A high-quality Buxus austro-yunnanensis (Buxales) genome provides new insights into karyotype evolution in early eudicots.

BACKGROUND: Eudicots are the most diverse group of flowering plants that compromise five well-defined lineages: core eudicots, Ranunculales, Proteales, Trochodendrales, and Buxales. However, the phylogenetic relationships between these five lineages and their chromosomal evolutions remain unclear, and a lack of high-quality genome analyses for Buxales has hindered many efforts to address this knowledge gap. RESULTS: Here, we present a high-quality chromosome-level genome of Buxus austro-yunnanensis (Buxales). Our phylogenomic analyses revealed that Buxales and Trochodendrales are genetically similar and classified as sisters. Additionally, both are sisters to the core eudicots, while Ranunculales was found to be the first lineage to diverge from these groups. Incomplete lineage sorting and hybridization were identified as the main contributors to phylogenetic discordance (34.33%) between the lineages. In fact, B. austro-yunnanensis underwent only one whole-genome duplication event, and collinear gene phylogeny analyses suggested that separate independent polyploidizations occurred in the five eudicot lineages. Using representative genomes from these five lineages, we reconstructed the ancestral eudicot karyotype (AEK) and generated a nearly gapless karyotype projection for each eudicot species. Within core eudicots, we recovered one common chromosome fusion event in asterids and malvids, respectively. Further, we also found that the previously reported fused AEKs in Aquilegia (Ranunculales) and Vitis (core eudicots) have different fusion positions, which indicates that these two species have different karyotype evolution histories. CONCLUSIONS: Based on our phylogenomic and karyotype evolution analyses, we revealed the likely relationships and evolutionary histories of early eudicots. Ultimately, our study expands genomic resources for early-diverging eudicots.

Developmental regulation of leaf venation patterns: monocot versus eudicots and the role of auxin.

Organisation and patterning of the vascular network in land plants varies in different taxonomic, developmental and environmental contexts. In leaves, the degree of vascular strand connectivity influences both light and CO2 harvesting capabilities as well as hydraulic capacity. As such, developmental mechanisms that regulate leaf venation patterning have a direct impact on physiological performance. Development of the leaf venation network requires the specification of procambial cells within the ground meristem of the primordium and subsequent proliferation and differentiation of the procambial lineage to form vascular strands. An understanding of how diverse venation patterns are manifest therefore requires mechanistic insight into how procambium is dynamically specified in a growing leaf. A role for auxin in this process was identified many years ago, but questions remain. In this review we first provide an overview of the diverse venation patterns that exist in land plants, providing an evolutionary perspective. We then focus on the developmental regulation of leaf venation patterns in angiosperms, comparing patterning in eudicots and monocots, and the role of auxin in each case. Although common themes emerge, we conclude that the developmental mechanisms elucidated in eudicots are unlikely to fully explain how parallel venation patterns in monocot leaves are elaborated.

Understanding the root xylem plasticity for designing resilient crops.

Xylem is the main route for transporting water, minerals and a myriad of signalling molecules within the plant. With its onset during early embryogenesis, the development of the xylem relies on hormone gradients, the activity of unique transcription factors, the distribution of mobile microRNAs, and receptor-ligand pathways. These regulatory mechanisms are often interconnected and together contribute to the plasticity of this water-conducting tissue. Environmental stresses, such as drought and salinity, have a great impact on xylem patterning. A better understanding of how the structural properties of the xylem are regulated in normal and stress conditions will be instrumental in developing crops of the future. In addition, vascular wilt pathogens that attack the xylem are becoming increasingly problematic. Further knowledge of xylem development in response to these pathogens will bring new solutions against these diseases. In this review, we summarize recent findings on the molecular mechanisms of xylem formation that largely come from Arabidopsis research with additional insights from tomato and monocot species. We emphasize the impact of abiotic factors and pathogens on xylem plasticity and the urgent need to uncover the underlying mechanisms. Finally, we discuss the multidisciplinary approach to model xylem capacities in crops.

Molecular phylogeny of the phytoparasitic mite family Phytoptidae (Acariformes: Eriophyoidea) identified the female genitalic anatomy as a major macroevolutionary factor and revealed multiple origins of gall induction.

Phytoptidae s.str. is a lineage of eriophyoid mites associated with angiosperms. Based on representative taxon sampling and four gene markers (COI, HSP70, 18S, and 28S), we inferred the molecular phylogeny of this group and performed comparative analyses of cuticle-lined female internal genitalia. Although basal relationships were unclear, several well supported clades were recovered. These clades were supported by geography, host associations, and female genital anatomy, but contradicted the current morphology-based systematics. The monophyly of each of five conventional supraspecific groupings (Fragariocoptes, Phytoptus, Phytoptinae, Sierraphytoptinae, and Sierraphytoptini) is rejected based on a series of statistical tests. Additionally, four morphological characters (the absence of tibial solenidion φ and opisthosomal seta c1, presence of telosomal pseudotagma, and 'morphotype') were found to be homoplasies that cannot be used to confidently delimit supraspecific lineages of phytoptids. However, our molecular topology was highly congruent with female genital characters. Eight molecular clades were unambiguously supported by the shapes and topography of the spermathecal apparatus and genital apodemes. This suggests that the female genital anatomy could be an important factor affecting cladogenesis in Phytoptidae, a conclusion contrasting with the general expectation that host characteristics should be a major macroevolutionary force influencing the evolution of host-specific symbionts. Indeed, despite the high host-specificity, there were no apparent cophylogenetic patterns. Furthermore, we show that gall-inducing ability evolved multiple times in phytoptids. Because gall formation creates nearly instantaneous niche partitioning and the potential loss or reduction of gene flow, we hypothesize that it could be an important evolutionary factor affecting speciation within different host-associated clades of phytoptid mites.

Combined LM and SEM study of the middle Miocene (Sarmatian) palynoflora from the Lavanttal Basin, Austria: Part V. Magnoliophyta 3 - Myrtales to Ericales.

The continued investigation of the middle Miocene palynoflora from the Lavanttal Basin reveals numerous additional angiosperm taxa. The Myrtales to Ericales pollen record documented here comprises 46 different taxa belonging to Onagraceae (Ludwigia), Ericaceae (Craigia, Reevesia, Tilia), Anacardiaceae (Pistacia), Rutaceae (Zanthoxylum), Sapindaceae (Acer), Santalaceae (Arceuthobium), Amaranthaceae, Caryophyllaceae, Polygonaceae (Persicaria, Rumex), Cornaceae (Alangium, Cornus, Nyssa), Ebenaceae (Diospyros), Ericaceae (Andromeda, Arbutus, Empetrum, Erica), Sapotaceae (Pouteria, Sideroxylon), Styracaceae (Rehderodendron) and Symplocaceae (Symplocos). Köppen signatures of potential modern analogues of the additional fossil woody elements confirm the hypothesis of a subtropical (Cfa, Cwa) climate at lower elevations and subsequent transition into a temperate climate with altitudinal succession (Cfa → Cfb/Dfa → Dfb; Cwa → Cwb → Dwb-climate). The fossil plants represent different vegetation units, from wetland lowlands to well-drained montane forests. Many of the fossil taxa have potential modern analogues that can be classified as nemoral and/or meridio-nemoral and/or semihumid-meridional vegetation elements. New is the recognition of oreotropical elements, which are direct indicators for a substantial altitudinal gradient.

Cretaceous asterid evolution: fruits of Eydeia jerseyensis sp. nov. (Cornales) from the upper Turonian of eastern North America.

BACKGROUND AND AIMS: The asterids (>80 000 extant species) appear in the fossil record with considerable diversity near the Turonian-Coniacian boundary (~90 Ma; Late Cretaceous) and are strongly represented in the earliest diverging lineage, Cornales. These early asterid representatives have so far been reported from western North America and eastern Asia. In this study, we characterize a new cornalean taxon based on charcoalified fruits from the upper Turonian of eastern North America, a separate landmass from western North America at the time, and identify early palaeobiogeographical patterns of Cornales during the Cretaceous. METHODS: Fossils were studied and imaged using scanning electron microscopy and micro-computed tomography (micro-CT) scanning. To assess the systematic affinities of the fossils, phylogenetic analyses were conducted using maximum parsimony. KEY RESULTS: The charcoalified fruits are represented by tri-locular woody endocarps with dorsal apically opening germination valves. Three septa intersect to form a robust central axis. Endocarp ground tissue consists of two zones: an outer endocarp composed of isodiametric sclereids and an inner endocarp containing circum-locular fibres. Central vasculature is absent; however, there are several small vascular bundles scattered within the septa. Phylogenetic analysis places the new taxon within the extinct genus Eydeia. DISCUSSION: Thick-walled endocarps with apically opening germination valves, no central vascular bundle and one seed per locule are indicative of the order Cornales. Comparative analysis suggests that the fossils represent a new species, Eydeia jerseyensis sp. nov. This new taxon is the first evidence of Cornales in eastern North America during the Cretaceous and provides insights into the palaeobiogeography and initial diversification of the order.

Characterization of Cellulose Synthase A (CESA) Gene Family in Eudicots.

Cellulose synthase A (CESA) is a key enzyme involved in the complex process of plant cell wall biosynthesis, and it remains a productive subject for research. We employed systems biology approaches to explore structural diversity of eudicot CESAs by exon-intron organization, mode of duplication, synteny, and splice site analyses. Using a combined phylogenetics and comparative genomics approach coupled with co-expression networks we reconciled the evolution of cellulose synthase gene family in eudicots and found that the basic forms of CESA proteins are retained in angiosperms. Duplications have played an important role in expansion of CESA gene family members in eudicots. Co-expression networks showed that primary and secondary cell wall modules are duplicated in eudicots. We also identified 230 simple sequence repeat markers in 103 eudicot CESAs. The 13 identified conserved motifs in eudicots will provide a basis for gene identification and functional characterization in other plants. Furthermore, we characterized (in silico) eudicot CESAs against senescence and found that expression levels of CESAs decreased during leaf senescence.

The NnCenH3 protein and centromeric DNA sequence profiles of Nelumbo nucifera Gaertn. (sacred lotus) reveal the DNA structures and dynamics of centromeres in basal eudicots.

Centromeres on eukaryotic chromosomes consist of large arrays of DNA repeats that undergo very rapid evolution. Nelumbo nucifera Gaertn. (sacred lotus) is a phylogenetic relict and an aquatic perennial basal eudicot. Studies concerning the centromeres of this basal eudicot species could provide ancient evolutionary perspectives. In this study, we characterized the centromeric marker protein NnCenH3 (sacred lotus centromere-specific histone H3 variant), and used a chromatin immunoprecipitation (ChIP)-based technique to recover the NnCenH3 nucleosome-associated sequences of sacred lotus. The properties of the centromere-binding protein and DNA sequences revealed notable divergence between sacred lotus and other flowering plants, including the following factors: (i) an NnCenH3 alternative splicing variant comprising only a partial centromere-targeting domain, (ii) active genes with low transcription levels in the NnCenH3 nucleosomal regions, and (iii) the prevalence of the Ty1/copia class of long terminal repeat (LTR) retrotransposons in the centromeres of sacred lotus chromosomes. In addition, the dynamic natures of the centromeric region showed that some of the centromeric repeat DNA sequences originated from telomeric repeats, and a pair of centromeres on the dicentric chromosome 1 was inactive in the metaphase cells of sacred lotus. Our characterization of the properties of centromeric DNA structure within the sacred lotus genome describes a centromeric profile in ancient basal eudicots and might provide evidence of the origins and evolution of centromeres. Furthermore, the identification of centromeric DNA sequences is of great significance for the assembly of the sacred lotus genome.

Complete plastome sequencing of both living species of Circaeasteraceae (Ranunculales) reveals unusual rearrangements and the loss of the ndh gene family.

BACKGROUND: Among the 13 families of early-diverging eudicots, only Circaeasteraceae (Ranunculales), which consists of the two monotypic genera Circaeaster and Kingdonia, lacks a published complete plastome sequence. In addition, the phylogenetic position of Circaeasteraceae as sister to Lardizabalaceae has only been weakly or moderately supported in previous studies using smaller data sets. Moreover, previous plastome studies have documented a number of novel structural rearrangements among early-divergent eudicots. Hence it is important to sequence plastomes from Circaeasteraceae to better understand plastome evolution in early-diverging eudicots and to further investigate the phylogenetic position of Circaeasteraceae. RESULTS: Using an Illumina HiSeq 2000, complete plastomes were sequenced from both living members of Circaeasteraceae: Circaeaster agrestis and Kingdonia uniflora . Plastome structure and gene content were compared between these two plastomes, and with those of other early-diverging eudicot plastomes. Phylogenetic analysis of a 79-gene, 99-taxon data set including exemplars of all families of early-diverging eudicots was conducted to resolve the phylogenetic position of Circaeasteraceae. Both plastomes possess the typical quadripartite structure of land plant plastomes. However, a large ~49 kb inversion and a small ~3.5 kb inversion were found in the large single-copy regions of both plastomes, while Circaeaster possesses a number of other rearrangements, particularly in the Inverted Repeat. In addition, infA was found to be a pseudogene and accD was found to be absent within Circaeaster, whereas all ndh genes, except for ndhE and ndhJ, were found to be either pseudogenized (ΨndhA, ΨndhB, ΨndhD, ΨndhH and ΨndhK) or absent (ndhC, ndhF, ndhI and ndhG) in Kingdonia. Circaeasteraceae was strongly supported as sister to Lardizabalaceae in phylogenetic analyses. CONCLUSION: The first plastome sequencing of Circaeasteraceae resulted in the discovery of several unusual rearrangements and the loss of ndh genes, and confirms the sister relationship between Circaeasteraceae and Lardizabalaceae. This research provides new insight to characterize plastome structural evolution in early-diverging eudicots and to better understand relationships within Ranunculales .

Resolution of deep eudicot phylogeny and their temporal diversification using nuclear genes from transcriptomic and genomic datasets.

Explosive diversification is widespread in eukaryotes, making it difficult to resolve phylogenetic relationships. Eudicots contain c. 75% of extant flowering plants, are important for human livelihood and terrestrial ecosystems, and have probably experienced explosive diversifications. The eudicot phylogenetic relationships, especially among those of the Pentapetalae, remain unresolved. Here, we present a highly supported eudicot phylogeny and diversification rate shifts using 31 newly generated transcriptomes and 88 other datasets covering 70% of eudicot orders. A highly supported eudicot phylogeny divided Pentapetalae into two groups: one with rosids, Saxifragales, Vitales and Santalales; the other containing asterids, Caryophyllales and Dilleniaceae, with uncertainty for Berberidopsidales. Molecular clock analysis estimated that crown eudicots originated c. 146 Ma, considerably earlier than earliest tricolpate pollen fossils and most other molecular clock estimates, and Pentapetalae sequentially diverged into eight major lineages within c. 15 Myr. Two identified increases of diversification rate are located in the stems leading to Pentapetalae and asterids, and lagged behind the gamma hexaploidization. The nuclear genes from newly generated transcriptomes revealed a well-resolved eudicot phylogeny, sequential separation of major core eudicot lineages and temporal mode of diversifications, providing new insights into the evolutionary trend of morphologies and contributions to the diversification of eudicots.

Characterization of CYCLOIDEA-like genes in Proteaceae, a basal eudicot family with multiple shifts in floral symmetry.

BACKGROUND AND AIMS: The basal eudicot family Proteaceae (approx. 1700 species) shows considerable variation in floral symmetry but has received little attention in studies of evolutionary development at the genetic level. A framework for understanding the shifts in floral symmetry in Proteaceae is provided by reconstructing ancestral states on an upated phylogeny of the family, and homologues of CYCLOIDEA (CYC), a key gene for the control of floral symmetry in both monocots and eudicots, are characterized. METHODS: Perianth symmetry transitions were reconstructed on a new species-level tree using parsimony and maximum likelihood. CYC-like genes in 35 species (31 genera) of Proteaceae were sequenced and their phylogeny was reconstructed. Shifts in selection pressure following gene duplication were investigated using nested branch-site models of sequence evolution. Expression patterns of CYC homologues were characterized in three species of Grevillea with different types of floral symmetry. KEY RESULTS: Zygomorphy has evolved 10-18 times independently in Proteaceae from actinomorphic ancestors, with at least four reversals to actinomorphy. A single duplication of CYC-like genes occurred prior to the diversification of Proteaceae, with putative loss or divergence of the ProtCYC1 paralogue in more than half of the species sampled. No shifts in selection pressure were detected in the branches subtending the two ProtCYC paralogues. However, the amino acid sequence preceding the TCP domain is strongly divergent in Grevillea ProtCYC1 compared with other species. ProtCYC genes were expressed in developing flowers of both actinomorphic and zygomorphic Grevillea species, with late asymmetric expression in the perianth of the latter. CONCLUSION: Proteaceae is a remarkable family in terms of the number of transitions in floral symmetry. Furthermore, although CYC-like genes in Grevillea have unusual sequence characteristics, they display patterns of expression that make them good candidates for playing a role in the establishment of floral symmetry.

Floral morphology and anatomy of Ophiocaryon, a paedomorphic genus of Sabiaceae.

Background and Aims: Ophiocaryon is a lesser known genus in Sabiaceae. This study examines flowers of six Ophiocaryon species in comparison with Meliosmaalba, to identify taxonomically informative characters for understanding relationships within the family Sabiaceae, to imply previously unknown pollination mechanisms of Ophiocaryon, and to contribute to the placement of Sabiaceae within the early-diverging eudicots. Methods: Floral morphology and anatomy of six Ophiocaryon species and M. alba were studied and described using scanning electron microscopy, clearing techniques and resin sectioning. Key Results: Novel characters of Ophiocaryon were identified, e.g. conical cells on petals, different kinds of orbicules in anthers, stomata on nectary appendage tips and ovary, two distinct surface patterns on stamens and ovary, tanniferous cell layers in the ovary wall, and acorn-shaped unitegmic ovules with very short integuments. Comparison of floral characters between Ophiocaryon and Meliosma found that the calyx, corolla, androecium and gynoecium of Ophiocaryon resemble an undeveloped state of the latter taxon, reflecting a paedomorphic regression of the flower of Ophiocaryon. The flower morphology and anatomy of Ophiocaryon was compared with its putative sister species M. alba, but no clear shared derived characters could be detected. Moreover, the findings of scent, presence of conical cells on petals and a nectary suggest flowers are pollinated by small insects with a secondary pollen presentation on the cupula of fertile stamens. Conclusions: We found that Ophiocaryon may be derived from ancestors that were similar to extant Meliosma in their flower structure and pollination mechanism. However, the lack of shared derived characters between Ophiocaryon and its phylogenetic sister group M. alba is puzzling and requires further investigations on the diversity of the latter species.

Floral development of Berberidopsis beckleri - can an additional species of the Berberidopsidaceae add evidence to floral evolution in the core eudicots?

Background and Aims: Berberidopsis beckleri is one of three species of the family Berberidopsidaceae. The flower of Berberidopsis is unusual for core eudicots in being spiral with an undifferentiated perianth. In a previous study of the sister species B. corallina , it was suggested that Berberidopsidaceae represent a prototype for the origin of the bipartite perianth and pentamery in core eudicots. Methods: The floral development of B. beckleri was investigated with a scanning electron microscope and compared with previous studies on B. corallina and Aextoxicon punctatum of Berberidopsidales. Key Results: Flowers are inserted at the end of short shoots, which are not distinguishable from a pedicel. The initiation of perianth parts is highly predictable and spiral with a divergence angle of 137·5°, in a progression of a variable number of bracts to weakly differentiated sepaloid and petaloid tepals. The androecium most often consists 11 stamens arising in a rapid sequence. Compared with B. corallina , the number of perianth parts and stamens is more variable and there is no evidence of an alternation of shorter and longer plastochrons leading to a whorled arrangement. However, the gynoecium is generally pentamerous and arises from five primordia. The carpels are laterally connected into massive intercarpellary ridges on which ovules are initiated. Conclusions: The position of Streptothamnus within Berberidopsidaceae is questioned. It is demonstrated that the floral development of Berberidopsis beckleri lies within a gradient from spiral flowers without perianth differentiation leading to flowers with differentiated sepals and petals. The arrangement of flowers in compact inflorescences in B. corallina and Aextoxicon leads to a more stabilized arrangement of organs in whorls. The inherent variability of the flower of Berberidopsis is well correlated with the limited canalization of flowers in taxa at the base of the core eudicots and could act as a prototype for the current eudicot floral Bauplan.

Evolution of the process underlying floral zygomorphy development in pentapetalous angiosperms.

PREMISE OF THE STUDY: Observations of floral ontogeny indicated that floral organ initiation in pentapetalous flowers most commonly results in a median-abaxial (MAB) petal during early development, a median-adaxial (MAD) petal being less common. Such different patterns of floral organ initiation might be linked with different morphologies of floral zygomorphy that have evolved in Asteridae. Here, we provide the first study of zygomorphy in pentapetalous angiosperms placed in a phylogenetic framework, the goal being to find if the different patterns of floral organ initiation are connected with particular patterns of zygomorphy. METHODS: We analyzed patterns of floral organ initiation and displays of zygomorphy, extracted from floral diagrams representing 405 taxa in 330 genera, covering 83% of orders (30 out of 36) and 37% of families (116 out of 313) in core eudicots in the context of a phylogeny using ancestral state reconstructions. KEY RESULTS: The MAB petal initiation is the ancestral state of the pattern of floral organ initiation in pentapetalous angiosperms. Taxa with MAD petal initiation represent ∼30 independent origins from the ancestral MAB initiation. There are distinct developmental processes that give rise to zygomorphy in different lineages of pentapetalous angiosperms, closely related lineages being likely to share similar developmental processes. CONCLUSIONS: We have demonstrated that development indeed constrains the processes that give rise to floral zygomorphy, while phylogenetic distance allows relaxation of these constraints, which provides novel insights on the role that development plays in the evolution of floral zygomorphy.

Root structure-function relationships in 74 species: evidence of a root economics spectrum related to carbon economy.

Although fine roots are important components of the global carbon cycle, there is limited understanding of root structure-function relationships among species. We determined whether root respiration rate and decomposability, two key processes driving carbon cycling but always studied separately, varied with root morphological and chemical traits, in a coordinated way that would demonstrate the existence of a root economics spectrum (RES). Twelve traits were measured on fine roots (diameter ≤ 2 mm) of 74 species (31 graminoids and 43 herbaceous and dwarf shrub eudicots) collected in three biomes. The findings of this study support the existence of a RES representing an axis of trait variation in which root respiration was positively correlated to nitrogen concentration and specific root length and negatively correlated to the root dry matter content, lignin : nitrogen ratio and the remaining mass after decomposition. This pattern of traits was highly consistent within graminoids but less consistent within eudicots, as a result of an uncoupling between decomposability and morphology, and of heterogeneity of individual roots of eudicots within the fine-root pool. The positive relationship found between root respiration and decomposability is essential for a better understanding of vegetation-soil feedbacks and for improving terrestrial biosphere models predicting the consequences of plant community changes for carbon cycling.

Phylogenomic and structural analyses of 18 complete plastomes across nearly all families of early-diverging eudicots, including an angiosperm-wide analysis of IR gene content evolution.

The grade of early-diverging eudicots includes five major lineages: Ranunculales, Trochodendrales, Buxales, Proteales and Sabiaceae. To examine the evolution of plastome structure in early-diverging eudicots, we determined the complete plastome sequences of eight previously unsequenced early-diverging eudicot taxa, Pachysandra terminalis (Buxaceae), Meliosma aff. cuneifolia (Sabiaceae), Sabia yunnanensis (Sabiaceae), Epimedium sagittatum (Berberidaceae), Euptelea pleiosperma (Eupteleaceae), Akebia trifoliata (Lardizabalaceae), Stephania japonica (Menispermaceae) and Papaver somniferum (Papaveraceae), and compared them to previously published plastomes of the early-diverging eudicots Buxus, Tetracentron, Trochodendron, Nelumbo, Platanus, Nandina, Megaleranthis, Ranunculus, Mahonia and Macadamia. All of the newly sequenced plastomes share the same 79 protein-coding genes, 4 rRNA genes, and 30 tRNA genes, except for that of Epimedium, in which infA is pseudogenized and clpP is highly divergent and possibly a pseudogene. The boundaries of the plastid Inverted Repeat (IR) were found to vary significantly across early-diverging eudicots; IRs ranged from 24.3 to 36.4kb in length and contained from 18 to 33 genes. Based on gene content, the IR was classified into six types, with shifts among types characterized by high levels of homoplasy. Reconstruction of ancestral IR gene content suggested that 18 genes were likely present in the IR region of the ancestor of eudicots. Maximum likelihood phylogenetic analysis of a 79-gene, 97-taxon data set that included all available early-diverging eudicots and representative sampling of remaining angiosperm diversity largely agreed with previous estimates of early-diverging eudicot relationships, but resolved Trochodendrales rather than Buxales as sister to Gunneridae, albeit with relatively weak bootstrap support, conflicting with what has been found for these three clades in most previous analyses. In addition, Proteales was resolved as sister to Sabiaceae with the highest support (bootstrap >90%) yet observed in plastome-scale phylogenetic analyses.

The sacred lotus genome provides insights into the evolution of flowering plants.

Sacred lotus (Nelumbo nucifera) is an ornamental plant that is also used for food and medicine. This basal eudicot species is especially important from an evolutionary perspective, as it occupies a critical phylogenetic position in flowering plants. Here we report the draft genome of a wild strain of sacred lotus. The assembled genome is 792 Mb, which is approximately 85-90% of genome size estimates. We annotated 392 Mb of repeat sequences and 36,385 protein-coding genes within the genome. Using these sequence data, we constructed a phylogenetic tree and confirmed the basal location of sacred lotus within eudicots. Importantly, we found evidence for a relatively recent whole-genome duplication event; any indication of the ancient paleo-hexaploid event was, however, absent. Genomic analysis revealed evidence of positive selection within 28 embryo-defective genes and one annexin gene that may be related to the long-term viability of sacred lotus seed. We also identified a significant expansion of starch synthase genes, which probably elevated starch levels within the rhizome of sacred lotus. Sequencing this strain of sacred lotus thus provided important insights into the evolution of flowering plant and revealed genetic mechanisms that influence seed dormancy and starch synthesis.

Functional and evolutionary analysis of the AP1/SEP/AGL6 superclade of MADS-box genes in the basal eudicot Epimedium sagittatum.

BACKGROUND AND AIMS: MADS-box transcriptional regulators play important roles during plant development. Based on phylogenetic reconstruction, the AP1/SEP/AGL6 superclade of floral MADS-box genes underwent one or two duplication events in the common ancestor of the core eudicots. However, the functional evolution of the AP1/SEP/AGL6 superclade in basal eudicots remains uncharacterized. Epimedium sagittatum is a basal eudicot species valued for its medicinal properties and showing unique floral morphology. In this study, structural and functional variation of FUL-like (AP1 subfamily), SEP-like and AGL6-like genes in this species was investigated to further our understanding of flower evolution in angiosperms. Detailed investigations into the microsynteny and evolutionary history of the floral A and E class MADS-box genes in eudicots were undertaken and used to trace their genomic rearrangements. METHODS: One AP1-like gene, two SEP-like genes and one AGL6-like gene were cloned from E. sagittatum. Their expression patterns were examined using quantitative RT-PCR in different vegetative and reproductive organs at two developmental stages. Yeast two-hybrid assays were carried out among AP1/SEP/AGL6 superclade, AP3/PI and AGAMOUS subfamily members for elucidation of dimerization patterns. In addition, possible formation of a ternary complex involving B class proteins with the A class protein EsFUL-like, the E class SEP-like protein EsAGL2-1 or the AGL6-class protein EsAGL6 were detected using yeast three-hybrid assays. Transgenic Arabidopsis or tobacco plants expressing EsFUL-like, EsAGL2-1 and EsAGL6-like under the cauliflower mosaic virus (CaMV) 35S promoter were generated and analysed. Genomic studies of AP1 syntenic regions in arabidopsis, columbine, strawberry, papaya, peach, grapevine and tomato were conducted for microsyntenic analyses. KEY RESULTS: Sequence and phylogenetic analyses showed that EsFUL-like is a member of the AP1 (A class) subfamily, EsAGL2-1 and EsAGL2-2 belong to the SEP-like (E class) subfamily, and EsAGL6-like belongs to the AGL6 (AGL6 class) subfamily. Quantitative RT-PCR analyses revealed that the transcripts of the four genes are absent, or minimal, in vegetative tissues and are most highly expressed in floral organs. Yeast two-hybrid results revealed that of the eight MADS-box proteins tested, only EsAGL6-like, EsAGL2-1 and EsAGL2 were able to form strong homo- and heterodimers, with EsAGL6-like and EsAGL2-1 showing similar interaction patterns. Yeast three-hybrid analysis revealed that EsFUL1-like, EsAGL6-like and EsAGL2-1 (representing the three major lineages of the Epimedium AGL/SEP/ALG6 superclade) could act as bridging proteins in ternary complexes with both EsAP3-2 (B class) and EsPI (B class), which do not heterodimerize themselves. Syntenic analyses of sequenced basal eudicots, rosids and asterids showed that most AP1-like and SEP-like genes have been tightly associated as neighbours since the origin of basal eudicots. Ectopic expression of EsFUL-like in arabidopsis caused early flowering through endogenous high-level expression of AP1 and formation of secondary flowers between the first and second whorls. Tobacco plants with ectopic expression of EsAGL2-1 showed shortened pistils and styles, as well as axillary and extra petals in the initial flower. CONCLUSIONS: This study provides a description of EsFUL-like, EsAGL2-1, EsAGL2-2 and EsAGL6-like function divergence and conservation in comparison with a selection of model core eudicots. The study also highlights how organization in genomic segments containing A and E class genes in sequenced model species has resulted in similar topologies of AP1 and SEP-like gene trees.

Genomic data and ecological niche modeling reveal an unusually slow rate of molecular evolution in the Cretaceous Eupteleaceae.

Living fossils are evidence of long-term sustained ecological success. However, whether living fossils have little molecular changes remains poorly known, particularly in plants. Here, we have introduced a novel method that integrates phylogenomic, comparative genomic, and ecological niche modeling analyses to investigate the rate of molecular evolution of Eupteleaceae, a Cretaceous relict angiosperm family endemic to East Asia. We assembled a high-quality chromosome-level nuclear genome, and the chloroplast and mitochondrial genomes of a member of Eupteleaceae (Euptelea pleiosperma). Our results show that Eupteleaceae is most basal in Ranunculales, the earliest-diverging order in eudicots, and shares an ancient whole-genome duplication event with the other Ranunculales. We document that Eupteleaceae has the slowest rate of molecular changes in the observed angiosperms. The unusually low rate of molecular evolution of Eupteleaceae across all three independent inherited genomes and genes within each of the three genomes is in association with its conserved genome architecture, ancestral woody habit, and conserved niche requirements. Our findings reveal the evolution and adaptation of living fossil plants through large-scale environmental change and also provide new insights into early eudicot diversification.

Differences in evolution rates among eudicotyledon species observed by analysis of protein divergence.

Genome evolution rates can vary considerably among plants. In particular, a correlation has often been reported between the evolution rate and annual/perennial habit, possibly associated with differences in generation time. For example, among the rosid species whose genome is fully sequenced, Vitis vinifera, a perennial species, was shown to have the genome that evolved the slowest. In order to extend knowledge of evolution rates to the asterid clade, one of the two major clades of core eudicotyledonous, the protein evolution rates in three asterid species, one perennial (Coffea canephora) and two annual species (Solanum lycopersicum and Mimulus guttatus), were investigated and compared with V. vinifera. Significant differences were observed among these species, and the proteins that evolved the most slowly were those of V. vinifera. Among the species belonging to the asterid clade, C. canephora appears to have evolved more slowly than the others. These findings are consistent with a correlation between perennial habit and slow evolution rates. The C. canephora genome seems to be an appropriate model for paleogenomic studies of asterids.