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Chloranthales remain the last major mesangiosperm lineage without a nuclear genome assembly. We therefore assemble a high-quality chromosome-level genome of Chloranthus spicatus to resolve enigmatic evolutionary relationships, as well as explore patterns of genome evolution among the major lineages of mesangiosperms (eudicots, monocots, magnoliids, Chloranthales, and Ceratophyllales). We find that synteny is highly conserved between genomic regions of Amborella, Vitis, and Chloranthus. We identify an ancient single whole-genome duplication (WGD) (κ) prior to the divergence of extant Chloranthales. Phylogenetic inference shows Chloranthales as sister to magnoliids. Furthermore, our analyses indicate that ancient hybridization may account for the incongruent phylogenetic placement of Chloranthales + magnoliids relative to monocots and eudicots in nuclear and chloroplast trees. Long genes and long introns are found to be prevalent in both Chloranthales and magnoliids compared to other angiosperms. Overall, our findings provide an improved context for understanding mesangiosperm relationships and evolution and contribute a valuable genomic resource for future investigations., Chloranthales remain the last lineage of core angiosperms that lacks a nuclear genome assembly. Here, the authors report the genome assembly of Chloranthus spicatus and show its contribution to deepen our understanding on diversification, phylogeny, and genome evolution in angiosperms.

Next-generation sequencing (NGS) has revolutionized molecular systematics as well as population and conservation genetics. It is now possible to obtain enormous amounts of gene sequence data from any species in a short time at low cost. More technological advances are on the horizon, ensuring that this trend will continue throughout the coming decade. These rapid advances provide unprecedented opportunities in systematics; they also pose new challenges, requiring that the next generation of systematists be well-versed in new skill sets (e.g., bioinformatics). As examples of the potential of NGS, it is now possible to develop genetic resources for any plant system that poses intriguing evolutionary questions. During the next decade many new "evolutionary model systems" will become available as systematists rapidly develop the necessary genetic/genomic frameworks for many previously unstudied plants. Phylogenetic reconstruction will be conducted at an unprecedented pace at both deep and fine scales with datasets of numerous taxa and genes—this includes rapid progress on assembling a more com- prehensive Tree of Life for green plants. For example, complete plastid genome sequencing is now routinely facilitating analyses of hundreds of taxa at deep levels, as well as enabling complete plastid genome phylogeographic analyses at the population level. Gene capture methods hold enormous promise for the rapid and inexpensive analyses of complete plastid genomes, as well as studies of hundreds of selected (targeted) nuclear loci. NGS has also had a big impact on population genetics, initially by dramatically simplifying microsatellite marker development, but more recently by opening new possibilities through vari- ous genotyping-by-sequencing (GBS) approaches that have great potential to expand on the types of questions that can be addressed at the population level. Transcriptome sequencing has enabled the construction of large datasets of nuclear genes while also providing a wealth of plastid and mitochondrial genes. NGS has also facilitated probe development for studies of chromosomes using FISH (fluorescence in situ hybridization). NGS is also making the rapid sequencing of complete nuclear genomes routine, thus transforming our field and opening up new avenues of systematic endeavor in comparative genomics. However, even as sequencing costs drop and technological advances make complete nuclear genome sequencing more com - monplace, genome assembly will remain a major challenge.

The origin of the flower was a key innovation in the history of complex organisms, dramatically altering Earth’s biota. Advances in phylogenetics, developmental genetics, and genomics during the past 25 years have substantially advanced our understanding of the evolution of flowers, yet crucial aspects of floral evolution remain, such as the series of genetic and morphological changes that gave rise to the first flowers; the factors enabling the origin of the pentamerous eudicot flower, which characterizes ∼70% of all extant angiosperm species; and the role of gene and genome duplications in facilitating floral innovations. A key early concept was the ABC model of floral organ specification, developed by Elliott Meyerowitz and Enrico Coen and based on two model systems, Arabidopsis thaliana and Antirrhinum majus. Yet it is now clear that these model systems are highly derived species, whose molecular genetic-developmental organization must be very different from that of ancestral, as well as early, angiosperms. In this article, we will discuss how new research approaches are illuminating the early events in floral evolution and the prospects for further progress. In particular, advancing the next generation of research in floral evolution will require the development of one or more functional model systems from among the basal angiosperms and basal eudicots. More broadly, we urge the development of “model clades” for genomic and evolutionary-developmental analyses, instead of the primary use of single “model organisms.” We predict that new evolutionary models will soon emerge as genetic/genomic models, providing unprecedented new insights into floral evolution.

Current understanding of floral developmental genetics comes primarily from the core eudicot model Arabidopsis thaliana. Here, we explore the floral transcriptome of the basal angiosperm, Nuphar advena (water lily), for insights into the ancestral developmental program of flowers. We identify several thousand Nuphar genes with significantly upregulated floral expression, including homologs of the well-known ABCE floral regulators, deployed in broadly overlapping transcriptional programs across floral organ categories. Strong similarities in the expression profiles of different organ categories in Nuphar flowers are shared with the magnoliid Persea americana (avocado), in contrast to the largely organ-specific transcriptional cascades evident in Arabidopsis, supporting the inference that this is the ancestral condition in angiosperms. In contrast to most eudicots, floral organs are weakly differentiated in Nuphar and Persea, with staminodial intermediates between stamens and perianth in Nuphar, and between stamens and carpels in Persea. Consequently, the predominantly organ-specific transcriptional programs that characterize Arabidopsis flowers (and perhaps other eudicots) are derived, and correlate with a shift towards morphologically distinct floral organs, including differentiated sepals and petals, and a perianth distinct from stamens and carpels. Our findings suggest that the genetic regulation of more spatially discrete transcriptional programs underlies the evolution of floral morphology.

The debate on the origin and evolution of flowers has recently entered the field of developmental genetics, with focus on the design of the ancestral floral regulatory program. Flowers can differ dramatically among angiosperm lineages, but in general, male and female reproductive organs surrounded by a sterile perianth of sepals and petals constitute the basic floral structure. However, the basal angiosperm lineages exhibit spectacular diversity in the number, arrangement, and structure of floral organs, whereas the evolutionarily derived monocot and eudicot lineages share a far more uniform floral ground plan. Here we show that broadly overlapping transcriptional programs characterize the floral transcriptome of the basal angiosperm Persea americana (avocado), whereas floral gene expression domains are considerably more organ specific in the model eudicot Arabidopsis thaliana . Our findings therefore support the “fading borders” model for organ identity determination in basal angiosperm flowers and extend it from the action of regulatory genes to downstream transcriptional programs. Furthermore, the declining expression of components of the staminal transcriptome in central and peripheral regions of Persea flowers concurs with elements of a previous hypothesis for developmental regulation in a gymnosperm “floral progenitor.” Accordingly, in contrast to the canalized organ-specific regulatory apparatus of Arabidopsis , floral development may have been originally regulated by overlapping transcriptional cascades with fading gradients of influence from focal to bordering organs.

A floral developmental series was determined for Persea americana (Lauraceae, avocado), and the floral morphology of this species was compared with available data for other members of Persea. We compared the structure of the inflorescence and flower with that of vegetative shoots with respect to phyllotaxy and leaf shape. The inflorescence is a determinate thyrse (panicle) with variable numbers of lateral branches. Staminal glands in Persea may represent abaxial‐marginal emergences rather than stamens. However, these glands are occasionally involved in transitions to pollen sacs and ovary margins. Stigmas, pollen sacs, staminal appendages, glands of staminodes, and margins of tepals share features that are subjectively associated with “androecia.” In the innermost androecial whorl, staminodial glands appear united because of the reduction of the middle portion to a staminodial apex. The apex of staminodes is homologous to the filament and anther, as well as to the stigma of the carpel, and corresponds to ...

LBD (lateral organ boundaries domain) genes are essential to the developmental programs of many fundamental plant organs and function in some of the basic metabolic pathways of plants. However, our historical perspective on the roles of LBD genes during plant evolution has, heretofore, been fragmentary. Here, we show that the LBD gene family underwent an initial radiation that established five gene lineages in the ancestral genome of most charophyte algae and land plants. By inference, the LBD gene family originated after the emergence of the green plants (Viridiplantae), but prior to the diversification of most extant streptophytes. After this initial radiation, we find limited instances of gene family diversification in land plants until successive rounds of expansion in the ancestors of seed plants and flowering plants. The most dynamic phases of LBD gene evolution, therefore, trace to the aquatic ancestors of embryophytes followed by relatively recent lineage-specific expansions on land.

The perianth of Persea americana (Lauraceae) consists of two whorls of morphologically similar laminar organs, termed tepals. Closely related Persea borbonia, however, produces a dimorphic perianth with smaller outer tepals. To assess whether homologues of floral organ identity genes in Persea may play a role in shaping this dimorphic perianth, we compared their expression patterns in the two species. A homologue of AP1 (A‐function) is expressed at low levels in both perianth types but was not tepal specific. Homologues of AGL6, however, show the tepal‐specific expression pattern expected of A‐function genes. Homologues of AP3 and PI (B‐function) are expressed in tepals of both perianth types, indicating that perianth dimorphism in Persea is not regulated by these genes. Differential expression across the dimorphic perianth as absence late in outer tepal development was evident for homologues of AG (C‐function) and SEP3 (E‐function). Genetic studies in model systems indicate a conserved role for AG homolo...

A salient feature of flowering plant diversification is the emergence of a novel suite of floral features coinciding with the origin of the most species-rich lineage, Pentapetalae. Advances in phylogenetics, developmental genetics and genomics, including new analyses presented here, are helping to reconstruct the specific evolutionary steps involved in the evolution of this clade. The enormous floral diversity among Pentapetalae appears to be built on a highly conserved ground plan of five-parted (pentamerous) flowers with whorled phyllotaxis. By contrast, lability in the number and arrangement of component parts of the flower characterize the early-diverging eudicot lineages subtending Pentapetalae. The diversification of Pentapetalae also coincides closely with ancient hexaploidy, referred to as thegammawhole-genome triplication, for which the phylogenetic timing, mechanistic details and molecular evolutionary consequences are as yet not fully resolved. Transcription factors regulating floral development often persist in duplicate or triplicate ingamma-derived genomes, and both individual genes and whole transcriptional programmes exhibit a shift from broadly overlapping to tightly defined expression domains in Pentapetalae flowers. Investigations of these changes associated with the origin of Pentapetalae can lead to a more comprehensive understanding of what is arguably one of the most important evolutionary diversification events within terrestrial plants.This article is part of the themed issue ‘Evo-devo in the genomics era, and the origins of morphological diversity’.

Background GSK3 (glycogen synthase kinase 3) genes encode signal transduction proteins with roles in a variety of biological processes in eukaryotes. In contrast to the low copy numbers observed in animals, GSK3 genes have expanded into a multi-gene family in land plants (embryophytes), and have also evolved functions in diverse plant specific processes, including floral development in angiosperms. However, despite previous efforts, the phylogeny of land plant GSK3 genes is currently unclear. Here, we analyze genes from a representative sample of phylogenetically pivotal taxa, including basal angiosperms, gymnosperms, and monilophytes, to reconstruct the evolutionary history and functional diversification of the GSK3 gene family in land plants. Results Maximum Likelihood phylogenetic analyses resolve a gene tree with four major gene duplication events that coincide with the emergence of novel land plant clades. The single GSK3 gene inherited from the ancestor of land plants was first duplicated along the ancestral branch to extant vascular plants, and three subsequent duplications produced three GSK3 loci in the ancestor of euphyllophytes, four in the ancestor of seed plants, and at least five in the ancestor of angiosperms. A single gene in the Amborella trichopoda genome may be the sole survivor of a sixth GSK3 locus that originated in the ancestor of extant angiosperms. Homologs of two Arabidopsis GSK3 genes with genetically confirmed roles in floral development, AtSK11 and AtSK12, exhibit floral preferential expression in several basal angiosperms, suggesting evolutionary conservation of their floral functions. Members of other gene lineages appear to have independently evolved roles in plant reproductive tissues in individual taxa. Conclusions Our phylogenetic analyses provide the most detailed reconstruction of GSK3 gene evolution in land plants to date and offer new insights into the origins, relationships, and functions of family members. Notably, the diversity of this “green” branch of the gene family has increased in concert with the increasing morphological and physiological complexity of land plant life forms. Expression data for seed plants indicate that the functions of GSK3 genes have also diversified during evolutionary time.

Recent advances in phylogeny reconstruction and floral genetics set the stage for new investigations of the origin and diversification of the flower. We review the current state of angiosperm phylogeny, with an emphasis on basal lineages. With the surprising inclusion of Hydatellaceae with Nymphaeales, recent studies support the topology of Amborella sister to all other extant angiosperms, with Nymphaeales and then Austrobaileyales as subsequent sisters to all remaining angiosperms. Notable modifications from most recent analyses are the sister relationships of Chloranthaceae with the magnoliids and of Ceratophyllaceae with eudicots. We review "trends" in floral morphology and contrast historical, intuitive interpretations with explicit character-state reconstructions using molecular-based trees, focusing on (1) the size, number, and organization of floral organs; (2) the evolution of the perianth; (3) floral symmetry; and (4) floral synorganization. We provide summaries of those genes known to affect floral features that contribute to much of floral diversity. Although most floral genes have not been investigated outside of a few model systems, sufficient information is emerging to identify candidate genes for testing specific hypotheses in nonmodel plants. We conclude with a set of evo-devo case studies in which floral genetics have been linked to variation in floral morphology.

The origin and rapid diversification of the angiosperms (Darwin's “Abominable Mystery”) has engaged generations of researchers. Here, we examine the floral genetic programs of phylogenetically pivotal angiosperms (water lily, avocado, California poppy, and Arabidopsis ) and a nonflowering seed plant (a cycad) to obtain insight into the origin and subsequent evolution of the flower. Transcriptional cascades with broadly overlapping spatial domains, resembling the hypothesized ancestral gymnosperm program, are deployed across morphologically intergrading organs in water lily and avocado flowers. In contrast, spatially discrete transcriptional programs in distinct floral organs characterize the more recently derived angiosperm lineages represented by California poppy and Arabidopsis . Deep evolutionary conservation in the genetic programs of putatively homologous floral organs traces to those operating in gymnosperm reproductive cones. Female gymnosperm cones and angiosperm carpels share conserved genetic features, which may be associated with the ovule developmental program common to both organs. However, male gymnosperm cones share genetic features with both perianth (sterile attractive and protective) organs and stamens, supporting the evolutionary origin of the floral perianth from the male genetic program of seed plants.

Background Recent phylogenetic analyses have identified Amborella trichopoda, an understory tree species endemic to the forests of New Caledonia, as sister to a clade including all other known flowering plant species. The Amborella genome is a unique reference for understanding the evolution of angiosperm genomes because it can serve as an outgroup to root comparative analyses. A physical map, BAC end sequences and sample shotgun sequences provide a first view of the 870 Mbp Amborella genome. Results Analysis of Amborella BAC ends sequenced from each contig suggests that the density of long terminal repeat retrotransposons is negatively correlated with that of protein coding genes. Syntenic, presumably ancestral, gene blocks were identified in comparisons of the Amborella BAC contigs and the sequenced Arabidopsis thaliana, Populus trichocarpa, Vitis vinifera and Oryza sativa genomes. Parsimony mapping of the loss of synteny corroborates previous analyses suggesting that the rate of structural change has been more rapid on lineages leading to Arabidopsis and Oryza compared with lineages leading to Populus and Vitis. The gamma paleohexiploidy event identified in the Arabidopsis, Populus and Vitis genomes is shown to have occurred after the divergence of all other known angiosperms from the lineage leading to Amborella. Conclusions When placed in the context of a physical map, BAC end sequences representing just 5.4% of the Amborella genome have facilitated reconstruction of gene blocks that existed in the last common ancestor of all flowering plants. The Amborella genome is an invaluable reference for inferences concerning the ancestral angiosperm and subsequent genome evolution.

Current understanding of floral developmental genetics comes primarily from the core eudicot model Arabidopsis thaliana. Here, we explore the floral transcriptome of the basal angiosperm, Nuphar advena (water lily), for insights into the ancestral developmental program of flowers. We identify several thousand Nuphar genes with significantly upregulated floral expression, including homologs of the well-known ABCE floral regulators, deployed in broadly overlapping transcriptional programs across floral organ categories. Strong similarities in the expression profiles of different organ categories in Nuphar flowers are shared with the magnoliid Persea americana (avocado), in contrast to the largely organ-specific transcriptional cascades evident in Arabidopsis, supporting the inference that this is the ancestral condition in angiosperms. In contrast to most eudicots, floral organs are weakly differentiated in Nuphar and Persea, with staminodial intermediates between stamens and perianth in Nuphar, and between stamens and carpels in Persea. Consequently, the predominantly organ-specific transcriptional programs that characterize Arabidopsis flowers (and perhaps other eudicots) are derived, and correlate with a shift towards morphologically distinct floral organs, including differentiated sepals and petals, and a perianth distinct from stamens and carpels. Our findings suggest that the genetic regulation of more spatially discrete transcriptional programs underlies the evolution of floral morphology.

Whole-genome duplication (WGD), or polyploidy, followed by gene loss and diploidization has long been recognized as an important evolutionary force in animals, fungi and other organisms, especially plants. The success of angiosperms has been attributed, in part, to innovations associated with gene or whole-genome duplications, but evidence for proposed ancient genome duplications pre-dating the divergence of monocots and eudicots remains equivocal in analyses of conserved gene order. Here we use comprehensive phylogenomic analyses of sequenced plant genomes and more than 12.6 million new expressed-sequence-tag sequences from phylogenetically pivotal lineages to elucidate two groups of ancient gene duplications-one in the common ancestor of extant seed plants and the other in the common ancestor of extant angiosperms. Gene duplication events were intensely concentrated around 319 and 192 million years ago, implicating two WGDs in ancestral lineages shortly before the diversification of extant seed plants and extant angiosperms, respectively. Significantly, these ancestral WGDs resulted in the diversification of regulatory genes important to seed and flower development, suggesting that they were involved in major innovations that ultimately contributed to the rise and eventual dominance of seed plants and angiosperms.

Shaping Plant Evolution Amborella trichopoda is understood to be the most basal extant flowering plant and its genome is anticipated to provide insights into the evolution of plant life on Earth (see the Perspective by Adams ). To validate and assemble the sequence, Chamala et al. (p. 1516 ) combined fluorescent in situ hybridization (FISH), genomic mapping, and next-generation sequencing. The Amborella Genome Project (p. 10.1126/science.1241089 ) was able to infer that a whole-genome duplication event preceded the evolution of this ancestral angiosperm, and Rice et al. (p. 1468 ) found that numerous genes in the mitochondrion were acquired by horizontal gene transfer from other plants, including almost four entire mitochondrial genomes from mosses and algae.

The avocado (Persea americana) is a major crop commodity worldwide. Moreover, avocado, a paleopolyploid, is an evolutionary "outpost" among flowering plants, representing a basal lineage (the magnoliid clade) near the origin of the flowering plants themselves. Following centuries of selective breeding, avocado germplasm has been characterized at the level of microsatellite and RFLP markers. Nonetheless, little is known beyond these general diversity estimates, and much work remains to be done to develop avocado as a major subtropical-zone crop. Among the goals of avocado improvement are to develop varieties with fruit that will "store" better on the tree, show uniform ripening and have better post-harvest storage. Avocado transcriptome sequencing, genome mapping and partial genomic sequencing will represent a major step toward the goal of sequencing the entire avocado genome, which is expected to aid in improving avocado varieties and production, as well as understanding the evolution of flowers from non-flowering seed plants (gymnosperms). Additionally, continued evolutionary and other comparative studies of flower and fruit development in different avocado strains can be accomplished at the gene expression level, including in comparison with avocado relatives, and these should provide important insights into the genetic regulation of fruit development in basal angiosperms.

Although the flower is the central feature of the angiosperms, little is known of its origin and subsequent diversification. The ABC model has long been the unifying paradigm for floral developmental genetics, but it is based on phylogenetically derived eudicot models. Synergistic research involving phylogenetics, classical developmental studies, genomics and developmental genetics has afforded valuable new insights into floral evolution in general, and the early flower in particular.Genomic studies indicate that basal angiosperms, and by inference the earliest angiosperms, had a rich tool kit of floral genes. Homologues of the ABCE floral organ identity genes are also present in basal angiosperm lineages; however, C-, E- and particularly B-function genes are more broadly expressed in basal lineages. There is no single model of floral organ identity that applies to all angiosperms; there are multiple models that apply depending on the phylogenetic position and floral structure of the group in question. The classic ABC (or ABCE) model may work well for most eudicots. However, modifications are needed for basal eudicots and, the focus of this paper, basal angiosperms. We offer 'fading borders' as a testable hypothesis for the basal-most angiosperms and, by inference, perhaps some of the earliest (now extinct) angiosperms.

Premise of the study: Here we present a series of protocols for RNA extraction across a diverse array of plants; we focus on woody, aromatic, aquatic, and other chemically complex taxa. Methods and Results: Ninety-one taxa were subjected to RNA extraction with three methods presented here: (1) TRIzol/TURBO DNA-free kits using the manufacturer’s protocol with the addition of sarkosyl; (2) a combination method using cetyltrimethylammonium bromide (CTAB) and TRIzol/sarkosyl/TURBO DNA-free; and (3) a combination of CTAB and QIAGEN RNeasy Plant Mini Kit. Bench-ready protocols are given. Conclusions: After an iterative process of working with chemically complex taxa, we conclude that the use of TRIzol supplemented with sarkosyl and the TURBO DNA-free kit is an effective, efficient, and robust method for obtaining RNA from 100 mg of leaf tissue of land plant species (Embryophyta) examined. Our protocols can be used to provide RNA of suitable stability, quantity, and quality for transcriptome sequencing.

The ABC‐model of floral organ identity explains the regular, sequential development of sepals, petals, stamens, and carpels in eudicot flowers. This general model, based on studies of the derived eudicots Arabidopsis and Antirrhinum, may apply to nearly all eudicots, most of which are characterized by discrete whorls of floral organs. However, floral morphology of basal angiosperms is typically characterized by variable numbers of floral parts and gradual transitions among floral organs, and it is unclear that the ABC‐model applies to such flowers. Here we explore the origin and evolution of ABC‐function through consideration of expression data for homologs of ABC‐genes for basal angiosperms and conclude that the ABC‐model represents an evolutionarily derived regulatory network that arose through spatial restriction of regulatory gene expression.

Phylogenetic relationships among 122 species of Lauraceae representing 44 of the 55 currently recognized genera are inferred from sequence variation in the chloroplast and nuclear genomes. The trnL-trnF, trnT-trnL, psbA-trnH, and rpll6 regions of cpDNA, and the 5' end of 26S rDNA resolved major lineages, while the ITS/5.8S region of rDNA resolved a large terminal lade. The phylogenetic estimate is used to assess morphology-based views of relationships and, with a temporal dimension added, to reconstruct the biogeographic history of the family. Results suggest Lauraceae radiated when trans-Tethyean migration was relatively easy, and basal lineages are established on either Gondwanan or Laurasian terrains by the Late Cretaceous. Most genera with Gondwanan histories place in Cryptocaryeae, but a small group of South American genera, the Chlorocardium-Mezilauruls lade, represent a separate Gondwanan lineage. Caryodaphnopsis and Neocinnamomum may be the only extant representatives of the ancient Lauraceae flora docu- mented in Mid- to Late Cretaceous Laurasian strata. Remaining genera place in a terminal Perseeae-Laureae lade that radiated in Early Eocene Laurasia. Therein, non-cupulate genera associate as the Persea group, and cupuliferous genera sort to Laureae of most classifications or Cinnamomeae sensu Kostermans. Laureae are Laurasian relicts in Asia. The Persea group and Cinnamomum group (of Cinnamomeae) show tropical amphi-Pacific disjunctions here credited to disruption of boreotropical ranges by Eocene-Oligocene climatic cooling. The Ocotea complex accommodates re- maining Cinnamomeae and shows a trans-Atlantic disjunction possibly derived from a Madrean-Tethyan ancestral distribution. These findings support Laurasian ancestry for most extant Lauraceae, with their considerable neotropical representation primarily derived from Early Miocene radiation of the Ocotea complex upon reaching South America.

Background Although it is agreed that a major polyploidy event, gamma, occurred within the eudicots, the phylogenetic placement of the event remains unclear. Results To determine when this polyploidization occurred relative to speciation events in angiosperm history, we employed a phylogenomic approach to investigate the timing of gene set duplications located on syntenic gamma blocks. We populated 769 putative gene families with large sets of homologs obtained from public transcriptomes of basal angiosperms, magnoliids, asterids, and more than 91.8 gigabases of new next-generation transcriptome sequences of non-grass monocots and basal eudicots. The overwhelming majority (95%) of well-resolved gamma duplications was placed before the separation of rosids and asterids and after the split of monocots and eudicots, providing strong evidence that the gamma polyploidy event occurred early in eudicot evolution. Further, the majority of gene duplications was placed after the divergence of the Ranunculales and core eudicots, indicating that the gamma appears to be restricted to core eudicots. Molecular dating estimates indicate that the duplication events were intensely concentrated around 117 million years ago. Conclusions The rapid radiation of core eudicot lineages that gave rise to nearly 75% of angiosperm species appears to have occurred coincidentally or shortly following the gamma triplication event. Reconciliation of gene trees with a species phylogeny can elucidate the timing of major events in genome evolution, even when genome sequences are only available for a subset of species represented in the gene trees. Comprehensive transcriptome datasets are valuable complements to genome sequences for high-resolution phylogenomic analysis.

Two new species of Endlicheria are described, illustrated, and discussed. Endlicheria canescens has hitherto been confused with Ocotea endlicheriopsis Mez, and Endlicheria chalisea was previously known only by the densely pubescent cupules of fruiting material. Two undescribed species were encountered dur- ing preparation of a revision of the Guianian mem- bers of Endlicheria. Described by Nees in 1833, this neotropical genus currently accommodates all dioecious Lauraceae that possess exinvolucrate thyrsoid or thyrso-paniculate inflorescences and two-thecous anthers. Although this generic concept has attracted recent criticism (Rohwer et al., 1991), it is maintained while the genus awaits monograph- ic attention. At the species level, taxonomic delim- itation in Endlicheria is complicated by dioecy. Species circumscription depends on the association of male and female specimens and must confront sexual dimorphism in floral structures. Fortunately, vegetative features are relatively constant between the sexes and may be used to support species con- cepts based on floral characters. Each of the new species here proposed is circumscribed by unique combinations of vegetative and reproductive fea- tures.

Background We have developed a simulation approach to help determine the optimal mixture of sequencing methods for most complete and cost effective transcriptome sequencing. We compared simulation results for traditional capillary sequencing with "Next Generation" (NG) ultra high-throughput technologies. The simulation model was parameterized using mappings of 130,000 cDNA sequence reads to the Arabidopsis genome (NCBI Accession SRA008180.19). We also generated 454-GS20 sequences and de novo assemblies for the basal eudicot California poppy (Eschscholzia californica) and the magnoliid avocado (Persea americana) using a variety of methods for cDNA synthesis. Results The Arabidopsis reads tagged more than 15,000 genes, including new splice variants and extended UTR regions. Of the total 134,791 reads (13.8 MB), 119,518 (88.7%) mapped exactly to known exons, while 1,117 (0.8%) mapped to introns, 11,524 (8.6%) spanned annotated intron/exon boundaries, and 3,066 (2.3%) extended beyond the end of annotated UTRs. Sequence-based inference of relative gene expression levels correlated significantly with microarray data. As expected, NG sequencing of normalized libraries tagged more genes than non-normalized libraries, although non-normalized libraries yielded more full-length cDNA sequences. The Arabidopsis data were used to simulate additional rounds of NG and traditional EST sequencing, and various combinations of each. Our simulations suggest a combination of FLX and Solexa sequencing for optimal transcriptome coverage at modest cost. We have also developed ESTcalc http://fgp.huck.psu.edu/NG_Sims/ngsim.pl, an online webtool, which allows users to explore the results of this study by specifying individualized costs and sequencing characteristics. Conclusion NG sequencing technologies are a highly flexible set of platforms that can be scaled to suit different project goals. In terms of sequence coverage alone, the NG sequencing is a dramatic advance over capillary-based sequencing, but NG sequencing also presents significant challenges in assembly and sequence accuracy due to short read lengths, method-specific sequencing errors, and the absence of physical clones. These problems may be overcome by hybrid sequencing strategies using a mixture of sequencing methodologies, by new assemblers, and by sequencing more deeply. Sequencing and microarray outcomes from multiple experiments suggest that our simulator will be useful for guiding NG transcriptome sequencing projects in a wide range of organisms.