A phylogeny of cycads (Cycadales) inferred from chloroplast matK gene, trnK intron, and nuclear rDNA ITS region

Phylogenetic relationships among the three families and 12 living genera of cycads were reconstructed by distance and parsimony criteria using three markers: the chloroplast matK gene, the chloroplast trnK intron and the nuclear ITS/5.8S rDNA sequence. All datasets indicate that Cycadaceae (including only the genus Cycas) is remotely related to other cycads, in which Dioon was resolved as the basal-most clade, followed by Bowenia and a clade containing the remaining nine genera. Encephalartos and Lepidozamia are closer to each other than to Macrozamia. The African genus Stangeria is embedded within the New World subfamily Zamiodeae. Therefore, Bowenia is an unlikely sister to Stangeria, contrary to the view that they form the Stangeriaceae. The generic status of Dyerocycas and Chigua is unsupportable as they are paraphyletic with Cycas and the Zamia, respectively. Nonsense mutations in the matK gene and indels in the other two datasets lend evidence to reinforce the above conclusions. According to the phylogenies, the past geography of the genera of cycads and the evolution of character states are hypothesized and discussed. Within the suborder Zamiieae, Stangeria, and the tribe Zamieae evolved significantly faster than other genera. The matK gene and ITS/5.8S region contain more useful information than the trnK intron in addressing phylogeny. Redelimitations of Zamiaceae, Stangeriaceae, subfamily Encephalartoideae and subtribe Macrozamiineae are necessary.     

Comparative analysis of cyanobacteria in the rhizosphere and as endosymbionts of cycads in drought-affected soils

Does the diversity of cyanobacteria in the cycad rhizosphere relate to the cyanobiont species found in the coralloid roots of these ancient plants? The aim of this study was to identify the diversity of soil cyanobacteria occurring in the immediate vicinity of 22 colonized coralloid roots belonging to members of the cycad genera: Macrozamia, Lepidozamia, Bowenia and Cycas. The majority of coralloid roots were sampled at depths >?10?cm below the soil surface. A total of 32 cyanobacterial isolates were cultured and their 16S rRNA gene partially sequenced. Phylogenetic analysis revealed nine operational taxonomic units of soil cyanobacteria comprising 30 Nostoc spp., a Tolypothrix sp. and a Leptolyngbya sp. Microscopy indicated that all isolates were unialgal and confirmed their genus identity. Rhizospheric diversity was compared to existing data on cyanobionts isolated at the same time from the cycad coralloid root. The same isolate was present in both the cycad coralloid root and rhizosphere at only six sites. Phylogenetic evidence indicates that most rhizosphere isolates were distinct from root cyanobionts. This weak relationship between the soil cyanobacteria and cycad cyanobionts might indicate that changes in the soil community composition are due to environmental factors.     

泽米科植物羽片脉序和解剖学及其系统学意义

研究了苏铁目泽米科Zamiaceae 2亚科的所有4族(Stevenson系统, 1992)共10种代表植物的羽片脉序及解剖学特征,结果显示泽米科羽片脉序为二歧分叉的平行脉,无中脉。小刺双子铁Dioon spinulosum、大头非洲铁Encephalartos friderici-guilielmii和摩尔大泽米Macrozamia moorei等的平行脉末端以不同的形式互相连接,而鳞木铁Lepidozamia peroffskyana、粗壮角果铁Ceratozamia mexicana var. robusta、竹叶角果铁C. hildae、佛州泽米Zamia　floridana、柔叶泽米Z. debilis、鳞秕泽米Z. furfuracea和短尖泽米Z. muricata等的平行脉末端不连接而直达叶缘,其中鳞木铁、粗壮角果铁和竹叶角果铁的脉达叶缘后逐渐消失。羽片的横切面结构通常由表皮、下皮厚壁细胞和叶肉组成,表皮层包括上、下表皮各一层,叶肉可能同时分化出近上表面的栅栏组织和近下表面的栅栏组织,或仅有近上表面的栅栏组织分化,或无栅栏组织分化而完全为海绵组织。然而,泽米科没有典型的海绵组织和传输组织分化。小刺双子铁、大头非洲铁、鳞叶木铁和摩尔大泽米的羽片具有粘液道而无工字厚壁组织,在小刺双子铁中粘液道与维管束对生,在另3种中则与维管束轮生;但粗壮角果铁、竹叶角果铁、佛州泽米、柔叶泽米、鳞秕泽米和短尖泽米的羽片则具有工字厚壁组织而没有粘液道,其中粗壮角果铁和竹叶角果铁的羽片工字厚壁组织仅与上表皮相连,而佛州泽米、柔叶泽米、鳞秕泽米和短尖泽米的羽片工字厚壁组织与上、下表皮都相连。羽片脉序和解剖学特征支持Stevenson将泽米铁科分为两亚科的观点。     

OBSERVATIONS ON PTYXIS,PHENOLOGY, AND TRICHOMES IN THE CYCADALES AND THEIR SYSTEMATIC IMPLICATIONS

Ptyxis, phenology, and leaf trichomes are described for 43 species representing all ten genera in the Cycadales. The typical annual growth sequence is: leaf flush production, cataphyll production, reproductive production, and finally cataphyll production in all taxa except Stangeria which does not have cataphylls and produces leaves one at a time throughout the year. The leaf and cataphyll bases are slightly winged except in Zamia and Ceratozamia, which have well developed stipules, and in Stangeria, which has a distinctive adaxial, stipular hood on the leaf bases. Longitudinal ptyxis of the whole leaf is of four types: circinate (only in Bowenia); erect (Cycas, Dioon, Encephalartos, Lepidozamia, Macrozamia, Microcycas, and some Zamia spp.); inflexed (Stangeria, Ceratozamia, and some Zamia spp.); and reflexed (rarely found in Cycas and Dioon). The pinnae are oriented so that the horizontal ptyxis is conduplicate in all taxa except Bowenia and Cycas where it is involute. The individual pinnae are circinate in Bowenia and Cycas, conduplicate in Stangeria, and flat in all other taxa. The pinnules of Bowenia are also flat. Leaf trichomes are of six types: transparent unbranched; transparent branched; colored unbranched; colored branched; colored idioblastic; and short colored curved. Cycas has only transparent branched (unequally) and unbranched. Ceratozamia, Dioon, Encephalartos, and Stangeria have transparent and colored trichomes, both unbranched. Bowenia, Lepidozamia and Macrozamia have short colored curved hairs and transparent unbranched hairs. Macrozamia is the only taxon with colored idioblastic trichomes. Zamia and Microcycas have transparent and colored hairs. Both trichome types occur branched and unbranched. Because of its decompound leaf, circinate ptyxis, cones on short determinate branches and other distinct characters the family Boweniaceae D. Stevenson fam. nov. is described. This family contains one genus: Bowenia.     

A biotechnology strategy for medium- and long-term conservation of cycads

It has been possible to regenerate a few cycad species in vitro by somatic embryogenesis, either from zygotic embryos (Ceratozamia hildae, C. mexicana, Encephalartos cycadifolius,E. dyerianus, E. natalensis, Zamia fischeri, Z. furfuracea, andZ. pumila) or from leaves of mature phase trees (C. euryphyllidia, Ceratozamia hildae, andC. mexicana). This strategy has great potential for the commercial vegetative propagation of certain highly endangered species (e.g.,C. euryphyllidia) and should indirectly protect wild populations of these species by discouraging collection in situ. Embryogenic cultures of several cycad species have grown vigorously and are highly morphogenic more than 11 years after induction. The long-term conservation of cycad genetic resources can also be addressed for species that can be regenerated by somatic embryogenesis. Preliminary studies indicate that embryogenic cultures that have been pretreated on plant growth medium containing 0.75 M sucrose for two days, encapsulated in sodium alginate, and desiccated for six hours can survive immersion in liquid nitrogen (−196°C).     

Comparison of DNA restriction fragment length polymorphisms of Nostoc strains in and from cycads

DNA was prepared from cyanobacteria freshly isolated from coralloid roots of natural populations of five cycad species: Ceratozamia mexicana mexicana (Mexico), C. mexicana robusta (Mexico), Dioon spinulosum (Mexico), Zamia furfuraceae (Mexico) and Z. skinneri (Costa Rica). Using the Southern blot technique and cloned Anabaena PCC 7120 nifK and glnA genes as probes, restriction fragment length polymorphisms of these cyanobacterial symbionts were compared. The five cyanobacterial preparations showed differences in the sizes of their DNA fragments hybridizing with both probes, indicating that different cyanobacterial species and/or strains were in the symbiotic associations. On the other hand, a similar comparison of cyanobacteria freshly collected from a single Encephalartos altensteinii coralloid root and from three independently subcultured isolates from the same coralloid root revealed that these were likely to be one and the same organism. Moreover, the complexity of restriction patterns shows that a mixture of Nostoc strains can associate with a single cycad species although a single cyanobacterial strain can predominate in the root of a single cycad plant. Thus, a wide range of Nostoc strains appear to associate with the coralloid roots of cycads.Non-standard abbreviations bp base pairs - kbp kilobase pairs - RFLP's restriction fragment length polymorphisms     

A phylogenetic analysis of American Zamiaceae (Cycadales) using chloroplast DNA restriction fragment length polymorphisms

A phylogenetic analysis of American cycad genera, all belonging to the family Zamiaceae, was attempted using chloroplast DNA restriction fragment polymorphisms.Ceratozamia mexicana Brongn.,Chigua restrepoi D. Stevenson,Dioon edule Lindley,Microcycas calocoma (Miq.) A. DC.,Zamia fischeri Miq., andZamia skinneri Warsz. ex A. Dietrich were used as representatives of the genera.Cycas revoluta Thunb., belonging to the family Cycadaceae, was used as an outgroup, following previous morphological works. One hundred and forty-one shared restriction fragments were scored for presence/absence and both Wagner and Dollo parsimony analyses were performed. The single, fully resolved, most parsimonious trees obtained from the analyses were topologically identical and perfectly matched previous morphology-based phylogenetic hypotheses. Statistical evaluation of the data showed a good reliability for the obtained phylogeny.Dioon edule, belonging to a different subfamily and more primitive on morphological grounds, proved to be the most primitive among American cycads as inferred from the molecular data;Chigua restrepoi, never analyzed before on cladistic grounds, was found to be the sister group of the genusZamia.     

泽米铁科三种植物的叶表皮特征

在光镜下观察了泽米铁科分属于3个属的3种植物叶表皮特征,结果表明：3种植物叶表皮细胞及气孔器差异显著,一定程度上证实了3属是自然的分类群。在鳞秕泽米铁(Zamia furfuracea)中发现了气孔簇。本文还对3种植物之间的系统演化关系及它们对环境的适应性进行了讨论。     

Formation of Phenolic Compounds in Apogeotropic Roots of Cycad Plants

Formation and location of phenolic compounds in apogeotropic roots (coralloid roots) were studied in six cycad species, which belong to the genera Cycas, Encephalartos, and Ceratozamia. Total contents of soluble phenolic compounds in coralloid roots in all species studied varied insignificantly, with except for Ceratozamia mexicana that accumulated three times higher amounts of phenolic compounds. Phenolic compounds were accumulated in cell walls of cortical parenchyma of coralloid roots, in intercellular spaces, and in specialized storage cells, found in all zones of apogeotropic roots. The greatest number of phenol-storing cells was situated in the cortical parenchyma of the central part of coralloid roots, adjacent to a zone where active symbiotic cyanobacteria were localized, and in the coralloid root basal region lacking viable forms of cyanobionts. It was suggested that phenolic compounds affect the formation of symbiosis between cyanobacteria and apogeotropic roots of cycad plants, as well as their metabolism.     

Occurrence of azoxyglycosides in Macrozamia riedlei and their effects on the free-living isolated Nostoc PCC 73102

The presence of azoxyglycosides in the Australian cycad Macrozamia reidlei was examined using high performance liquid chromatography. Cycasin and macrozamia were present in all tissues examined, cycasin being three to 17 times more abundant than macrozamin. The symbiotic organ, the coralloid root, contained 0.16% [g/g (fresh weight)] cycasin and 0.01 % (same unit) macrozamin. Addition of these azoxyglycoside concentrations to nitrogen-fixing Nostoc PCC 73102 cultures, a filamentous heterocystous cyanobacterium originally isolated from Macrozia, inhibited light and dark nitrogenase (acetylene reduction) activity. No effects were observed on in vitro glutamine synthetase activity or net in vivo CO₂ fixation. Cycasin (1.6%) and macrozamin (0.1%), i.e. 10 times the concentrations observed in the coralloid root, decreased phycobiliprotein content by 25 and 45%, 1 and 4 hr after the addition, respectively. The relative distribution of individual phycobiliproteins was not affected.     

八种苏铁植物叶轴的比较解剖研究

对苏铁科和泽米铁科8种苏铁植物,即台湾苏铁(Cycas taiwaniana)、海南苏铁(C·hainanensis)、元江苏铁(C·parvulus)、单羽苏铁(C·simplicipinna)、滇南苏铁(C·diannanensis)、越南篦齿苏铁(C·elon-gata)、长刺大泽米铁(Macrozamia longispina)、双子铁(Dioon edule)的叶轴横切面结构进行了比较研究,以期为苏铁植物的系统演化和分类进一步提供解剖学依据,并探讨其解剖结构与生态的适应机制。结果表明,较原始的苏铁科和较进化的泽米铁科叶轴的横切面结构无论在表皮、机械组织、基本组织和维管组织,还是在后含物方面都存在着明显的不同;泽米铁科的大泽米铁属与双子铁属差异也较大;苏铁科科内的6个种则相似性稍大,表明它们是亲缘关系较密切的类群,但每个种都有各自的结构特点。越南篦齿苏铁的维管束排列方式和数目的增加方式与泽米铁科两个种的相似,因此推断它处于较进化的地位。研究还发现苏铁科种类的叶轴的近轴面均像叶片那样具有栅栏组织状同化组织存在。苏铁类植物叶轴具有旱生植物的解剖结构特征:角质层较厚、表皮细胞壁厚、机械组织发达、晶体较多、分泌道和维管束数目较多等结构特征。这些对研究苏铁纲各科及种类之间的进化与亲缘关系提供了佐证,同时揭示了苏铁纲这类古老的植物类群其多个属的种类能够经历如此漫长的地质年代而幸存下来,是由于其在长期演化过程中均形成了这些独特的结构特征,因而具备了相对应的生理功能,能够在恶劣的自然环境下,具备耐干旱、贫瘠、高温及耐盐碱等逆境的结果。     

RADIAL GROWTH IN THE CYCADALES

The development of radial growth which leads to the pachycaulous form was investigated in eight of the 10 genera of the Cycadales; i.e., Ceratozamia, Cycas, Dioon, Encephalartos, Macrozamia, Microcycas, Stangeria, and Zamia. In all taxa, development of radial growth is essentially the same: a primary thickening meristem is differentiated in the stelar region of the cotyledonary node of the seedling at germination and produces derivatives mainly centrifugally. This primary thickening meristem (PTM) then differentiates acropetally and becomes continuous with the peripheral zone of the shoot apex. At first the PTM is a vertical cylinder, but as the seedling continues to grow into an adult plant, the PTM shows a more horizontal orientation (like an open umbrella) and produces the broad cortex. Secondary growth is by a vascular cambium which produces secondary xylem to the inside and secondary phloem to the outside. The broad pith originates from derivatives of the rib meristem of the massive shoot apex. The seedling and young plant is composed of a shortened shoot (i.e., no internodes) produced by the PTM and rib meristem, and a large fleshy primary root which results from a diffuse growth pattern. Individual cells in both the pith and cortex of the root divide. Their derivatives divide at right angles to the original division plane. Thus, quartets and even octets of cells are recognizable and can be traced to individual parent cells.     

Oscillatory Heat-Production in the Male Cones of Cycads

Microcalorimetric analysis of heat-production by single intactsporophylls from thermogenic male cones of two cycad species(Ceratozamia miqueliana H. Wendl. and Zamia furfuracea L. f.)showed an oscillatory behaviour for three consecutive days duringpollen maturation and shedding. In sporophylls from C. rumphiiMiq., a weakly thermogenic species with no maximum of heat-production,such oscillations were not detected. The presence of the alternativerespiratory pathway in the sporophylls of the male cones ofZamia pumila L. has been demonstrated. Key words: Alternative pathway, cycads, heat-production, microcalorimetry, oscillation     

ACC oxidase is found in seedlings of two (Coniferales, Gnetales) of the four gymnosperm orders

Leaf material and seedlings of species representing all gymnosperm orders were tested for 1-aminocyclopropane-1-carboxylate (ACC) oxidase activity. Seedlings of Pinus nigra , Pinus radiata , Pseudotsuga menziesii , Cupressocyparis leylandii , Ephedra major and E. nevadensis showed high level in vitro ACC oxidase activity. The enzyme from seedlings of Pinus nigra var. nigra (Arnold) was shown to resemble the angiosperm enzyme in a requirement for ascorbate, carbon dioxide and Fe(II). In contrast, seedlings of Ginkgo biloba , Dioon edule , Zamia furfuraceae and Cycas revoluta showed no detectable ACC oxidase activity. Leaf material from species representing all orders of gymnosperms was also tested for ACC oxidase activity in vitro, but none could be demonstrated. The results presented here support an origin of ACC oxidase in a common ancestor of the angiosperms, Gnetales and Coniferales.     

Beetle pollination of the fruit-scented cones of the South African cycad Stangeria eriopus

There has been considerable uncertainty about the importance of wind vs. insects in cycad pollination, but recent studies in several cycad genera have indicated that these are pollinated primarily, if not exclusively, by insects. Stangeria represents an isolated southern African cycad lineage previously thought to be wind-pollinated. Unlike in most other cycads, there is no evidence of cone thermogenesis in Stangeria. We found that the scent of both male and female Stangeria cones mimics that of fermented fruit, the main volatiles being esters of acetic acid, ketones, and aldehydes. We found a large variety of insect visitors on the cones, the most common ones being sap and rove beetles (Coleoptera: Nitidulidae, Staphylinidae) and fruit flies (Diptera: Drosophilidae). Of these, only sap beetles (Nitidulidae) were able to effect pollination under experimental conditions. Because sap beetles are also pollinators of Cycas and members of several ancient angiosperm families, their role in the pollination of Stangeria adds interesting details to the role this group of insects has played in the history of plant-pollinator interactions.     

Atypical Cell Forms Overproducing Extracellular Substances in Populations of Cycad Cyanobionts

The ultrastructure of the cyanobionts of the greenhouse-grown cycads Cycas circinalis, Ceratozamia mexicana, and Encephalartos villosus was studied. The cyanobiont microcolonies grown in the intercellular space of the cyanobacterial zone of cortical parenchyma in the cycad coralloid roots contained two specific forms of vegetative cells with a reduced cell wall, namely, protoplasts and spheroplasts. The protoplasts and spheroplasts exhibited ultrastructural properties indicating the overproduction of two extracellular substances, one of which resembled the mucilage polysaccharides and the other was protein-like. The substances were likely to be synthesized intracellularly and then be excreted with the aid of surface vesicles or by ruptures in the cytoplasmic membrane to form, respectively, a mucilagious extracellular matrix and an additional electron-opaque envelope around the cell. At the late developmental stages, the excretion of these substances was accompanied by degradative changes in the cells, leading eventually to cell death. The physiological role of these specific cell forms and the factors that induce their development and death in the cell populations of cyanobionts are discussed.     

Aboriginal preparation ofCycas seeds in Australia1

The seeds of cycad plants are a toxic food used by many Aboriginal groups in northern Australia. Acute symptoms produced after consumption of untreated Cycas seeds are due to azoxyglycosides, especially cycasin, although the toxic dose depends on the animal species tested. There are three traditional methods used to treat these seeds: brief leaching in water; prolonged leaching in water; and aging. Aboriginal people living at Donydji outstation in northeast Arnhem Land, most regularly consume aged seeds ofCycas angulata R.Br. Analyses of fresh seeds and seeds prepared at Donydji and in the laboratory indicate that cycasin is effectively removed by all the traditional preparation techniques, although each technique has an end product with different storage and handling properties. The social implications of processing need further elaboration, but these techniques have a long history and archaeological remains of seeds in Australia may date back to the Pleistocene.     

Atypical cell forms overproducing extracellular substances in population of cycad cyanobionts

The ultrastructure of the cyanobionts of the greenhouse-grown cycads Cycas circinalis, Ceratozamia mexicana, and Encephalartos villosus was studied. The cyanobiont microcolonies grown in the intercellular space of the cyanobacterial zone of cortical parenchyma in the cycad coralloid roots contained two specific forms of vegetative cells with a reduced cell wall, namely, protoplasts and spheroplasts. The protoplasts and spheroplasts exhibited ultrastructural changes indicating the overproduction of two extracellular substances, one of which resembled the mucilage polysaccharides and the other was proteinous. The substances were likely to be synthesized intracellularly and then be excreted with the aid of surface vesicles or by channels in the cytoplasmic membrane to form, respectively, a slimy extracellular matrix and an additional electron-opaque envelope around the cell. At the late developmental stages, the excretion of these substances was accompanied by degradative changes in the cells, leading eventually to cell death. The physiological role of these specific cell forms and the factors that induce their development and death in the cell populations of cyanobionts are discussed.     

A new species of Leptocycas(Zamiaceae)from the Upper Triassic sediments of Liaoning Province,China

A new cycad,Leptocycas yangcaogouensis sp.nov.,was found in sediments from the Late Triassic in western Liaoning,China.The pinnately compound leaves(Pseudoctenis type)are screwed in a crown on the stem top.The leaflets are linear,with parallel veins and decurrent bases on the rachis.The leaf bases are persistent.The cataphylls intermix with the leaves.The female cone is ovoid in shape.The characteristics of the new plant are more similar to those of Leptocycas gracilis,a Triassic cycad from North America,but the new species differs from L.gracilis in the size of its stem(7-8 vs.3-5 cm in diameter,respectively),leaves(length × width 100 × 16 vs.30 × 7 cm,respectively)and leaf density along the stem(4-6 vs.1-2 bases/1 cm length,respectively).Both L.gracilis and L.yangcaogouensis,having leaves of the Pseudoctenis type,show a closer relationship to the extant Dioon of Zamiaceae.The present study provides evidence for the origin of the genus Dioon,which may have come from Leptocycas plants of the Triassic.It would be assumed that the extent cycads in Zamiaceae originate from the pteridosperms in the Late Paleozoic and have evolved through the stage of L.gracilis and L.yangcaogouensis in Late Triassic,and reaching the extant Dioon.     

Studies on Cycasin,a New Toxic Glycoside,of Cycas revoluta Thunb

The enzymatic hydrolysis of cycasin with cycad-emulsin, prepared from the seeds of Cycas revoluta, is described. As the complete degradation products we obtained about one mole each of formaldehyde, nitrogen gas, and methanol per mole of cycasin, in addition to glucose, the sugar component. These products are the same as those found in the acid hydrolysis of cycasin which has been reported previously. Therefore, it is concluded that the aglycone of cycasin can not be liberated intact as a single component, but decomposes into those smaller molecules as above even by means of the enzymatic hydrolysis.