Plastome phylogenomics of Cephalotaxus (Cephalotaxaceae) and allied genera

Background and Aims Cephalotaxus is a paleo-endemic genus in East Asia that consists of about 7–9 conifer species. Despite its great economic and ecological importance, the relationships between Cephalotaxus and related genera, as well as the interspecific relationships within Cephalotaxus, have long been controversial, resulting in contrasting taxonomic proposals in delimitation of Cephalotaxaceae and Taxaceae. Based on plastome data, this study aims to reconstruct a robust phylogeny to infer the systematic placement and the evolutionary history of Cephalotaxus.MethodsA total of 11 plastomes, representing all species currently recognized in Cephalotaxus and two Torreya species, were sequenced and assembled. Combining these with previously published plastomes, we reconstructed a phylogeny of Cephalotaxaceae and Taxaceae with nearly full taxonomic sampling. Under a phylogenetic framework and molecular dating, the diversification history of Cephalotaxus and allied genera was explored.Key ResultsPhylogenetic analyses of 81 plastid protein-coding genes recovered robust relationships between Cephalotaxus and related genera, as well as providing a well-supported resolution of interspecific relationships within Cephalotaxus, Taxus, Torreya and Amentotaxus. Divergence time estimation indicated that most extant species of these genera are relatively young, although fossil and other molecular evidence consistently show that these genera are ancient plant lineages.ConclusionsOur results justify the taxonomic proposal that recognizes Cephalotaxaceae as a monotypic family, and contribute to a clear-cut delineation between Cephalotaxaceae and Taxaceae. Given that extant species of Cephalotaxus are derived from recent divergence events associated with the establishment of monsoonal climates in East Asia and Pleistocene climatic fluctuations, they are not evolutionary relics.     

Interspecific relationships and origins of Taxaceae and Cephalotaxaceae revealed by partitioned Bayesian analyses of chloroplast and nuclear DNA sequences

The precise delimitation of Taxaceae and Cephalotaxaceae is not totally resolved. Some contradicting taxonomic proposals have been published, which demonstrates the difficulties in establishing a natural classification of the families and especially in proposing a relevant treatment within the genera Taxus and Cephalotaxus. The aims of this study are to contribute to the phylogeny and specific delineation of the two conifer families on the basis of molecular data. A cladistic analysis of the sequences of five chloroplast (matK, rbcL, trnL, trnL-trnF spacer, and psbA-trnH spacer) and one nuclear (ITS) molecular markers was carried out, both individually and in combination, by distance, parsimony, likelihood, and Bayesian methods. The results confirm that the two families are monophyletic. In the genus Taxus, T. floridana is the first-branching taxon; T. brevifolia and T. globosa cluster together and are sister to T. baccata; the endemic T. yunnanensis clusters with T. wallichiana in subclade B and is only distantly related with the other four Taxus species in China (subclade A); T. fuana is closer to T. baccata than to other Taxus species. Torreya jackii and A. formosana are the first-branching species within Torreya and Amentotaxus, respectively. C. koreana and C. wilsoniana could be treated as two varieties of C. harringtonia. The ancestral distribution area of Taxaceae and Cephalotaxaceae is restricted either to Southwest China or Southeast China by DIVA analysis. The relaxed molecular clock indicates that the deepest divergences in Taxus go back to the late-Cretaceous. psbA-trnH, rbcL third codon position, and matK first codon position contributed most to the separation of taxa in Discriminant function analysis. Our results confirm, on a basis of multiple molecular markers and a complete sampling of basic species, the suggested monophyly of Taxaceae and Cephalotaxaceae and propose interspecific relationships within each group, with profound nomenclatural and taxonomic implications. Combination of partitioned Bayesian analysis and likelihood-based methods produced a more robust phylogenetic hypothesis for the two studied families.     

Cladistic analysis of Taxaceae s. l.

Taxaceae s. l. is a wider concept of classification treating five genera of Taxaceae s. str. and Cephalotaxus together. Cephalotaxus is morphologically very similar to the five genera of Taxaceae s. str. Various models of classification for six genera have already been published. However, the phylogenetic position and genuine relationships of these genera and species are still confusing. A cladistic analysis of Taxaceae s. l. has been carried out to resolve the problem existing in their phylogeny and to provide a new approach regarding the relationships of these six genera. Parsimony analyses were based on 28 characters and eight genera including two outgroups Agathis and Sciadopitys. The most parsimonious tree retained with branch length 38 and 194 rearrangement trials. Consistency index was 0.68 and retention index was 0.66. Principally, two main clades were found: one represented by Austrotaxus forming the base of the tree and another by the remaining five genera. Taxus + Pseudotaxus clade split after Austrotaxus, and Cephalotaxus was sister to Torreya + Amentotaxus clade. Taxus + Pseudotaxus clade was supported by the highest bootstrap value. Finally, cladistic analysis does not support existence of Cephalotaxaceae. Therefore, it would be better to classify Cephalotaxus within Taxaceae s. l. with the other five genera.     

Comparative palynology and wood anatomy of <Emphasis Type="Italic">Taxodium distichum</Emphasis> (L.) Rich. and <Emphasis Type="Italic">Taxodium mucronatum</Emphasis> Ten.

A comparative study of Taxodium distichum (L.) Rich. and Taxodium mucronatum Ten. was carried out on the basis of pollen morphology and wood anatomy by light and scanning electron microscopy. We describe a detailed analysis of the anatomical characteristics of the wood, including the tracheids, ray parenchyma, axial parenchyma and number of cross-field pits. Palynological characters were also studied to reveal the shape, size and ultrastructure of the pollen grains. These studies give taxonomic support for the recognition of T. distichum and T. mucronatum as two different species.     

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).     

部分裸子植物茎次生韧皮部和木材的比较解剖

茎次生韧皮部和木材的比较解剖支持红豆杉科、三尖杉科和罗汉松科自然类群、不主张建立穗花杉科和竹柏科     

Characteristics of Wood Structure in Gymnosperms and Their systematic Significance

The systematic positions and taxonomic ranks of orders and families in Gymnosperms, especially those in Coniferopsida, are analysed and discussed in this paper based on the evolutionary trends in the wood structure. The opinions of the present authors are as follow: 1. The separation of the Araucariaceae from the Coniferae and establishment of the Araucariales are reasonable,because the intertracheid pitting in the wood is the Araucarioid type and there are no pits on both horizontal and end walls of ray parenchyma cells in the family. 2. The position of the genus Ginkgo in Cheng’s system is acceptable. Ginkgo is more similar to Coniferae than to Cycadaceae in the wood structure. 3. According to the characteristics of wood structure, arrangement of the Podocarpaceae, Cephalotaxaceae and Taxaceae between the Araucariaceae and the Pinaceae is reasonable. Among these families, the Cephalotaxaceae and Taxaceae are more closely related to each other in the view of the spiral thickenings which often appear on the inner wall of wood tracheids. 4. Further evidence for the establishment of the Sciadopitysaceae is provided. For example, most of cross-field pits in the wood of the Sciadopitysaceae are window like, while some of them are of the Lemon type or the Subtaxodioid type; bordered pits are of the Araucaria B type. 5. The characteristics of wood structure in the genus Platycladus differ greatly from Thuja. The former has cross field pits of the Cupressoid type, bordered pits of Araucaria B type and warty layer on the inner surface of tracheids. All of these characteristics have added further evidence for the separation of Platycladus from Thuja. 6. Based on the structural characteristics of woody rays in the Pinaceae, the most primitive genera are Abies, Keteleeria and Pseudolarix, while more advanced ones are Cedrus and Tsuga, and even more advanced ones are Pseudotsuga, Cathaya, Picea and Larix, all of which share normal resin canals. The most advanced genus is Pinus which is also of normal resin canals. Pinus can be divided into three subgenera, Haploxylon, Parry and Diploxylon, according to the presence or absence of dentation and warty layer in wood tracheids. 7. It is reasonable to place the genus Amentotaxus in the Taxaceae, because membrane of bordered pits in the genus is similar to that in the other four genera of the Taxaceae, both of the Araucaria type. 8. The present authors agree with Cheng’s (1978) treatment of Sect. Heopeuce in Tsuga, based on the fact that Tsuga longibracteata has traumatic resin canals and warty layer. Reducing Pinus hwangshanensis into P. taiwanensis, made by Cheng, is reasonable because of the similarities between P. hwangshanensis and P. taiwanensis in the wood structure. The establishment of a new subgenus, Patty, for Pinus bungeana is suitable based on chemotaxonomy, morphology and the distinct warty layer on the innersurface of wood tracheids.     

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).     

Comparative Anatomy of the Secondary Phloem of the Stem in Cephalotaxus

The family Cephalotaxaceae contains only one genus, Cephalotaxus, with 9 species and some varieties, mostly endemic to China. The present paper deals with the comparative anatomy of secondary phloem of stems in 4 species and I cultivar, of this genus under light microscope and scanning electron microscope. The main results are as follows: (1) The structure of secondary phloem is rather uniform in the various species of this genus. In cross section, sieve cells, phloem parenchyma cells form continuous tangential rows of one cell in width respectively, which occur alternately. Sclerenchyma cells also form continuous tangential rows, each with a radial width of 1-4 cells. The interval between the rows is rather wide. (2) The type of phloem fiber and the quantity of sclereids can be served as the characters for identification of species and the evidence for the separation of two sections of Cephalotaxus, namely: Cephalotaxus and Pectinatae. (3) The secondary phloem of this genus contains more or less crystalliferous parenchyma cells, in the inner tangential walls of which calcium oxalate crystals are embedded. So far this character has not been reported in the other families and the genera of conifers. Our conclusion, therefore, agrees with the opinion that Cephalotaxaceae is a natural taxon and includes only one genus, Cephalotaxus.     

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     

Evolution of the Chloroplast trnL-trnF Region in the Gymnosperm Lineages Taxaceae and Cephalotaxaceae

The trnL-trnF region is located in the large single-copy region of the chloroplast genome. It consists of the trnL gene, a group I intron, and the trnL-F intergenic spacer. We analyzed the evolution of the region in three gymnosperm families, Taxaceae, Cephalotaxaceae, and Podocarpaceae, with especially dense sampling in Taxaceae and Cephalotaxaceae, for which we sequenced 43 accessions, representing all species. The trnL intron has a conserved secondary structure and contains elements that are homologous across land plants, and the spacer is highly variable in length and composition. The spatial distribution of nucleotide diversity along the trnL-F region suggests that different portions of this region have different evolutionary patterns. Tandem repeats that form stem–loop structures were detected in both the trnL intron and the trnL-F spacer, and the spacer sequences contain promoter elements for the trnF gene. The presence of promoters and stem–loop structures in the trnL-F spacer and high sequence variation in this region suggest that trnL and trnF are independently transcribed. Stem–loop regions P6, P8, and P9 of the trnL intron and the trnL-F spacer (except the promoter elements) might undergo neutral evolution with respect to their escape from functional constraints.     

红豆杉科、三尖杉科和罗汉松科的实验分类学研究进展

红豆杉科、三尖杉科和罗汉松科的系统位置及系统发育关系长期以来都存有争议,本文对此三科在实验分类学方面的研究进展及趋势作了综述。     

A study of the alkaloids in Cephalotaxus and their bearing on the chemotaxonomic problems of the genus

The family Cephalotaxaceae contains so far known only 1 genus with 8 species and possibly 2-3 varieties mostly native in China. In recent years, some ester alkaloids of Cephalotaxus have been shown significant activity in a variety of experimental leukemia systems. 6 species (including varieties) of the genus have been investigated for their alkaloids. The detected alkaloids in Cephalotaxaceous plants, from the chemotaxonomic point of view, may be of some significance to the plant systemstics. Our present study has indicated that: (1) The species C. sinensis and C. fortunei (C. oliveri ?) are chemically considered to be more primitive than others, while C. harringtonia, C. hainanensis and C. wilsoniana (including C. mannii?) seem to be derived. (2) Chemical evidence has suggested that the genus Cephalotaxus be accorded a family rank. (3) On the basis of morphology, anatomy, embryogeny, geographical distribution etc., the recent findings in the chemistry of Cephalotaxaceae support the inclusion of Cephalotaxaceae, Taxaceae and Podocarpaceae in Taxineae of Coniferales. (4) C. fortunei, C. hainanensis and C. oliveri (C. harringtonia) contain richer ester alkaloids than other species of the genus. C. fortunei which has a wide distribution and great amount in many provinces of China, may prove to be a good new resource for antitumor medicine.     

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.     

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。     

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.     

The phylogenetic positions of the conifer genera Amentotaxus,Phyllocladus, and Nageia inferred from 18s rRNA sequences

To determine the evolutionary positions of the conifer genera Amentotaxus, Phyllocladus, and Nageia, we obtained 18S rRNA sequences from 11 new taxa representing the major living orders and families of gymnosperms. With the published Chlamydomonas as an outgroup, phylogenetic analyses of our new data and available sequences indicate that (1) the Gnetales form a monophyletic group, which is an outgroup to the conifers, (2) the conifers are monophyletic, (3) Taxaceae, Cephalotaxaceae, Cupressaceae, and Taxodiaceae form a monophyletic group, (4) Amentotaxus is closer to Torreya than to Cephalotaxus, suggesting that Amentotaxus is better to be classified as a member of Taxaceae, (5) Phyllocladus, Dacrycarpus, Podocarpus, and Nageia form a monophyletic group, and (6) Pinaceae is an outgroup to the other families of conifers. Our finding that Phyllocladus is a sister group of the Podocarpaceae disagrees with the suggestion that the phylloclade of the genus is an ancient structure and that the genus is a terminal taxon within the Podocarpaceae. The genus Nageia is more closely related to Podocarpus than to Dacrycarpus and was derived from within the Podocarpaceae. In conclusion, our data indicate that in conifers, the uniovulate cone occurred independently in Taxacaeae and Cephalotaxaceae, and in Podocarpaceae after the three families separated from Pinaceae, and support the hypothesis that the uniovulate cone is derived from reduction of a multiovulate cone.Correspondence to: S.-M. Chaw     

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。