Eudicots Research Articles

Virus-induced gene silencing as a tool for functional analyses in the emerging model plant Aquilegia (columbine, Ranunculaceae).

BackgroundThe lower eudicot genus Aquilegia, commonly known as columbine, is currently the subject of extensive genetic and genomic research aimed at developing this taxon as a new model for the study of ecology and evolution. The ability to perform functional genetic analyses is a critical component of this development process and ultimately has the potential to provide insight into the genetic basis for the evolution of a wide array of traits that differentiate flowering plants. Aquilegia is of particular interest due to both its recent evolutionary history, which involves a rapid adaptive radiation, and its intermediate phylogenetic position between core eudicot (e.g., Arabidopsis) and grass (e.g., Oryza) model species.ResultsHere we demonstrate the effective use of a reverse genetic technique, virus-induced gene silencing (VIGS), to study gene function in this emerging model plant. Using Agrobacterium mediated transfer of tobacco rattle virus (TRV) based vectors, we induce silencing of PHYTOENE DESATURASE (AqPDS) in Aquilegia vulgaris seedlings, and ANTHOCYANIDIN SYNTHASE (AqANS) and the B-class floral organ identity gene PISTILLATA in A. vulgaris flowers. For all of these genes, silencing phenotypes are associated with consistent reduction in endogenous transcript levels. In addition, we show that silencing of AqANS has no effect on overall floral morphology and is therefore a suitable marker for the identification of silenced flowers in dual-locus silencing experiments.ConclusionOur results show that TRV-VIGS in Aquilegia vulgaris allows data to be rapidly obtained and can be reproduced with effective survival and silencing rates. Furthermore, this method can successfully be used to evaluate the function of early-acting developmental genes. In the future, data derived from VIGS analyses will be combined with large-scale sequencing and microarray experiments already underway in order to address both recent and ancient evolutionary questions.

Two AGAMOUS‐like MADS‐box genes from Taihangia rupestris (Rosaceae) reveal independent trajectories in the evolution of class C and class D floral homeotic functions

Duplicate genes may be retained by sub- and/or neofunctionalization through changes in gene expression and/or coding sequence, and therefore have the potential to contribute to the genetic robustness and diversification of an organism. In this study, two MADS-box genes were isolated from Taihangia rupestris, a core eudicot species belonging to the Rosaceae. Sequence and phylogenetic analyses revealed that they are clade members of the euAG and PLE lineages, respectively, and hence the two genes are named TrAG (Taihangia rupestris AGAMOUS) and TrSHP (Taihangia rupestris SHATTERPROOF). Southern blot analysis shows that TrSHP is a single-copy gene in the T. rupestris genome. In situ hybridization analyses show that both TrAG and TrSHP are mainly expressed in the stamens, carpels, and ovules. When the stamen primordia are firstly observed, TrAG is initially expressed in the floral meristem domain that will initiate stamens and carpels. In contrast, no TrSHP signal is observed at this developmental stage. At late stages of carpel development, TrAG expression is detected in the ovules, ovaries, and developing styles and stigmas, whereas TrSHP expression is tightly restricted to the ovules. The transgenic Arabidopsis plants containing 35S::TrAG and 35S::TrSHP, respectively, showed similar phenotypes, including homeotic conversions of sepals into carpelloid structures bearing ovules and petals into staminoid organs, and the fruits shattering prematurely along the dehiscence zone. In addition, the phenotype of the transgenic 35S::TrSHP Arabidopsis plants revealed that perianth abscission was inhibited. Yeast two-hybrid assays indicated that TrAG can interact with TrSEP3, whereas TrSHP cannot. The data suggest that the euAG and PLE paralogs, TrAG and TrSHP, may have subfunctionalized and/or neofunctionalized through changes in expression patterns and accumulating variations in the coding regions. Taking these findings together with those available expression and functional data from Arabidopsis and other species, we conclude that the compensatory ways vary among the euAG and PLE lineage pairs in eudicot species.

Evolution of endoapertures in early-divergent eudicots, with particular reference to pollen morphology in Sabiaceae

Endoapertures, the inner openings of compound apertures in pollen grains, are common in eudicots, but occur infrequently in early-divergent eudicot lineages, in which they are restricted to three families: Menispermaceae, Sabiaceae and Buxaceae. Pollen of Sabiaceae was examined using light, scanning and transmission electron microscopy. The endoapertures are large and lalongate, and intine onci are associated with their development. Optimisation of endoapertures onto an existing angiosperm phylogeny indicates that endoapertures have evolved at least three times independently: in Menispermaceae, in Sabiaceae plus Buxaceae (or possibly separately in these two families), and in the core eudicot clade. Sabiaceae are unusual among early-divergent eudicots in that they possess some characters that are more common in core eudicots, including pollen with endoapertures and pentamerous flowers. This indicates either that they are more closely related to core eudicots than is indicated by current molecular evidence, or that these characters are homoplastic. The latter would suggest a high degree of experimentation prior to evolutionary canalisation of some key morphological features in eudicots. The evolution of endoapertures in early-divergent eudicots is probably associated with possession of endexine sculpture (endosculpture) such as endocracks; endoapertures may have been retained in eudicots as a harmomegathic mechanism.

Diversity and Evolution of CYCLOIDEA-Like TCP Genes in Relation to Flower Development in Papaveraceae

Monosymmetry evolved several times independently during flower evolution. In snapdragon (Antirrhinum majus), a key gene for monosymmetry is CYCLOIDEA (CYC), which belongs to the class II TCP gene family encoding transcriptional activators. We address the questions of the evolutionary history of this gene family and of possible recruitment of genes homologous to CYC in floral development and symmetry in the Papaveraceae. Two to three members of the class II TCP family were found in each species analyzed, two of which were CYC-like genes, on the basis of the presence of both the TCP and R conserved domains. The duplication that gave rise to these two paralogous lineages (named PAPACYL1 and PAPACYL2) probably predates the divergence of the two main clades within the Papaveraceae. Phylogenetic relationships among angiosperm class II TCP genes indicated that (1) PAPACYL genes were closest to Arabidopsis (Arabidopsis thaliana) AtTCP18, and a duplication at the base of the core eudicot would have given rise to two supplementary CYC-like lineages; and (2) at least three class II TCP genes were present in the ancestor of monocots and eudicots. Semiquantitative reverse transcription-polymerase chain reaction and in situ hybridization approaches in three species with different floral symmetry indicated that both PAPACYL paralogs were expressed during floral development. A pattern common to all three species was observed at organ junctions in inflorescences and flowers. Expression in the outer petals was specifically observed in the two species with nonactinomorphic flowers. Hypotheses concerning the ancestral pattern of expression and function of CYC-like genes and their possible role in floral development of Papaveraceae species leading to bisymmetric buds are discussed.

Duplication and paralog sorting of RPB2 and RPB1 genes in core eudicots

RPB1 and RPB2, which encode the largest and second largest subunits of RNA polymerase II, respectively, are essential single copy genes in fungi, animals and most plants. Two paralogs of the RPB2 gene have been found in some groups of angioperms [Oxelman, B., Yoshikawa, N., McConaughy, B.L., Luo, J., Denton, A.L., Hall, B.D., 2004. RPB2 gene phylogeny in flowering plants, with particular emphasis on asterids. Mol. Phylogenet. Evol. 32, 462–479]. Here, we report the results of experiments designed to identify the evolutionary origin of the RPB2 duplicate copies. Through careful sampling and phylogenetic analysis, we were able to construct the RPB2 gene tree in angiosperms and infer the phylogenetic positions of the gene duplication and gene loss events that occurred. Our study shows that an RPB2 gene duplication occurred early in core eudicot evolution, at or near the time of the Buxaceae/Trochodendraceae divergence. Subsequently, multiple gene duplication and paralog sorting events happened independently in different core eudicot taxa. Differential expression of the two RPB2 gene paralogs may explain the preservation of both paralogs in the asterids. One gene ( RPB2-i) accounts for most of the RPB2 mRNA made in the flower organs while the other gene ( RPB2-d) is predominantly used in the vegetative tissues. We also found two paralogs of the RPB1 gene in some core eudicot species. The RPB1 gene duplication occurred before core eudicot divergence, around the time of RPB2 gene duplication. Several independent RPB1 paralog sorting events happened in different core eudicot taxa; their occurrence was independent of the RPB2 paralog sorting events. Our results suggest that a polyploidization event happened at or near the time of the Buxaceae/Trochodendraceae divergence. We propose that this polyploidization and the partial diploidization processes thereafter may have been the driving force of core eudicot radiation.

Functional Diversification of B MADS-Box Homeotic Regulators of Flower Development: Adaptive Evolution in Protein–Protein Interaction Domains after Major Gene Duplication Events

B-class MADS-box genes have been shown to be the key regulators of petal and stamen specification in several eudicot model species such as Arabidopsis thaliana, Antirrhinum majus, and Petunia hybrida. Orthologs of these genes have been found across angiosperms and gymnosperms, and it is thought that the basic regulatory function of B proteins is conserved in seed plant lineages. The evolution of B genes is characterized by numerous duplications that might represent key elements fostering the functional diversification of duplicates with a deep impact on their role in the evolution of the floral developmental program. To evaluate this, we performed a rigorous statistical analysis with B gene sequences. Using maximum likelihood and Bayesian methods, we estimated molecular substitution rates and determined the selective regimes operating at each residue of B proteins. We implemented tests that rely on phylogenetic hypotheses and codon substitution models to detect significant differences in substitution rates (DSRs) and sites under positive adaptive selection (PS) in specific lineages before and after duplication events. With these methods, we identified several protein residues fixed by PS shortly after the origin of PISTILLATA-like and APETALA3-like lineages in angiosperms and shortly after the origin of the euAP3-like lineage in core eudicots, the 2 main B gene duplications. The residues inferred to have been fixed by positive selection lie mostly within the K domain of the protein, which is key to promote heterodimerization. Additionally, we used a likelihood method that accommodates DSRs among lineages to estimate duplication dates for AP3-PI and euAP3-TM6, calibrating with data from the fossil record. The dates obtained are consistent with angiosperm origins and diversification of core eudicots. Our results strongly suggest that novel multimer formation with other MADS proteins could have been crucial for the functional divergence of B MADS-box genes. We thus propose a mechanism of functional diversification and persistence of gene duplicates by the appearance of novel multimerization capabilities after duplications. Multimer formation in different combinations of regulatory proteins can be a mechanistic basis for the origin of novel regulatory functions and a gene regulatory mechanism for the appearance of morphological innovations.

Rosales sister to Fabales: Towards resolving the rosid puzzle

Rosids represent the largest of the eight major clades of core eudicots comprising of 140 families and approximately one-third of all angiosperm species (Soltis and Soltis, 2004). Other than the traditional Rosidae, it encompasses families from Magnoliidae, Dilleniidae and Hamamelidae (Cronquist, 1981; Takhtajan, 1980, 1997). Relationships within the rosids still remain unclear, but with addition of new data in the form of complete plastome sequences, pieces of the puzzle seem to be falling in the right places. With the completion of the grape chloroplast genome (Jansen et al., 2006), doubts regarding “Vitis sister to the rosids” have been eliminated. However, relationships within the two large subclades of rosids, eurosids I (fabids) and II (malvids), remain uncertain and poorly resolved (Savolainen et al., 2000a,b; Soltis et al., 2000). Eurosids I comprises of orders Celastrales, Oxalidales, Malpighiales, Zygophyllales and the nitrogen-Wxing clade comprising of orders Cucurbitales, Fabales, Fagales and Rosales (Soltis et al., 1995). Eurosids II comprises of orders Brassicales, Malvales and Sapindales. The rosids remain one of the most challenging problems for molecular phylogeneticists within the angiosperms (Soltis and Soltis, 2004). Single/few gene analyses have not been able to provide robust support to these problematic areas. In a study on Urticalean rosids, Sytsma et al. (2002), using rbcL, trnL-F and ndhF sequences, revealed that Fagales was sister to Rosales. Fabales appeared sister to Fagales + Rosales and Cucurbitales sister to (Fabales, (Fagales, Rosales)). However, the branch points received low bootstrap support

Petaloidy and petal identity MADS‐box genes in the balsaminoid genera Impatiens and Marcgravia

Impatiens and Marcgravia have striking morphological innovations associated with the flowers. One of the sepals in Impatiens is spurred and petaloid, while in Marcgravia the petals are fused into a cap and nectary cups are associated with the inflorescence. Balsaminaceae (Impatiens) and Marcgraviaceae have surprisingly been shown to be closely related, since both belong to the balsaminoid clade of Ericales (basal asterids). However, several thorough morphological studies thus far have not revealed shared derived characters (synapomorphies) that support a close relationship between these families. In the balsaminoid clade, transitions from entirely green flowers to flowers with heterotopic petaloid organs can be observed. The primary role of class B genes in core eudicots is to specify the identity of petal and stamen floral organs. E-class genes, of which SEP3 is a representative, have been identified as redundant mediators that confer transcriptional activation potential on protein complexes that specify organ identity. Given the conserved function of organ-identity MADS-box genes in model plants, but the rapid molecular evolution in angiosperms, it remains controversial whether these genes have been involved in shaping floral diversity. We have identified a SEP3-like gene and a total of five class B genes from Impatiens hawkeri and Marcgravia umbellata and report their quantitative expression in the floral organs. In Impatiens, two AP3/DEF-like genes were identified with strongly divergent C-terminal domains, one truncated and one unusually long. Both genes show a gradual decrease in expression towards the outer perianth organs, but no GLO-like gene expression is observed in the petaloid sepal. Remarkably, SEP3-like gene expression in the Impatiens perianth is absent from the green sepals but present in the petaloid sepal and in the petals. Dimeric protein interactions of the cloned Impatiens genes were studied in yeast and by using gel retardation. In Marcgravia, strong overlapping class B gene expression is limited to the stamens, but a SEP3-like gene is strongly expressed in the Marcgravia nectary, indicating that both Impatiens and Marcgravia show heterotopic expression of a SEP3-like gene. We discuss several candidate mechanisms for heterotopic petaloidy involving modified gene expression and protein interaction of SEP3-like and class B genes.

Functional Analyses of Two TomatoAPETALA3Genes Demonstrate Diversification in Their Roles in Regulating Floral Development

The floral homeotic APETALA3 (AP3) gene in Arabidopsis thaliana encodes a MADS box transcription factor required for specifying petal and stamen identities. AP3 is a member of the euAP3 lineage, which arose by gene duplication coincident with radiation of the core eudicots. Although Arabidopsis lacks genes in the paralogous Tomato MADS box gene 6 (TM6) lineage, tomato (Solanum lycopersicum) possesses both euAP3 and TM6 genes, which have functionally diversified. A loss-of-function mutation in Tomato AP3 (TAP3) resulted in homeotic transformations of both petals and stamens, whereas RNA interference-induced reduction in TM6 function resulted in flowers with homeotic defects primarily in stamens. The functional differences between these genes can be ascribed partly to different expression domains. When overexpressed in an equivalent domain, both genes can partially rescue the tap3 mutant, indicating that relative levels as well as spatial patterns of expression contribute to functional differences. Our results also indicate that the two proteins have differing biochemical capabilities. Together, these results suggest that TM6 and TAP3 play qualitatively different roles in floral development; they also support the ideas that the ancestral role of AP3 lineage genes was in specifying stamen development and that duplication and divergence in the AP3 lineage allowed for the acquisition of a role in petal specification in the core eudicots.

Analysis of thePetunia TM6MADS Box Gene Reveals Functional Divergence within theDEF/AP3Lineage

Antirrhinum majus DEFICIENS (DEF) and Arabidopsis thaliana APETALA3 (AP3) MADS box proteins are required to specify petal and stamen identity. Sampling of DEF/AP3 homologs revealed two types of DEF/AP3 proteins, euAP3 and TOMATO MADS BOX GENE6 (TM6), within core eudicots, and we show functional divergence in Petunia hybrida euAP3 and TM6 proteins. Petunia DEF (also known as GREEN PETALS [GP]) is expressed mainly in whorls 2 and 3, and its expression pattern remains unchanged in a blind (bl) mutant background, in which the cadastral C-repression function in the perianth is impaired. Petunia TM6 functions as a B-class organ identity protein only in the determination of stamen identity. Atypically, Petunia TM6 is regulated like a C-class rather than a B-class gene, is expressed mainly in whorls 3 and 4, and is repressed by BL in the perianth, thereby preventing involvement in petal development. A promoter comparison between DEF and TM6 indicates an important change in regulatory elements during or after the duplication that resulted in euAP3- and TM6-type genes. Surprisingly, although TM6 normally is not involved in petal development, 35S-driven TM6 expression can restore petal development in a def (gp) mutant background. Finally, we isolated both euAP3 and TM6 genes from seven solanaceous species, suggesting that a dual euAP3/TM6 B-function system might be the rule in the Solanaceae.

Phylogenetic analysis of the "ECE" (CYC/TB1) clade reveals duplications predating the core eudicots.

Flower symmetry is of special interest in understanding angiosperm evolution and ecology. Evidence from the Antirrhineae (snapdragon and relatives) indicates that several TCP gene-family transcription factors, especially CYCLOIDEA (CYC) and DICHOTOMA (DICH), play a role in specifying dorsal identity in the corolla and androecium of monosymmetric (bilateral) flowers. Studies of rosid and asterid angiosperms suggest that orthologous TCP genes may be important in dorsal identity, but there has been no broad phylogenetic context to determine copy number or orthology. Here, we compare published data from rosids and asterids with newly collected data from ranunculids, caryophyllids, Saxifragales, and Asterales to ascertain the phylogenetic placement of major duplications in the "ECE" (CYC/TB1) clade of TCP transcription factors. Bayesian analyses indicate that there are three major copies of "CYC" in the ECE clade, and that duplications leading to these copies predate the core eudicots. CYC1 contains no subsequent duplications and may not be expressed in floral tissue. CYC3 exhibits similar patterns of duplication to CYC2 in several groups. Using RT-PCR, we show that, in flowers of Lonicera morrowii (Caprifoliaceae), DipsCYC2B is expressed in the four dorsal petals and not in the ventral petal. DipsCYC3B is expressed in flower and petal primordia, possibly most strongly in the ventral petal.

Widespread genome duplications throughout the history of flowering plants

Genomic comparisons provide evidence for ancient genome-wide duplications in a diverse array of animals and plants. We developed a birth-death model to identify evidence for genome duplication in EST data, and applied a mixture model to estimate the age distribution of paralogous pairs identified in EST sets for species representing the basal-most extant flowering plant lineages. We found evidence for episodes of ancient genome-wide duplications in the basal angiosperm lineages including Nuphar advena (yellow water lily: Nymphaeaceae) and the magnoliids Persea americana (avocado: Lauraceae), Liriodendron tulipifera (tulip poplar: Magnoliaceae), and Saruma henryi (Aristolochiaceae). In addition, we detected independent genome duplications in the basal eudicot Eschscholzia californica (California poppy: Papaveraceae) and the basal monocot Acorus americanus (Acoraceae), both of which were distinct from duplications documented for ancestral grass (Poaceae) and core eudicot lineages. Among gymnosperms, we found equivocal evidence for ancient polyploidy in Welwitschia mirabilis (Gnetales) and no evidence for polyploidy in pine, although gymnosperms generally have much larger genomes than the angiosperms investigated. Cross-species sequence divergence estimates suggest that synonymous substitution rates in the basal angiosperms are less than half those previously reported for core eudicots and members of Poaceae. These lower substitution rates permit inference of older duplication events. We hypothesize that evidence of an ancient duplication observed in the Nuphar data may represent a genome duplication in the common ancestor of all or most extant angiosperms, except Amborella.

A simplified explanation for the frameshift mutation that created a novel C-terminal motif in the APETALA3 gene lineage.

BackgroundThe evolution of type II MADS box genes has been extensively studied in angiosperms. One of the best-understood subfamilies is that of the Arabidopsis gene APETALA3 (AP3). Previous work has demonstrated that the ancestral paleoAP3 lineage was duplicated at some point within the basal eudicots to give rise to the paralogous TM6 and euAP3 lineages. This event was followed in euAP3 orthologs by the replacement of the C-terminal paleoAP3 motif with the derived euAP3 motif. It has been suggested that the new motif was created by an eight-nucleotide insertion that produced a translational frameshift.ResultsThe addition of 25 eudicot AP3 homologs to the existing dataset has allowed us to clarify the process by which the euAP3 motif evolved. Phylogenetic analysis indicates that the euAP3/TM6 duplication maps very close to the base of the core eudicots, associated with the families Trochodendraceae and Buxaceae. We demonstrate that although the transformation of paleoAP3 into euAP3 was due to a frameshift mutation, this was the result of a single nucleotide deletion. The use of ancestral character state reconstructions has allowed us to demonstrate that the frameshift was accompanied by few other nucleotide changes. We further confirm that the sequence is evolving as coding region.ConclusionThis study demonstrates that the simplest of genetic changes can result in the remodeling of protein sequence to produce a kind of molecular 'hopeful monster.' Moreover, such a novel protein motif can become conserved almost immediately on the basis of what appears to be a rapidly generated new function. Given that the existing data on the function of such C-terminal motifs are somewhat disparate and contradictory, we have sought to synthesize previous findings within the context of the current analysis and thereby highlight specific hypotheses that require further investigation before the significance of the euAP3 frameshift event can be fully understood.

Molecular Basis of Development in Petaloid Monocot Flowers

and several studies have confirmed the extended ABC model as the molecular background of flower development in this plant group. The core eudicots are characterized as having one copy of each of the B-class genes and at least two copies of A-class genes: one is expressed in floral meristems, the other in inflorescence meristems. In monocots and non-core eudicots the validity of the ABC model is under discussion. Generally, more than one functional copy is found of at least one of the B-class genes. The A-class genes apparently are expressed in meristems of both flower and inflorescence. Morphologically petaloid stamens and styles are well known within the petaloid monocots, whereas the phenomenon is rare in core eudicots. A simple model based on the extra copies of B-class genes can explain the molecular background of petaloid stamens in the monocots; the only requirement is that two copies of the same gene have different expression patterns and are responsible for development of petals and stamens, respectively. The formation of petaloid styles can be explained in the same way, but this hypothesis requires that A- and C-class gene expression is not mutually exclusive in monocots. The difference in expression of the A-class genes outside the floral organs shows a fundamental difference between monocot and core eudicot flowers.

Conservation and divergence in the AGAMOUS subfamily of MADS‐box genes: evidence of independent sub‐ and neofunctionalization events

The MADS-box gene AGAMOUS (AG) plays a key role in determining floral meristem and organ identities. We identified three AG homologs, EScaAG1, EScaAG2, and EScaAGL11 from the basal eudicot Eschscholzia californica (California poppy). Phylogenetic analyses indicate that EScaAG1 and EScaAG2 are recent paralogs within the AG clade, independent of the duplication in ancestral core eudicots that gave rise to the euAG and PLENA (PLE) orthologs. EScaAGL11 is basal to core eudicot AGL11 orthologs in a clade representing an older duplication event after the divergence of the angiosperm and gymnosperm lineages. Detailed in situ hybridization experiments show that expression of EScaAG1 and EScaAG2 is similar to AG; however, both genes appear to be expressed earlier in floral development than described in the core eudicots. A thorough examination of available expression and functional data in a phylogenetic context for members of the AG and AGL11 clades reveals that gene expression has been quite variable throughout the evolutionary history of the AG subfamily and that ovule-specific expression might have evolved more than twice. Although sub- and neofunctionalization are inferred to have occurred following gene duplication, functional divergence among orthologs is evident, as is convergence, among paralogs sampled from different species. We propose that retention of multiple AG homologs in several paralogous lineages can be explained by the conservation of ancestral protein activity combined with evolutionarily labile regulation of expression in the AG and AGL11 clades such that the collective functions of the AG subfamily in stamen and carpel development are maintained following gene duplication.

The Gunnera Flower: Key to Eudicot Diversification or Response to Pollination Mode?

Recent molecular phylogenetic studies have shown that the Gunnerales, including Gunnera and Myrothamnus, form a clade, sister to other core eudicots. This systematic position puts Gunnera in a pivotal position regarding the evolution of morphological characters in eudicots. In particular, the dimerous flower organization of Gunnera has recently been considered precursor to the pentamerous condition found in most core eudicots. In order to understand the role of Gunnera in the flower evolution of core eudicots, new detailed studies of the flowers of Gunnera are needed. In this study, the flower ontogeny and morphology of Gunnera monoica and G. dentata as well as of G. macrophylla were studied with SEM. The new observations are compared with previous studies of G. manicata and G. herteri. The first four species have dimerous perfect or staminate flowers with two small median sepals, followed by two large petals opposite the two stamens. In G. herteri, the staminate flowers are reduced to single stamens. In the pistillate flowers, there are two sepals, alternate to the two stylodia above the ovary. In G. monoica, two petal primordia initiate but abort early in floral development; in G. dentata, petal primordia arise after the sepals but remain smaller in the flower; in G. macrophylla petals are lacking. We focus on the floral reductions found in Gunnera and how these are connected to wind pollination common to the entire genus. Clear reductive trends in the perianth can be demonstrated in Gunnera, culminating in the completely perianthless G. herteri. A comparison is also made between the flowers of Gunnera and other simplified flowers such as those of Myrothamnus and other early‐diverging eudicots. The floral reductions of dimerous Gunnerales cannot be related to the pentamerous core eudicots. Floral evolution of Gunneraceae, as well as that of other wind‐pollinated, basally placed eudicots, is best seen as a unique specialization linked to this pollination mode.

Recruitment of CRABS CLAW to promote nectary development within the eudicot clade

Nectaries are secretory organs that are widely present in flowering plants that function to attract floral pollinators. Owing to diversity in nectary positions and structures, they are thought to have originated multiple times during angiosperm evolution, with their potential contribution to the diversification of flowering plants and pollinating animals being considerable. We investigated the genetic basis of diverse nectary forms in eudicot angiosperm species using CRABS CLAW (CRC), a gene required for nectaries in Arabidopsis. CRC expression is conserved in morphologically different nectaries from several core eudicot species and is required for nectary development in both rosids and asterids, two major phylogenetic lineages of eudicots. However, in a basal eudicot species, no evidence of CRC expression in nectaries was found. Considering the phylogenetic distribution of nectary positions and CRC expression analyses in eudicots, we propose that diverse nectaries in core eudicots share conserved CRC gene regulation, and that derived nectary positions in eudicots have altered regulation of CRC. As the ancestral function of CRC lies in the regulation of carpel development, it may have been co-opted as a regulator of nectary development within the eudicots, concomitant with the association of nectaries with reproductive organs in derived lineages.

Dating phylogenetically basal eudicots using rbcL sequences and multiple fossil reference points

A molecular dating of the phylogenetically basal eudicots (Ranunculales, Proteales, Sabiales, Buxales and Trochodendrales sensu Angiosperm Phylogeny Group II) has been performed using several fossils as minimum age constraints. All rbcL sequences available in GenBank were sampled for the taxa in focus. Dating was performed using penalized likelihood, and results were compared with nonparametric rate smoothing. Fourteen eudicot fossils, all with a Cretaceous record, were included in this study for age constraints. Nine of these are assigned to basal eudicots and the remaining five taxa represent core eudicots. Our study shows that the choice of methods and fossil constraints has a great impact on the age estimates, and that removing one single fossil change the results in the magnitude of tens of million years. The use of several fossil constraints increase the probability of approaching the true ages. Our results suggest a rapid diversification during the late Early Cretaceous, with all the lineages of basal eudicots emerging during the latest part of the Early Cretaceous. The age of Ranunculales was estimated to 120 my, Proteales to 119 my, Sabiales to 118 my, Buxales to 117 my, and Trochodendrales to 116 my.

Floral and Vegetative Morphogenesis in California Poppy (Eschscholzia californicaCham.)

For studies of the evolution of development in angiosperms, early‐diverging eudicot taxa are of particular interest for comparisons with established core eudicot model plants, such as Arabidopsis. Here we provide a detailed description of shoot and floral development of the basal eudicot California poppy (Eschscholzia californica). Rosette formation in the vegetative phase is accompanied by increased leaf complexity and shoot apex size. The flowering phase is characterized by internode elongation, formation of dissected cauline leaves, terminal flowers, and basipetal inflorescence branching. For developing flowers and fruits, we have defined 14 stages according to important landmark events, from inflorescence primordium initiation through seed dispersal. Floral organ initiation, morphogenesis, increase in floral meristem size, and the surface structure of mature floral organs are recorded in detail. The duration of the later floral stages, as well as the path of pollen tube growth in the gynoecium, is documented. Comparison of California poppy floral development with that of Arabidopsis indicates considerable differences in terms of organ fusion, whorl proliferation, and variability of size and organ number between the two species. Transitions in meristem identity from germination to floral organogenesis were monitored using expression of the developmental control gene EcFLO, the Eschscholzia ortholog of FLORICAULA/LEAFY. We found that the pattern of expression of EcFLO in the flanks of the shoot apex is maintained from late embryogenesis to flower initiation, indicating a continuous role for this gene in meristem function. As flower organs develop, EcFLO expression becomes more restricted to petal and stamen primordia. Development of the gynoecium occurs without EcFLO expression, indicating that EcFLO may not be necessary for the activation of C‐class genes.

APETALA3 and PISTILLATA homologs exhibit novel expression patterns in the unique perianth of Aristolochia (Aristolochiaceae)

Several lines of evidence suggest that sterile floral organs, collectively known as the perianth, have evolved multiple times during the evolution of the angiosperms. In the family Aristolochiaceae, the perianth is formed by two whorls of organs in the genus Saruma but by only one whorl in the remaining genera, including Aristolochia. Although the morphology of Saruma is similar in appearance to the core eudicot perianth, with leaf-like sepals and showy colored petals, the unipartite perianth of Aristolochia combines morphological aspects of both calyx and corolla. To investigate the organ identity program functioning in the novel perianth of Aristolochia, we identified homologs of the B-class genes APETALA3 (AP3) and PISTILLATA (PI) in both Saruma and Aristolochia. The expression patterns of these genes in Saruma indicate they are functioning in the development of the second whorl petaloid organs and third whorl stamens. In Aristolochia, however, the expression of AP3 and PI homologs in the perianth does not suggest a role in organ identity but, rather, in promoting late aspects of cell differentiation. The implications of these findings for the evolution of both petaloidy and B gene function are discussed.