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Encephalartos eugene-maraisii is an endangered, endemic plant in the Waterberg Mountain range of South Africa. This is the first report of herbivory by Zerenopsis lepida (Lepidoptera: Geometridae) on natural populations of E. eugene-maraisii. The impact of herbivory by folivorous arthropods on this cycad species has not been studied before. This study aimed to determine the incidence and intensity of Z. lepida herbivory on E. eugenemaraisii under field conditions. Local plant dynamics were studied by recording the plant location, sex, size and phenological stage for each plant. Damage surveys were carried out over two consecutive seasons (2020–2021 and 2021–2022). Leaf damage was assessed on 227 plants comprising 692 stems. Approximately 40% of the stems had signs of leaf herbivory by Z. lepida and 10% of the total leaf area was removed by Z. lepida larvae. Less herbivory was recorded in areas where plants synchronously produced new leaf flushes compared to areas with asynchronous leaf flushes. Greater apparency of plants led to a greater incidence of herbivory and level of damage. Repeated damage to plants may delay future leaf and cone flushing events and contribute to the threat against this cycad species

Encephalartos eugene-maraisii is an endangered, endemic plant in the Waterberg Mountain range of South Africa. This is the first report of herbivory by Zerenopsis lepida (Lepidoptera: Geometridae) on natural populations of E. eugene-maraisii. The impact of herbivory by folivorous arthropods on this cycad species has not been studied before. This study aimed to determine the incidence and intensity of Z. lepida herbivory on E. eugenemaraisii under field conditions. Local plant dynamics were studied by recording the plant location, sex, size and phenological stage for each plant. Damage surveys were carried out over two consecutive seasons (2020–2021 and 2021–2022). Leaf damage was assessed on 227 plants comprising 692 stems. Approximately 40% of the stems had signs of leaf herbivory by Z. lepida and 10% of the total leaf area was removed by Z. lepida larvae. Less herbivory was recorded in areas where plants synchronously produced new leaf flushes compared to areas with asynchronous leaf flushes. Greater apparency of plants led to a greater incidence of herbivory and level of damage. Repeated damage to plants may delay future leaf and cone flushing events and contribute to the threat against this cycad species

The beetle genus Pharaxonotha Reitter (Coleoptera: Erotylidae: Pharaxonothinae) is found in the cones of cycad genera in the New World, including species of Dioon Lindl., Ceratozamia Brongn., Microcycas (Miq.) A.DC and Zamia L. In this paper nine new species found in Dioon are described by Skelley, Tang and Pérez-Farrera: Pharaxonotha bicolor, P. dimorpha, P. fawcettae, P. gigantea, P. novoai, P. occidentalis, P. sclerotiza, P. woodruffi, P. vovidesi. A key to described species of Pharaxonotha inhabiting Dioon is presented, along with an account of Pharaxonotha kirschii Reitter for comparison. The New World beetle genus Pharaxonotha Reitter (Erotylidae: Pharaxonothinae) has a close association with cycads (Cycadales), an ancient lineage of gymnosperms (Norstog and Nichols 1997). Except for the type species of Pharaxonotha, P. kirschii Reitter, which is a minor stored products pest, all other species of the genus that have been described are inhabitants of cycad cones (Pakaluk 1988; Chaves and Genaro 2005; Franz and Skelley 2008; Xu et al. 2015; Skelley et al. 2017; Santiago-Jiménez et al. 2019; Skelley and Segalla 2019; Skelley and Tang 2020). In the New World, almost all cycad populations studied have an associated species or two of Pharaxonotha, rang­ing from the southern US and Caribbean to Bolivia and Brazil. Exclusion experiments on the cycad genus Zamia L. indicate that they are pollinators (Tang 1987; Valencia-Montoya et al. 2017; Segalla et al. 2021). As part of a series of papers describing species on New World cycads, here we describe nine species of Pharaxonotha inhabiting cones of the cycad genus Dioon Lindl. Although Vovides (1991) and Navarrete-Heredia (2018) reported on the presence of Pharaxonotha in Dioon cones, this is the first paper to our knowledge, where species inhabiting Dioon have been scientifically described. To aid with their identification, an addendum to the key of Skelley and Tang (2020) for species of Pharaxonotha found on Dioon is pr

Two species of the early-diverging lineages of Pharaxonotha Reitter (Coleoptera: Erotylidae: Pharaxonothinae) are described: Pharaxonotha taylori Skelley and Tang, new species, and Pharaxonotha thomasi Skelley and Tang, new species. A new key to described species of Pharaxonotha, based on previously unused characters, is presented. In recent phylogenetic analyses of beetle diversity based on a large nuclear data set (McKenna et al. 2019) and on nuclear and mitochondrial legacy loci (Powell, pers. comm. 2020), the New World genus Pharaxonotha Reitter (Erotylidae: Pharaxonothinae) was shown to be sister to all remaining Erotylidae in a clade comprised of the Erotylidae+Phytophaga, which includes Cucujoidea, Chrysomeloidea and Curculionoidea. Since these three superfamilies of Coleoptera contain the majority of beetle pest taxa on human plant crops, a closer examina­tion of this genus may provide some insight into the origin and evolution of these insect groups of agricultural concern. Except for the type species of Pharaxonotha, P. kirschii Reitter, which is a minor stored products pest, all other species of the genus that have been studied are inhabitants of cycad cones (Pakaluk 1988; Chaves and Genaro 2005; Franz and Skelley 2008; Skelley and Segalla 2019). Exclusion experiments on two of these species indicate that they are pollinators of cycads (Tang 1987; Valencia-Montoya et al. 2017), and it is probable that these ancient plant and beetle lineages have coevolved for many millions of years. Leschen and Buckley (2007) hypothesize that fungivory may be the ancestral state in the Erotylidae, however, adult and early instar larvae of Pharaxonotha floridana (Casey) inhabiting cycad cones appear to be pollen feeders, while late instar larvae feed on cone sporophyll and axis tissue (Norstog et al. 1992). Tang et al. (2018b, 2020) presented preliminary relationships of species within Pharaxonotha, based on analysis of the 16S rRNA gene, recognizing three distinct radiati

Over the years studies on sporopollenin have reported a wide variety of structures. However, the methods and techniques used to elucidate sporopollenin structures are highly diverse so that much is still unclear with respect to the nature and structural diversity of sporopollenins. In order to investigate the structural diversity in sporopollenin between different taxa, extant sporomorphs of ten different species ranging from a mushroom to a cycad were examined using a relatively simple and fast analytical procedure. Sporomorphs, before and after saponification, were analysed for sporopollenin composition by Thermally assisted Hydrolysis and Methylation (THM) using [13C]tetramethylammonium hydroxide ([13C]TMAH). The sporomorp chemical composition differed markedly between the groups of organisms analysed. Moreover, we not only identified the nature and relative quantities of the well-known sporopollenin constituents p-coumaric acid and ferulic acid but also many other phenolic moieties, such as caffeic acid, which appeared to be the most abundant phenolic constituent in spores of Equisetum palustre, Salvinia molesta, Cyrtomium falcatum and Anemia phyllitidis. Within the two Equisetum species analysed as well as in the closely related Azolla and Salvinia species the same suite of phenolic constituents were observed, but their relative distribution varied largely. We thus demonstrate the existence of a high structural diversity, both qualitatively and quantitatively in sporopollenins enabling future studies related to the evolution, phylogeny and (palaeo)environment of sporopollenin-producing organisms. Furthermore, a better knowledge of sporopollenin and its structural variety is of relevance to the rapidly growing application of spores and pollen as a drug delivery agent in medicine.

The bioaccumulation of environmental toxins as possible risk factors in the etiology ofamyotrophic lateral sclerosis and parkinsonism-dementia complex (ALS/PDC) is studied inthree foci of the Western Pacific: Guam, the Kii Peninsula, and West Papua New Guinea. Theobjective of this study was to evaluate the best evidence on the exogenous causes of ALS/PDC, with emphasis on the role of cycads, iron, and manganese in the Western Pacific foci,by performing a systematic review of major electronic databases using predefined criteria, 68of which met the selection criteria. Two major environmental hypotheses are associated withthis enigmatic disease: the vegetal hypothesis, which focuses on the neurotoxic and genotoxicproperties of the cycad, and the mineral hypothesis, which focuses on the neurotoxic propertiesof metals. Although typically studied independently, environmental data suggests these twohypotheses may, in fact, converge. Epidemiologic research investigating the association betweenexposure to environmental toxins and ALS/PDC has proven inconclusive. Nevertheless,possible causal links indicate a need for more holistic research to not only better understandALS/PDC, but also glean new insights regarding the associated neurodegenerative diseases.

The bioaccumulation of environmental toxins as possible risk factors in the etiology ofamyotrophic lateral sclerosis and parkinsonism-dementia complex (ALS/PDC) is studied inthree foci of the Western Pacific: Guam, the Kii Peninsula, and West Papua New Guinea. Theobjective of this study was to evaluate the best evidence on the exogenous causes of ALS/PDC, with emphasis on the role of cycads, iron, and manganese in the Western Pacific foci,by performing a systematic review of major electronic databases using predefined criteria, 68of which met the selection criteria. Two major environmental hypotheses are associated withthis enigmatic disease: the vegetal hypothesis, which focuses on the neurotoxic and genotoxicproperties of the cycad, and the mineral hypothesis, which focuses on the neurotoxic propertiesof metals. Although typically studied independently, environmental data suggests these twohypotheses may, in fact, converge. Epidemiologic research investigating the association betweenexposure to environmental toxins and ALS/PDC has proven inconclusive. Nevertheless,possible causal links indicate a need for more holistic research to not only better understandALS/PDC, but also glean new insights regarding the associated neurodegenerative diseases.

Exposure to beta-N-methylamino-l-alanine (BMAA) might be linked to the incidence of amyotrophic lateral sclerosis, Alzheimer's disease and Parkinson's disease. Analytical chemistry plays a crucial role in determining human BMAA exposure and the associated health risk, but the performance of various analytical methods currently employed is rarely compared. A CYANOCOST initiated workshop was organized aimed at training scientists in BMAA analysis, creating mutual understanding and paving the way towards interlaboratory comparison exercises. During this workshop, we tested different methods (extraction followed by derivatization and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis, or directly followed by LC-MS/MS analysis) for trueness and intermediate precision. We adapted three workup methods for the underivatized analysis of animal, brain and cyanobacterial samples. Based on recovery of the internal standard D(3)BMAA, the underivatized methods were accurate (mean recovery 80%) and precise (mean relative standard deviation 10%), except for the cyanobacterium Leptolyngbya. However, total BMAA concentrations in the positive controls (cycad seeds) showed higher variation (relative standard deviation 21%-32%), implying that D(3)BMAA was not a good indicator for the release of BMAA from bound forms. Significant losses occurred during workup for the derivatized method, resulting in low recovery (<10%). Most BMAA was found in a trichloroacetic acid soluble, bound form and we recommend including this fraction during analysis.

Exposure to beta-N-methylamino-l-alanine (BMAA) might be linked to the incidence of amyotrophic lateral sclerosis, Alzheimer's disease and Parkinson's disease. Analytical chemistry plays a crucial role in determining human BMAA exposure and the associated health risk, but the performance of various analytical methods currently employed is rarely compared. A CYANOCOST initiated workshop was organized aimed at training scientists in BMAA analysis, creating mutual understanding and paving the way towards interlaboratory comparison exercises. During this workshop, we tested different methods (extraction followed by derivatization and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis, or directly followed by LC-MS/MS analysis) for trueness and intermediate precision. We adapted three workup methods for the underivatized analysis of animal, brain and cyanobacterial samples. Based on recovery of the internal standard D(3)BMAA, the underivatized methods were accurate (mean recovery 80%) and precise (mean relative standard deviation 10%), except for the cyanobacterium Leptolyngbya. However, total BMAA concentrations in the positive controls (cycad seeds) showed higher variation (relative standard deviation 21%-32%), implying that D(3)BMAA was not a good indicator for the release of BMAA from bound forms. Significant losses occurred during workup for the derivatized method, resulting in low recovery (<10%). Most BMAA was found in a trichloroacetic acid soluble, bound form and we recommend including this fraction during analysis.

Exposure to beta-N-methylamino-l-alanine (BMAA) might be linked to the incidence of amyotrophic lateral sclerosis, Alzheimer's disease and Parkinson's disease. Analytical chemistry plays a crucial role in determining human BMAA exposure and the associated health risk, but the performance of various analytical methods currently employed is rarely compared. A CYANOCOST initiated workshop was organized aimed at training scientists in BMAA analysis, creating mutual understanding and paving the way towards interlaboratory comparison exercises. During this workshop, we tested different methods (extraction followed by derivatization and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis, or directly followed by LC-MS/MS analysis) for trueness and intermediate precision. We adapted three workup methods for the underivatized analysis of animal, brain and cyanobacterial samples. Based on recovery of the internal standard D(3)BMAA, the underivatized methods were accurate (mean recovery 80%) and precise (mean relative standard deviation 10%), except for the cyanobacterium Leptolyngbya. However, total BMAA concentrations in the positive controls (cycad seeds) showed higher variation (relative standard deviation 21%-32%), implying that D(3)BMAA was not a good indicator for the release of BMAA from bound forms. Significant losses occurred during workup for the derivatized method, resulting in low recovery (<10%). Most BMAA was found in a trichloroacetic acid soluble, bound form and we recommend including this fraction during analysis.

Cyanobacteria are a tantalising prospect for research into biofertilisers and plant growth promotion. With photolithic autotrophy, broadly distributed capacities for biological N2 fixation and phytohormone production, cyanobacteria have all the necessary requirements for enhancing plant growth in cultivated soils. Research into the use of cyanobacterial inoculants for wheat cultivation, and particularly for salt stress amelioration, has never been assessed in Australia. Therefore, this thesis aimed to explore the potential of Australian cyanobacteria to ameliorate the effects of irrigation salinity on Australian wheat cultivars. This thesis describes the first investigation into the diversity of cyanobacteria from Australian wheat fields and cycad rhizospheres. It also characterises the nitrogenase activity and salt physiology of selected isolates and evaluates their application in ameliorating the salt stress of Australian wheat. The results of this study indicate that there is only a weak relationship between cycad cyanobionts and their associated rhizosphere cyanobacteria. A Cylindrospermum wheat field isolate had the highest nitrogenase activity, being almost double that of Nostoc. Microcoleus vaginatus was also isolated from a wheat field and was found to have nitrogenase activity and to contain cyanobacterial nifH. Analysis of cyanobacterial salt physiology determined that salinity generally lowered O2 evolution and nitrogenase activity of cyanobacteria though the relationships between salinity, O2 evolution and nitrogenase activity were more complicated for the individual isolates studied. The chlorophyll fluorescence parameters failed to explain the observed effects of salinity on O2 evolution. In pot trials, inoculation with cyanobacteria negatively affected the biomass and nutrient concentrations of wheat seedlings at all salinities, though the effects were fewer as salinity increased. The most likely explanation of these results is the sorption of nutrien

The genertic diversity of cyanobacteria associated with cycads was examined using the tRNA Leu (UAA) intron as a genetic marker. Coralloid roots of both natural populations of the cycad Macrozamia riedlei (Fischer ex Gaudichaud-Beaupré) C.A. Gardner growing in Perth, Australia and cycads growing in greenhouses, also in Perth, were used and their respective cyanobionts analyzed. Several Nostoc strains were found to be involved in this symbiosis, both in natural populations and greenhouse-orginated cycads. However, only one strain was present in individual coralloid roots and in individual plants, even when analyzing different coralloid roots from the same plant. Moreover, when examining plants growing close to each other (female plants and their respective offspring) the same cyanobacterium was consistently present in the different coralloid roots. Whether this reflects a selective mechanism or merely the availability of Nostoc strains remains to be ascertained. The high cyanobacterial diversity in coralloid roots of cycads revealed by PCR fingerprinting is, therefore, contested. In this study, the potential probems of using different methods (e.g. PCR fingerprinting) to study the genetic diversity of symbiotic cyanobacteria, is also addressed.

The genertic diversity of cyanobacteria associated with cycads was examined using the tRNA Leu (UAA) intron as a genetic marker. Coralloid roots of both natural populations of the cycad Macrozamia riedlei (Fischer ex Gaudichaud-Beaupré) C.A. Gardner growing in Perth, Australia and cycads growing in greenhouses, also in Perth, were used and their respective cyanobionts analyzed. Several Nostoc strains were found to be involved in this symbiosis, both in natural populations and greenhouse-orginated cycads. However, only one strain was present in individual coralloid roots and in individual plants, even when analyzing different coralloid roots from the same plant. Moreover, when examining plants growing close to each other (female plants and their respective offspring) the same cyanobacterium was consistently present in the different coralloid roots. Whether this reflects a selective mechanism or merely the availability of Nostoc strains remains to be ascertained. The high cyanobacterial diversity in coralloid roots of cycads revealed by PCR fingerprinting is, therefore, contested. In this study, the potential probems of using different methods (e.g. PCR fingerprinting) to study the genetic diversity of symbiotic cyanobacteria, is also addressed.

Exposure to β-Ν-methylamino-l-alanine (BMAA) might be linked to the incidence of amyotrophic lateral sclerosis, Alzheimer's disease and Parkinson's disease. Analytical chemistry plays a crucial role in determining human BMAA exposure and the associated health risk, but the performance of various analytical methods currently employed is rarely compared. A CYANOCOST initiated workshop was organized aimed at training scientists in BMAA analysis, creating mutual understanding and paving the way towards interlaboratory comparison exercises. During this workshop, we tested different methods (extraction followed by derivatization and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis, or directly followed by LC-MS/MS analysis) for trueness and intermediate precision. We adapted three workup methods for the underivatized analysis of animal, brain and cyanobacterial samples. Based on recovery of the internal standard D3BMAA, the underivatized methods were accurate (mean recovery 80%) and precise (mean relative standard deviation 10%), except for the cyanobacterium Leptolyngbya. However, total BMAA concentrations in the positive controls (cycad seeds) showed higher variation (relative standard deviation 21%-32%), implying that D3BMAA was not a good indicator for the release of BMAA from bound forms. Significant losses occurred during workup for the derivatized method, resulting in low recovery ( < 10%). Most BMAA was found in a trichloroacetic acid soluble, bound form and we recommend including this fraction during analysis.

Exposure to ?-?-methylamino-l-alanine (BMAA) might be linked to the incidence of amyotrophic lateral sclerosis, Alzheimer's disease and Parkinson's disease. Analytical chemistry plays a crucial role in determining human BMAA exposure and the associated health risk, but the performance of various analytical methods currently employed is rarely compared. A CYANOCOST initiated workshop was organized aimed at training scientists in BMAA analysis, creating mutual understanding and paving the way towards interlaboratory comparison exercises. During this workshop, we tested different methods (extraction followed by derivatization and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis, or directly followed by LC-MS/MS analysis) for trueness and intermediate precision. We adapted three workup methods for the underivatized analysis of animal, brain and cyanobacterial samples. Based on recovery of the internal standard D3BMAA, the underivatized methods were accurate (mean recovery 80%) and precise (mean relative standard deviation 10%), except for the cyanobacterium Leptolyngbya. However, total BMAA concentrations in the positive controls (cycad seeds) showed higher variation (relative standard deviation 21%-32%), implying that D3BMAA was not a good indicator for the release of BMAA from bound forms. Significant losses occurred during workup for the derivatized method, resulting in low recovery ( < 10%). Most BMAA was found in a trichloroacetic acid soluble, bound form and we recommend including this fraction during analysis.