Evolutionary trends of reproductive phenotype in Cycadales: an analysis of morphological evolution in Ceratozamia.

BACKGROUND AND AIMS: The size and shape of reproductive structures is especially relevant in evolution because these characters are directly related to the capacity of pollination and seed dispersal, a process that plays a basic role in evolutionary patterns. The evolutionary trajectories of reproductive phenotypes in gymnosperms have received special attention in terms of pollination and innovations related to the emergence of the Spermatophytes. However, variability of reproductive structures, evolutionary trends and the role of environment in the evolution of cycad species have not been well documented and explored. This study considered this topic under an explicitly phylogenetic and evolutionary approach that included a broad sampling of reproductive structures in the genus Ceratozamia. METHODS: We sampled 1400 individuals of 36 Ceratozamia species to explore evolutionary pattern and identify and evaluate factors that potentially drove their evolution. We analyzed characters for both pollen and ovulate strobili within a phylogenetic framework using different methods and characters (i. e., molecular and both quantitative and qualitative morphological) to infer phylogenetic relationships. Using this phylogenetic framework, evolutionary models of trait evolution for strobilar size were evaluated. In addition, quantitative morphological variation and its relation to environmental variables across species were analyzed. KEY RESULTS: We found contrasting phylogenetic signals between characters of pollen and ovulate strobili. These structures exhibited high morphological disparity in several characters related to size. Results of analyses of evolutionary trajectories suggested a stabilizing selection model. In regards to phenotype-environment, the analysis produced mixed results and differences for groups in the vegetation type where the species occur; however, a positive relationship with climatic variables was found. CONCLUSIONS: The integrated approach synthesized reproductive phenotypic variation with current phylogenetic hypotheses and provided explicit statements of character evolution. The characters of volume for ovulate strobili were the most informative, which could provide a reference for further study of the evolutionary complexity in Ceratozamia. Finally, heterogeneous environments, which are under changing weather conditions, promote variability of reproductive structures.

Leaf Endophytes Relationship with Host Metabolome Expression in Tropical Gymnosperms.

Plant-microbe interactions play a pivotal role in shaping host fitness, especially concerning chemical defense mechanisms. In cycads, establishing direct correlations between specific endophytic microbes and the synthesis of highly toxic defensive phytochemicals has been challenging. Our research delves into the intricate relationship between plant-microbe associations and the variation of secondary metabolite production in two closely related Zamia species that grow in distinct habitats; terrestrial and epiphytic. Employing an integrated approach, we combined microbial metabarcoding, which characterize the leaf endophytic bacterial and fungal communities, with untargeted metabolomics to test if the relative abundances of specific microbial taxa in these two Zamia species were associated with different metabolome profiles. The two species studied shared approximately 90% of the metabolites spanning diverse biosynthetic pathways: alkaloids, amino acids, carbohydrates, fatty acids, polyketides, shikimates, phenylpropanoids, and terpenoids. Co-occurrence networks revealed positive associations among metabolites from different pathways, underscoring the complexity of their interactions. Our integrated analysis demonstrated to some degree that the intraspecific variation in metabolome profiles of the two host species was associated with the abundance of bacterial orders Acidobacteriales and Frankiales, as well as the fungal endophytes belonging to the orders Chaetothyriales, Glomerellales, Heliotiales, Hypocreales, and Sordariales. We further associate individual metabolic similarity with four specific fungal endophyte members of the core microbiota, but no specific bacterial taxa associations were identified. This study represents a pioneering investigation to characterize leaf endophytes and their association with metabolomes in tropical gymnosperms, laying the groundwork for deeper inquiries into this complex domain.

Cretaceous pollen cone with three-dimensional preservation sheds light on the morphological evolution of cycads in deep time.

The Cycadales are an ancient and charismatic group of seed plants. However, their morphological evolution in deep time is poorly understood. While molecular divergence time analyses estimate a Cretaceous origin for most major living cycad clades, much of the extant diversity is inferred to be a result of Neogene diversifications. This leads to long branches throughout the cycadalean phylogeny that, with few exceptions, have yet to be rectified by unequivocal fossil cycads. We report a permineralized pollen cone from the Campanian Holz Shale located in Silverado Canyon, CA, USA (c. 80 million yr ago). This fossil was studied via serial sectioning, SEM, 3D reconstruction and phylogenetic analyses. Microsporophyll and pollen morphology indicate this cone is assignable to Skyttegaardia, a recently described genus based on disarticulated lignitized microsporophylls from the Early Cretaceous of Denmark. Data from this new species, including a simple cone architecture, anatomical details and vasculature organization, indicate cycadalean affinities for Skyttegaardia. Phylogenetic analyses support this assignment and recover Skyttegaardia as crown-group Cycadales, nested within Zamiaceae. Our findings support a Cretaceous diversification for crown-group Zamiaceae, which included the evolution of morphological divergent extinct taxa with unique traits that have yet to be widely identified in the fossil record.

Correlations between leaf economics, mechanical resistance and drought tolerance across 41 cycad species.

BACKGROUND AND AIMS: We conducted a comprehensive analysis of the functional traits of leaves (leaflets) of cycads. The aim of this study was to clarify the functional divergence between the earlier origin Cycadaceae and the later differentiated Zamiaceae, and the differences in trait associations between cycads and angiosperms. METHODS: We selected 20 Cycadaceae species and 21 Zamiaceae species from the same cycad garden in South China, and measured their leaf structure, economic traits, mechanical resistance (Fp) and leaf water potential at the turgor loss point (πtlp). In addition, we compiled a dataset of geographical distribution along with climatic variables for these cycad species, and some leaf traits of tropical-sub-tropical angiosperm woody species from the literature for comparison. KEY RESULTS: The results showed significantly contrasting leaf trait syndromes between the two families, with Zamiaceae species exhibiting thicker leaves, higher carbon investments and greater Fp than Cycadaceae species. Leaf thickness (LT) and πtlp were correlated with mean climatic variables in their native distribution ranges, indicating their evolutionary adaptation to environmental conditions. Compared with the leaves of angiosperms, the cycad leaves were thicker and tougher, and more tolerant to desiccation. Greater Fp was associated with a higher structural investment in both angiosperms and cycads; however, cycads showed lower Fp at a given leaf mass per area or LT than angiosperms. Enhancement of Fp led to more negative πtlp in angiosperms, but the opposite trend was observed in cycads. CONCLUSIONS: Our results reveal that variations in leaf traits of cycads are mainly influenced by taxonomy and the environment of their native range. We also demonstrate similar leaf functional associations in terms of economics, but different relationships with regard to mechanics and drought tolerance between cycads and angiosperms. This study expands our understanding of the ecological strategies and likely responses of cycads to future climate change.

Insights from an ancient gymnosperm lineage: ambient temperature and light and the timing of thermogenesis in cycad cones.

PREMISE: Although maintaining the appropriate mid-day timing of the diel thermogenic events of cones of the dioecious cycads Macrozamia lucida and M. macleayi is central to the survival of both plant and pollinator in this obligate pollination mutualism, the nature of the underlying mechanism remains obscure. We investigated whether it is under circadian control. Circadian mechanisms control the timing of many ecologically important processes in angiosperms, yet only a few gymnosperms have been studied in this regard. METHODS: We subjected cones to different ambient temperature and lighting regimens (constant temperature and darkness; stepwise cool/warm ambient temperatures in constant darkness; stepwise dark/light exposures at constant temperature) to determine whether the resulting timing of their thermogenic events was consistent with circadian control. RESULTS: Cones exposed to constant ambient temperature and darkness generated multiple temperature peaks endogenously, with an average interpeak-temperature period of 20.7 (±0.20) h that is temperature-compensated (Q10 = 1.02). Exposure to 24-h ambient temperature cycles (12 h cool/12 h warm, constant darkness) yielded an interpeak-temperature period of 24.0 (±0.05) h, accurately and precisely replicating the ambient temperature period. Exposure to 24-h photo-cycles (12 h light/12 h dark, constant ambient temperature) yielded a shorter, more variable interpeak-temperature period of 23 (±0.23) h. CONCLUSIONS: Our results indicate that cycad cone thermogenesis is under circadian clock control and differentially affected by ambient temperature and light cycles. Our data from cycads (an ancient gymnosperm lineage) adds to what little is known about circadian timing in gymnosperms, which have rarely been studied from the circadian perspective.

Stomatal development in the cycad family Zamiaceae.

BACKGROUND AND AIMS: The gymnosperm order Cycadales is pivotal to our understanding of seed-plant phylogeny because of its phylogenetic placement close to the root node of extant spermatophytes and its combination of both derived and plesiomorphic character states. Although widely considered a 'living fossil' group, extant cycads display a high degree of morphological and anatomical variation. We investigate stomatal development in Zamiaceae to evaluate variation within the order and homologies between cycads and other seed plants. METHODS: Leaflets of seven species across five genera representing all major clades of Zamiaceae were examined at various stages of development using light microscopy and confocal microscopy. KEY RESULTS: All genera examined have lateral subsidiary cells of perigenous origin that differ from other pavement cells in mature leaflets and could have a role in stomatal physiology. Early epidermal patterning in a 'quartet' arrangement occurs in Ceratozamia, Zamia and Stangeria. Distal encircling cells, which are sclerified at maturity, are present in all genera except Bowenia, which shows relatively rapid elongation and differentiation of the pavement cells during leaflet development. CONCLUSIONS: Stomatal structure and development in Zamiaceae highlights some traits that are plesiomorphic in seed plants, including the presence of perigenous encircling subsidiary cells, and reveals a clear difference between the developmental trajectories of cycads and Bennettitales. Our study also shows an unexpected degree of variation among subclades in the family, potentially linked to differences in leaflet development and suggesting convergent evolution in cycads.

Niche conservatism promotes speciation in cycads: the case of Dioon merolae (Zamiaceae) in Mexico.

Niche conservatism is the tendency of lineages to retain the same niche as their ancestors. It constrains biological groups and prevents ecological divergence. However, theory predicts that niche conservatism can hinder gene flow, strengthen drift and increase local adaptation: does it mean that it also can facilitate speciation? Why does this happen? We aim to answer these questions. We examined the variation of chloroplast DNA, genome-wide single nucleotide polymorphisms, morphological traits and environmental variables across the Dioon merolae cycad populations. We tested geographical structure, scenarios of demographic history, and niche conservatism between population groups. Lineage divergence is associated with the presence of a geographical barrier consisting of unsuitable habitats for cycads. There is a clear genetic and morphological distinction between the geographical groups, suggesting allopatric divergence. However, even in contrasting available environmental conditions, groups retain their ancestral niche, supporting niche conservatism. Niche conservatism is a process that can promote speciation. In D. merolae, lineage divergence occurred because unsuitable habitats represented a barrier against gene flow, incurring populations to experience isolated demographic histories and disparate environmental conditions. This study explains why cycads, despite their ancient lineage origin and biological stasis, have been able to diversify into modern ecosystems worldwide.

Evolution of Ceratozamia cycads: A proximate-ultimate approach.

Evidence suggests that past climatic fluctuations affected speciation of extant cycads. However, empirical genetic and morphological evidence explaining patterns and processes of species diversification are scarce. There are some explanations for the origin and evolution of the genus Ceratozamia, but with inconclusive results. To elucidate the evolution of Ceratozamia, we used genetic and phenotypic sources as empirical data, which were applied in a 'proximate-ultimate' framework (ecological and evolutionary scale, respectively). Our results suggested that the evolutionary mechanisms of speciation were shaped by deterministic (natural selection-adaptation) driven by climatic conditions associated to water stress, and probably enhanced by stochastic processes (gene drift and inbreeding). In general terms, punctuated evolution models were those that best explained the patterns of speciation throughout the phylogenetic history of the lineages encompassed in the genus Ceratozamia. Finally, we provide empirical evidence on the tempo and mode of the evolution of a 'living plant fossil'.

Aridification as a driver of biodiversity: a case study for the cycad genus Dioon (Zamiaceae).

Background and Aims: Aridification is considered a selective pressure that might have influenced plant diversification. It is suggested that plants adapted to aridity diversified during the Miocene, an epoch of global aridification (≈15 million years ago). However, evidence supporting diversification being a direct response to aridity is scarce, and multidisciplinary evidence, besides just phylogenetic estimations, is necessary to support the idea that aridification has driven diversification. The cycad genus Dioon (Zamiaceae), a tropical group including species occurring from humid forests to arid zones, was investigated as a promising study system to understand the associations among habitat shifts, diversification times, the evolution of leaf epidermal adaptations, and aridification of Mexico. Methods: A phylogenetic tree was constructed from seven chloroplast DNA sequences and the ITS2 spacer to reveal the relationships among 14 Dioon species from habitats ranging from humid forests to deserts. Divergence times were estimated and the habitat shifts throughout Dioon phylogeny were detected. The epidermal anatomy among Dioon species was compared and correlation tests were performed to associate the epidermal variations with habitat parameters. Key Results: Events of habitat shifts towards arid zones happened exclusively in one of the two main clades of Dioon. Such habitat shifts happened during the species diversification of Dioon, mainly during the Miocene. Comparative anatomy showed epidermal differences between species from arid and mesic habitats. The variation of epidermal structures was found to be correlated with habitat parameters. Also, most of the analysed epidermal traits showed significant phylogenetic signals. Conclusions: The diversification of Dioon has been driven by the aridification of Mexico. The Miocene timing corresponds to the expansion of arid zones that embedded the ancestral Dioon populations. As response, species in arid zones evolved epidermal traits to counteract aridity stress. This case study provides a robust body of evidence supporting the idea that aridification is an important driver of biodiversity.

Pleistocene diversification in an ancient lineage: a role for glacial cycles in the evolutionary history of Dioon Lindl. (Zamiaceae).

PREMISE OF THE STUDY: Recent estimates of crown ages for cycad genera (Late Miocene) challenge us to consider what processes have produced the extant diversity of this ancient group in such relatively little time. Pleistocene climate change has driven major shifts in species distributions in Mexico and may have led to speciation in the genus Dioon by forcing populations to migrate up in elevation, thereby becoming separated by topography. METHODS: We inferred orthologs from transcriptomes of five species and sequenced these in 42 individuals representing all Dioon species. From these data and published plastid sequences, we inferred dated species trees and lineage-specific diversification rates. KEY RESULTS: Analyses of 84 newly sequenced nuclear orthologs and published plastid data confirm four major clades within Dioon, all of Pleistocene age. Gene tree analysis, divergence dates, and an increase in diversification rate support very recent and rapid divergence of extant taxa. CONCLUSIONS: This study confirms the Pleistocene age of Dioon species and implicates Pleistocene climate change and established topography in lineage spitting. These results add to our understanding of the cycads as evolutionarily dynamic lineages, not relicts or evolutionary dead ends. We also find that well-supported secondary calibration points can be reliable in the absence of fossils. Our hypothesis of lineage splitting mediated by habitat shifts may be applicable to other taxa that are restricted to elevation specific ecotones.

Shifting Quaternary migration patterns in the Bahamian archipelago: Evidence from the Zamia pumila complex at the northern limits of the Caribbean island biodiversity hotspot.

PREMISE OF THE STUDY: The Bahamas archipelago is formed by young, tectonically stable carbonate banks that harbor direct geological evidence of global ice-volume changes. We sought to detect signatures of major changes on gene flow patterns and reconstruct the phylogeographic history of the monophyletic Zamia pumila complex across the Bahamas. METHODS: Nuclear molecular markers with both high and low mutation rates were used to capture two different time scale signatures and test several gene flow and demographic hypotheses. KEY RESULTS: Single-copy nuclear genes unveiled apparent ancestral admixture on Andros, suggesting a significant role of this island as main hub of diversity of the archipelago. We detected demographic and spatial expansion of the Zamia pumila complex on both paleo-provinces around the Piacenzian (Pliocene)/Gelasian (Pleistocene). Populations evidenced signatures of different migration models that have occurred at two different times. Populations on Long Island (Z. lucayana) may either represent a secondary colonization of the Bahamas by Zamia or a rapid and early-divergence event of at least one population on the Bahamas. CONCLUSIONS: Despite changes in migration patterns with global climate, expected heterozygosity with both marker systems remains within the range reported for cycads, but with significant levels of increased inbreeding detected by the microsatellites. This finding is likely associated with reduced gene flow between and within paleo-provinces, accompanied by genetic drift, as rising seas enforced isolation. Our study highlights the importance of the maintenance of the predominant direction of genetic exchange and the role of overseas dispersion among the islands during climate oscillations.

Spatiotemporal features of microsporogenesis in the cycad species Macrozamia communis.

UNLABELLED: • PREMISE OF THE STUDY: Spatiotemporal features of microsporogenesis may provide important clues about the evolution of microsporogenesis in seed plants. One cellular feature that attracts special attention is advance cell wall ingrowths (ACWIs) at future cytokinetic sites in microsporocytes since they have been found only in species of an ancient lineage of angiosperms, Magnolia, and in much less detail, of an ancient lineage of gymnosperms, cycads. Further investigation into microsporogenesis in a cycad species may yield knowledge critical to understanding the establishment of ACWIs as an important feature for comparative studies of microsporogenesis in seed plants.• METHODS: Bright-field and epifluorescence microscopy, confocal laser scanning microscopy, and transmission electron microscopy were used to investigate the microsporogenic process in Macrozamia communis, a species in the Zamiaceae family of cycads.• KEY RESULTS: In prophase-II microsporocytes in M. communis, ACWIs form as a callose ring between the newly formed nuclei and are not accompanied by cytokinetic apparatuses such as mini-phragmoplasts, wide tubules, or wide tubular networks. Shortly after the second nuclear division, new ACWIs, albeit thinner than the previous ACWIs, form between the newly formed nuclei. Subsequent cell plate formation in the planes of the ACWIs typically results in tetragonal tetrads.• CONCLUSIONS: Cytokinesis at the cell periphery is initiated earlier than cell plate formation in the cell interior in microsporogenesis in M. communis. The cellular features uncovered in M. communis may serve as useful reference features for comparative studies of microsporogenesis in plants.

Leaf traits and herbivory levels in a tropical gymnosperm, Zamia stevensonii (Zamiaceae).

PREMISE OF THE STUDY: Slow-growing understory cycads invest heavily in defenses to protect the few leaves they produce annually. The Neotropical cycad Zamia stevensonii has chemical and mechanical barriers against insect herbivores. Mechanical barriers, such as leaf toughness, can be established only after the leaf has expanded. Therefore, chemical defenses may be important during leaf expansion. How changes in leaf traits affect the feeding activity of cycad specialist insects is unknown. We investigated leaf defenses and incidence of specialist herbivores on Z. stevensonii during the first year after leaf flush. METHODS: Herbivore incidence, leaf production, and leaf traits that might affect herbivory-including leaf age, lamina thickness, resistance-to-fracture, work-to-fracture, trichome density, and chlorophyll, water, and toxic azoxyglycoside (AZG) content-were measured throughout leaf development. Principal component analysis and generalized linear models identified characteristics that may explain herbivore incidence. KEY RESULTS: Synchronized leaf development in Z. stevensonii is characterized by quick leaf expansion and delayed greening. Specialist herbivores feed on leaves between 10 and 100 d after flush and damage ∼37% of all leaflets produced. Young leaves are protected by AZGs, but these defenses rapidly decrease as leaves expand. Leaves older than 100 d are protected by toughness. CONCLUSIONS: Because AZG concentrations drop before leaves become sufficiently tough, there is a vulnerable period during which leaves are susceptible to herbivory by specialist insects. This slow-growing gymnosperm invests heavily in constitutive defenses against highly specialized herbivores, underlining the convergence in defensive syndromes by major plant lineages.

Cytotoxic and antimicrobial mycophenolic acid derivatives from an endophytic fungus Penicillium sp. MNP-HS-2 associated with Macrozamia communis.

Macrozamia communis and its associated endophytic fungi are untapped sources of bioactive metabolites with great potential for medicinal exploitation. Chemical investigation of the mycelial extract derived from an endophytic fungus Penicillium sp. MNP-HS-2 associated with M. communis fruit afforded four mycophenolic acid derivatives recognized as previously undescribed natural products (1-4), together with nine known metabolites (5-13). Chemical structures of isolated compounds were determined based on extensive spectroscopic analyses, including 1D/2D NMR and HRESIMS. The absolute stereochemistry of alternatain E (1) was unambiguously established by comparing its experimental and calculated time-dependent density functional theory electronic circular dichroism spectra (TDDFT-ECD). All isolated compounds were assessed for their antimicrobial and cytotoxic activities, where mycophenolic acid methyl ester (7), displayed significant cytotoxic activity against seven different cell lines with IC50 values in the low micromolar to nanomolar range. Mycophenolene A (3) exhibited significant antibacterial activity against Staphylococcus aureus (MIC = 2.1 µg/mL).

Patterns of odour emission, thermogenesis and pollinator activity in cones of an African cycad: what mechanisms apply?

BACKGROUND AND AIMS: Ontogenetic patterns of odour emissions and heating associated with plant reproductive structures may have profound effects on insect behaviour, and consequently on pollination. In some cycads, notably Macrozamia, temporal changes in emission of specific odour compounds and temperature have been interpreted as a 'push-pull' interaction in which pollinators are either attracted or repelled according to the concentration of the emitted volatiles. To establish which mechanisms occur in the large Encephalartos cycad clade, the temporal patterns of volatile emissions, heating and pollinator activity of cones of Encephalartos villosus in the Eastern Cape (EC) and KwaZulu Natal (KZN) of South Africa were investigated. METHODS AND KEY RESULTS: Gas chromatography-mass spectrometry (GC-MS) analyses of Encephalartos villosus cone volatiles showed that emissions, dominated by eucalyptol and 2-isopropyl-3-methoxypyrazine in EC populations and (3E)-1,3-octadiene and (3E,5Z)-1,3,5-octatriene in the KZN populations, varied across developmental stages but did not vary significantly on a daily cycle. Heating in male cones was higher at dehiscence than during pre- and post-dehiscence, and reached a maximum at about 1830 h when temperatures were between 7·0 and 12·0 °C above ambient. Daily heating of female cones was less pronounced and reached a maximum at about 1345 h when it was on average between 0·9 and 3·0 °C above ambient. Insect abundance on male cones was higher at dehiscence than at the other stages and significantly higher in the afternoon than in the morning and evening. CONCLUSIONS: There are pronounced developmental changes in volatile emissions and heating in E. villosus cones, as well as strong daily changes in thermogenesis. Daily patterns of volatile emissions and pollinator abundance in E. villosus are different from those observed in some Macrozamia cycads and not consistent with the push-pull pattern as periods of peak odour emission do not coincide with mass exodus of insects from male cones.

Seed dispersal of the Australian cycad Macrozamia miquelii (Zamiaceae): are cycads megafauna-dispersed "grove forming" plants?

PREMISE OF THE STUDY: Plants that invest in large, heavy seeds and colorful, fleshy fruits or analogous structures seem adapted for dispersal by large vertebrates. Some such plants, like Australian cycads in the genus Macrozamia, do not disperse well, which could be explained by seed-dispersal relationships with megafauna that are rare or extinct in contemporary ecosystems. Such plants provide an opportunity to investigate the ecological consequences of low seed-dispersal distances. • METHODS: We investigated seed dispersal of Macrozamia miquelii in Central Queensland by tracking the fate of marked seeds, identifying the dispersal fauna and quantifying population demography and spatial structure. • KEY RESULTS: We found that 70-100% of marked seeds remained within 1 m of maternal females (cycads are dioecious). Of the 812 seeds recovered (from 840 originally marked) only 24 dispersed >1 m from maternal females, the greatest observed dispersal being 5 m. We found an average of 2.2 seedlings and 0.7 juveniles within 1.5 m of mature females, which suggests that most seeds that remain in the vicinity of maternal females perish. Within-stand densities ranged between 1000 and 5000 plants/ha. The brushtail possum Trichosurus vulpecula was the only animal observed to move the seeds. • CONCLUSIONS: Macrozamia are adapted for dispersal by megafauna that are rare or absent in contemporary ecosystems. We argue that Macrozamia are "grove forming" plants that derive ecological benefit from existing as high-density, spatially discrete populations, the function of megafaunal dispersal adaptations being the infrequent dispersal of seeds en masse to establish new such groves in the landscape.

Unique chemistry associated with diversification in a tightly coupled cycad-thrips obligate pollination mutualism.

Cycad cone thermogenesis and its associated volatiles are intimately involved in mediating the behavior of their obligate specialist pollinators. In eastern Australia, thrips in the Cycadothrips chadwicki species complex are the sole pollinators of many Macrozamia cycads. Further, they feed and reproduce entirely in the pollen cones. M. miquelii, found only in the northern range of this genus, is pollinated only by a C. chadwicki cryptic species that is the most distantly related to others in the complex. We examined the volatile profile from M. miquelii pollen and ovulate (receptive and non-receptive) cones to determine how this mediates pollination mechanistically, using GC-MS (gas chromatography-mass spectrometry) and behavioral tests. Monoterpenes comprise the bulk of M. miquelii volatile emissions, as in other Macrozamia species, but we also identified compounds not reported previously in any cycad, including three aliphatic esters (prenyl acetate and two of uncertain identity) and two aliphatic alcohols. The two unknown esters were confirmed as prenyl (3-methylbut-2-enyl) esters of butyric and crotonic ((E))-but-2-enoic) acids after chemical synthesis. Prenyl crotonate is a major component in emissions from pollen and receptive ovulate cones, is essentially absent from non-receptive cones, and has not been reported from any other natural source. In field bioassays, Cycadothrips were attracted only to those volatile treatments containing prenyl crotonate. We discuss M. miquelii cone odorants relative to those of other cycads, especially with respect to prenyl crotonate being a species-specific signal to this northern C. chadwicki cryptic species, and how this system may have diversified.

The complete chloroplast genome of Microcycas calocoma (Miq.) A. DC. (Zamiaceae, Cycadales) and evolution in Cycadales.

Cycadales is an extant group of seed plants occurring in subtropical and tropical regions comprising putatively three families and 10 genera. At least one complete plastid genome sequence has been reported for all of the 10 genera except Microcycas, making it an ideal plant group to conduct comprehensive plastome comparisons at the genus level. This article reports for the first time the plastid genome of Microcycas calocoma. The plastid genome has a length of 165,688 bp with 134 annotated genes including 86 protein-coding genes, 47 non-coding RNA genes (39 tRNA and eight rRNA) and one pseudogene. Using global sequence variation analysis, the results showed that all cycad genomes share highly similar genomic profiles indicating significant slow evolution and little variation. However, identity matrices coinciding with the inverted repeat regions showed fewer similarities indicating that higher polymorphic events occur at those sites. Conserved non-coding regions also appear to be more divergent whereas variations in the exons were less discernible indicating that the latter comprises more conserved sequences. Phylogenetic analysis using 81 concatenated protein-coding genes of chloroplast (cp) genomes, obtained using maximum likelihood and Bayesian inference with high support values (>70% ML and = 1.0 BPP), confirms that Microcycas is closest to Zamia and forms a monophyletic clade with Ceratozamia and Stangeria. While Stangeria joined the Neotropical cycads Ceratozamia, Zamia and Microcyas, Bowenia grouped with the Southern Hemisphere cycads Encephalartos, Lepidozamia and Macrozamia. All Cycas species formed a distinct clade separated from the other genera. Dioon, on the other hand, was outlying from the rest of Zamiaceae encompassing two major clades-the Southern Hemisphere cycads and the Neotropical cycads. Analysis of the whole cp genomes in phylogeny also supports that the previously recognized family-Stangeriaceae-which contained Bowenia and Stangeria, is not monophyletic. Thus, the cp genome topology obtained in our study is congruent with other molecular phylogenies recognizing only a two-family classification (Cycadaceae and Zamiaceae) within extant Cycadales.

Ethnobotany of Mexican and northern Central American cycads (Zamiaceae).

BACKGROUND: This study documents cycad-human relationships in Mexico, Belize, Guatemala, El Salvador, and Honduras over the last 6000 years. The impetus was acute need for a better understanding of previously undocumented uses of cycads in this region, and the need to improve cycad conservation strategies using ethnobotanical data. We hypothesized that cycads are significant dietary items with no long-term neurological effects, are important to religious practice, and contribute to cultural identity and sense of place, but that traditional knowledge and uses are rapidly eroding. Guiding questions focused on nomenclature, food and toxicity, relationships to palms and maize, land management issues, roles in religious ceremony, and medicinal uses, among others, and contributions of these to preservation of cycads. METHODS: From 2000 to 2017, the authors conducted 411 semi-structured ethnographic interviews, engaged in participant-observation in Mexican and Honduran communities, and carried out archival research and literature surveys. RESULTS: We documented 235 terms and associated uses that 28 ethnic groups have for 57 species in 19 languages across 21 Mexican states and 4 Central American nations. Carbohydrate-rich cycads have been both famine foods and staples for at least six millennia across the region and are still consumed in Mexico and Honduras. Certain parts are eaten without removing toxins, while seed and stem starches are detoxified via several complex processes. Leaves are incorporated into syncretic Roman Catholic-Mesoamerican religious ceremonies such as pilgrimages, Easter Week, and Day of the Dead. Cycads are often perceived as ancestors and protectors of maize, revealing a close relationship between both groups. Certain beliefs and practices give cycads prominent roles in conceptions of sense of place and cultural heritage. CONCLUSIONS: Cycads are still used as foods in many places. Though they do not appear to cause long-term neurological damage, their health effects are not fully understood. They are often important to religion and contribute to cultural identity and sense of place. However, because most traditional knowledge and uses are rapidly eroding, new community-based biocultural conservation efforts are needed. These should incorporate tradition where possible and seek inspiration from existing successful cases in Honduras and Mexico.

Best in the company of nearby males: female success in the threatened cycad, Zamia portoricensis.

Variation in plant reproductive success is affected by ecological conditions including the proximity of potential mates. We address the hypothesis that spatial distribution of sexes affects female reproductive success (RS) in the dioecious cycad, Zamia portoricensis. Are the frequencies of males, operational sex ratios, and distances to the nearest mate associated with RS in females? We studied the spatial distribution of sexes in two populations in Puerto Rico and compared RS of target females with the number of males and operational sex ratios. Population structure suggests regular successful recruitment. Adults, males, and females were randomly distributed with respect to one another. Reproductive success of females was highly variable, but was higher in neighborhoods with more males than females and generally decreased with increasing distance to the nearest male, becoming statistically significant beyond 190 cm. This possible mate-finding Allee effect indicates that pollinator movement among plants may be limited for this mutually dependent plant-pollinator interaction. Yet being close to male plants is a matter of chance, perhaps a factor generating the high intra-population genetic diversity in Z. portoricensis.