上,特别是对于那些经历了适应辐射或快速进化的属来说,分辨率较低。而DNA条形码的应用主要集中在属内物种水平的鉴别,因此只有针对具体类群进行探索研究,发现进化速率较快、分辨率高且通用性好的条形码,才可能为建立完整的条形码数据库起到积极有效的作用。     

Root character evolution and systematics in Cranichidinae, Prescottiinae and Spiranthinae (Orchidaceae, Cranichideae)

Background and Aims: Previous studies have suggested that velamen characteristicsare useful as taxonomic markers in Orchidaceae. Members of tribeCranichideae have been assigned to two velamen types constructedbased on combinations of characters such as the presence ofsecondary cell-wall thickenings and pores. However, such charactershave not been analysed on an individual basis in explicit cladisticanalyses. Methods: The micromorphology of roots of 26 species of Cranichideae wasexamined through scanning electron microscopy and light microscopy,scoring the variation and distribution of four characters: numberof velamen cell layers, velamen cell-wall thickenings, presenceand type of tilosomes, and supraendodermal spaces. The lastthree characters were analysed cladistically in combinationwith DNA sequence data of plastid trnK/matK and nuclear ribosomalinternal transcribed spacer (ITS) regions and optimized on theresulting phylogenetic tree. Key Results: Thickenings of velamen cell walls group Prescottiinae with Spiranthinae,whereas tilosomes, documented here for the first time in Cranichideae,provide an unambiguous synapomorphy for subtribe Spiranthinae.Supraendodermal spaces occur mostly in species dwelling in seasonallydry habitats and appear to have evolved three times. Conclusions: Three of the four structural characters assessed are phylogeneticallyinformative, marking monophyletic groups recovered in the combinedmolecular–morphological analysis. This study highlightsthe need for conducting character-based structural studies toovercome analytical shortcomings of the typological approach.     

Molecular Phylogeny of Casuarinaceae Based on rbcL and matK Gene Sequences

rbcL (1310 bp) and matK (1014 bp), using 15 species representing the family. The study included analyses of Ticodendron (Ticodendraceae) and three species of Betulaceae as close relatives, and one species each of Juglandaceae and Myricaceae as outgroups. Analyses based on matK gene sequences, which provided a much better resolution than the analyses based on rbcL gene sequences alone, resulted in a single most parsimonious tree whose topology is almost identical with the strict consensus tree generated by the combined data set of rbcL and matK gene sequences. Results showed that Casuarinaceae are monophyletic, comprising four distinct genera, Allocasuarina, Casuarina, Ceuthostoma and Gymnostoma, which were not recognized until recently. Within the family, Gymnostoma is positioned at the most basal position and sister to the remainder. Within the remainder Ceuthostoma is sister to the Allocasuarina-Casuarina clade. Morphologically the basalmost position of Gymnostoma is supported by plesiomorphies such as exposed stomata in the shallow longitudinal furrows of the branchlets, a basic chromosome number x=8 and the gynoecium composed of two fertile, biovulate carpels. The three other genera, Allocasuarina, Casuarina, and Ceuthostoma, have invisible stomata in the deep longitudinal furrows of the branchlets, a higher basic chromosome number x=9 or 10–14 (unknown in Ceuthostoma), the gynoecium composed of one fertile and one sterile carpel with a single ovule (unknown in Ceuthostoma). The diversity of infructescence morphology found in the latter three genera suggests that they may have evolved in close association with the elaboration of fruit dispersal mechanisms. Received 14 September 2001/ Accepted in revised form 12 October 2001     

Out of Africa: molecular phylogenetics and biogeography of Wolffiella (Lemnaceae)

The monophyletic genus Wolffiella (Lemnaceae) comprises 10 species divided taxonomically into three sections. Relative to other genera of Lemnaceae, Wolffiella has a restricted range, with species distributed in warm temperate to tropical areas of Africa and the Americas, with only one species occurring in both areas. Sequence data from coding (rbcL and matK) and non‐coding (trnK and rpl16 introns) regions of cpDNA were analyzed phylogenetically to resolve relationships within Wolffiella, and these results were compared to earlier allozyme and morphological studies. Allozymes, cpDNA and morphology all supported the recognition of three sections. Relationships among species were similar in most respects between the allozyme and cpDNA trees, as well as among the different plastid partitions. In Wolffiella, both non‐synonymous and synonymous substitutions were greater in matK than in rbcL, as observed in other taxa. The synonymous substitution rate in matK was similar to the substitution rate of the non‐coding regions. All partitions, including coding regions, exhibited some homoplasy. Biogeographical reconstructions from a combination of cpDNA partitions indicated that Wolffiella originated in Africa with early movement to and radiation in the Americas. The one species found in both Africa and the Americas, W. welwitschii, likely originated in the Americas and subsequently dispersed to Africa. Using the SOWH test, the cpDNA data could reject two alternative biogeographical hypotheses suggested from analyses of morphological and allozyme data. The present distribution of Wolffiella can be explained by two major dispersal events and this contrasts with the more complex species distributions in other Lemnaceae genera. Limited dispersal in Wolffiella relative to other Lemnaceae genera may be due to more recent origins of species, lower dispersibility and poorer colonizing ability. © 2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 79 , 565–576.     

Phylogenetic Relationships of Amaryllidaceae Based on matK Sequence Data

matK gene, which is located in the chloroplast genome and evolves more quickly than the rbcL gene. A total of 31 species representing 31 of the 59 genera in the family were examined in this study. We also used 21 species from another ten families of Asparagales, four species from three families of Liliales and Acorus as outgroups. We obtained partial sequences of matK with lengths of 1,109–1,148 bp, corresponding to positions 230 to 1,343 of the Oryza sativa matK gene. The pairwise percentage sequence divergence ranged from 0 to 19.1% for all the species examined except Acorus, and 0 to 4.6% within Amaryllidaceae. Two methods of phylogenetic analysis, the Maximum Parsimony and Neighbor-Joining methods, were used. The trees obtained from these two analyses were fundamentally consistent. In both trees, the Amaryllidaceae sensu Dahlgren et al. formed a well-supported monophyletic clade with 100% bootstrap support. Amaryllidaceae were included in the Asparagales; however, its phylogenetic position within the Asparagales was not clearly resolved. Judging from the NJ tree, Agapanthus might be a sister group of the Amaryllidaceae, although bootstrap support for this was low. Character-state mapping was used to infer a center of origin and the biogeographic history of Amaryllidaceae. The result supports the hypothesis that the family evolved in Africa and subsequently spread to other continents, further suggesting that South America is the center of secondary diversification. Received 6 January 1999/ Accepted in revised form 8 April 1999     

金粉蕨属的分子系统学研究——基于5种叶绿体DNA序列片段

金粉蕨属（Onychium Kaulfuss）隶属广义凤尾蕨科中的凤尾蕨亚科。迄今为止,该属属下分组及种间界定等仍有诸多问题亟待解决。本研究选取5个叶绿体DNA 序列片段 （rbcL/atpA/matK/trnL-trnF/trnG-trnR）,采用最大似然法（ML）和贝叶斯法（BI）构建金粉蕨属的系统发育树。结果表明：（1）金粉蕨属的9个成员被分置于两大支上。其中野雉尾金粉蕨（Onychium japonicum(Thunberg) Kunze）、西藏金粉蕨（O.tibeticum Ching & S.K.Wu）、木坪金粉蕨（O.moupinense Ching）、湖北金粉蕨（O.moupinense var. ipii(Ching) K.H.Shing）、栗柄金粉蕨（O.japonicum var. lucidum(D.Don) Christ）、黑足金粉蕨（O.cryptogrammoides Christ）、繁羽金粉蕨（O.plumosum Ching）聚为一支;而金粉蕨（O.siliculosum(Desvaux) C.Christensen）和蚀盖金粉蕨（O.tenuifrons Ching）则聚为另一支,可为该属的属下分组提供分子系统学证据;（2）野雉尾金粉蕨与栗柄金粉蕨在系统树中并没有聚在一起,而是被其它类群分割开来,不支持将后者作为野雉尾金粉蕨的变种,建议将栗柄金粉蕨提升为种的等级;（3）系统树上木坪金粉蕨与湖北金粉蕨的样本聚在一个细支上,支持《中国植物志》将湖北金粉蕨作为木坪金粉蕨变种的分类处理;（4）西藏金粉蕨与野雉尾金粉蕨聚在一起,并得到较高的支持,说明两者的关系近缘。     

连香树科及其近缘植物matK序列分析和系统学意义

测定和分析了连香树科(Cercidiphyllaeeae)、交让木科(Daplmiphyllaceac)、金缕梅科(Hamamelidaceae)代表植物的叶绿体marK序列(5′端31bps除外),以木兰属作为外类群,应用邻接法构建分子系统树,结果表明：连香树科与水青树科的亲缘关系较远。连香树科、交让木科和金缕梅科形成了一个自展数据支持率(bootstrap)为100％的单系类群,其中金缕梅科枫香属(Liquidambar)、红花荷属(Rhodoleia)和金缕梅属(Hamamelis)虽构成了一个单系类群,但自展数据支持率仅为68％;连香树科与交让木科构成的单系分支自展数据支持率仅为53％。由于连香树科、交让木科、金缕梅科之间的进化距离相当短,表明这3个科之间亲缘关系密切,内部分支的自展数据支持率不高,表明它们之间准确的亲缘关系有待进一步研究。本研究结果与rbcL、aptB、18S rDNA序列分析结果相似,但自展数据支持率更高,表明marK序列分析可应用于较高等级分类群系统发育关系的研究。     

Chloroplast diversity in the genus Malus: new insights into the relationship between the European wild apple (Malus sylvestris (L.) Mill.) and the domesticated apple (Malus domestica Borkh.)

To unravel the relationship between the European wild apple, Malus sylvestris (L.) Mill., and its domesticated relative M. domestica Borkh., we studied chloroplast DNA variation in 634 wild and 422 domesticated accessions originating from different regions. Hybridization between M. sylvestris and M. domestica was checked using 10 nuclear microsatellites and a Bayesian assignment approach. This allowed us to identify hybrids and feral plants escaped from cultivation. Sixty-eight genotypes belonging to 12 other wild Malus species, including 20 M. sieversii (Ledeb.) Roem. accessions were also included in the analysis of chloroplast diversity. Marker techniques were developed to type a formerly described duplication and a newly detected transversion in the matK gene. Chloroplast DNA variation was further investigated using PCR-RFLP (Polymerase Chain Reaction-Random Fragment Length Polymorphism), and haplotypes were constructed based on all mutational combinations. A closer relationship than presently accepted between M. sylvestris and M. domestica was established at the cytoplasmic level, with the detection of eight chloroplast haplotypes shared by both species. Hybridization between M. sylvestris and M. domestica was also apparent at the local level with sharing of rare haplotypes among local cultivars and sympatric wild trees. Indications of the use of wild Malus genotypes in the (local) cultivation process of M. domestica and cytoplasmic introgression of chloroplast haplotypes into M. sylvestris from the domesticated apple were found. Only one of the M. sieversii trees studied displayed one of the three main chloroplast haplotypes shared by M. sylvestris and M. domestica. This is surprising as M. sieversii has formerly been described as the main maternal progenitor of the domesticated apple. This study hereby reopens the exciting discussion on the origin of M. domestica.     

Testing four proposed barcoding markers for the identification of species within Ligustrum L. (Oleaceae)

DNA barcoding is a biological technique that uses short and standardized genes or DNA regions to facilitate species identification. DNA barcoding has been used successfully in several animal and plant groups. Ligustrum (Oleaceae) species occur widely throughout the world and are used as medicinal plants in China. Therefore, the accurate identification of species in this genus is necessary. Four potential DNA barcodes, namely the nuclear ribosomal internal transcribed spacer (ITS) and three chloroplast (cp) DNA regions (rbcL, matK, and trnH–psbA), were used to differentiate species within Ligustrum. BLAST, character-based method, tree-based methods and TAXONDNA analysis were used to investigate the molecular identification capabilities of the chosen markers for discriminating 92 samples representing 20 species of this genus. The results showed that the ITS sequences have the most variable information, followed by trnH–psbA, matK, and rbcL. All sequences of the four regions correctly identified the species at the genus level using BLAST alignment. At the species level, the discriminating power of rbcL, matK, trnH–psbA, and ITS based on neighbor-joining (NJ) trees was 36.8%, 38.9%, 77.8%, and 80%, respectively. Using character-based and maximum parsimony (MP) tree methods together, the discriminating ability of trnH–psbA increased to 88.9%. All species could be differentiated using ITS when combining the NJ tree method with character-based or MP tree methods. Overall, the results indicate that DNA barcoding is an effective molecular identification method for Ligustrum species. We propose the nuclear ribosomal ITS as a plant barcode for plant identification and trnH–psbA as a candidate barcode sequence.