Two new species of Pharaxonothinae beetles (Coleoptera: Erotylidae) inhabiting cones of the cycad Ceratozamia santillanii Prez-Farr. amp; Vovides (Cycadales: Zamiaceae) in Mexico.

Two beetles, Ceratophila (Vovidesa) chipjonesi, new species, and Pharaxonotha perezi, new species, (Coleoptera: Erotylidae: Pharaxonothinae) are described from the male cones of the New World cycad Ceratozamia santillanii Prez-Farr. Vovides (Cycadales: Zamiaceae). Morphological analysis and keys are provided for distinguishing these from related beetles inhabiting Ceratozamia in Mexico.

Phylotranscriptomics reveal the spatio-temporal distribution and morphological evolution of Macrozamia, an Australian endemic genus of Cycadales.

BACKGROUND AND AIMS: Cycads are regarded as an ancient lineage of living seed plants, and hold important clues to understand the early evolutionary trends of seed plants. The molecular phylogeny and spatio-temporal diversification of one of the species-rich genera of cycads, Macrozamia, have not been well reconstructed. METHODS: We analysed a transcriptome dataset of 4740 single-copy nuclear genes (SCGs) of 39 Macrozamia species and two outgroup taxa. Based on concatenated (maximum parsimony, maximum likelihood) and multispecies coalescent analyses, we first establish a well-resolved phylogenetic tree of Macrozamia. To identify cyto-nuclear incongruence, the plastid protein coding genes (PCGs) from transcriptome data are extracted using the software HybPiper. Furthermore, we explore the biogeographical history of the genus and shed light on the pattern of floristic exchange between three distinct areas of Australia. Six key diagnostic characters are traced on the phylogenetic framework using two comparative methods, and infra-generic classification is investigated. KEY RESULTS: The tree topologies of concatenated and multi-species coalescent analyses of SCGs are mostly congruent with a few conflicting nodes, while those from plastid PCGs show poorly supported relationships. The genus contains three major clades that correspond to their distinct distributional areas in Australia. The crown group of Macrozamia is estimated to around 11.80 Ma, with a major expansion in the last 5-6 Myr. Six morphological characters show homoplasy, and the traditional phenetic sectional division of the genus is inconsistent with this current phylogeny. CONCLUSIONS: This first detailed phylogenetic investigation of Macrozamia demonstrates promising prospects of SCGs in resolving phylogenetic relationships within cycads. Our study suggests that Macrozamia, once widely distributed in Australia, underwent major extinctions because of fluctuating climatic conditions such as cooling and mesic biome disappearance in the past. The current close placement of morphologically distinct species in the phylogenetic tree may be related to neotenic events that occurred in the genus.

Discriminatory of some morphometry and biological aspects of the Cycad blue butterfly, Chilades pandava reared on Cycas and Zamia ornamental palms.

The serious blue butterfly, Chilades pandava -Horsfield, 1829- (Lepidoptera: Lycaenidae) is consider one of the main destructive insect pests for ornamental palms Cycas and Zamia. Biological and morphological measurements were carried out of C. pandava stages reared on Cycas revoluta (Cycadaceae) and Zamia encephalartoides (Zamiaceae). In description details, non-significant variations were recorded between the two gender of cycad blue butterfly in the obtained data, but the male adult was more densely blue or violet than female adult. By the aid of SEM, C. pandava all stages were distinct by long and thick hairs covered all the body. The morphometric characters namely, length, width and venation of wings, body length, forewing, hindwing could be as a guide for taxonomic discrimination. The data showed that the life cycle duration of C. pandava was ranged between 20.64 to 21.7 days. The developmental periods of different C. pandava stages are slightly higher on zamia than cycas palms. This investigation detected that a high survival rate was found on Cycas palms (86%) than the survived rate recording on Zamia palms (82%). In the present study, the described morphometric characters could be used as a guide for taxonomic discrimination of this pest. Consequently, this study added a valuable knowledge about C. pandava to have sound decisions for proposal of its management and conservation in Egypt.

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.

Histological assessment, anti-quorum sensing, and anti-biofilm activities of Dioon spinulosum extract: in vitro and in vivo approach.

Pseudomonas aeruginosa is an opportunistic bacterium causing several health problems and having many virulence factors like biofilm formation on different surfaces. There is a significant need to develop new antimicrobials due to the spreading resistance to the commonly used antibiotics, partly attributed to biofilm formation. Consequently, this study aimed to investigate the anti-biofilm and anti-quorum sensing activities of Dioon spinulosum, Dyer Ex Eichler extract (DSE), against Pseudomonas aeruginosa clinical isolates. DSE exhibited a reduction in the biofilm formation by P. aeruginosa isolates both in vitro and in vivo rat models. It also resulted in a decrease in cell surface hydrophobicity and exopolysaccharide quantity of P. aeruginosa isolates. Both bright field and scanning electron microscopes provided evidence for the inhibiting ability of DSE on biofilm formation. Moreover, it reduced violacein production by Chromobacterium violaceum (ATCC 12,472). It decreased the relative expression of 4 quorum sensing genes (lasI, lasR, rhlI, rhlR) and the biofilm gene (ndvB) using qRT-PCR. Furthermore, DSE presented a cytotoxic activity with IC50 of 4.36 ± 0.52 µg/ml against human skin fibroblast cell lines. For the first time, this study reports that DSE is a promising resource of anti-biofilm and anti-quorum sensing agents.

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.

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.

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.

Arabinogalactan-proteins from non-coniferous gymnosperms have unusual structural features.

Arabinogalactan-proteins (AGPs), important signalling molecules of the plant cell wall, are structurally extensively investigated in angiosperms, but information on AGPs in gymnosperms is still limited. We characterized AGPs from the gymnosperms Ginkgo biloba, Ephedra distachya, Encephalartos longifolius and Cycas revoluta. The protein contents are comparable to that of angiosperm AGPs. Hydroxyproline is the site of linking the carbohydrate part and was detected in all AGPs with highest concentration in Cycas AGP (1.1 % of the AGP). Interestingly, with the exception of Cycas, all AGPs contained the monosaccharide 3-O-methylrhamnose not present in angiosperm polysaccharides. The carbohydrate moieties of Cycas and Ephredra showed the main components 1,3,6-linked galactose and terminal arabinose typical of angiosperm AGPs, whereas that of Ginkgo AGP was unique with 1,4-linked galactose as dominant structural element. Bioinformatic search for glycosyltransferases in Ginkgo genome also revealed a lower number of galactosyltransferases responsible for biosynthesis of the 1,3-Gal/1,6-Gal AGP backbone.

Hepatoprotective, cytotoxic, antimicrobial and antioxidant activities of Dioon spinulosum leaves Dyer Ex Eichler and its isolated secondary metabolites.

Given the lack of adequate research on Dioon spinulosum (D. spinulosum) Dyer Ex Eichler, this study was conducted focusing on different biological activities and phytochemical investigation of D. spinulosum for the first time. D. spinulosum showed strong protective activity against DNA damage and potent activity against VERO cell line. It also presented antimicrobial and hepatoprotective activity. Phytochemical investigation of the leaves resulted in isolation of two new flavonoids, apigenin 7-O-α-d-glucopyranoside (15) and amentoflavone 7-O-α-l-rhamnopyranoside (16), in addition to fifteen known compounds: phytone (1), trans-phytol (2), ß-sitosterol (3), stigmasterol (4), oliveriflavone (5), 7,4',7″,4″'-tetramethylamentoflavone (6), 7,4',7''-trimethylamentoflavone (7), scaidopitysin (8), bilobetin (9), isoginkgetin (10), aromadendrin (11), sotusflavone (12), engeletin (14) and eriocitrin (17) for the first time together with amentoflavone (13). Compounds (11) and (13) displayed very strong cytotoxic activity and showed the highest protective activity against DNA damage.

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'.

Unlocking a high bacterial diversity in the coralloid root microbiome from the cycad genus Dioon.

Cycads are among the few plants that have developed specialized roots to host nitrogen-fixing bacteria. We describe the bacterial diversity of the coralloid roots from seven Dioon species and their surrounding rhizosphere and soil. Using 16S rRNA gene amplicon sequencing, we found that all coralloid roots are inhabited by a broad diversity of bacterial groups, including cyanobacteria and Rhizobiales among the most abundant groups. The diversity and composition of the endophytes are similar in the six Mexican species of Dioon that we evaluated, suggesting a recent divergence of Dioon populations and/or similar plant-driven restrictions in maintaining the coralloid root microbiome. Botanical garden samples and natural populations have a similar taxonomic composition, although the beta diversity differed between these populations. The rhizosphere surrounding the coralloid root serves as a reservoir and source of mostly diazotroph and plant growth-promoting groups that colonize the coralloid endosphere. In the case of cyanobacteria, the endosphere is enriched with Nostoc spp and Calothrix spp that are closely related to previously reported symbiont genera in cycads and other early divergent plants. The data reported here provide an in-depth taxonomic characterization of the bacterial community associated with coralloid root microbiome. The functional aspects of the endophytes, their biological interactions, and their evolutionary history are the next research step in this recently discovered diversity within the cycad coralloid root microbiome.

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.

A Study on Infestation Factors of Cycas and Zamia Palms with Butterfly, Chilades pandava and its Control in Egypt.

BACKGROUND AND OBJECTIVE: Cycas revoluta and Zamia encephalartoides were commercially ornamental palms. Butterfly, Chilades pandava was an important pest of ornamental palms either cycas or zamia. Impact factors on C. pandava infestations on cycas and zamia palms were studied. MATERIALS AND METHOD: Two field experiments were carried out during the period from 1st January-15th December, 2018 in a private palm nursery at Abu-Ghaleb village, Giza, Egypt to study the infestation of C. pandava on cycas and zamia palms and also, provided its control strategies. RESULTS: The infested percent of C. pandava was recorded the highest values at 1st week of May and September, 2018 with 63.89 % on cycas palms. Whereas, the high value of the infestation percent was 66.67% on zamia palms. A positive effect was reported with maximum and minimum temperatures but a negative effect was recorded with average RH% on C. pandava infestations. The increasing of the C. pandava infestations decreased these 2 plant enzymes, peroxidase and phenoloxidase. The average reduction percentages of the tested 9 pesticides against C. pandava infestations on cycas palms were markedly higher in case of sulfur 70% SC and fipronil 80% WG being 69.88 and 61.30% reductions than other treatments after 3 sequential applications throughout 3 months, respectively. CONCLUSION: Chilades pandava infestation was higher on cycas palms than zamia palms. Sulfur and Fipronil were more efficacy pesticides against this pest.

Ceratophila, a new genus of erotylid beetles (Erotylidae: Pharaxonothinae) inhabiting male cones of the cycad Ceratozamia (Cycadales: Zamiaceae).

Ceratophila new genus (Erotylidae: Pharaxonothinae) inhabiting male cones of the Mesoamerican cycad genus Ceratozamia Brongniart (Zamiaceae) is described for seven new species and compared to other genera of the Pharaxonothinae. Ceratophila differs from other genera of the Pharaxonothinae in many characters, most notable of which are: lateral pronotal carina thicker anteriorly in lateral view; head with supraocular and transverse occipital lines; elytra lacking a basal bead; and stridulatory files at base of head narrowly separated. Ceratophila has the male genitalia laterally compressed with a short median lobe and flagellum, similar to other Erotylidae. In comparison, the most superficially similar pharaxonothine genus Pharaxonotha Reitter has: lateral pronotal carina thin along entire length; head lacking supraocular lines, but with transverse occipital lines; elytra with distinct basal bead; stridulatory files at base of head widely separated; and unique male genitalia that are dorsoventrally flattened with a long median lobe and flagellum. Ceratophila contains two subgenera, which differ most notably in external characters and sexual dimorphism. Members of Ceratophila (Ceratophila), the nominal subgenus, have: more cylindrical bodies, pronotum evenly convex dorsally; the pronotum lacking basal longitudinal grooves on disc; metatibiae that are distinctly triangularly dilated toward apex; and no apparent external sexual dimorphism. In comparison, members of Ceratophila (Vovidesa) new subgenus have: dorsoventrally flattened bodies; the pronotum explanate (concave) along lateral margins; the pronotum with basal longitudinal grooves on disc; metatibiae weakly or not dilated toward apex; and with all tibiae in males showing strong sexual dimorphism. Four new species are described within the subgenus Ceratophila: C. (C.) chemnicki, C. (C.) gregoryi, C. (C.) picipennis, C. (C.) sanchezae; and three new species are described within the subgenus Vovidesa: C. (V.) chiapensis, C. (V.) mixeorum, C. (V.) vazquezi. Keys to genera, subgenera and species of known adults based on morphology are provided.

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.

The phylogeography of the cycad genus Dioon (Zamiaceae) clarifies its Cenozoic expansion and diversification in the Mexican transition zone.

Background and Aims: Biogeographic transition zones are promising areas to study processes of biogeographic evolution and its influence on biological groups. The Mexican transition zone originated due to the overlap of Nearctic and Neotropical biota, which promoted great biological diversification. However, since most previous studies in this area were focused on revealing the phylogeography of Nearctic plants, how historical biogeographic configuration influenced the expansion and diversification of the Neotropical flora remains almost unknown. Using the cycad genus Dioon (Zamiaceae), this study aimed to test whether the biogeographic provinciality of the Mexican transition zone reflects the history of diversification of Neotropical plants. Methods: Two chloroplast DNA (cpDNA) regions were analysed from 101 specimens of 15 Dioon species to reveal the distribution of haplogroups. In addition, genome-wide single nucleotide polymorphisms (SNPs) from 84 specimens were used to test the concordance between phylogenetic clusters and the biogeographic provinces. An ultrametric tree was constructed from the sequences containing SNPs to reconstruct the biogeographic events of vicariance and dispersal of Dioon across the Neotropical biogeographic provinces. Key Results: Four Dioon lineages with strong phylogeographic structures were recognized using both cpDNA and SNP data. The lineages correspond to two clades that originated from a common ancestor in Eastern Mexico. One clade expanded and diversified in South-east Mexico and Central America. Another clade diversified into three lineages that dispersed to North-east, South and North-west Mexico. Each lineage was biogeographically delimitated. Biogeographic provinces might have provided disparate ecological conditions that facilitated speciation in Dioon since the Miocene. Conclusions: The current genetic structure and species diversity of Dioon depict the history of expansion and diversification of the northernmost Neotropical provinces. Past biogeographic connectivities were favoured by elevated topographies, since mountain systems served as corridors for the migration of Dioon and as refugia of tropical communities that diversified during the formation of modern Neotropical forests.

Geographic variation in cone volatiles and pollinators in the thermogenic African cycad Encephalartos ghellinckii Lem.

Heat and odour production can have profound effects on pollination in cycads. It is therefore expected that these traits would co-vary geographically with pollinator assemblages. Such intraspecific variation, may lead to the evolution of pollination ecotypes, which can be an early stage of pollinator-mediated speciation. We measured cone temperatures using miniature temperature data loggers and examined the composition of cone volatile odours using headspace sampling and analysis with gas chromatography-mass spectrometry in four populations spanning the range of the African cycad Encephalartos ghellinckii. Pollinator assemblages were also investigated in three populations. Male and female cones were thermogenic at pollen shed and receptive stages, respectively, but patterns of thermogenesis did not vary among populations. Scent emissions from cones in populations in the Drakensberg Mountains were characterised by cis-ß-ocimene, ß-myrcene and (3E)-1,3-octadiene, while camphene and α-pinene were characteristic of scent emissions from cones in populations closer to the coast. These differences in volatile blends corresponded with differences in insect assemblages. These results confirm intraspecific variation in volatile emissions of E. ghellinckii and support the predictions that intraspecific variation in volatile emissions will be associated with shifts in pollinator assemblages. While further work needs to be done to test for local adaptation in this system, this preliminary evidence is consistent with the formation of pollination ecotypes in the E. ghellinckii species complex.

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.