Two draft genomes of fungal leaf endophytes from tropical gymnosperms.

Two ascomycetes, Neofusicoccum sp. and Xylaria sp., were isolated from healthy leaves of the tropical gymnosperms Zamia pseudoparasitica (Z2) and Zamia nana (Z50) from Panama. The two draft genomes possess a broad predicted repertoire of carbohydrate-degrading CAZymes, peptidases, and secondary metabolites, with more secondary metabolite clusters in the Xylaria isolate.

Phylometagenomics of cycad coralloid roots reveals shared symbiotic signals.

Cycads are known to host symbiotic cyanobacteria, including Nostocales species, as well as other sympatric bacterial taxa within their specialized coralloid roots. Yet, it is unknown if these bacteria share a phylogenetic origin and/or common genomic functions that allow them to engage in facultative symbiosis with cycad roots. To address this, we obtained metagenomic sequences from 39 coralloid roots sampled from diverse cycad species and origins in Australia and Mexico. Culture-independent shotgun metagenomic sequencing was used to validate sub-community co-cultures as an efficient approach for functional and taxonomic analysis. Our metanalysis shows a host-independent microbiome core consisting of seven bacterial orders with high species diversity within the identified taxa. Moreover, we recovered 43 cyanobacterial metagenome-assembled genomes, and in addition to Nostoc spp., symbiotic cyanobacteria of the genus Aulosira were identified for the first time. Using this robust dataset, we used phylometagenomic analysis to reveal three monophyletic cyanobiont clades, two host-generalist and one cycad-specific that includes Aulosira spp. Although the symbiotic clades have independently arisen, they are enriched in certain functional genes, such as those related to secondary metabolism. Furthermore, the taxonomic composition of associated sympatric bacterial taxa remained constant. Our research quadruples the number of cycad cyanobiont genomes and provides a robust framework to decipher cyanobacterial symbioses, with the potential of improving our understanding of symbiotic communities. This study lays a solid foundation to harness cyanobionts for agriculture and bioprospection, and assist in conservation of critically endangered cycads.

A new distribution and host record for the rare moth, Callioratis millari (Lepidoptera: Geometridae), and some ecological observations.

Callioratis millari Hampson (Lepidoptera: Geometridae) is a Critically Endangered moth endemic to South Africa. Despite extensive searches, it was previously known only from the Entumeni Nature Reserve in KwaZulu-Natal, where its larvae exclusively feed on the cycad Stangeria eriopus (Kunze) Baill (Cycadales: Stangeriaceae). In July 2022, a new population of C. millari was discovered in the Kabouga section of Addo Elephant National Park in the Eastern Cape. Larvae of C. millari were feeding on the cycad Encephalartos caffer (Thunb.) Lehm (Cycadales: Zamiaceae), which also constitutes a new host record. In June 2023, we determined larval incidence and herbivory at this new locality, offering insights into the ecological requirements of C. millari. Known C. millari localities, although ecologically different, share low altitudes (700-950 m a.s.l.), moderate to high rainfall, and grassy habitats with sparse woody cover. A total of 59 larvae were counted in Kabouga, mostly in the fifth and sixth (final) instars. Herbivory incidence was lower on smaller plants and those covered by other vegetation. The flight period of adult C. millari likely occurs between mid-March and April in Kabouga, but further investigation is needed to clarify this. The peak period of larval occurrence in Kabouga occurs during the driest and coldest months of the year (May-July). Considering limited habitat availability, host plant poaching, and the risk of untimely fires, the species should be considered highly threatened. This study adds to our understanding of the biology of C. millari and provides information on its ecological requirements and may contribute to making informed management decisions.

Museomics unveil systematics, diversity and evolution of Australian cycad-pollinating weevils.

Weevils have been shown to play significant roles in the obligate pollination of Australian cycads. In this study, we apply museomics to produce a first molecular phylogeny estimate of the Australian cycad weevils, allowing an assessment of their monophyly, placement and relationships. Divergence dating suggests that the Australian cycad weevils originated from the Late Oligocene to the Middle Miocene and that the main radiation of the cycad-pollinating groups occurred from the Middle to the Late Miocene, which is congruent with the diversification of the Australian cycads, thus refuting any notion of an ancient ciophilous system in Australia. Taxonomic studies reveal the existence of 19 Australian cycad weevil species and that their associations with their hosts are mostly non-species-specific. Co-speciation analysis shows no extensive co-speciation events having occurred in the ciophilous system of Australian cycads. The distribution pattern suggests that geographical factors, rather than diversifying coevolution, constitute the overriding process shaping the Australian cycad weevil diversity. The synchronous radiation of cycads and weevil pollinators is suggested to be a result of the post-Oligocene diversification common in Australian organisms.

Ghost introgression facilitates genomic divergence of a sympatric cryptic lineage in Cycas revoluta.

A cryptic lineage is a genetically diverged but morphologically unrecognized variant of a known species. Clarifying cryptic lineage evolution is essential for quantifying species diversity. In sympatric cryptic lineage divergence compared with allopatric divergence, the forces of divergent selection and mating patterns override geographical isolation. Introgression, by supplying preadapted or neutral standing genetic variations, can promote sympatric cryptic lineage divergence via selection. However, most studies concentrated on extant species introgression, ignoring the genetic legacy of introgression from extinct or unsampled lineages ("ghost introgression"). Cycads are an ideal plant for studying the influence of ghost introgression because of their common interspecific gene flow and past high extinction rate. Here, we utilized reference-based ddRADseq to clarify the role of ghost introgression in the evolution of a previously identified sympatric cryptic lineage in Cycas revoluta. After re-evaluating the evolutionary independency of cryptic lineages, the group-wise diverged single-nucleotide polymorphisms among sympatric and allopatric lineages were compared and functionally annotated. Next, we employed an approximate Bayesian computation method for hypothesis testing to clarify the cryptic lineage evolution and ghost introgression effect. SNPs with the genomic signatures of ghost introgression were further annotated. Our results reconfirmed the evolutionary independency of cryptic lineage among C. revoluta and demonstrated that ghost introgression to the noncryptic lineage facilitated their divergence. Gene function related to heat stress and disease resistance implied ecological adaptation of the main extant populations of C. revoluta.

Characterization of starch extracted from seeds of Cycas revoluta.

Introduction: Starch is major component in the big seeds of Cycas revoluta, however the characteristics of Cycas revoluta remain unknown. Methods: In this study, the physicochemical and structural properties of two starch samples extracted from Cycad revoluta seeds were systematically investigated, using various techniques. Results: The amylose contents of the two samples were 34.3 % and 35.5%, respectively. The spherical-truncated shaped starch granules possessed A-type crystallinity, and had an average diameter less than 15 µm. Compared to most commonly consumed cereal and potato starch, Cycad revoluta starch showed distinctive characteristics. For physicochemical properties, in the process of gelatinization, the Cycad revoluta starch showed similar viscosity profile to starches of some potato varieties, but Cycad revoluta starch had higher gelatinization temperature. Upon cooling, Cycad revoluta starch formed harder gels than rice starch. For structure, the molecular weight (indexed by Mw, Mn and Rz values), branching degree and the branch chain length distribution were determined. Discussion: The results suggested that Cycad revoluta starch were different in structure from the main-stream starches. Notable differences in some starch traits between the two samples were recorded, which could be attributed to environmental factors. In general, this study provides useful information on the utilization of Cycad revoluta starch in both food and non-food industries.

Cone humidity is a strong attractant in an obligate cycad pollination system.

Studies of pollination biology often focus on visual and olfactory aspects of attraction, with few studies addressing behavioral responses and morphological adaptation to primary metabolic attributes. As part of an in-depth study of obligate nursery pollination of cycads, we find that Rhopalotria furfuracea weevils show a strong physiological response and behavioral orientation to the cone humidity of the host plant Zamia furfuracea in an equally sensitive manner to their responses to Z. furfuracea-produced cone volatiles. Our results demonstrate that weevils can perceive fine-scale differences in relative humidity (RH) and that individuals exhibit a strong behavioral preference for higher RH in binary choice assays. Host plant Z. furfuracea produces a localized cloud of higher than ambient humidity around both pollen and ovulate cones, and R. furfuracea weevils preferentially land at the zone of maximum humidity on ovulate cones, i.e., the cracks between rows of megasporophylls that provide access to the ovules. Moreover, R. furfuracea weevils exhibit striking antennal morphological traits associated with RH perception, suggesting the importance of humidity sensing in the evolution of this insect lineage. Results from this study suggest that humidity functions in a signal-like fashion in this highly specialized pollination system and help to characterize a key pollination-mediating trait in an ancient plant lineage.

Composition of Sexual Fluids in Cycas revoluta Ovules During Pollination and Fertilization.

The composition of fluids that mediate fertilization in cycads is described for the first time. Using tandem mass spectrometry, proteomes of two stages of fluid production, megagametophyte fluid and archegonial chamber fluid production, are compared in Cycas revoluta. These were compared with the proteome of another sexual fluid produced by ovules, the pollination drop proteins. Cycad ovules produce complex liquids immediately prior fertilization. Compared with the pollination drops that mainly had few proteins in classes involved in defense and carbohydrate modification, megagametophyte fluid and archegonial chamber fluid had larger proteomes with many more protein classes, e.g. proteins involved in programmed cell death. Using high-performance liquid chromatography, megagametophyte fluid and archegonial chamber fluid were shown to have elevated concentrations of smaller molecular weight molecules including glucose, pectin and glutamic acid. Compared to megagametophyte fluid, archegonial chamber fluid had elevated pH as well as higher osmolality. Supplementary Information: The online version contains supplementary material available at 10.1007/s12229-021-09271-1.

Are legumes different? Origins and consequences of evolving nitrogen fixing symbioses.

Nitrogen fixing symbioses between plants and bacteria are ancient and, while not numerous, are formed in diverse lineages of plants ranging from microalgae to angiosperms. One symbiosis stands out as the most widespread one is that between legumes and rhizobia, leading to the formation of nitrogen-fixing nodules. The legume family is one of the largest and most diverse group of plants and legumes have been used by humans since the beginning of agriculture, both as high nitrogen food, as well as pastures and rotation crops. One open question is whether their ability to form a nitrogen-fixing symbiosis has contributed to legumes' success, and whether legumes have any unique characteristics that have made them more diverse and widespread than other groups of plants. This review examines the evolutionary journey that has led to the diversification of legumes, in particular its nitrogen-fixing symbiosis, and asks four questions to investigate which legume traits might have contributed to their success: 1. In what ways do legumes differ from other plant groups that have evolved nitrogen-fixing symbioses? In order to answer this question, the characteristics of the symbioses, and efficiencies of nitrogen fixation are compared between different groups of nitrogen fixing plants. 2. Could certain unique features of legumes be a reason for their success? This section examines the manifestations and possible benefits of a nitrogen-rich 'lifestyle' in legumes. 3. If nitrogen fixation was a reason for such a success, why have some species lost the symbiosis? Formation of symbioses has trade-offs, and while these are less well known for non-legumes, there are known energetic and ecological reasons for loss of symbiotic potential in legumes. 4. What can we learn from the unique traits of legumes for future crop improvements? While exploiting some of the physiological properties of legumes could be used to improve legume breeding, our increasing molecular understanding of the essential regulators of root nodule symbioses raise hope of creating new nitrogen fixing symbioses in other crop species.

Taxonomic Revision of the Genus Miltotranes Zimmerman, 1994 (Coleoptera: Curculionidae: Molytinae), the Bowenia-Pollinating Cycad Weevils in Australia, with Description of a New Species and Implications for the Systematics of Bowenia.

The Australian endemic weevils of the genus Miltotranes Zimmerman, 1994 (Curculionidae: Molytinae: Tranes group), comprising two species, M. prosternalis (Lea, 1929) and M. subopacus (Lea, 1929), are highly host-specific and the only known pollinators of Bowenia cycads, which comprise two CITES-protected species restricted to Tropical Queensland in Australia. In the present study, the taxonomy of Miltotranes is reviewed, a lectotype for the name Tranes prosternalis Lea, 1929 is designated and a new species associated with the Bowenia population in the McIlwraith Range is described as M. wilsoni sp. n. The descriptions and diagnoses of all species are supplemented with illustrations of their habitus and salient structures, and an identification key to all species and a distribution map are provided. Potential implications of the new species and of the taxonomy and biogeography of Miltotranes overall on the systematics and conservation of Bowenia are discussed.

A modified diet to support conservation of the Atala hairstreak butterfly (Eumaeus atala Poey).

Raising insects in a laboratory for release into the wild is a common conservation practice, but maintaining breeding colonies year-round can be limited by seasonal food availability. Food availability is particularly challenging for insects which depend on specific host plants. For example, our early efforts to rear the imperiled Atala hairstreak butterfly (Eumaeus atala Poey) resulted in colony failure during winter due to lack of food. To overcome this barrier, we developed a modified freeze-dried host plant diet to support the colony. The diet consisted of reconstituted freeze-dried leaves and stems from fresh-growth coontie (Zamia integrifolia), the host plant for the Atala butterflies. We fed larvae less than 9 mm on this freeze-dried diet and transferred them to live coontie plants after they were more than 9 mm. We reared a colony of Atala butterflies using these methods for 859 days, resulting in more than 3400 animals released into the wild. Comparing colony counts during that time period to the 548 days we reared them without modified freeze-dried diet showed a clear benefit in using freeze-dried diet. A growth trial (N = 40) of larvae fed on only freeze-dried diet compared to larvae fed on fresh coontie cuttings found no significant difference in larval or pupal development between groups (p = 0.71 and p = 0.47, respectively). We, therefore, conclude that the freeze-dried diet provided an appropriate alternative for Atala colonies when fresh growth from the host plant is unavailable, and we recommend use of this technique for raising other host plant-dependent insect species of conservation concern.

Height increment of Cycas micronesica informs conservation decisions.

Growth dynamics of pachycaulous stems of arborescent cycad plants are not well understood, and most observations have been made in cultivated garden plants. We studied Cycas micronesica plants in Guam, Tinian, and Yap to understand the influences of geography, plant size, sex, and herbivory on stem growth. We also determined the changes in demography of Guam's population after 15 years of damage by non-native insect herbivores. The height increment (HI) was similar for plants within the height range from 100 cm to more than 600 cm, so the relative growth rate declined with height. Female tree HI was 68% of male tree HI, and Yap tree HI was 87% of Guam tree HI. Chronic herbivory by non-native insect herbivores caused a mean 44% decline in HI. Plants in managed gardens grew more rapidly than plants in a wild habitat. The HI was used to estimate that Guam has experienced a complete loss of ≈70 y of demographic depth resulting from the selective mortality of small plants since 2005. When future conservation interventions successfully mitigate the ubiquitous biological threats, our HI may be useful for empirically quantifying recovery of plant health.

Presumptive Cycad Toxicosis in a Dog. Clinical and Magnetic Resonance Imaging Findings: A Case Report.

Cases of cycad toxicosis have been described in dogs that have presented with gastrointestinal, hematologic, hepatic, neurological, and carcinogenic signs. This case report describes brain magnetic resonance imaging (MRI) lesions in a dog with gastrointestinal and neurological signs secondary to cycad toxicosis. A 5-year-old neutered female Jack Russell terrier presented with a 2-days history of gastroenteric signs, progressive generalized tremors, and altered mentation after possible ingestion of Cycad revoluta. Neurologic examinations revealed disorientation, a wide-based stance, severe spasticity of the four limbs, intention tremors, severe cerebellar ataxia, decreased postural reactions in all four limbs, and intermittent decreased menace response in both eyes-all of which are consistent with a multifocal intracranial disorder involving the forebrain and cerebellum. A brain MRI showed diffuse/ill-defined, intra-axial bilateral and symmetrical changes, predominantly affecting the white matter of the cerebral hemispheres, thalamus, hippocampus, and cerebellum. A presumptive diagnosis of toxic-metabolic encephalopathy was made. Medical management of the clinical signs was performed, and the dog was discharged 7 days after presentation with no neurological abnormalities. Two and 8 weeks later, complete blood count (CBC), chemistry, electrolytes, and 8 weeks later brain MRI were performed, revealing no abnormalities. To the best of the authors' knowledge, this is the first case report describing lesions detected by brain MRI secondary to cycad toxicosis as well as a complete resolution of brain lesions on a follow-up MRI 8 weeks later.

The characterization of cycad palm toxicosis and treatment effects in 130 dogs.

The purpose of this retrospective study is to characterize the clinical signs, laboratory values, treatment modalities and mortality outcomes related to cycad palm toxicosis in dogs and to identify medical interventions that potentially correlate with mortality. Dogs with confirmed cycad palm ingestion were identified by reviewing medical records from two private veterinary referral centres for key terms and phrases indicative of cycad palm toxicosis. Of 130 dogs included, 107 (82.3%) presented with clinical signs. A total of 2 (1.5%) died and 14 (10.8%) were euthanized. Diarrhea, lethargy and mortality were all associated with an elevated initial alanine transaminase (ALT) concentration. Overall mortality was 12.3%, with a significantly higher proportion of dogs with an ALT concentration <125 U/L surviving compared to dogs with an elevated ALT concentration. Treatment with activated charcoal reduced the odds of death among all dogs by 82% and was even more protective among dogs with an elevated initial ALT concentration. Among dogs that had an initial platelet count performed, the presence of thrombocytopenia (<200,000/µL) was a negative prognostic indicator and was associated with a significant increase in mortality. Given the high mortality rates reported for cycad palm toxicosis, it is imperative to identify early clinical indicators of mortality as well as treatments that reduce mortality.

Etiology of Retinal and Cerebellar Pathology in Western Pacific Amyotrophic Lateral Sclerosis and Parkinsonism-Dementia Complex.

PURPOSE: To reexamine the etiology of a unique retinal pathology (linear and vermiform sub-retinal tubular structures) described among subjects with and without neurodegenerative disease in former high-incidence foci of Western Pacific amyotrophic lateral sclerosis and parkinsonism-dementia complex (ALS/PDC) in Guam (USA) and the Kii peninsula of Honshu island (Japan). METHODS: Analysis of published and unpublished reports of 1) ALS/PDC and the retinal and cerebellar pathology associated therewith and 2) exogenous neurotoxic factors associated with ALS/PDC and the developing retina and cerebellum. RESULTS: ALS/PDC retinal and cerebellar pathology matches persistent retinal and cerebellar dysplasia found in laboratory animals given single in utero or postnatal systemic treatment with cycasin, the principal neurotoxic component in the seed of cycad plants traditionally used for food (Guam) or oral medicine (Kii-Japan), both of which have been linked to the human neurodegenerative disease. CONCLUSION: ALS/PDC-associated retinal and cerebellar dysplasia could arise from in utero exposure to methylazoxymethanol, the genotoxic metabolite of cycasin that results from maternal ingestion of this azoxyglucoside. These results support the environmental toxic etiology of retinal and brain pathology in ALS/PDC.

Artifleck: The Study of Artifactual Responses to Light Flecks with Inappropriate Leaves.

Methods in sunfleck research commonly employ the use of experimental leaves which were constructed in homogeneous light. These experimental organs may behave unnaturally when they are challenged with fluctuating light. Photosynthetic responses to heterogeneous light and leaf macronutrient relations were determined for Cycas micronesica, Glycine max, and Zea mays leaves that were grown in homogeneous shade, heterogeneous shade, or full sun. The speed of priming where one light fleck increased the photosynthesis during a subsequent light fleck was greatest for the leaves grown in heterogeneous shade. The rate of induction and the ultimate steady-state photosynthesis were greater for the leaves that were grown in heterogeneous shade versus the leaves grown in homogeneous shade. The leaf mass per area, macronutrient concentration, and macronutrient stoichiometry were also influenced by the shade treatments. The amplitude and direction in which the three developmental light treatments influenced the response variables were not universal among the three model species. The results indicate that the historical practice of using experimental leaves which were constructed under homogeneous light to study leaf responses to fluctuating light may produce artifacts that generate dubious interpretations.

Kampo medicine and Muro disease (Amyotrophic Lateral Sclerosis and Parkinsonism-Dementia Complex).

Western Pacific Amyotrophic Lateral Sclerosis and Parkinsonism-dementia Complex (ALS/PDC) is a disappearing neurodegenerative disease in three former high-incidence foci of Guam-USA, Papua-Indonesia and Kii Peninsula, Honshu Island, Japan. The latter includes two distinct ALS/PDC-affected regions (Hohara and Kozagawa), where the disorder is known as Muro disease. In Hohara, oral exposure to plant (cycad) neurotoxins used in traditional medical practice has been linked previously to Muro disease. We report new observations that link Kampo medicine to Muro disease in the southern Kozagawa focus. Oral exposure to cycad seed toxins is associated with all three foci of Western Pacific ALS/PDC.

Cycas revoluta (sago cycad) exposures reported to Texas poison centers.

BACKGROUND: Cycas revoluta (sago cycad or palm) is a popular ornamental plant in the United States. All parts of the plant contain toxins such cycasin and beta-methylamino-l-alanine, the ingestion of which can be harmful to humans and animals. The objective of this study was to characterize C. revoluta exposures reported to poison centers. METHODS: Cases were C. revoluta exposures reported to Texas poison centers during 2000-2018. The distribution of cases by selected variables was determined. RESULTS: Of 192 total C. revoluta exposures, the most common exposure routes were ingestion (55.7%) and dermal (34.4%). The patient age distribution was 28.1% 5 years or less, 15.1% 6-12 years, 4.7% 13-19 years, and 50.5% 20 years or more; 55.2% were male. The exposure was unintentional in 92.2% of the cases and occurred at the patient's own residence in 94.8%. The patient was managed on site in 78.6% of the cases, already at/en route to a healthcare facility in 12.0%, and referred to a healthcare facility in 8.9%. The most common reported clinical effects were dermal (23.4%), particularly puncture/wound (16.1%), dermal irritation/pain (14.6%), and edema (8.9%), followed by gastrointestinal (13.0%), particularly vomiting (8.9%) and nausea (7.8%). CONCLUSION: Most of the C. revoluta exposures tended to be unintentional and occurred at home. Although most of the C. revoluta exposures involved ingestion, 35% were dermal, and the most frequently reported clinical effects were dermal followed by gastrointestinal. Most of the exposures were not serious and were managed outside of a healthcare facility.

Hypothesis: Etiologic and Molecular Mechanistic Leads for Sporadic Neurodegenerative Diseases Based on Experience With Western Pacific ALS/PDC.

Seventy years of research on Western Pacific amyotrophic lateral sclerosis and Parkinsonism-dementia Complex (ALS/PDC) have provided invaluable data on the etiology, molecular pathogenesis and latency of this disappearing, largely environmental neurodegenerative disease. ALS/PDC is linked to genotoxic chemicals (notably methylazoxymethanol, MAM) derived from seed of the cycad plant (Cycas spp.) that were used as a traditional food and/or medicine in all three disease-affected Western Pacific populations. MAM, nitrosamines and hydrazines generate methyl free radicals that damage DNA (in the form of O 6-methylguanine lesions) that can induce mutations in cycling cells and degenerative changes in post-mitotic cells, notably neurons. This paper explores exposures to naturally occurring and manmade sources of nitrosamines and hydrazines in association with sporadic forms of ALS (with or without frontotemporal degeneration), progressive supranuclear palsy, and Alzheimer disease. Research approaches are suggested to examine whether these associations might have etiological significance. LAY SUMMARY: Unknown environmental exposures are thought to be risk factors for non-inherited forms of certain progressive brain diseases, such as sporadic forms of amyotrophic lateral sclerosis (sALS), progressive supranuclear palsy (sPSP), and Alzheimer's disease (sAD). Related progressive and fatal brain disorders coalesce in a single neurodegenerative disease of largely environmental origin (ALS-Parkinsonism-dementia Complex) that has affected three genetically distinct populations residing in islands of the Western Pacific region. Prolonged study of this prototypical neurodegenerative disease has provided invaluable information on the probable environmental cause (specific chemical genotoxins) and molecular mechanisms (unrepaired nerve cell DNA-damage) by which brain degeneration begins, evolves and, years or decades later, clinical signs appear, and progress. This information is used as a foundation to explore whether chemically related genotoxins (nitrosamines, hydrazines) are possible risk factors for sALS, sPSP, and sAD. Methods to test this hypothesis in the field and laboratory are proposed.