Taxus yunnanensis genome offers insights into gymnosperm phylogeny and taxol production.

Taxol, a natural product derived from Taxus, is one of the most effective natural anticancer drugs and the biosynthetic pathway of Taxol is the basis of heterologous bio-production. Here, we report a high-quality genome assembly and annotation of Taxus yunnanensis based on 10.7 Gb sequences assembled into 12 chromosomes with contig N50 and scaffold N50 of 2.89 Mb and 966.80 Mb, respectively. Phylogenomic analyses show that T. yunnanensis is most closely related to Sequoiadendron giganteum among the sampled taxa, with an estimated divergence time of 133.4-213.0 MYA. As with most gymnosperms, and unlike most angiosperms, there is no evidence of a recent whole-genome duplication in T. yunnanensis. Repetitive sequences, especially long terminal repeat retrotransposons, are prevalent in the T. yunnanensis genome, contributing to its large genome size. We further integrated genomic and transcriptomic data to unveil clusters of genes involved in Taxol synthesis, located on the chromosome 12, while gene families encoding hydroxylase in the Taxol pathway exhibited significant expansion. Our study contributes to the further elucidation of gymnosperm relationships and the Taxol biosynthetic pathway.

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.

Revealing the diversity of amber source plants from the Early Cretaceous Crato Formation, Brazil.

BACKGROUND: Amber has been reported from the Early Cretaceous Crato Formation, as isolated clasts or within plant tissues. Undescribed cones of uncertain gymnosperm affinity have also been recovered with amber preserved in situ. Here, we provide multiple lines of evidence to determine the botanical affinity of this enigmatic, conspicuous cone type, and to better understand the diversity of amber-source plants present in the Crato Formation and beyond. RESULTS: A new taxon of amber-bearing pollen cone Araripestrobus resinosus gen. nov. et sp. nov. is described here from complete cones and characteristic disarticulated portions. The best-preserved cone portion has both in situ amber infilling the resin canals inside the preserved microsporophyll tissues and pollen of the Eucommiidites-type. This places this genus within the Erdtmanithecales, an incompletely known gymnosperm group from the Mesozoic. FTIR analysis of the in situ amber indicates a potential araucariacean conifer affinity, although affinity with cupressacean conifers cannot be definitely ruled out. Pyr-GC-MS analysis of the Araripestrobus resinosus gen. nov. et sp. nov. in situ fossil resin shows that it is a mature class Ib amber, thought to indicate affinities with araucariacean and cupressacean, but not pinaceous, conifers. This is the first confirmed occurrence of this class of amber in the Crato Formation flora and in South America, except for an archaeological sample from Laguna Guatavita, Colombia. CONCLUSIONS: The combined results of the cones' novel gross morphology and the analyses of the in situ amber and pollen clearly indicate that the new taxon of resinous gymnosperm pollen cones from the Crato Formation is affiliated with Erdtmanithecales. The cone morphology is very distinct from all known pollen cone types of this extinct plant group. We therefore assume that the plant group that produced Eucommiidites-type pollen is much more diverse in habits than previously thought. Moreover, the diversity of potential amber source plants from the Crato Formation is now expanded beyond the Araucariaceae and the Cheirolepidiaceae to include this member of the Erdtmanithecales. Despite dispersed Eucommiidites pollen being noted from the Crato Formation, this is the first time macrofossils of Erdtmanithecales have been recognized from the Early Cretaceous of South America.

Two Independent Plastid accD Transfers to the Nuclear Genome of Gnetum and Other Insights on Acetyl-CoA Carboxylase Evolution in Gymnosperms.

Acetyl-CoA carboxylase (ACCase) is the key regulator of fatty acid biosynthesis. In most plants, ACCase exists in two locations (cytosol and plastids) and in two forms (homomeric and heteromeric). Heteromeric ACCase comprises four subunits, three of them (ACCA-C) are nuclear encoded (nr) and the fourth (ACCD) is usually plastid encoded. Homomeric ACCase is encoded by a single nr-gene (ACC). We investigated the ACCase gene evolution in gymnosperms by examining the transcriptomes of newly sequenced Gnetum ula, combined with 75 transcriptomes and 110 plastomes of other gymnosperms. AccD-coding sequences are elongated through the insertion of repetitive DNA in four out of five cupressophyte families (except Sciadopityaceae) and were functionally transferred to the nucleus of gnetophytes and Sciadopitys. We discovered that, among the three genera of gnetophytes, only Gnetum has two copies of nr-accD. Furthermore, using protoplast transient expression assays, we experimentally verified that the nr-accD precursor proteins in Gnetum and Sciadopitys can be delivered to the plastids. Of the two nr-accD copies of Gnetum, one dually targets plastids and mitochondria, whereas the other potentially targets plastoglobuli. The distinct transit peptides, gene architectures, and flanking sequences between the two Gnetum accDs suggest that they have independent origins. Our findings are the first account of two distinctly targeted nr-accDs of any green plants and the most comprehensive analyses of ACCase evolution in gymnosperms to date.

No mass extinction for land plants at the Permian-Triassic transition.

The most severe mass extinction among animals took place in the latest Permian (ca. 252 million years ago). Due to scarce and impoverished fossil floras from the earliest Triassic, the common perception has been that land plants likewise suffered a mass extinction, but doubts remained. Here we use global occurrence data of both plant macro- and microfossils to analyse plant biodiversity development across the Permian-Triassic boundary. We show that the plant fossil record is strongly biased and that evidence for a mass extinction among plants in the latest Permian is not robust. The taxonomic diversities of gymnosperm macrofossils and of the pollen produced by this group are particularly incongruent. Our results indicate that gymnosperm macrofossils are considerably undersampled for the Early Triassic, which creates the impression of increased gymnosperm extinction in the latest Permian.

Phylogenomics resolves the deep phylogeny of seed plants and indicates partial convergent or homoplastic evolution between Gnetales and angiosperms.

After decades of molecular phylogenetic studies, the deep phylogeny of gymnosperms has not been resolved, and the phylogenetic placement of Gnetales remains one of the most controversial issues in seed plant evolution. To resolve the deep phylogeny of seed plants and to address the sources of phylogenetic conflict, we conducted a phylotranscriptomic study with a sampling of all 13 families of gymnosperms and main lineages of angiosperms. Multiple datasets containing up to 1 296 042 sites across 1308 loci were analysed, using concatenation and coalescence approaches. Our study generated a consistent and well-resolved phylogeny of seed plants, which places Gnetales as sister to Pinaceae and thus supports the Gnepine hypothesis. Cycads plus Ginkgo is sister to the remaining gymnosperms. We also found that Gnetales and angiosperms have similar molecular evolutionary rates, which are much higher than those of other gymnosperms. This implies that Gnetales and angiosperms might have experienced similar selective pressures in evolutionary histories. Convergent molecular evolution or homoplasy is partially responsible for the phylogenetic conflicts in seed plants. Our study provides a robustly reconstructed backbone phylogeny that is important for future molecular and morphological studies of seed plants, in particular gymnosperms, in the light of evolution.

Palyno-morphological characteristics of gymnosperm flora of pakistan and its taxonomic implications with LM and SEM methods.

The present study is intended to assess gymnosperms pollen flora of Pakistan using Light Microscope (LM) and Scanning Electron Microscopy (SEM) for its taxonomic significance in identification of gymnosperms. Pollens of 35 gymnosperm species (12 genera and five families) were collected from its various distributional sites of gymnosperms in Pakistan. LM and SEM were used to investigate different palyno-morphological characteristics. Five pollen types (i.e., Inaperturate, Monolete, Monoporate, Vesiculate-bisaccate and Polyplicate) were observed. Six In equatorial view seven types of pollens were observed, in which ten species were sub-angular, nine species were Traingular, six species were Perprolate, three species were Rhomboidal, three species were semi-angular, two species were rectangular and two species were prolate. While five types of pollen were observed in polar view, in which ten species were Spheroidal, nine species were Angular, eight were Interlobate, six species were Circular, two species were Elliptic. Eighteen species has rugulate and 17 species has faveolate ornamentation. Eighteen species has verrucate and 17 have gemmate type sculpturing. The data was analysed through cluster analysis. The study showed that these palyno-morphological features have significance value in classification and identification of gymnosperms. Based on these different palyno-morphological features, a taxonomic key was proposed for the accurate and fast identifications of gymnosperms from Pakistan.

Evidence for an ancient whole genome duplication in the cycad lineage.

Contrary to the many whole genome duplication events recorded for angiosperms (flowering plants), whole genome duplications in gymnosperms (non-flowering seed plants) seem to be much rarer. Although ancient whole genome duplications have been reported for most gymnosperm lineages as well, some are still contested and need to be confirmed. For instance, data for ginkgo, but particularly cycads have remained inconclusive so far, likely due to the quality of the data available and flaws in the analysis. We extracted and sequenced RNA from both the cycad Encephalartos natalensis and Ginkgo biloba. This was followed by transcriptome assembly, after which these data were used to build paralog age distributions. Based on these distributions, we identified remnants of an ancient whole genome duplication in both cycads and ginkgo. The most parsimonious explanation would be that this whole genome duplication event was shared between both species and had occurred prior to their divergence, about 300 million years ago.

Single-Copy Genes as Molecular Markers for Phylogenomic Studies in Seed Plants.

Phylogenetic relationships among seed plant taxa, especially within the gymnosperms, remain contested. In contrast to angiosperms, for which several genomic, transcriptomic and phylogenetic resources are available, there are few, if any, molecular markers that allow broad comparisons among gymnosperm species. With few gymnosperm genomes available, recently obtained transcriptomes in gymnosperms are a great addition to identifying single-copy gene families as molecular markers for phylogenomic analysis in seed plants. Taking advantage of an increasing number of available genomes and transcriptomes, we identified single-copy genes in a broad collection of seed plants and used these to infer phylogenetic relationships between major seed plant taxa. This study aims at extending the current phylogenetic toolkit for seed plants, assessing its ability for resolving seed plant phylogeny, and discussing potential factors affecting phylogenetic reconstruction. In total, we identified 3,072 single-copy genes in 31 gymnosperms and 2,156 single-copy genes in 34 angiosperms. All studied seed plants shared 1,469 single-copy genes, which are generally involved in functions like DNA metabolism, cell cycle, and photosynthesis. A selected set of 106 single-copy genes provided good resolution for the seed plant phylogeny except for gnetophytes. Although some of our analyses support a sister relationship between gnetophytes and other gymnosperms, phylogenetic trees from concatenated alignments without 3rd codon positions and amino acid alignments under the CAT + GTR model, support gnetophytes as a sister group to Pinaceae. Our phylogenomic analyses demonstrate that, in general, single-copy genes can uncover both recent and deep divergences of seed plant phylogeny.

Exposing the illegal trade in cycad species (Cycadophyta: Encephalartos) at two traditional medicine markets in South Africa using DNA barcoding.

Species in the cycad genus Encephalartos are listed in CITES Appendix I and as Threatened or Protected Species in terms of South Africa's National Environmental Management: Biodiversity Act (NEM:BA) of 2004. Despite regulations, illegal plant harvesting for medicinal trade has continued in South Africa and resulted in declines in cycad populations and even complete loss of sub-populations. Encephalartos is traded at traditional medicine markets in South Africa in the form of bark strips and stem sections; thus, determining the species traded presents a major challenge due to a lack of characteristic plant parts. Here, a case study is presented on the use of DNA barcoding to identify cycads sold at the Faraday and Warwick traditional medicine markets in Johannesburg and Durban, respectively. Market samples were sequenced for the core DNA barcodes (rbcLa and matK) as well as two additional regions: nrITS and trnH-psbA. The barcoding database for cycads at the University of Johannesburg was utilized to assign query samples to known species. Three approaches were followed: tree-based, similarity-based, and character-based (BRONX) methods. Market samples identified were Encephalartos ferox (Near Threatened), Encephalartos lebomboensis (Endangered), Encephalartos natalensis (Near Threatened), Encephalartos senticosus (Vulnerable), and Encephalartos villosus (Least Concern). Results from this study are crucial for making appropriate assessments and decisions on how to manage these markets.

Comparison of intraspecific, interspecific and intergeneric chloroplast diversity in Cycads.

Cycads are among the most threatened plant species. Increasing the availability of genomic information by adding whole chloroplast data is a fundamental step in supporting phylogenetic studies and conservation efforts. Here, we assemble a dataset encompassing three taxonomic levels in cycads, including ten genera, three species in the genus Cycas and two individuals of C. debaoensis. Repeated sequences, SSRs and variations of the chloroplast were analyzed at the intraspecific, interspecific and intergeneric scale, and using our sequence data, we reconstruct a phylogenomic tree for cycads. The chloroplast was 162,094 bp in length, with 133 genes annotated, including 87 protein-coding, 37 tRNA and 8 rRNA genes. We found 7 repeated sequences and 39 SSRs. Seven loci showed promising levels of variations for application in DNA-barcoding. The chloroplast phylogeny confirmed the division of Cycadales in two suborders, each of them being monophyletic, revealing a contradiction with the current family circumscription and its evolution. Finally, 10 intraspecific SNPs were found. Our results showed that despite the extremely restricted distribution range of C. debaoensis, using complete chloroplast data is useful not only in intraspecific studies, but also to improve our understanding of cycad evolution and in defining conservation strategies for this emblematic group.

Evolution of the YABBY gene family in seed plants.

Members of the YABBY gene family of transcription factors in angiosperms have been shown to be involved in the initiation of outgrowth of the lamina, the maintenance of polarity, and establishment of the leaf margin. Although most of the dorsal-ventral polarity genes in seed plants have homologs in non-spermatophyte lineages, the presence of YABBY genes is restricted to seed plants. To gain insight into the origin and diversification of this gene family, we reconstructed the evolutionary history of YABBY gene lineages in seed plants. Our findings suggest that either one or two YABBY genes were present in the last common ancestor of extant seed plants. We also examined the expression of YABBY genes in the gymnosperms Ephedra distachya (Gnetales), Ginkgo biloba (Ginkgoales), and Pseudotsuga menziesii (Coniferales). Our data indicate that some YABBY genes are expressed in a polar (abaxial) manner in leaves and female cones in gymnosperms. We propose that YABBY genes already acted as polarity genes in the last common ancestor of extant seed plants.

The complete plastome sequence of Gnetum ula (Gnetales: Gnetaceae).

This study reports the complete plastome sequence of Gnetum ula, a gymnosperm species of Gnetaceae (Gnetophyta). The plastome is 113 249 bp long. It has a quadripartite structure containing a pair of large inverted repeat regions of 19 772 bp each, a large single-copy region of 64 914 bp, and a small single-copy region of 8791 bp. One hundred sixteen genes were predicted in the plastome, including 68 protein-coding genes, eight ribosomal RNA genes, and 40 transfer RNA genes. The gene density is 1.024 (genes/kb). Similar to other known Gnetum plastomes, the G.ula plastome has lost 20 protein-coding genes commonly present in other seed plant plastomes. Our phylogenetic analyses indicate that the four sampled Gnetum species are monophyletic and that G. ula is close to the two other lianas rather than the only small tree species, G. gnemon. Our phylogenetic trees also indicate that gnetophytes have the fastest evolutionary rates among gymnosperms.

A Survey of Plant Iron Content-A Semi-Systematic Review.

Iron is an essential mineral nutrient for all living organisms, involved in a plurality of biological processes. Its deficit is the cause of the most common form of anemia in the world: iron deficiency anemia (IDA). This paper reviews iron content in various parts of 1228 plant species and its absorption from herbal products, based on data collected from the literature in a semi-systematic manner. Five hundred genera randomly selected from the Angiosperms group, 215 genera from the Pteridophytes groups and all 95 Gymnosperm genera as listed in the Plant List version 1.1 were used as keywords together with the word "iron" in computerized searches. Iron data about additional genera returned by those searches were extracted and included in the analysis. In total, iron content values for a number of 1228 species, 5 subspecies, and 5 varieties were collected. Descriptive and inferential statistics were used to compare iron contents in various plant parts (whole plant, roots, stems, shoots, leaves, aerial parts, flowers, fruits, seeds, wood, bark, other parts) and exploratory analyses by taxonomic groups and life-forms were carried out. The absorption and potential relevance of herbal iron for iron supplementation are discussed.

Origin and diversification of living cycads: a cautionary tale on the impact of the branching process prior in Bayesian molecular dating.

BACKGROUND: Bayesian relaxed-clock dating has significantly influenced our understanding of the timeline of biotic evolution. This approach requires the use of priors on the branching process, yet little is known about their impact on divergence time estimates. We investigated the effect of branching priors using the iconic cycads. We conducted phylogenetic estimations for 237 cycad species using three genes and two calibration strategies incorporating up to six fossil constraints to (i) test the impact of two different branching process priors on age estimates, (ii) assess which branching prior better fits the data, (iii) investigate branching prior impacts on diversification analyses, and (iv) provide insights into the diversification history of cycads. RESULTS: Using Bayes factors, we compared divergence time estimates and the inferred dynamics of diversification when using Yule versus birth-death priors. Bayes factors were calculated with marginal likelihood estimated with stepping-stone sampling. We found striking differences in age estimates and diversification dynamics depending on prior choice. Dating with the Yule prior suggested that extant cycad genera diversified in the Paleogene and with two diversification rate shifts. In contrast, dating with the birth-death prior yielded Neogene diversifications, and four rate shifts, one for each of the four richest genera. Nonetheless, dating with the two priors provided similar age estimates for the divergence of cycads from Ginkgo (Carboniferous) and their crown age (Permian). Of these, Bayes factors clearly supported the birth-death prior. CONCLUSIONS: These results suggest the choice of the branching process prior can have a drastic influence on our understanding of evolutionary radiations. Therefore, all dating analyses must involve a model selection process using Bayes factors to select between a Yule or birth-death prior, in particular on ancient clades with a potential pattern of high extinction. We also provide new insights into the history of cycad diversification because we found (i) periods of extinction along the long branches of the genera consistent with fossil data, and (ii) high diversification rates within the Miocene genus radiations.

Molecular evolution and sequence divergence of plant chalcone synthase and chalcone synthase-Like genes.

Plant chalcone synthase (CHS) and CHS-Like (CHSL) proteins are polyketide synthases. In this study, we evaluated the molecular evolution of this gene family using representative types of CHSL genes, including stilbene synthase (STS), 2-pyrone synthase (2-PS), bibenzyl synthase (BBS), acridone synthase (ACS), biphenyl synthase (BIS), benzalacetone synthase, coumaroyl triacetic acid synthase (CTAS), and benzophenone synthase (BPS), along with their CHS homologs from the same species of both angiosperms and gymnosperms. A cDNA-based phylogeny indicated that CHSLs had diverse evolutionary patterns. STS, ACS, and 2-PS clustered with CHSs from the same species (late diverged pattern), while CTAS, BBS, BPS, and BIS were distant from their CHS homologs (early diverged pattern). The amino-acid phylogeny suggested that CHS and CHSL proteins formed clades according to enzyme function. The CHSs and CHSLs from Polygonaceae and Arachis had unique evolutionary histories. Synonymous mutation rates were lower in late diverged CHSLs than in early diverged ones, indicating that gene duplications occurred more recently in late diverged CHSLs than in early diverged ones. Relative rate tests proved that late diverged CHSLs had unequal rates to CHSs from the same species when using fatty acid synthase, which evolved from the common ancestor with the CHS superfamily, as the outgroup, while the early diverged lineages had equal rates. This indicated that late diverged CHSLs experienced more frequent mutation than early diverged CHSLs after gene duplication, allowing obtaining new functions in relatively short period of time.

Evolution and biogeography of gymnosperms.

Living gymnosperms comprise only a little more than 1000 species, but represent four of the five main lineages of seed plants, including cycads, ginkgos, gnetophytes and conifers. This group has huge ecological and economic value, and has drawn great interest from the scientific community. Here we review recent advances in our understanding of gymnosperm evolution and biogeography, including phylogenetic relationships at different taxonomic levels, patterns of species diversification, roles of vicariance and dispersal in development of intercontinental disjunctions, modes of molecular evolution in different genomes and lineages, and mechanisms underlying the formation of large nuclear genomes. It is particularly interesting that increasing evidence supports a sister relationship between Gnetales and Pinaceae (the Gnepine hypothesis) and the contribution of recent radiations to present species diversity, and that expansion of retrotransposons is responsible for the large and complex nuclear genome of gymnosperms. In addition, multiple coniferous genera such as Picea very likely originated in North America and migrated into the Old World, further indicating that the center of diversity is not necessarily the place of origin. The Bering Land Bridge acted as an important pathway for dispersal of gymnosperms in the Northern Hemisphere. Moreover, the genome sequences of conifers provide an unprecedented opportunity and an important platform for the evolutionary studies of gymnosperms, and will also shed new light on evolution of many important gene families and biological pathways in seed plants.

Conserved genetic regions across angiosperms as tools to develop single-copy nuclear markers in gymnosperms: an example using cycads.

Several individuals of the Caribbean Zamia clade and other cycad genera were used to identify single-copy nuclear genes for phylogeographic and phylogenetic studies in Cycadales. Two strategies were employed to select target loci: (i) a tblastX search of Arabidopsis conserved ortholog sequence (COS) set and (ii) a tblastX search of Arabidopsis-Populus-Vitis-Oryza Shared Single-Copy genes (APVO SSC) against the EST Zamia databases in GenBank. From the first strategy, 30 loci were selected, and from the second, 16 loci. In both cases, the matching GenBank accessions of Zamia were used as a query for retrieving highly similar sequences from Cycas, Picea, Pinus species or Ginkgo biloba. After retrieving and aligning all the sequences in each locus, intron predictions were completed to assist in primer design. PCR was carried out in three rounds to detect paralogous loci. A total of 29 loci were successfully amplified as a single band of which 20 were likely single-copy loci. These loci showed different diversity and divergence levels. A preliminary screening allowed us to select 8 promising loci (40S, ATG2, BG, GroES, GTP, LiSH, PEX4 and TR) for the Zamia pumila complex and 4 loci (COS26, GroES, GTP and HTS) for all other cycad genera.

New evidence of the reproductive organs of Glossopteris based on permineralized fossils from Queensland, Australia. II: pollen-bearing organ Ediea gen. nov.

Ediea homevalensis H. Nishida, Kudo, Pigg & Rigby gen. et sp. nov. is proposed for permineralized pollen-bearing structures from the Late Permian Homevale Station locality of the Bowen Basin, Queensland, Australia. The taxon represents unisexual fertile shoots bearing helically arranged leaves on a central axis. The more apical leaves are fertile microsporophylls bearing a pair of multi-branched stalks on their adaxial surfaces that each supports a cluster of terminally borne pollen sacs. Proximal to the fertile leaves there are several rows of sterile scale-like leaves. The pollen sacs (microsporangia) have thickened and dark, striate walls that are typical of the Arberiella type found in most pollen organs presumed to be of glossopterid affinity. An examination of pollen organs at several developmental stages, including those containing in situ pollen of the Protohaploxypinus type, provides the basis for a detailed analysis of these types of structures, which bear similarities to both compression/impression Eretmonia-type glossopterid microsporangiate organs and permineralized Eretmonia macloughlinii from Antarctica. These fossils demonstrate that at least some Late Permian pollen organs were simple microsporophyll-bearing shoot systems and not borne directly on Glossopteris leaves.

Phylogeny of the cycads based on multiple single-copy nuclear genes: congruence of concatenated parsimony, likelihood and species tree inference methods.

BACKGROUND AND AIMS: Despite a recent new classification, a stable phylogeny for the cycads has been elusive, particularly regarding resolution of Bowenia, Stangeria and Dioon. In this study, five single-copy nuclear genes (SCNGs) are applied to the phylogeny of the order Cycadales. The specific aim is to evaluate several gene tree-species tree reconciliation approaches for developing an accurate phylogeny of the order, to contrast them with concatenated parsimony analysis and to resolve the erstwhile problematic phylogenetic position of these three genera. METHODS: DNA sequences of five SCNGs were obtained for 20 cycad species representing all ten genera of Cycadales. These were analysed with parsimony, maximum likelihood (ML) and three Bayesian methods of gene tree-species tree reconciliation, using Cycas as the outgroup. A calibrated date estimation was developed with Bayesian methods, and biogeographic analysis was also conducted. KEY RESULTS: Concatenated parsimony, ML and three species tree inference methods resolve exactly the same tree topology with high support at most nodes. Dioon and Bowenia are the first and second branches of Cycadales after Cycas, respectively, followed by an encephalartoid clade (Macrozamia-Lepidozamia-Encephalartos), which is sister to a zamioid clade, of which Ceratozamia is the first branch, and in which Stangeria is sister to Microcycas and Zamia. CONCLUSIONS: A single, well-supported phylogenetic hypothesis of the generic relationships of the Cycadales is presented. However, massive extinction events inferred from the fossil record that eliminated broader ancestral distributions within Zamiaceae compromise accurate optimization of ancestral biogeographical areas for that hypothesis. While major lineages of Cycadales are ancient, crown ages of all modern genera are no older than 12 million years, supporting a recent hypothesis of mostly Miocene radiations. This phylogeny can contribute to an accurate infrafamilial classification of Zamiaceae.