Encephalartos Natalensis Research Articles

Encephalartos natalensis, Their Nutrient-Cycling Microbes and Enzymes: A Story of Successful Trade-Offs.

Encephalartos spp. establish symbioses with nitrogen (N)-fixing bacteria that contribute to soil nutrition and improve plant growth. Despite the Encephalartos mutualistic symbioses with N-fixing bacteria, the identity of other bacteria and their contribution to soil fertility and ecosystem functioning is not well understood. Due to Encephalartos spp. being threatened in the wild, this limited information presents a challenge in developing comprehensive conservation and management strategies for these cycad species. Therefore, this study identified the nutrient-cycling bacteria in Encephalartos natalensis coralloid roots, rhizosphere, and non-rhizosphere soils. Additionally, the soil characteristics and soil enzyme activities of the rhizosphere and non-rhizosphere soils were assayed. The coralloid roots, rhizosphere, and non-rhizosphere soils of E. natalensis were collected from a population of >500 E. natalensis in a disturbed savanna woodland at Edendale in KwaZulu-Natal (South Africa) for nutrient analysis, bacterial identification, and enzyme activity assays. Nutrient-cycling bacteria such as Lysinibacillus xylanilyticus; Paraburkholderia sabiae, and Novosphingobium barchaimii were identified in the coralloid roots, rhizosphere, and non-rhizosphere soils of E. natalensis. Phosphorus (P) cycling (alkaline and acid phosphatase) and N cycling (β-(D)-Glucosaminidase and nitrate reductase) enzyme activities showed a positive correlation with soil extractable P and total N concentrations in the rhizosphere and non-rhizosphere soils of E. natalensis. The positive correlation between soil enzymes and soil nutrients demonstrates that the identified nutrient-cycling bacteria in E. natalensis coralloid roots, rhizosphere, and non-rhizosphere soils and associated enzymes assayed may contribute to soil nutrient bioavailability of E. natalensis plants growing in acidic and nutrient-poor savanna woodland ecosystems.

Evidence for an ancient whole genome duplication in the cycad lineage.

Contrary to the many whole genome duplication events recorded for angiosperms (flowering plants), whole genome duplications in gymnosperms (non-flowering seed plants) seem to be much rarer. Although ancient whole genome duplications have been reported for most gymnosperm lineages as well, some are still contested and need to be confirmed. For instance, data for ginkgo, but particularly cycads have remained inconclusive so far, likely due to the quality of the data available and flaws in the analysis. We extracted and sequenced RNA from both the cycad Encephalartos natalensis and Ginkgo biloba. This was followed by transcriptome assembly, after which these data were used to build paralog age distributions. Based on these distributions, we identified remnants of an ancient whole genome duplication in both cycads and ginkgo. The most parsimonious explanation would be that this whole genome duplication event was shared between both species and had occurred prior to their divergence, about 300 million years ago.

Exposing the illegal trade in cycad species (Cycadophyta: Encephalartos) at two traditional medicine markets in South Africa using DNA barcoding.

Species in the cycad genus Encephalartos are listed in CITES Appendix I and as Threatened or Protected Species in terms of South Africa's National Environmental Management: Biodiversity Act (NEM:BA) of 2004. Despite regulations, illegal plant harvesting for medicinal trade has continued in South Africa and resulted in declines in cycad populations and even complete loss of sub-populations. Encephalartos is traded at traditional medicine markets in South Africa in the form of bark strips and stem sections; thus, determining the species traded presents a major challenge due to a lack of characteristic plant parts. Here, a case study is presented on the use of DNA barcoding to identify cycads sold at the Faraday and Warwick traditional medicine markets in Johannesburg and Durban, respectively. Market samples were sequenced for the core DNA barcodes (rbcLa and matK) as well as two additional regions: nrITS and trnH-psbA. The barcoding database for cycads at the University of Johannesburg was utilized to assign query samples to known species. Three approaches were followed: tree-based, similarity-based, and character-based (BRONX) methods. Market samples identified were Encephalartos ferox (Near Threatened), Encephalartos lebomboensis (Endangered), Encephalartos natalensis (Near Threatened), Encephalartos senticosus (Vulnerable), and Encephalartos villosus (Least Concern). Results from this study are crucial for making appropriate assessments and decisions on how to manage these markets.

Embryo cell wall properties in relation to development and desiccation in the recalcitrant-seeded Encephalartos natalensis (Zamiaceae) Dyer and Verdoorn.

Plant cell walls are dynamic entities that may change with development, differ between plant species and tissue type and play an important role in responses to various stresses. In this regard, the present investigation employed immunocytochemistry to determine wall composition and possible changes during development of immature and mature embryos of the recalcitrant-seeded cycad Encephalartos natalensis. Fluorescent and gold markers, together with cryo-scanning and transmission electron microscopy (TEM) were also used to analyse potential changes in the cell walls of mature embryos upon desiccation. Immature cell walls were characterised by low- and high methyl-esterified epitopes of pectin, rhamnogalacturonan-associated arabinan, and the hemicellulose xyloglucan. Arabinogalactan protein recognised by the LM2 antibody, along with rhamnogalacturonan-associated galactan and the hemicellulose xylan, were not positively localised using immunological probes, suggesting that the cell walls of the embryo of E. natalensis do not possess these epitopes. Interestingly, mature embryos appeared to be identical to immature ones with respect to the cell wall components investigated, implying that these may not change during the protracted post-shedding embryogenesis of this species. Drying appeared to induce some degree of cell wall folding in mature embryos, although this was limited by the abundant amyloplasts, which filled the cytomatrical space. Folding, however, was correlated with relatively high levels of wall plasticisers typified by arabinose polymers. From the results of this study, it is proposed that the embryo cell walls of E. natalensis are constitutively prepared for the flexibility required during cell growth and expansion, which may also facilitate the moderate cell wall folding observed in mature embryos upon drying. This, together with the abundant occurrence of amyloplasts in the cytomatrix, may provide sufficient mechanical stabilisation if water is lost, even though the seeds of this species are highly desiccation-sensitive.

From fragments to figures: Estimating the number of Encephalartos stems in a muthi market

There is an active trade in South African Encephalartos species in traditional medicine (muthi) outlets throughout the country. No attempt has been made to date to estimate the number of individual stems damaged by harvesters supplying stem fragments (including bark strips) to the markets. To progress from stem fragments to stem figures, a plausible technique of enumerating the number of stems from which the fragments were derived is proposed. The method considered the physical condition, post-harvest age and stem diameter of fragments identified to species level. From the samples of 133 cycad fragments purchased in the Johannesburg and Durban muthi markets in 2009, it was estimated that they originated from 81 different damaged stems (66% of which were likely to have been from Encephalartos natalensis). This estimate is a significant advance in quantifying this hitherto unknown aspect of the cycad trade.

Development of cycad ovules and seeds. 2. Histological and ultrastructural aspects of ontogeny of the embryo in Encephalartos natalensis (Zamiaceae)

Development of the embryo of Encephalartos natalensis from a rudimentary meristematic structure approximately 700 μm in length extends over 6 months after the seed is shed from the strobilus. Throughout its development, the embryo remains attached to a long suspensor. Differentiation of the shoot meristem flanked by two cotyledonary protuberances occurs over the first 2 months, during which peripheral tannin channels become apparent. Tannins, apparently elaborated by the endoplasmic reticulum, first accumulate in the large central vacuole and ultimately fill the channel. By the fourth month of development, the root meristem is apparent and procambial tissue forming discrete vascular bundles can be discerned in the elongating cotyledons. Between 4 and 6 months, mucilage ducts differentiate; after 6 months, when the seed becomes germinable, the embryo is characterised by cotyledons far longer than the axis. Shoot and root meristem cells remain ultrastructurally similar throughout embryo ontogeny, containing small vacuoles, many well-differentiated mitochondria and endoplasmic reticulum (ER) profiles, abundant polysomes, plastids containing small starch deposits and Golgi bodies. Unusually, however, Golgi bodies are infrequent in other cells including those elaborating mucilage which is accumulated in distended ER and apparently secreted into the duct lumen directly by ER-derived vesicles. The non-meristematic cells accumulate massive starch deposits to the exclusion of any protein bodies and only very sparse lipid, features which are considered in terms of the prolonged period of embryo development and the high atmospheric oxygen content of the Carboniferous Period, when cycads are suggested to have originated.

Uncovering the cycad taxa ( Encephalartos species) traded for traditional medicine in Johannesburg and Durban, South Africa

The bark and stems of Encephalartos species are used for traditional medicine across South Africa, and some species are traded in traditional medicine markets. The absence of characteristic plant parts such as leaves and cones in the chopped up market material presents a major challenge to the identification of the species traded. In this study, South Africa's two largest traditional medicine markets, Faraday in Johannesburg and Warwick in Durban, were surveyed to ascertain the source areas and species of Encephalartos in trade. Samples of stem fragments were purchased from vendors, identified to probable species and their stem diameter size class distributions determined using a specially designed size class chart. Species identification was undertaken by comparing trader citations of harvesting areas with the distributions of Encephalartos species in the province of KwaZulu-Natal (since all the specimens originated from KZN). The species most commonly recorded in the markets were identified as Encephalartos natalensis, E. villosus and E. ghellinckii; small quantities of what are likely to be E. ferox and E. senticosus were also observed. Following this study, the total number of South Africa's 37 Encephalartos species recorded as being used for traditional medicine is 25. Stem diameter size class distributions showed that most stem fragments came from sub-adult and adult cycads. Large arborescent species appear to be harvested by removing bark strips from adult individuals, while smaller arborescent and subterranean species are harvested by removing the entire plant. Regression relationships between stem tissue type and stem diameter for E. natalensis and E. ghellinckii indicate a strong, positive linear relationship of leaf base length and pith radius with stem diameter. Overall, this is the first known study that attempts to identify the cycad species traded in South African traditional medicine markets in conjunction with the size classes of the specimens in trade.

Scent chemistry and patterns of thermogenesis in male and female cones of the African cycad Encephalartos natalensis (Zamiaceae)

Most, if not all, extant cycads are pollinated by insects which use the cones as larval brood sites. These interactions appear to be mediated by cone volatiles, and, in some species, by patterns of thermogenesis. We investigated the chemical composition of volatile emissions and patterns of thermogenesis in cones of the South African cycad Encephalartos natalensis, using a gas chromatograph–mass spectrometer (GC–MS) and miniature temperature data loggers (ibuttons), respectively. This was done during various developmental stages (before and during receptivity and pollen release) for both female and male cones. A total of 31 compounds were identified in headspace samples; 17 of which were common to both sexes, 12 found only in male cones, and two found only in female cones. The major volatiles in pollen and female cones are (3 E)-1,3-octadiene (averaging 54.25% and 15.82% of total emissions), (3 E,5 Z)-1,3,5-octatriene (averaging 13.37% and 47.66%), and α-pinene (averaging 16.29% and 12.24%). Female cones were not thermogenic before and during receptivity whereas pollen cones were thermogenic during pollen shedding. Thermogenesis of male cones occurred between 1400 h and 1530 h on successive afternoons, reaching an average of c. 10.5 °C above ambient temperature. Volatile emissions and thermogenesis occurred in association with insect activity on the cones suggesting that they both play a role in regulating insect behaviour.

Development of cycad ovules and seeds. 1. Implication of the ER in primary cellularisation of the megagametophyte in Encephalartos natalensis Dyer and Verdoorn

Very little is known about the pre- and post-shedding megagametophyte development and utilisation of accumulated reserves, respectively, in cycads (Zamiaceae). In the present study on developing ovules of the recalcitrant-seeded species, Encephalartos natalensis, cells of the megagametophyte were found to become progressively packed with starch and protein as the two main storage reserves, a limited number of discrete lipid bodies, and occasional mitochondria all of which appeared to be embedded in a homogeneous matrix. ER-derived vesicles (and not Golgi-derived vesicles) appeared to be the principal contributors of the primary cell wall components, pectin and xylan, during megagametophyte cellularisation. This was confirmed by the use of enzyme-gold localisation. High-pressure freezing (HPF) and freeze substitution (FS) of samples the following season showed that while the apparently featureless cytomatrix of the megaspore was an artefact of conventional fixation, there was still an insignificant occurrence of Golgi bodies during primary wall formation. When enzyme-gold localisation was employed on the HPF-FS material, label for pectin and xylan was found only in the regions of ER and vesicles and not in any of the few Golgi bodies or their associated vesicles. Immunocytochemistry revealed that pectin and xylan were restricted to the ER and not to any vesicles or to the occasional Golgi body that was found. This suggests that the ER exclusively, is involved in the deposition of these primary cell wall components during the cellularisation of the E. natalensis megagametophyte. While cellularisation took place over approximately 1–2 weeks, subsequent development of the megagametophyte cells involved the accumulation of storage reserves, this phase lasting approximately 8 months—after which the seeds were shed whether pollination/fertilisation had recently occurred, or not.

The genetic relationship between Encephalartos natalensis and E. woodii determined using RAPD fingerprinting

Abstract The second largest cycad genus Encephalartos, consists of 50 living species all endemic to Africa. Encephalartos natalensis, a common variety found in KwaZulu-Natal is similar in morphology to E. woodii of which only a single clump has been found in the wild. This enigmatic species is represented by a single male plant. Although the phylogenetic relationship between E. natalensis and E. woodii is unknown, the ability of these species to hybridise suggests a close relationship. The aim of this study was to determine the genetic variability between E. natalensis and E. woodii. Genetic fingerprints were generated using the RAPD technique and data analysed using distance methods. Based on RAPD fingerprints, the intraspecific genetic variation among different E. natalensis plants is similar to the interspecific variation between E. natalensis and E. woodii. These data confirm the close relationship between E. natalensis and E. woodii.

Sex identification in Encephalartos natalensis (Dyer and Verdoorn) using RAPD markers

All cycads are strictly dioecious with a long juvenile stage. Currently, there is no method available to determine the sexuality of seedlings prior to the onset of cone formation. This study aimed to develop a sex specific Random Amplified Polymorphic DNA (RAPD) marker for Encephalartos natalensis. Initially, 140 primers were used to amplify the bulk DNA of five individuals each of known male and female sexuality. While a high degree of polymorphism was observed in the amplification profiles of male and female plants, only primer OPD-20 generated a specific band (∼850 bp) in female DNA. To validate this observation, this primer was re-tested with 69 individuals of E. natalensis. The 850 bp DNA band was present in all 38 female individuals tested and it was consistently absent in all 31 male plants tested. The result offers a rapid and simple test to determine sexuality of E. natalensis seedlings at early stages of development, before the onset of reproductive maturity thereby saving time and economic resources when cultivating these specimens.

A Comparison of the Endogenous Cytokinins in the Leaves of Four Gymnosperms

Compounds that are active in the soybean bioassay and which cochromatograph with Z and its derivatives were detected in the mature leaves of Ginkgo biloba, Podocar pus henkelii, Welwitschia mirabilis, and Encephalartos natalensis. While 2iP was not found in the leaves of the first two species, it appears to be present in the latter two plants.