Application of the Serological Method for Evaluation of Relations between Gymnospermous and Dicotyledonous Plants

An attempt has been undertaken to evaluate interrelations of gymnospermous and dicotyledonous plants on the basis of immunochemical studies of seed proteins. For this purpose, 12 antisera were raised to proteins of taxa representing four gymnosperm classes: Ginkgoopsida, Cycadopsida, Coniferopsida, and Gnetopsida. Seed proteins of eight dicotyledonous subclasses (after Takhtadzhyan, 1987) were used. The representatives of all dicotyledonous subclasses gave immunochemical reactions with those of all gymnospermous classes. The data obtained suggest the presence of sufficiently close immunochemical relations between gymnosperms and dicotyledons. Samples were found among the representatives of subclasses Dilleniidae, Hamamelididae, and Rosidae, which gave satisfactory reactions with eight to ten antisera to proteins of dicotyledonous seeds. Analysis of the data we obtained suggests that gymnospermous and dicotyledonous plants took their origin from a common pragymnospermous ancestor and later evolved independently or that dicotyledons separated from gymnosperms at an early stage of their evolution before divergence of the latter into several phyletic lineages.     

The evolution of embryo size in angiosperms and other seed plants: implications for the evolution of seed dormancy

Abstract.— Seed dormancy plays an important role in germination ecology and seed plant evolution. Morphological seed dormancy is caused by an underdeveloped embryo that must mature prior to germination. It has been suggested that the presence of an underdeveloped embryo is plesiomorphic among seed plants and that parallel directional change in embryo morphology has occurred separately in gymnosperms and in angiosperms. We test these hypotheses using original data on embryo morphology of key basal taxa, a published dataset, and the generalized least squares (GLS) method of ancestral character state reconstruction. Reconstructions for embryo to seed ratio (E:S) using family means for 179 families showed that E:S has increased between the ancestral angiosperm and almost all extant angiosperm taxa. Species in the rosid clade have particularly large embryos relative to the angiosperm ancestor. Results for the gymnosperms show a similar but smaller increase. There were no statistically significant differences in E:S between basal taxa and any derived group due to extremely large standard errors produced by GLS models. However, differences between reconstructed values for the angiosperm ancestor and more highly nested nodes are large and these results are robust to topological and branch-length manipulations. Our analysis supports the idea that the underdeveloped embryo is primitive among seed plants and that there has been a directional change in E:S within both angiosperms and gymnosperms. Our analysis suggests that dormancy enforced by an underdeveloped embryo is plesiomorphic among angiosperms and that nondormancy and other dormancy types probably evolved within the angiosperms. The shift in E:S was likely a heterochronic change, and has important implications for the life history of seed plants.     

Ferulic acid is bound to the primary cell walls of all gymnosperm families

Unlignified primary cell walls containing ester-linked ferulic acid fluoresce blue in ultraviolet radiation which changes to green with increased intensity on treatment with ammonium hydroxide. Using this fluorescence behaviour, we detected ester-linked ferulic acid in the primary cell walls of all 41 species of gymnosperms we examined. These species were in 17 families representing all four extant classes of gymnosperms. In addition, we obtained cell-wall preparations containing >95% primary cell walls from nine gymnosperm species in nine families, representing all four extant classes. These preparations were analysed for ester-linked monomeric phenolic acids. We found ferulic acid (mostly trans) (88-1,561μg/g cell walls) in all of the preparations and p-coumaric acid (mostly trans) (0-106μg/g cell walls) in all except one of them. Ferulic acid ester-linked to primary cell walls has previously been found in angiosperms: in the commelinoid monocotyledons and in the dicotyledon order Caryophyllales, both monophyletic groups. From the present results, we postulate that the extant classes of gymnosperms are monophyletic and no class is sister to the angiosperms.     

A cladistic classification of green plants

A cladistic classification comprises strictly monophyletic taxa only. Groups such as the green algae and the gymnosperms are not monophyletic and should not be recognized taxonomically. In this proposal the green plants are recognized as a sub–kingdom, Chlorobionta. One of the divisions is Slreptophyta with some green algae and the higher plants, the latter group being recognized as a subdivision, Em–bryophylina. The (extant) higher plants consist of eight classes, one of them being Pinatae , the seed plants. The angiosperms constitute one of the subclasses, viz Mag–noliidae.     

The late pollen-specific actins in angiosperms

The actin gene family of Arabidopsis has eight functional genes that are grouped into two ancient classes, vegetative and reproductive, and into five subclasses based on their phylogeny and mRNA expression patterns. Progress in deciphering the functional significance of this diversity is hindered by the lack of tools that can distinguish the highly conserved subclasses of actin proteins at the biochemical and cellular level. In order to address the functional diversity of actin isovariants, we have used Arabidopsis recombinant actins as immunogens and produced several new anti-actin monoclonal antibodies. One of them, MAb45a, specifically recognizes two closely related reproductive subclasses of actins. On immunoblots, MAb45a reacts strongly with actins expressed in mature pollen but not with actins in other Arabidopsis tissues. Moreover, immunocytochemical studies show that this antibody can distinguish actin filaments in pollen tubes from those in most vegetative tissues. Peptide competition analyses demonstrate that asparagine at position 79 (Asn79) within an otherwise conserved sequence is essential for MAb45a specificity. Actins with the Asn79 epitope are also expressed in the mature pollen from diverse angiosperms and Ephedra but not from lower gymnosperms, suggesting that this epitope arose in an ancestor common to angiosperms and advanced gymnosperms more than 220 million years ago. During late pollen development in angio- sperms there is a switch in expression of actins from vegetative to predominantly reproductive subclasses, perhaps to fulfil the unique functions of pollen in fertilization.     

晋东南山地种子植物区系研究

晋东南山地有野生种子植物974种,隶属于456属125科,其中裸子植物7科11属15种,被子植物118科445属959种(其中双子叶植物104科396属833种,单子叶植物14科,49属126种)。种子植物属的区系成分复杂多样,以温带成分占绝对优势(温带属250属,占总属数的60．98％)。种子植物种的区系成分中,中国特有种占绝对优势,共有383种,占总种数的39．32％,它们是构成晋东南山地森林植被和灌丛植被的建群种和优势种的主要成分。     

Phylogeny and Divergence Times of Gymnosperms Inferred from Single-Copy Nuclear Genes

Phylogenetic reconstruction is fundamental to study evolutionary biology and historical biogeography. However, there was not a molecular phylogeny of gymnosperms represented by extensive sampling at the genus level, and most published phylogenies of this group were constructed based on cytoplasmic DNA markers and/or the multi-copy nuclear ribosomal DNA. In this study, we use LFY and NLY, two single-copy nuclear genes that originated from an ancient gene duplication in the ancestor of seed plants, to reconstruct the phylogeny and estimate divergence times of gymnosperms based on a complete sampling of extant genera. The results indicate that the combined LFY and NLY coding sequences can resolve interfamilial relationships of gymnosperms and intergeneric relationships of most families. Moreover, the addition of intron sequences can improve the resolution in Podocarpaceae but not in cycads, although divergence times of the cycad genera are similar to or longer than those of the Podocarpaceae genera. Our study strongly supports cycads as the basal-most lineage of gymnosperms rather than sister to Ginkgoaceae, and a sister relationship between Podocarpaceae and Araucariaceae and between Cephalotaxaceae-Taxaceae and Cupressaceae. In addition, intergeneric relationships of some families that were controversial, and the relationships between Taxaceae and Cephalotaxaceae and between conifers and Gnetales are discussed based on the nuclear gene evidence. The molecular dating analysis suggests that drastic extinctions occurred in the early evolution of gymnosperms, and extant coniferous genera in the Northern Hemisphere are older than those in the Southern Hemisphere on average. This study provides an evolutionary framework for future studies on gymnosperms.     

Lineage-specific group II intron gains and losses of the mitochondrial rps3 gene in gymnosperms

According to PCR assays and sequencing, we now report the shared presence of two rps3 introns, namely the rps3i74 and the rps3i249, in the mitochondria of all the classes representing the surviving lineages of gymnosperms, and unveil several lineages experiencing intron loss.Interestingly, the rps3 intron gains and losses within the four groups of gymnosperms let us sort out the Pinaceae and the non-Pinaceae into intron (+)- and intron (?)-lineages, respectively. Worthy of mention is also the finding that only Gnetum within the Gnetales harbours both the rps3 introns.This intron distribution pattern is consistent with the hypothesis that the two rps3 introns were likely present in the common ancestor of the seed plants and, then, independently lost in the non-Pinaceae during gymnosperm evolution.The derived secondary structural model of the novel group IIA intron improves our understanding of the significance and origin of the extraordinary length polymorphisms observed among rps3i249 orthologs.Despite the remarkable structural plasticity to adopt and reject introns, the rps3 mRNAs undergo accurate processing by splicing and extensive editing in gymnosperm mitochondria.This study provides additional insights into the evolutionarily high dynamics of mitochondrial introns which may come and go in closely related plant species.The turnover of the mitochondrial rps3 group II introns seen among lineages of seed plants further suggests that these introns might be an additional signature to discriminate between particularly cryptical taxonomic groups for which there is a need of a further evaluation of their evolutionary affiliation.     

湖北利川市种子植物区系研究

根据调查和统计,利川市共有野生种子植物145科750属2028种,其中裸子植物6科19属24种,被子植物139科731属2004种,木本植物1002种,草本植物1026种;本区系具有古老、孑遗、原始性,单种属和寡种属数量多;分布类型多样,地理成分复杂,仅属有15个分布类型和21个变型,科的热带成分占61.62%、温带成分占36.36%,属的热带成分占39.05%,温带成分占55.62%。利川种子植物区系具有以温带为主的亚热带性质。     

The molecular characterization of PaHB2, a homeobox gene of the HD-GL2 family expressed during embryo development in Norway spruce

PaHB1 (for Picea abies Homeobox1), an evolutionarily conserved HD-GL2 homeobox gene, specifically expressed in the protoderm during somatic embryogenesis in the gymnosperm Norway spruce has been reported previously. An additional HD-GL2 gene designated PaHB2 is reported here. During somatic embryogenesis, the PaHB2 gene is uniformly ex pressed in proembryogenic masses and in early somatic embryos, but it is not detectably transcribed at the beginning of maturation. In mature embryos, PaHB2 expression was essentially detected in the outermost layer of the cortex and the root cap. A similar PaHB2 expression is detected post-embryonically in both the primary root and the hypocotyl. Phylogenetic reconstructions and intron pattern analyses revealed that the PAHB proteins fall within two distinct subclasses comprising highly similar angiosperm homologues. The PAHB1 subclass consists of protoderm/epiderm-specific members. By contrast, the PAHB2 subclass gathers homologues with a subepidermal and protodermal/epidermal activity. This study suggests that at least two distinct HD-GL2 genes with a layer-specific expression already existed in the last common ancestor of angiosperms and gymnosperms. The conserved protodermal/epidermal and subepidermal expression of HD-GL2 genes could be used to study embryo radial pattern formation across seed plants.     

High universality of matK primers for barcoding gymnosperms

DNA barcoding is a tool to provide rapid and accurate taxonomic identification using a standard DNA region. A two-marker combination of rnatK+rbcL was formally proposed as the core barcode for land plants by the Consortium for the Barcode of Life Plant Working Group. However, there are currently no barcoding primers for matK showing high universality in gymnosperms. We used 57 gymnosperm species representing 40 genera, 11families and four subclasses to evaluate the universality of nine candidate matK primers and one rbcL primer in this study. Primer (1F/724R) of rbcL is proposed here as a universal primer for gymnosperms due to high universality. One of the nine candidate matK primers (Gym_F1A/Gym_R1A) is proposed as the best "universal" matK primer for gynnosperms because of high polymerase chain reaction success and routine generation of high quality bidirectional sequences. A specific matK primer for Ephedra was newly designed in this study, which performed well on the sampled species. The primers proposed here for rbcL and matK can be easily and successfully amplified for most gymnosperms.     

Chloroplast genome (cpDNA) of Cycas taitungensis and 56 cp protein-coding genes of Gnetum parvifolium: insights into cpDNA evolution and phylogeny of extant seed plants

Phylogenetic relationships among the 5 groups of extant seed plants are presently unsettled. To reexamine this long-standing debate, we determine the complete chloroplast genome (cpDNA) of Cycas taitungensis and 56 protein-coding genes encoded in the cpDNA of Gnetum parvifolium. The cpDNA of Cycas is a circular molecule of 163,403 bp with 2 typical large inverted repeats (IRs) of 25,074 bp each. We inferred phylogenetic relationships among major seed plant lineages using concatenated 56 protein-coding genes in 37 land plants. Phylogenies, generated by the use of 3 independent methods, provide concordant and robust support for the monophylies of extant seed plants, gymnosperms, and angiosperms. Within the modern gymnosperms are 2 highly supported sister clades: Cycas-Ginkgo and Gnetum-Pinus. This result agrees with both the "gnetifer" and "gnepines" hypotheses. The sister relationships in Cycas-Ginkgo and Gnetum-Pinus clades are further reinforced by cpDNA structural evidence. Branch lengths of Cycas-Ginkgo and Gnetum were consistently the shortest and the longest, respectively, in all separate analyses. However, the Gnetum relative rate test revealed this tendency only for the 3rd codon positions and the transversional sites of the first 2 codon positions. A PsitufA located between psbE and petL genes is here first detected in Anthoceros (a hornwort), cycads, and Ginkgo. We demonstrate that the PsitufA is a footprint descended from the chloroplast tufA of green algae. The duplication of ycf2 genes and their shift into IRs should have taken place at least in the common ancestor of seed plants more than 300 MYA, and the tRNAPro-GGG gene was lost from the angiosperm lineage at least 150 MYA. Additionally, from cpDNA structural comparison, we propose an alternative model for the loss of large IR regions in black pine. More cpDNA data from non-Pinaceae conifers are necessary to justify whether the gnetifer or gnepines hypothesis is valid and to generate solid structural evidence for the monophyly of extant gymnosperms.     

Expression patterns of conserved microRNAs in the male gametophyte of loblolly pine (Pinus taeda)

MicroRNAs (miRNAs) are small RNAs that regulate genes involved in various aspects of plant development, but their presence and expression patterns in the male gametophytes of gymnosperms have not yet been established. Therefore, this study identified and compared the expression patterns of conserved miRNAs from two stages of the male gametophyte of loblolly pine (Pinus taeda), which are the mature (ungerminated) and germinated pollen. Microarray was used to identify conserved miRNAs that varied in expression between these two stages of the loblolly pine male gametophyte. Forty-seven conserved miRNAs showed significantly different expression levels between mature and germinated loblolly pine pollen. In particular, miRNAs representing 14 and 8 families were up- and down-regulated in germinated loblolly pine pollen, respectively. qRT-PCR was used to validate their expression patterns using representative miRNAs. Target genes and proteins were identified using psRNATarget program. Predicted targets of the 22 miRNA families belong mostly to classes of genes involved in defense/stress response, metabolism, regulation, and signaling. qRT-PCR was also used to validate the expression patterns of representative target genes. This study shows that conserved miRNAs are expressed in mature and germinated loblolly pine pollen. Many of these miRNAs are differentially expressed, which indicates that the two stages of the male gametophyte examined are regulated at the miRNA level. This study also expands our knowledge of the male gametophytes of seed plants by providing insights on some similarities and differences in the types and expression patterns of conserved miRNAs between loblolly pine with those of rice and Arabidopsis.     

Transfer of chloroplast genomic DNA to mitochondrial genome occurred at least 300 MYA

With the completion of the first gymnosperm mitochondrial genome (mtDNA) from Cycas taitungensis and the availability of more mtDNA taxa in the past 5 years, we have conducted a systematic analysis of DNA transfer from chloroplast genomes (cpDNAs) to mtDNAs (mtpts) in 11 plants, including 2 algae, 1 liverwort, 1 moss, 1 gymnosperm, 3 monocots, and 3 eudicots. By using shared gene order and boundaries between different mtpts as the criterion, the timing of cpDNA transfer during plant evolution was estimated from the phylogenetic tree reconstructed independently from concatenated protein-coding genes of 11 available mtDNAs. Several interesting findings emerged. First, frequent DNA transfer from cpDNA to mtDNA occurred at least as far back as the common ancestor of extant gymnosperms and angiosperms, about 300 MYA. The oldest mtpt is trnV(uac)-trnM(cau)-atpE-atpB-rbcL. Three other mtpts--psaA-psaB, rps19-trnH(gug)-rpl2-rpl23, and psbE-psbF--were dated to the common ancestor of extant angiosperms, at least 150 MYA. However, all protein-coding genes of mtpts have degenerated since their first transfer. Therefore, mtpts contribute nothing to the functioning of mtDNA but junk sequences. We discovered that the cpDNA transfers have occurred randomly at any positions of the cpDNAs. We provide strong evidence that the cp-derived tRNA-trnM(cau) is the only mtpt (1 out of 3 cp-derived tRNA shared by seed plants) truly transferred from cpDNA to mtDNA since the time of the common ancestor of extant gymnosperms and angiosperms. Our observations support the proposition of Richly and Leister (2004) that "primary insertions of organellar DNAs are large and then diverge and fragment over evolutionary time."     

湖北星斗山国家级自然保护区种子植物区系研究

于2013-2015年对湖北星斗山国家级自然保护区进行多次全面野外调查,采集植物标本3441号,并对其种子植物区系特点进行了研究。结果显示,该保护区共有野生种子植物138科678属1617种,其中裸子植物6科15属19种,被子植物132科663属1598种。区系分析表明,热带科(66.06%)多于温带科(33.94%),这与该区曾经历过古热带的历史背景相吻合;属级水平上则为温带属性(58.45%)强于热带(41.55%);中国特有种623个(占总种数的38.53%),均与四周区域所共有,反映出东西南北植物区系在此交汇渗透的态势;这些均表明该区种子植物区系成分不但复杂多样,而且还具有过渡区域的特点;保护区内植物区系还具有古老、残遗的特性,一些古老的残遗植物在该地有良好的原生群落分布;保护区内特有树种和珍稀濒危植物也很丰富,具有很大的保护价值。     

Structure and Evolution of the Actin Gene Family in Arabidopsis Thaliana

Higher plants contain families of actin-encoding genes that are divergent and differentially expressed. Progress in understanding the functions and evolution of plant actins has been hindered by the large size of the actin gene families. In this study, we characterized the structure and evolution of the actin gene family in Arabidopsis thaliana. DNA blot analyses with gene-specific probes suggested that all 10 of the Arabidopsis actin gene family members have been isolated and established that Arabidopsis has a much simpler actin gene family than other plants that have been examined. Phylogenetic analyses suggested that the Arabidopsis gene family contains at least two ancient classes of genes that diverged early in land plant evolution and may have separated vegetative from reproductive actins. Subsequent divergence produced a total of six distinct subclasses of actin, and five showed a distinct pattern of tissue specific expression. The concordance of expression patterns with the phylogenetic structure is discussed. These subclasses appear to be evolving independently, as no evidence of gene conversion was found. The Arabidopsis actin proteins have an unusually large number of nonconservative amino acid substitutions, which mapped to the surface of the actin molecule, and should effect protein-protein interactions.     

太行山脉东坡中南段种子植物区系初步分析

太行山脉东坡中南段分布有种子植物106科、464属、942种。对该地区种子植物区系进行了统计分析,结果表明:(1)本地区种子植物种类不丰富,有裸子植物2科, 4属, 5种;单子叶植物16科, 88属, 173种;双子叶植物88科, 372属, 764种。(2)属的地理分布类型多样,有15个分布类型及12个变型。其中温带成分占73.25%,具有明显的优势。热带成分占23.75%。(3)古老、残遗、原始成分较多,反映该地区植物区系具有一定的古老性。(4)有中国种子植物特有属12属,国家级保护植物11种。分析认为该地区是华北植物区系的重要组成部分。     

Distinct developmental mechanisms reflect the independent origins of leaves in vascular plants

Vascular plants diverged more than 400 million years ago into two lineages, the lycophytes and the euphyllophytes . Leaf-like organs evolved independently in these two groups . Microphylls in lycophytes are hypothesized to have originated as lateral outgrowths of tissue that later became vascularized (the enation theory) or through the sterilization of sporangia (the sterilization hypothesis) . Megaphylls in euphyllophytes are thought to represent modified lateral branches . The fossil record also indicates that the seed plant megaphyll evolved uniquely in the ancestor of seed plants, independent of megaphylls in ferns, because seed plants evolved from leafless progymnosperm ancestors . Surprisingly, a recent study of KNOX and ARP gene expression in a lycophyte was reported to indicate recruitment of a similar mechanism for determinacy in both types of leaves . We examined the expression of Class III HD-Zip genes in the lycophyte Selaginella kraussiana and in two gymnosperms, Ginkgo and Pseudotsuga. Our data indicate that mechanisms promoting leaf initiation, vascularization, and polarity are quite different in lycophytes and seed plants, consistent with the hypotheses that megaphylls originated as lateral branches whereas microphylls originated as tissue outgrowths.     

秦岭南坡中段种子植物区系初步分析

秦岭南坡中段有种子植物152科,815属,2184种,对该地区植物区系进行了统计分析,结果表明,（1）本地区种子植物种类丰富,共有裸子植物5科,15属,30种,单子叶植物23科,169属,390种;双子叶植物124科,631属,1764种。（2）属的地理分布类型种类多,有15个分布类型及19个变型,其中温带成分占64.17%,具一定的优势。但同时表现亚热带成分向温带成分过渡的特点。（3）本地区植物古老、残遗、原始成分多,反映了该地区植物区系古老的性质。（4）有中国种子植物特有属41属。国家级保护植物32种,分析结果认为本地区是秦岭种子植物区系的重要组成部分。