Diversity and evolution of leaf anatomical characters in Taxaceae s.l.-fluorescence microscopy reveals new delimitating characters.

Taxaceae s.l. comprise six genera (including Cephalotaxus) and about 35 species; The present study aims to give new insights into the evolution of this family, especially into the phylogenetic position of Cephalotaxus. Moreover, only little is known about comparative leaf anatomy of this family and this study aims to expose and interpret the diversity and evolution of leaf anatomical characters and to assess their applicability to identify taxa at the generic and species level. A detailed phylogeny was reconstructed, using both maximum likelihood and Bayesian inference, with a combined dataset of four molecular markers from the plastid and nuclear genomes. Leaf sections from 132 specimens, representing 32 species and four varieties (fresh and herbarium material) were inspected, using fluorescence microscopy. Ancestral characters were reconstructed using Mesquite. The phylogenetic analyses provided full support for Cephalotaxus as sister group to Taxaceae s.str. Within the latter, two monophyletic tribes Taxeae (comprising Austrotaxus, Pseudotaxus, and Taxus) and Torreyeae (comprising Amentotaxus and Torreya) were fully supported. Fluorescence microscopy was shown to be very useful for identifying leaf tissues and their constitution. We were able to show that particularly sclerified tissues have highest potential for the discrimination of both freshly collected samples and rehydrated herbarium specimens at the generic and species level. A correlation between the presence of different sclereid types could be shown and sclereids were hypothesized to pose a primitive trait in the evolution of Taxaceae s.l. New identification keys were generated on the basis of leaf anatomical characters. The microscopic method presented here is applicable for further studies within gymnosperms and probably in angiosperms, as well.

Cross-Species, Amplifiable EST-SSR Markers for Amentotaxus Species Obtained by Next-Generation Sequencing.

Amentotaxus, a genus of Taxaceae, is an ancient lineage with six relic and endangered species. Four Amentotaxus species, namely A. argotaenia, A. formosana, A. yunnanensis, and A. poilanei, are considered a species complex because of their morphological similarities. Small populations of these species are allopatrically distributed in Asian forests. However, only a few codominant markers have been developed and applied to study population genetic structure of these endangered species. In this study, we developed and characterized polymorphic expressed sequence tag-simple sequence repeats (EST-SSRs) from the transcriptome of A. formosana. We identified 4955 putative EST-SSRs from 68,281 unigenes as potential molecular markers. Twenty-six EST-SSRs were selected for estimating polymorphism and transferability among Amentotaxus species, of which 23 EST-SSRs were polymorphic within Amentotaxus species. Among these, the number of alleles ranged from 1-4, the polymorphism information content ranged from 0.000-0.692, and the observed and expected heterozygosity were 0.000-1.000 and 0.080-0.740, respectively. Population genetic structure analyses confirmed that A. argotaenia and A. formosana were separate species and A. yunnanensis and A. poilanei were the same species. These novel EST-SSRs can facilitate further population genetic structure research of Amentotaxus species.

Leaf anatomy and its implications for phylogenetic relationships in Taxaceae s. l.

The comparative study on leaf anatomy and stomata structures of six genera of Taxaceae s. l. was conducted. Leaf anatomical structures were very comparable to each other in tissue shape and their arrangements. Taxus, Austrotaxus, and Pseudotaxus have no foliar resin canal, whereas Amentotaxus, Cephalotaxus, and Torreya have a single resin canal located below the vascular bundle. Among them, Torreya was unique with thick-walled, almost round sclerenchymatous epidermal cells. In addition, Amentotaxus and Torreya were comprised of some fiber cells around the vascular bundle. Also, Amentotaxus resembled Cephalotaxus harringtonia and its var. nana because they have discontinuous fibrous hypodermis. However, C. fortunei lacked the same kind of cells. Stomata were arranged in two stomatal bands separated by a mid-vein. The most unique stomatal structure was of Taxus with papillose accessory cells forming stomatal apparatus and of Torreya with deeply seated stomata covered with a special filament structure. Some morphological and molecular studies have already been discussed for the alternative classification of taxad genera into different minor families. The present study is also similar to these hypotheses because each genus has their own individuality in anatomical structure and stomata morphology. In conclusion, these differences in leaf and stomata morphology neither strongly support the two tribes in Taxaceae nor fairly recognize the monogeneric family, Cephalotaxaceae. Rather, it might support an alternative classification of taxad genera in different minor families or a single family Taxaceae including Cephalotaxus. In this study, we would prefer the latter one because there is no clear reason to separate Cephalotaxus from the rest genera of Taxaceae. Therefore, Taxaceae should be redefined with broad circumscriptions including Cephalotaxus.

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.

The complete chloroplast genome sequence of Amentotaxus argotaenia (Taxaceae).

The complete chloroplast genome sequence of Amentotaxus argotaenia was determined in this study. The genome is 136 657 bp in length and lacks one inverted repeat region. The overall GC content of the genome is 35.85% (protein-coding genes, 36.90%; tRNA genes, 53.31%; rRNA genes, 52.99%; introns, 36.10%; intergenic spacers, 31.03%). The A. argotaenia chloroplast genome contains 118 unique genes, including 83 protein-coding genes, 31 tRNA genes, and four rRNA genes. Ten protein-coding genes and six tRNA genes have one intron, while ycf3 contains two. The coding regions occupy 60.27% of the genome length and the gene density is estimated to be 0.88 genes/kb. A maximum likelihood phylogenetic analysis suggested that Amentotaxus is sister to Taxus within the Taxaceae family.

Chemical components of cold pressed kernel oils from different Torreya grandis cultivars.

The chemical compositions of cold pressed kernel oils of seven Torreya grandis cultivars from China were analyzed in this study. The contents of the chemical components of T. grandis kernels and kernel oils varied to different extents with the cultivar. The T. grandis kernels contained relatively high oil and protein content (45.80-53.16% and 10.34-14.29%, respectively). The kernel oils were rich in unsaturated fatty acids including linoleic (39.39-47.77%), oleic (30.47-37.54%) and eicosatrienoic acid (6.78-8.37%). The kernel oils contained some abundant bioactive substances such as tocopherols (0.64-1.77mg/g) consisting of α-, ß-, γ- and δ-isomers; sterols including ß-sitosterol (0.90-1.29mg/g), campesterol (0.06-0.32mg/g) and stigmasterol (0.04-0.18mg/g) in addition to polyphenols (9.22-22.16µgGAE/g). The results revealed that the T. grandis kernel oils possessed the potentially important nutrition and health benefits and could be used as oils in the human diet or functional ingredients in the food industry.

[Morphological and commercial identification on semen torreyae].

The morphological characters and commercial identification on Semen Torreyae has been reported. The results of identification of commercial samples indicated that Torreya grandis was mainly used; T. fargesii, T. yunnanensis and T. jackii were used in their producing areas.

[Determination of amino acids in seeds of genus Torreya in China].

OBJECTIVE: To determine the contents and analyze the ingredients of the amino acids in seeds of genus Torreya. METHODS: The amino acid contents and ingredients were determined by amino acid analyzer. RESULTS: There were 19 amino acids in the seeds of T. grandis, T. grandis cv. merrillii, T. yunnanensis, T. jackii and T. fargesii, and their amino acid contents are 9.10%, 9.90%, 5.03%, 7.26% and 6.07% respectively. There were 17 amino acids in the seeds of T. jiulongshanensis, T. nucifera and T. grandis cv. merrillii (baked) and their amino acid contents are 14.11%, 9.87% and 9.22% respectively. tively.

[Genetic diversity of endangered plant Torreya jackii: a study with RAPD markers].

With random amplified polymorphic DNA (RAPD) techniques, this paper studied the genetic diversity and genetic differentiation of Torreya jackii, an endangered plant endemic to China. In the 180 individuals of 9 T. jackii natural populations, 180 repetitive loci were detected by using 12 random primers, among which, 119 were polymorphic. At species level, the percentage of polymorphic loci (P) was 66.11%, and the genetic diversity estimated by Shannon information index (I) and Nei's index (h) was 0.3087 and 0.2015, respectively, suggesting that the genetic diversity at species level was relatively high. However, at population level, the genetic diversity was relatively low (P = 23.76% , I = 0.1221, and h = 0.0813). The analysis of molecular variance (AMOVA) showed that 42.57% of genetic variance was found within populations, and 57.43% of it was resided among populations. The coefficient of gene differentiation (G(st)) was 0.5965, indicating the high genetic differentiation among the populations of T. jackii, and the gene flow among the populations was quite low, being 0.3382. The bottleneck effect, population isolation, and low gene flow among populations would have contributed to such a population genetic structure in T. jackii. The averaged genetic distance among 9 T. jackii populations was 0.1630. By using the unweighted pair group method with arithmetic mean (UPGMA), the 9 populations could be divided into two groups, i. e., Zhejiang group and Fujian group. It was proposed that in the ex situ conservation of T. jackii, the gcrmplasm transferring among populations should be avoided.

[Population structure and community characteristics of Pseudotaxus chienii in Fengyangshan National Natural Reserve].

Pseudotaxus chienii, an endemic plant in China, is one of the second grade state protection wild plants, and distributes in Fengyangshan Natural Reserve as one of its concentrative dwelling places. A survey in the region was carried out on 10 communities, which were dominated by P. chienii. The analysis on its size structure, spatial distribution pattern and community characteristics showed that the populations of P. chienii could grow in the communities dominated by Rhododendron simiarum, Fokienia hodginsii-R. simiarum, and evergreen broad-leaved forests. In R. simiarum communities, the size structure, survival curve, and overwhelming community distribution pattern of P. chienii showed a sustaining development, while in communities dominated by F. hodginsii-R. simiarum, though the size structure was declining, the survival curve was Deevy- III type. Plenty of plantlets were still existed, and the populations kept steady. Two types were considered to be the most suitable ones for P. chienii populations. The important value of P. chienii reached 5% - 10%. In evergreen broad-leaved forests, the populations of P. chienii showed to be a declining type, and the survival curve was Deevy- II type. The distribution pattern was random, indicating that P. chienii populations could not fit for survive in this kind of communities. Correlation analysis showed that P. chienii populations had a positive correlation with R. simiarum and F. hodginsii, but a negative correlation with Cyclobalanopsis stewardiana and Schima superba. P. chienii could associate to the habits with the community canopy density of about 0.6 - 0.8. From the results mentioned above, the populations of P. chienii could be able to sustain and develop, and the existing habitat in Fengyangshan should be protected effectively. The protection of P. chienii requires more basic work to establish efficient measures to protect its habitat.

Low genetic diversity and significant population structuring in the relict Amentotaxus argotaenia complex (Taxaceae) based on ISSR fingerprinting.

Amentotaxus, a genus of the Taxaceae, represents an ancient lineage that has long existed in Eurasia. All Amentotaxus species experienced frequent population expansion and contraction over periodical glaciations in Tertiary and Quaternary. Among them, Amentotaxus argotaenia complex consists of three morphologically alike species, A. argotaenia, Amentotaxus yunnanensis, and Amentotaxus formosana. This complex is distributed in the subtropical region of mainland China and Taiwan where many Pleistocene refugia have been documented. In this study, genetic diversity and population structuring within and between species were investigated based on the inter-simple sequence repeats (ISSR) fingerprinting. Mean genetic diversity within populations was estimated in three ways: (1) the percentage of polymorphic loci out of all loci (P) (2) Nei's unbiased expected heterozygosity (He), and (3) Shannon's index of phenotypic diversity. For a total of 310 individuals of 15 populations sampled from the three species, low levels of ISSR genetic variation within populations were detected, with P=4.66-16.58%, He=0.0176-0.0645 and Hpop=0.0263-0.0939, agreeing with their seriously threatened status. AMOVA analyses revealed that the differences between species only accounted for 27.38% of the total variation, whereas differences among populations and within populations were 57.70 and 14.92%, respectively, indicating substantial isolation between the patch-like populations. A neighbor-joining tree identified a close affinity between A. yunnanensis and A. formosana. Genetic drift due to small population size, plus limited current gene flow, resulted in significant genetic structuring. Low levels of intra-populational genetic variation and considerable inter-populational divergence were also attributable to demographic bottlenecks during and/or after the Pleistocene glaciations.