Phylogeny of taxaceae and cephalotaxaceae genera inferred from chloroplast matK gene and nuclear rDNA ITS region

Phylogeny of the Taxaceae genera and the monotypic family Cephalotaxaceae has been extraordinarily controversial. In this paper chloroplast matK genes and nuclear ITS sequences were determined for all six genera of the two families and representatives of other conifer families. Analysis using either the nonsynonymous sites or the deduced amino acid sequences of matK genes strongly indicates that taxad genera and Cephalotaxaceae are monophyletic, with the Taxodiaceae/Cupressaceae clade as their sister group. Cephalotaxus is basal to the taxad genera, among which two clades, Torreya/Amentotaxus and Taxus/Pseudotaxus/Austrotaxus, are resolved. They correspond to Janchen's two tribes, Torreyeae and Taxeae. In Taxeae, Austrotaxus is the first to branch off. Analyses of the nuclear ITS sequence data corroborated the topology of the matK gene tree. These results refute the views that Cephalotaxaceae has no alliance with Taxaceae and that Austrotaxus and Amentotaxus should be excluded from the Taxaceae. We estimated the divergence time between the Taxodiaceae/Cupressaceae and the Cephalotaxaceae/Taxaceae clades to be 192-230 Myr ago and the divergence time between taxads and Cephalotaxus to be 149-179 Myr ago. Soon after the latter divergence event, within 6-8 Myr, the two taxad tribes originated. In conclusion, our data do not support Florin's claim that taxads could be traced to Devonian psilophytes (359-395 Myr ago).     

红豆杉科及相关类群rbcL基因PCR—RFLP分析

运用RFLP方法对红豆杉科及相关类群14种植物叶绿体rbcL基因PCR产物进行限制酶酶切分析,共获29个酶切变异位点。采用PHYLIP软件包对限制位点变异数据进行极大简约法分析得到18个步长为6的最简约树并求得一致树,结果显示：⑴红豆杉科和三尖杉科属单系群;⑵穗花杉属Amentotaxus以置于红豆杉科内为宜,不支持将穗花杉属独立成科的处理方式;⑶白豆杉应为红豆杉科内一个属Pseudotaxus;⑷三尖杉属内篦子三尖杉地位特殊,可设篦子三尖杉组;⑸不赞同将竹柏类从罗汉松属中分离出去成立新科;⑹红豆杉科、三尖杉科和罗汉松科三者间,前两者的关系更为接近。     

白豆杉的叶绿体基因组结构与系统进化分析

以红豆杉科单种属植物白豆杉(Pseudotaxus chienii(W.C.Cheng)W.C.Cheng)为材料,进行叶绿体全基因组测序,并对其基因含量、结构及重复序列进行分析.结果显示:白豆杉叶绿体基因组不包含典型的反向重复区,基因组全长为130427 bp,共编码116个基因,包含83个蛋白编码基因、4个rRNA...     

红豆杉科及相关类群rbc L基因PCR-RFLP分析

运用RFLP方法对红豆杉科及相关类群14种植物叶绿体rbcL基因PCR产物进行限制酶酶切分析,共获29个酶切变异位点。采用PHYLIP 软件包对限制位点变异数据进行极大简约法分析得到18个步长为6的最简约树并求得一致树,结果显示：（1）红豆杉科和三尖杉科属单系群;(2) 穗花杉属Amentotaxus以置于红豆杉科内为宜,不支持将穗花杉属独立成科的处理方式;(3)白豆杉应为红豆杉科内一个属Pseudotaxus;(4) 三尖杉属内篦子三尖杉地位特殊,可设篦子三尖杉组;(5) 不赞同将竹柏类从罗汉松属中分离出去成立新科;(6) 红豆杉科、三尖杉科和罗汉松科三者间,前两者的关系更为接近。     

部分裸子植物叶片总蛋白分析

采用ＳＤＳ－ ＰＡＧＥ 技术, 分析了红豆杉科（Ｔａｘａｃｅａｅ） 植物南方红豆杉（ Ｔａｘｕｓ ｃｈｉｎｅｎｓｉｓｖａｒ－ ｍａｉｒｅｉ （Ｌｅｍｅｅ ｅｔ Ｌｅｖｌ－） Ｃｈｅｎｇ ｅｔ Ｌ－Ｋ－Ｆｕ） 、穗花杉（ Ａｍｅｎｔｏｔａｘｕｓ ａｒｇｏｔａｅｎｉａ （Ｈａｎｃｅ） Ｐｉｌ ｇｅｒ） 、云南穗花杉（ Ａ－ ｙｕｎｎａｎｅｎｓｉｓ Ｌｉ） 、白豆杉（ Ｐｓｅｕｄｏｔａｘｕｓｃｈｉｅｎｉｉ（Ｃｈｅｎｇ） Ｃｈｅｎｇ） 以及三尖杉科（Ｃｅｐｈａｌｏｔａｘａｃｅａｅ） 、植物三尖杉（ Ｃｅｐｈａｌｏｔａｘｕｓ ｆｏｒｔｕｎｅｉ Ｈｏｏｋ－ｆ－） 、粗榧（ Ｃ－ｓｉｎｅｎｓｉｓ （Ｒｅｈｄ－ｅｔＷｉｌｓ－） Ｌｉ） 、海南粗榧（ Ｃ－ｈａｉｎａｎｅｎｓｉｓ Ｌｉ） 、篦子三尖杉（ Ｃ－ｏｌｉｖｅｒｉ Ｍａｓｔ－） 和罗汉松科（Ｐｏｄｏｃａｒｐａｃｅａｅ） 、植 物罗汉松 （ Ｐｏｄｏｃａｒｐｕｓ ｍａｃｒｏｐｈｙｌｌｕｓ （ Ｔｈｕｎｂ－ ） Ｄ－Ｄｏｎ） 、鸡毛 松（ Ｐ－ｉｍｂｒｉｃａｔｕｓ Ｂｌ－） 、竹柏（ Ｐ－ ｎａｇｉ（Ｔｈｕｎｂ－） Ｚｏｌｌ） 、陆均松（ Ｄａｃｒｙｄｉｕｍ ｐｉｅｒｒｅｉ Ｈｉｃｋｅｌ） 共１２ 种植物的叶片蛋白, 在蛋白质水平上采用     

红豆杉科、三尖杉科和罗汉松科植物叶片结构的比较观察

扫描电镜和光镜下的叶表皮特征以及叶片解剖结构的研究结果,支持在红豆杉科内建立白豆杉属的处理方式;赞同在三尖杉属内建立篦子三尖杉组,叶片解剖结构方面,三尖杉科和红豆杉科的穗花杉属的相似之处颇多。而叶表皮形态特征方面又表现为三尖杉科和罗汉松科更加接近。反映了出了红豆杉科、三尖杉科和罗汉松科之间的密切而又复杂的系统关系。     

Physicochemical evolution and positive selection of the gymnosperm matK proteins

It is not clear whether matK evolves under Darwinian selection. In this study, the gymnosperm Taxaceae, Cephalotaxaceae and Pinaceae were used to illustrate the physicochemical evolution, molecular adaptation and evolutionary dynamics of gene divergence in matKs. matK sequences were amplified from 27 Taxaceae and 12 Cephalotaxaceae species. matK sequences of 19 Pinaceae species were retrieved from GenBank. The phylogenetic tree was generated using conceptual-translated amino acid sequences. Selective influences were investigated using standard d _N/d _S ratio methods and more sensitive techniques investigating the amino acid property changes resulting from nonsynonymous replacements in a phylogenetic context. Analyses revealed the presence of positive selection in matKs (N-terminal region, RT domain and domain X) of Taxaceae and Pinaceae, and found positive destabilizing selection in N-terminal region and RT domain of Cephalotaxaceae matK. Moreover, various amino acid properties were found to be influenced by destabilizing positive selection. Amino acid sites relating to these properties and to different secondary structures were found and have the potential to affect group II intron maturase function. Despite the evolutionary constraint on the rapidly evolving matK, this protein evolves under positive selection in gymnosperm. Several regions of matK have experienced molecular adaptation which fine-tunes maturase performance.     

红豆杉科植物RAPD分析及其系统学意义

利用随机扩增多态性ＤＮＡ（ＲＡＰＤ）技术分析了红豆杉科（Ｔａｘａｃｅａｅ）红豆杉属（Ｔａｘｕｓ）、白豆杉属（Ｐｓｅｕｄｏｔａｘｕｓ）、穗花杉属（Ａｍｅｎｔｏｔａｘｕｓ）和榧树属（Ｔｏｒｒｅｙａ）的６种植物：红豆杉（Ｔａｘｕｓ ｃｈｉｎｅｎｓｉｓ （Ｐｉｌｇｅｒ）Ｒｅｈｄ）、南方红豆杉（Ｔａｘｕｓ ｃｈｉｎｅｎｓｉｓ ｖａｒ．ｍａｉｒｅｉ（Ｌｅｍｅｅ ｅｔＬｅｖｌ．）Ｃｈｅｎｇ ｅｔＭａｉｒｅＹｅｗ     

On the Systematic Position of Amentotaxus from Its Embryological Investigation

The present paper deals with the embryological study and the systematic position of Amentotaxus argotaenia (Hance) Pilger. The material used was collected during 1980-1981 from Jin-fo Shan, 1400-1600 m, Sichuan Province, China. The species is dioecious. The male cone sheds its pollen during the period from the end of May to the middle of June. The pollen at mature stage is 2-celled. Pollen chamber appears obvious at the end of the nucellus. When pollen grains are dispersed, megaspore mother cell, which is situated deep in the nucellus, is in meiosis. The megaspore divides mitotically after pollination and the free nuclei of female gametophyte divide for the last time at the end of June. The wall formation takes place at the stage of 256 free nuclei. The development of archegonia takes place at the beginning of July and the fertilization occurs about July 20-23. The fertilized egg divides successively four times and results in a 16-nucleate proembryo. The young embryo is developing in August. It is interesting to note that the development of the young embryo is very slow. When the seed reaches the mature stage from June to July in the following year, the multicellular masses of the young embryos resulted from simple polyembryony remain immature within the female gametophyte. No cleavage polyembryony has been found. The subsequent embryogeny takes place after the seed has shed. Keng (1975) considers that Amentotaxus links the Taxaceae with Cephalotaxaceae. Our embryological data support Keng’s conclusion since they share (1) compound microstrobilus, (2) 2-celled pollen grains at shedding stage and (3) the rather long life cycle. Keng (1975) also mentions that Podocarpaceae may connect with Taxaceae through Phyllocladus. According to Keng the Podocarpaceae is related to Taxaceae to certain degree. It is obvious that the primitive spike-like male strobilus like the one in Cordaitales is obviously retained in Podocarpus spicatus and P. andinus of Podocarpaceae and Amentotaxus of Taxaceae. In addition, like in Amentotaxus there are 16 nuclei before wall formation in the proembryo of Podocarpus nivalis. These facts may well indicate that at least the Podocarpaceae and the Taxaceae were derived from a common stock. As far as the Taxaceae is concerned the authors tend to support the view of Koidzumi (1932) that Amentotaxus and Austrotaxus should be put in the same tribe since both possess the spike-like strobilus, the long life cycle and the seed maturation in the following year. They are probably rather primitive genera in the Taxaceae. The proembryogeny of Torreya is more or less specialized. It may be placed in a rather advanced tribe and the tribe Taxeae (including Taxus and Pseudotaxus)may be between the above two tribes. In conclusion, the Taxaceae is related to the Coniferales in certain respects and, as Keng (1975), Harri (1976) and Wang et al. (1979) have pointed out recently, placing of the Taxaceae in Coniferales is rather justifiable.     

Phylogeny of Taxaceae and Cephalotaxaceae Genera Inferred from Chloroplast matK Gene and Nuclear rDNA ITS Region

Phylogeny of the Taxaceae genera and the monotypic family Cephalotaxaceae has been extraordinarily controversial. In this paper chloroplast matK genes and nuclear ITS sequences were determined for all six genera of the two families and representatives of other conifer families. Analysis using either the nonsynonymous sites or the deduced amino acid sequences of matK genes strongly indicates that taxad genera and Cephalotaxaceae are monophyletic, with the Taxodiaceae/Cupressaceae clade as their sister group. Cephalotaxus is basal to the taxad genera, among which two clades, Torreya/Amentotaxus and Taxus/Pseudotaxus/Austrotaxus, are resolved. They correspond to Janchen's two tribes, Torreyeae and Taxeae. In Taxeae, Austrotaxus is the first to branch off. Analyses of the nuclear ITS sequence data corroborated the topology of the matK gene tree. These results refute the views that Cephalotaxaceae has no alliance with Taxaceae and that Austrotaxus and Amentotaxus should be excluded from the Taxaceae. We estimated the divergence time between the Taxodiaceae/Cupressaceae and the Cephalotaxaceae/Taxaceae clades to be 192–230 Myr ago and the divergence time between taxads and Cephalotaxus to be 149–179 Myr ago. Soon after the latter divergence event, within 6–8 Myr, the two taxad tribes originated. In conclusion, our data do not support Florin's claim that taxads could be traced to Devonian psilophytes (359–395 Myr ago).     

Biochemical Systematics of Gymnosperms (3)—On the Systematic Position of Tax aceae from Their Seed Protein Peptides and Needle Peroxidases

The major peptides in the seed of Taxus and Pseudotaxus have molecular weight about 31, 22 and 20 kilodaltons (Kd) shown by SDS polyacrylamide gel electrophoresis. The seed protein peptides of Cephalotaxus is very similar to those of Taxus and Pseudotaxus except a few bands of high molecular weight. Some considerable differences in peptide pattern exist between Amentotaxus and the three genera cited above. The former has a new major peptide, 33K, but lacks 22 K. The seed of Torreya does not contain peptide 44 K, although Torreya and Amentotaxus have many bands in common. To a certain extent, the seed protein peptides of Podocarpus nagi are similar to those of the above taxa. A great range of divergency in needle peroxidases among different genera of Taxaceae has been observed by using electrophoresis, while the zymogram of Cephalotaxus is somewhat similar to that of Taxus. Two series of protein data demonstrate, that there is an evolutionary tendency from Taxus to Torreya through Pseudotaxus and Amentotaxus within Taxaceae. And the Taxaceae is closely related to Cephalotaxus by way of Taxus. The systematic positionof the Taxaceae, therefore, should be placed under the Coniferales.     

A Contribution to Pollination and Fertilization of Amentotaxus argotaenta

The present investigation was conducted during 1980–1982, and mater- ials collected from Jin-Fo shan (Golden Buddha Mountain), at a height of 1400-1600 m, Sichuan province, China. Pollination of Amentotaxus argotaenia began to proceed last week of May, and came into bloom the first week of June. The male strobiles were almost entirely wilting at June 12–15. Thus, florescence of Amentotaxus spread over a period of 3 weeks. While the pollen grains approaching to maturity, most of the microspores divide to form a larger tube cell and a smaller antheridial initial. In this case the mature pollen grains of Amentotaxus consist of two cells. Then pollen grains are attracted down into the pollen chamber in the apex of the nucellus after pollination. The pollen chamber of Amentotaxus in longitudinal section looks like a flask in shape and is very much similar to that of Ginkgo biloba. As pollen grains at pollen chamber begin to germinate, the antheridial initials divide again to give rise to a spermatogenous cell and a sterile cell. At first, the spermatogenous cell is of a size only 11–13 μ in diameter. When the pollen tube reaches the middle part of the nucellus, the spermatogeneous cell is of a size about 30 μ. In the middle of July, pollen tube approaches the top of the female gametophyte. In this time, the spermatogenous cell has already been mature enough and is of 58–85 μ in diameter. The nuclei of spermatogenous ceils, 30–36 μ in size, are usually lying in the lateral side of the cytoplasm at its micropylar end. From the middle to the end of July, spermatogenous cells divide to form two unequal sperms, one of which is larger than the other and is the functional one. The large sperm is almost round in shape and about 56 μ in diameter. The small sperm is elliptic in shape, non-functional, and about 33 μ in diameter. The nuclei of the large and small sperms are about 40 μ and 26 μ, respectively. In some cases there are lateral pollen tube and sperms in the ovules of Amentotaxus, or the pollen tube even grows toward the lower part of female gametophyte in the chalazal end and there are well developed sperms in such a case. In the middle of July, nucleus of the central cell divides to form a ventral canal nucleus and an egg nucleus. The former then breaks down quickly and the latter continues to develope and moves toward the central part of the egg cell gradually. It is interesting to note that there are a number of nucleolus-like grains in the cytoplasm of the egg cell in Amentotaxus. The large nueleolus-like grains contain a larger central vacuole with several smaller vacuoles surrounding it. These grains show a positive reaction and blue colour by PAS and aniline blue black or coomassie brilliant blue, respectively. The above facts show that the nucleolus-like grains contain not only po- lysaccharides, but also protein. Similar grains may also found in the developing pollen tube. This is a unique feature in Amentotaxus and even in Gymnosperms. Otherwise, there are often two groups of the dense cytoplasm under the egg nucleus in Amentotaxus. Fertilization of Amentotaxus took place around July 20–29 (1980–1982). Interval between pollination and fertilization was about two months. After male nucleus fuses entirely with the female nucleus, the zygote begins to divide by mitosis. During fertilization, in addition that the large sperm enters the egg cell and fuses with the egg nucleus, the small sperm, tube nucleus, and sterile cell are often delivered into the egg cell. But they are disintegrated gradual]y and eventually. It is worthy to note that the nucleolus-like grains and the starches in pollen tube are also released into the egg cell. Then enlargement, fusion, and budding in the nucleolus-like grains may be found within the cytoplasm of the egg cell after fertilization. The history of investigating Amentotaxus found in 1883 has been lasting a long period of 100 years. But researches in sex production has never been studied before. The present work has shown that fertilization in Araentotaxus is very much similar to that in Taxus, Pseudotaxus, and Torreya. In other words, they all belong to the same type, that is, mitosis of zygote taking place after fusion of the two sexual nuclei. This condition constitutes one of the features of Taxaceae. But fertilization in Cephalotaxaceae is different from that of Taxaceae in having mitosis taking place before fusion of the two sexual nuclei. Pollination of Amentotaxus is similar to that of Cephalotaxus with dual-cell pollen grains at shedding stage. On the other hand, interval between pollination and fertilization in Austrotaxus lasts for 13.5 months, and this is the longest one in Taxaceae, and it is similar to that of Cephalotaxus proceeding for 14 months. To sum up, from the point of view of pollination, fertilization, and embryogenesis, Amentotaxus could be considered a primitive type in Taxaceae. Perhaps an order of systematic position of the genera belonging to Taxaceae can be arranged thus: Amentotaxus, Austrotaxus, Taxus, Pseudotaxus, and Torreya. And Cephalotaxaceae may be related to Taxaceae by way of Amentotaxus.     

Biflavone of Torreya jackii Chun and Its Taxonomical Significance

Kayaflavone has been isolated from leaves of Torreya jackii Chun, a species endemic to China. Seven species, belonging to Taxus, Pseudotaxus, Amentotaxus as well as Torreya, in Taxaceae were examined for kayaflavone, which has been detected only in the genus Torreya. The result seems to justify the separation of the genus from the others and the establishment of the monotypic tribe, Torreyeae.     

四种裸子植物叶精油化学成分的研究

采用气相色谱—质谱—计算机联用仪对穗花杉、南方红豆杉、三尖杉和罗汉松叶精油化学成分的研究发现,三尖杉和穗花杉叶精油的组成特点极为相似,相同成分１３个,占各自精油组成的４８．０７％和３３．３２％．三尖杉和南方红豆杉叶精油的相同成分４个,占各自精油组成的１６．１４％和４０．５９％．在一定程度上支持三尖杉科和红豆杉科的亲缘关系接近,红豆杉科可能是通过穗花杉属和三尖杉科相联系的观点．罗汉松和穗花杉叶精油的相同成分４个,占各自叶精油组成的比例为２４．０９％和２０．８２％,比罗汉松和南方红豆杉、三尖杉之间组分的相似性要高．反映出罗汉松科和红豆杉科之间有一定联系,穗花杉属是认识红豆杉科、三尖杉科和罗汉松科之间系统关系的关键属     

Studies on Pollen Morphology of Taxaceae of China

The present study deals with pollen morphology of 4 genera and l0 species of Taxaceae in gymnosperms. Pollen grains of the family are spheroidal or subspheroidal, 20.8μm in diameter and with laptoma or papilla in the distal face. Exine is two-layered, with sexine equal to nexine in thickness, but sometimes the stratification is indistinct. The surface is scabrous or slightly granular under LM. Coarse verrucae and fine tuberculae on pollen surface are observed under SEM. From thin section, endexine is shown to have lamellate structure, and ectexine is made of verrucate elements. In Amentotaxus argotaenia, some pollen grains show remnant saccate. According to pollen morphology, this family may be divided into two tribes: 1, Pseudotaxeae (including Pseudotaxus only), and 2, Taxeae (including Taxus and Torreya). Owirg to the special feature of pollen grains in Amentotaxus the present author suggests that the genus be separated from Taxaceae and raised to the level of family, Amentotaxaceae.     

A Study on the Genus Cephalotaxus Sieb. et Zucc.

The genus Cephalotaxus contains a small number of species. It is adequately appreciated as a newly discovered cancerresistant medicament for the alkaloids obtained from its branches leaves and barks are of curative effect. This paper deals with the classificatory revision based on the morphological features, with the reference to the anatomical characters of leaves, types of alkaloids and pollen morphology observed. Two new combinations are proposed, and 4 species and varieties are reduced in the paper. The genus Cephalotaxus is thus suggested to consist of 2 sections and 9 species. The trees occur in East Asia and the north of Indo-China, with 88% found in China where is the distribution centre and refuge of the genus. The genus in discussion is of unique morphological features which are distinctly different from these of Amentotaxus, Cephalotaxaceae, containing a single genus of Cephalotaxus, is closely related to Taxaceae, and therefore the Cephalotaxaceae is best placed in the Taxinieae of Coniferales.     

阴地蕨属药用植物matK基因编码区全序列测定及编码产物研究

以阴地蕨属(Botrychium)5种药用植物的matK基因为对象,分析matK基因编码区全序列和其编码产物MATK蛋白的氨基酸序列特征,并比较他们在用于阴地蕨属药用植物系统发育关系研究中的差异。结果显示,阴地蕨属5种植物matK基因全长为1500~1503 bp,共有153个变异位点,其编码产物均为不稳定的亲水性蛋白,无跨膜结构,二级结构以α-螺旋和无规则卷曲为主。系统发育分析结果表明,基于matK基因序列的系统发育分析更适合于阴地蕨属种间亲缘关系的鉴定,说明matK基因在阴地蕨属植物的鉴定中具有一定的应用价值。     

穗花杉染色体的研究

穗花杉为国家三级重点保护的珍稀濒危植物,雌雄异株。根尖细胞染色体分析表明：雌株和雄株的染色体数目为２ｎ＝４０,其中第１对和第２对为中部着丝粒染色体,第３－２０对为端部着丝粒染色体。核型为２ｎ＝４０＝４ｍ＋３６Ｔ。雌雄株除第２对的长度稍有差异外,其余各对的相对长度和臂比都较近似,可能尚无性染色体分化。Ｇｉｅｍｓａ Ｃ带显示,间期核有３个较大的染色中心,最长的３条染色体的中央缢痕有深染色带纹,可能是着     

红豆杉科次生韧皮部的比较解剖

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