Eco-evolutionary evidence for the global diversity pattern of Cycas (Cycadaceae).

The evolution of the latitudinal diversity gradient (LDG), characterized by a peak in diversity toward the tropics, has captured significant attention in evolutionary biology and ecology. However, the inverse LDG (i-LDG) mechanism, wherein species richness increases toward the poles, remains inadequately explored. Cycads are among one of the oldest lineages of extant seed plants and have undergone extensive diversification in the tropics. Intriguingly, the extant cycad abundance exhibits an i-LDG pattern, and the underlying causes for this phenomenon remain largely elusive. Here, using 1,843 nuclear genes from a nearly complete sampling, we conducted comprehensive phylogenomic analyses to establish a robust species-level phylogeny for Cycas, the largest genus within cycads. We then reconstructed the spatial-temporal dynamics and integrated global environmental data to evaluate the roles of species ages, diversification rates, contemporary environment, and conservatism to ancestral niches in shaping the i-LDG pattern. We found Cycas experienced decreased diversification rates, coupled with the cooling temperature since its origin in the Eocene from continental Asia. Different regions have distinctively contributed to the formation of i-LDG for Cycas, with the northern hemisphere acting as evolutionary museums and the southern hemisphere serving as cradles. Moreover, water-related climate variables, specifically precipitation seasonality and potential evapotranspiration, were identified as paramount factors constraining Cycas species richness in the rainforest biome near the equator. Notably, the adherence to ancestral monsoonal climates emerges as a critical factor in sustaining the diversity pattern. This study underscores the imperative of integrating both evolutionary and ecological approaches to comprehensively unravel the mechanisms underpinning global biodiversity patterns.

The Cycas genome and the early evolution of seed plants.

Cycads represent one of the most ancient lineages of living seed plants. Identifying genomic features uniquely shared by cycads and other extant seed plants, but not non-seed-producing plants, may shed light on the origin of key innovations, as well as the early diversification of seed plants. Here, we report the 10.5-Gb reference genome of Cycas panzhihuaensis, complemented by the transcriptomes of 339 cycad species. Nuclear and plastid phylogenomic analyses strongly suggest that cycads and Ginkgo form a clade sister to all other living gymnosperms, in contrast to mitochondrial data, which place cycads alone in this position. We found evidence for an ancient whole-genome duplication in the common ancestor of extant gymnosperms. The Cycas genome contains four homologues of the fitD gene family that were likely acquired via horizontal gene transfer from fungi, and these genes confer herbivore resistance in cycads. The male-specific region of the Y chromosome of C. panzhihuaensis contains a MADS-box transcription factor expressed exclusively in male cones that is similar to a system reported in Ginkgo, suggesting that a sex determination mechanism controlled by MADS-box genes may have originated in the common ancestor of cycads and Ginkgo. The C. panzhihuaensis genome provides an important new resource of broad utility for biologists.

Towards the plastome evolution and phylogeny of Cycas L. (Cycadaceae): molecular-morphology discordance and gene tree space analysis.

BACKGROUND: Plastid genomes (plastomes) present great potential in resolving multiscale phylogenetic relationship but few studies have focused on the influence of genetic characteristics of plastid genes, such as genetic variation and phylogenetic discordance, in resolving the phylogeny within a lineage. Here we examine plastome characteristics of Cycas L., the most diverse genus among extant cycads, and investigate the deep phylogenetic relationships within Cycas by sampling 47 plastomes representing all major clades from six sections. RESULTS: All Cycas plastomes shared consistent gene content and structure with only one gene loss detected in Philippine species C. wadei. Three novel plastome regions (psbA-matK, trnN-ndhF, chlL-trnN) were identified as containing the highest nucleotide variability. Molecular evolutionary analysis showed most of the plastid protein-coding genes have been under purifying selection except ndhB. Phylogenomic analyses that alternatively included concatenated and coalescent methods, both identified four clades but with conflicting topologies at shallow nodes. Specifically, we found three species-rich Cycas sections, namely Stangerioides, Indosinenses and Cycas, were not or only weakly supported as monophyly based on plastomic phylogeny. Tree space analyses based on different tree-inference methods both revealed three gene clusters, of which the cluster with moderate genetic properties showed the best congruence with the favored phylogeny. CONCLUSIONS: Our exploration in plastomic data for Cycas supports the idea that plastid protein-coding genes may exhibit discordance in phylogenetic signals. The incongruence between molecular phylogeny and morphological classification reported here may largely be attributed to the uniparental attribute of plastid, which cannot offer sufficient information to resolve the phylogeny. Contrasting to a previous consensus that genes with longer sequences and a higher proportion of variances are superior for phylogeny reconstruction, our result implies that the most effective phylogenetic signals could come from loci that own moderate variation, GC content, sequence length, and underwent modest selection.

Not that young: combining plastid phylogenomic, plate tectonic and fossil evidence indicates a Palaeogene diversification of Cycadaceae.

BACKGROUND AND AIMS: Previous molecular dating studies revealed historical mass extinctions and recent radiations of extant cycads, but debates still exist between palaeobotanists and evolutionary biologists regarding the origin and evolution of Cycadaceae. METHODS: Using whole plastomic data, we revisited the phylogeny of this family and found the Palawan endemic Cycas clade was strongly related to all lineages from Southeast Eurasia, coinciding with a plate drift event occurring in the Early Oligocene. By integrating fossil and biogeographical calibrations as well as molecular data from protein-coding genes, we established different calibration schemes and tested competing evolutionary timelines of Cycadaceae. KEY RESULTS: We found recent dispersal cannot explain the distribution of Palawan Cycas, yet the scenario including the tectonic calibration yielded a mean crown age of extant Cycadaceae of ~69-43 million years ago by different tree priors, consistent with multiple Palaeogene fossils assigned to this family. Biogeographical analyses incorporating fossil distributions revealed East Asia as the ancestral area of Cycadaceae. CONCLUSIONS: Our findings challenge the previously proposed Middle-Late Miocene diversification of cycads and an Indochina origin for Cycadaceae and highlight the importance of combining phylogenetic clades, tectonic events and fossils for rebuilding the evolutionary history of lineages that have undergone massive extinctions.

Congruence between ocean-dispersal modelling and phylogeography explains recent evolutionary history of Cycas species with buoyant seeds.

Ocean currents play a significant role in driving the long-distance dispersal (LDD), spatial distribution and phylogeographic patterns of many organisms. Integrating phylogeographic analyses and mechanistic ocean current modelling can provide novel insights into the evolutionary history of terrestrial littoral species but has been rarely applied in this context. We focused on a group of Cycas that have buoyant seeds and occupy coastal habitats. By integrating evidence from mechanistic simulations and whole plastomic data, we examined the role of ocean circulation in shaping the phylogeography of these Cycas species. Plastomes of the studied Cycas species showed extreme conservatism, following a post-Pleistocene divergence. Phylogenies revealed three subclades, corresponding to the Pacific Ocean, Sunda Shelf and Indian Ocean. The ocean modelling results indicate that hotspots of seed stranding coincide well with the contemporary distribution of the Cycas species and that drifting trajectories from the three subclades are largely confined to separate regions. These findings suggest that ocean current systems, by driving long-distance dispersal, have shaped the distribution and phylogeography for Cycas with buoyant seeds. This study highlights how the combination of genomic data and ocean drift modelling can help explain phylogeographic patterns and diversity in terrestrial littoral ecosystems.

Diel patterns of stem CO2 efflux vary among cycads, arborescent monocots, and woody eudicots and gymnosperms.

The diel patterns of stem carbon dioxide efflux (Es) were determined for cycads, monocots, and woody eudicot and gymnosperm tree species. Stem Es at a height of 30-40 cm was measured every 2 h throughout 31-h campaigns. Our range of Es was 1.5-4.0 µmol·m-2·s-1 for cycads, 1.0-3.5 µmol·m-2·s-1 for arborescent monocots, and 1.5-4.5 µmol·m-2·s-1 for woody eudicot and gymnosperm trees species. Time of day did not influence Es of cycads or monocots. In contrast, the woody stems of eudicots and gymnosperms exhibited diurnal Es that was 36% to 40% greater than nocturnal Es. The established literature based on Es of woody tree species cannot be used to estimate habitat carbon cycles in habitats which contain cycad or monocot trees. Time of day must be included for accuracy of research on Es of woody tree species. Failures to account for the spatiotemporal differences of Es may explain some of the disparity in outcomes of published stem respiration studies.

Characterization of the complete chloroplast genome of Microcycas calocoma (Zamiaceae), an Endangered monotypic cycad species from Cuba.

Microcycas calocoma is the monotypic species from the critical endangered and endemic cycad genus Microcycas in Cuba, an important taxon to study the evolution of extant gymnosperms. Here we report the complete chloroplast sequences of M. calocoma and characterize the genome structure of this species. The genome size of M. calocoma is 165,667 bp in length which contains 135 genes, including 88 protein-coding genes, eight ribosomal RNA genes and 39 transfer RNA genes. The GC content of this genome is 39.6%. Phylogenomic study shows M. calocoma is mostly closely related to the cycad genus Zamia, which corresponds to previous studies based on multiple nuclear genes and combined plastid and nuclear evidence. The plastome information of M. calocoma offered by this study can contribute to further comparative chloroplast genome in cycads/gymnosperms as well as conservation genetic studies of M. calocoma.

Inserting cycads into global nutrient relations data sets.

Global research agendas on plant nutrient relations attempt to illuminate biotic and abiotic factors that mediate nutrient relations. We contend that cycad species are not adequately represented in these global agendas. Little is known about how various cycad traits such as phylogenetics, growth form, latitudinal range, or ecological niche influence concentration, stoichiometry, and resorption dynamics of leaf nutrients. The addition of cycad species data to the global research dataset will address a critical knowledge gap and benefit global research needs to improve our systemic understanding of biotic and abiotic influences on plant nutrition.

Phylogeny of the gymnosperm genus Cycas L. (Cycadaceae) as inferred from plastid and nuclear loci based on a large-scale sampling: Evolutionary relationships and taxonomical implications.

The gymnosperm genus Cycas is the sole member of Cycadaceae, and is the largest genus of extant cycads. There are about 115 accepted Cycas species mainly distributed in the paleotropics. Based on morphology, the genus has been divided into six sections and eight subsections, but this taxonomy has not yet been tested in a molecular phylogenetic framework. Although the monophyly of Cycas is broadly accepted, the intrageneric relationships inferred from previous molecular phylogenetic analyses are unclear due to insufficient sampling or uninformative DNA sequence data. In this study, we reconstructed a phylogeny of Cycas using four chloroplast intergenic spacers and seven low-copy nuclear genes and sampling 90% of extant Cycas species. The maximum likelihood and Bayesian inference phylogenies suggest: (1) matrices of either concatenated cpDNA markers or of concatenated nDNA lack sufficient informative sites to resolve the phylogeny alone, however, the phylogeny from the combined cpDNA-nDNA dataset suggests the genus can be roughly divided into 13 clades and six sections that are in agreement with the current classification of the genus; (2) although with partial support, a clade combining sections Panzhihuaenses + Asiorientales is resolved as the earliest diverging branch; (3) section Stangerioides is not monophyletic because the species resolve as a grade; (4) section Indosinenses is not monophyletic as it includes Cycas macrocarpa and C. pranburiensis from section Cycas; (5) section Cycas is the most derived group and its subgroups correspond with geography.

First, do no harm.

Conservation agencies charged with care of threatened plant species should be governed by the concepts that conservation actions should do no harm. Adaptive management research progresses in imperfect situations due to incomplete knowledge. Interpreting new experimental or observational evidence for inclusion in conservation plans should first consider the big picture by identifying collateral quandaries before scaling up the approach to large-scale implementation. We discuss a case study of Cycas micronesica conservation activities on the island of Guam. The use of large stem cuttings has been shown to be a viable approach for rescuing trees from planned construction sites. However, this artificial means of producing transplants exhibits shortcomings, some of which may add new threats to the existing plant population. Moreover, devoting funds for use of the new technique in tree rescue projects does not address the primary threats that have led to listing under the United States Endangered Species Act (ESA). Transplanted trees will likely succumb to those ubiquitous threats shortly after the completion of a successful rescue project. Alternatively, investing conservation funds into mitigation of the primary threats could lead to removal of the species from the ESA.

Diversity in Cycas (Cycadales: Cycadaceae) Species Offered as Larval Food Influences Fecundity of Chilades pandava (Lepidoptera: Lycaenidae) Adults.

Chilades pandava (Lepidoptera: Lycaenidae) larval food quality was studied to determine its influence on adult life history traits. A wild population from Cycas nongnoochiae (Cycadales: Cycadaceae) endemic habitat behaved similarly to the population collected from a garden setting. Cycas micronesica, Cycas revoluta, and Cycas seemannii leaves were used as high-quality food, whereas C nongnoochiae, Cycas taitungensis, and Cycas condaoensis leaves were used as low-quality food. The daily oviposition rate was not influenced by food quality, but longevity and lifetime fecundity of females were increased by high-quality larval food. These results indicate that in situ Cycas species impose a physiological constraint on the genetic capacity to produce offspring by C pandava. The removal of that constraint by high-quality novel Cycas species may be one reason this butterfly can increase in population rapidly after an invasion event and express greater herbivory of Cycas species within invaded regions.

Review of Cycadophila Xu, Tang & Skelley (Coleoptera: Erotylidae: Pharaxonothinae) inhabiting Cycas (Cycadaceae) in Asia, with descriptions of a new subgenus and thirteen new species.

The genus Cycadophila Xu, Tang & Skelley (Coleoptera: Erotylidae: Pharaxonothinae) associated with Cycas L. (Cycadacaeae) in Asia is reviewed. Strobilophila, new subgenus, with five species is described: Cycadophila (Strobilophila) assamensis new species, C. (S.) hiepi new species, C. (S.) kwaiensis new species, C. (S.) tansachai new species and C. (S.) yangi new species, all associated with Cycas. For the nominate subgenus Cycadophila eight new species are described, Cycadophila (Cycadophila) abyssa new species, C. (C.) collina new species,C. (C.) samara new species, C. (C.) convexa new species, C. (C.) cyclochasma new species, C. (C.) eurynota new species, C. (C.) papua new species, and C. (C.) torquata new species and four new generic combinations are proposed: C. (C.) vittata (Arrow) new combination, C. (C.) discimaculata (Mader) new combination, C. (C.) intermedia (Chûjô) new combination, and C. (C.) lata (Grouvelle) new combination. Only the first three listed species of the nominate subgenus have known associations with Cycas. Species are distinguished on the basis of morphology and/or by analysis of 16S rRNA gene sequences. The larva of subgenus Strobilophila is described based on individuals collected together with adults and matched with analysis of 16S rRNA gene sequences. Keys to subgenera and species of known adults and larvae are provided.

Chilades pandava mothers discriminate among Cycas species during oviposition choice tests, but only in an endemic naïve population.

Host and non-host plant species communicate with insect herbivores to influence oviposition decisions. We studied if Chilades pandava female adults expressed oviposition preferences among host Cycas species in 2 choice tests, counting 39,420 eggs among assays from 4 butterfly populations. A naïve butterfly population from Cycas nongnoochiae habitat oviposited 2.2-fold more eggs on leaves of Cycas species that are susceptible to butterfly herbivory than on leaves of its native host Cycas nongnoochiae. In contrast, Chilades pandava populations experienced with novel Cycas species in Thailand, Philippines, and Guam exhibited no preference in choice tests between leaves of susceptible versus leaves of minimally damaged Cycas species. The results indicated that oviposition deterrents and/or stimulants partly mediate the sustainable relationship between an endemic Cycas species and the naïve Chilades pandava population from its habitat. Alternatively, differences in infochemicals among Cycas species do not enable discrimination in oviposition choices for Chilades pandava populations that have experienced Cycas species exhibiting no evolutionary history with Chilades pandava.

Carbohydrates, pollinators, and cycads.

Cycad biology, ecology, and horticulture decisions are not supported by adequate research, and experiments in cycad physiology in particular have been deficient. Our recent report on free sugar content in a range of cycad taxa and tissues sets the stage for developing continued carbohydrate research. Growth and development of cycad pollen, mediation of the herbivory traits of specialist pollinators, and support of expensive strobilus behavioral traits are areas of cycad pollination biology that would benefit from a greater understanding of the role of carbohydrate relations.

Free sugar profile in cycads.

The sugars fructose, glucose, maltose, and sucrose were quantified in seven tissues of Zamia muricata Willd. to determine their distribution throughout various organs of a model cycad species, and in lateral structural roots of 18 cycad species to determine the variation in sugar concentration and composition among species representing every cycad genus. Taproot and lateral structural roots contained more sugars than leaf, stem, female strobilus, or coralloid roots. For example, taproot sugar concentration was 6.4-fold greater than stem sugar concentration. The dominant root sugars were glucose and fructose, and the only detected stem sugar was sucrose. Sucrose also dominated the sugar profile for leaflet and coralloid root tissue, and fructose was the dominant sugar in female strobilus tissue. Maltose was a minor constituent of taproot, leaflet, and female strobilus tissue, but absent in other tissues. The concentration of total free sugars and each of the four sugars did not differ among genera or families. Stoichiometric relationships among the sugars, such as the quotient hexoses/disaccharides, differed among organs and families. Although anecdotal reports on cycad starch have been abundant due to its historical use as human food and the voluminous medical research invested into cycad neurotoxins, this is the first report on the sugar component of the non-structural carbohydrate profile of cycads. Fructose, glucose, and sucrose are abundant in cycad tissues, with their relative abundance highly contrasting among organs. Their importance as forms of carbon storage, messengers of information, or regulators of cycad metabolism have not been determined to date.