Flower morphology as a predictor of pollination mode in a biotic to abiotic pollination continuum.

BACKGROUND AND AIMS: Wind pollination has evolved repeatedly in flowering plants, yet the identification of a wind pollination syndrome as a set of integrated floral traits can be elusive. Thalictrum (Ranunculaceae) comprises temperate perennial herbs that have transitioned repeatedly from insect to wind pollination while also exhibiting mixed pollination, providing an ideal system to test for evolutionary correlation between floral morphology and pollination mode in a biotic to abiotic continuum. Moreover, the lack of floral organ fusion across this genus allows testing for specialization to pollination vectors in the absence of this feature. METHODS: We expanded phylogenetic sampling in the genus from a previous study using six chloroplast loci, which allowed us to test whether species cluster into distinct pollination syndromes based on floral morphology. We then used multivariate analyses on floral traits followed by ancestral state reconstruction of the emerging flower morphotypes and determined whether these traits are evolutionarily correlated under a Bayesian framework with Brownian motion. KEY RESULTS: Floral traits fell into five distinct clusters, which were reduced to three after considering phylogenetic relatedness and were largely consistent with flower morphotypes and associated pollination vectors. Multivariate evolutionary analyses found a positive correlation between the lengths of floral reproductive structures (styles, stigmas, filaments and anthers). Shorter reproductive structures tracked insect-pollinated species and clades in the phylogeny, whereas longer structures tracked wind-pollinated ones, consistent with selective pressures exerted by biotic vs. abiotic pollination vectors, respectively. CONCLUSIONS: Although detectable suites of integrated floral traits across Thalictrum were correlated with wind or insect pollination at the extremes of the morphospace distribution, a presumed intermediate, mixed pollination mode morphospace was also detected. Thus, our data broadly support the existence of detectable flower morphotypes from convergent evolution underlying the evolution of pollination mode in Thalictrum, presumably via different paths from an ancestral mixed pollination state.

The symmetry spectrum in a hybridising, tropical group of rhododendrons.

Many diverse plant clades possess bilaterally symmetrical flowers and specialised pollination syndromes, suggesting that these traits may promote diversification. We examined the evolution of diverse floral morphologies in a species-rich tropical radiation of Rhododendron. We used restriction-site associated DNA sequencing on 114 taxa from Rhododendron sect. Schistanthe to reconstruct phylogenetic relationships and examine hybridisation. We then captured and quantified floral variation using geometric morphometric analyses, which we interpreted in a phylogenetic context. We uncovered phylogenetic conflict and uncertainty caused by introgression within and between clades. Morphometric analyses revealed flower symmetry to be a morphological continuum without clear transitions between radial and bilateral symmetry. Tropical Rhododendron species that began diversifying into New Guinea c. 6 million years ago expanded into novel floral morphological space. Our results showed that the evolution of tropical Rhododendron is characterised by recent speciation, recurrent hybridisation and the origin of floral novelty. Floral variation evolved via changes to multiple components of the corolla that are only recognised in geometric morphometrics with both front and side views of flowers.

Gene Duplication and Differential Expression of Flower Symmetry Genes in Rhododendron (Ericaceae).

Bilaterally symmetric flowers have evolved over a hundred times in angiosperms, yet orthologs of the transcription factors CYCLOIDEA (CYC), RADIALIS (RAD), and DIVARICATA (DIV) are repeatedly implicated in floral symmetry changes. We examined these candidate genes to elucidate the genetic underpinnings of floral symmetry changes in florally diverse Rhododendron, reconstructing gene trees and comparing gene expression across floral organs in representative species with radial and bilateral flower symmetries. Radially symmetric R. taxifolium Merr. and bilaterally symmetric R. beyerinckianum Koord. had four and five CYC orthologs, respectively, from shared tandem duplications. CYC orthologs were expressed in the longer dorsal petals and stamens and highly expressed in R. beyerinckianum pistils, whereas they were either ubiquitously expressed, lost from the genome, or weakly expressed in R. taxifolium. Both species had two RAD and DIV orthologs uniformly expressed across all floral organs. Differences in gene structure and expression of Rhododendron RAD compared to other asterids suggest that these genes may not be regulated by CYC orthologs. Our evidence supports CYC orthologs as the primary regulators of differential organ growth in Rhododendron flowers, while also suggesting certain deviations from the typical asterid gene regulatory network for flower symmetry.

The Rhododendron Genome and Chromosomal Organization Provide Insight into Shared Whole-Genome Duplications across the Heath Family (Ericaceae).

The genus Rhododendron (Ericaceae), which includes horticulturally important plants such as azaleas, is a highly diverse and widely distributed genus of >1,000 species. Here, we report the chromosome-scale de novo assembly and genome annotation of Rhododendron williamsianum as a basis for continued study of this large genus. We created multiple short fragment genomic libraries, which were assembled using ALLPATHS-LG. This was followed by contiguity preserving transposase sequencing (CPT-seq) and fragScaff scaffolding of a large fragment library, which improved the assembly by decreasing the number of scaffolds and increasing scaffold length. Chromosome-scale scaffolding was performed by proximity-guided assembly (LACHESIS) using chromatin conformation capture (Hi-C) data. Chromosome-scale scaffolding was further refined and linkage groups defined by restriction-site associated DNA (RAD) sequencing of the parents and progeny of a genetic cross. The resulting linkage map confirmed the LACHESIS clustering and ordering of scaffolds onto chromosomes and rectified large-scale inversions. Assessments of the R. williamsianum genome assembly and gene annotation estimate them to be 89% and 79% complete, respectively. Predicted coding sequences from genome annotation were used in syntenic analyses and for generating age distributions of synonymous substitutions/site between paralgous gene pairs, which identified whole-genome duplications (WGDs) in R. williamsianum. We then analyzed other publicly available Ericaceae genomes for shared WGDs. Based on our spatial and temporal analyses of paralogous gene pairs, we find evidence for two shared, ancient WGDs in Rhododendron and Vaccinium (cranberry/blueberry) members that predate the Ericaceae family and, in one case, the Ericales order.

Partial redundancy and functional specialization of E-class SEPALLATA genes in an early-diverging eudicot.

Plant MADS-box genes have duplicated extensively, allegedly contributing to the immense diversity of floral form in angiosperms. In Arabidopsis thaliana (a core eudicot model plant), four SEPALLATA (SEP) genes comprise the E-class from the extended ABCE model of flower development. They are redundantly involved in the development of the four types of floral organs (sepals, petals, stamens and carpels) and in floral meristem determinacy. E-class genes have been examined in other core eudicots and monocots, but have been less investigated in non-core eudicots. Our goal was to functionally characterize the E-class genes in the early-diverging eudicot Thalictrum thalictroides (Ranunculaceae), whose flowers are apetalous. We identified four SEP orthologs, which when placed in a phylogenetic context, resulted from a major gene duplication event before the origin of angiosperms and a subsequent duplication at the origin of the Ranunculales. We used Virus-Induced Gene Silencing (VIGS) to down-regulate the three expressed paralogs individually and in combination to investigate their function and to determine the degree of conservation versus divergence of this important plant transcription factor. All loci were partially redundant in sepal and stamen identity and in promoting petaloidy of sepals, yet the SEP3 ortholog had a more pronounced role in carpel identity and development. The two other paralogs appear to have subfunctionalized in their cadastral roles to keep the boundaries between either sepal and stamen zones or stamen and carpel zones. Double knockdowns had enhanced phenotypes and the triple knockdown had an even more severe phenotype that included partial to complete homeotic conversion of stamens and carpels to sepaloid organs and green sepals, highlighting a role of E-class genes in petaloidy of sepals in this species. While no floral meristem determinacy defects were observed, this could be due to residual amounts of gene expression in the VIGS experiments being sufficient to perform this function or to the masking role of a redundant gene.

Pattern and process in the evolution of the sole dioecious member of Brassicaceae.

BACKGROUND: Lepidium sisymbrioides, a polyploid New Zealand endemic, is the sole dioecious species in Brassicaceae and therefore the closest dioecious relative of the model plant Arabidopsis thaliana. The attractiveness of developing this system for future studies on the genetics of sex determination prompted us to investigate historical and developmental factors surrounding the evolution of its unisexual flowers. Our goal was to determine the evolutionary pattern of polyploidization of L. sisymbrioides and the timing and process of flower reproductive organ abortion. To that end, we used a combination of phylogenetics to place this species within the complex history of polyploidization events in Lepidium and histology to compare its floral ontogeny to that of its closest hermaphroditic relatives and to A. thaliana. RESULTS: Using a nuclear locus (PISTILLATA), we reconstructed the gene tree among Lepidium taxa and applied a phylogenetic network analysis to identify ancestral genomes that contributed to the evolution of L. sisymbrioides. Combining this phylogenetic framework with cytological and genome size data, we estimated L. sisymbrioides as an allo-octoploid resulting from three hybridization events. Our investigations of flower development showed that unisexual flowers appear to abort reproductive organs by programmed cell death in female flowers and by developmental arrest in male flowers. This selective abortion occurs at the same floral developmental stage in both males and females, corresponding to Arabidopsis stage nine. CONCLUSIONS: Dioecy in Brassicaceae evolved once in L. sisymbrioides following several allopolyploidization events, by a process of selective abortion of reproductive organs at intermediate stages of flower development. Different developmental processes, but similar timing of abortions, affect male versus female flower development. An increased understanding of how and when reproductive organs abort in this species, combined with our estimates of ancestral genome contributions, ploidy and genome size, lay the foundation for future efforts to examine the genetic mechanisms involved in the evolution of unisexual flowers in the closest dioecious relative of the best studied model plant.

Timing and consequences of recurrent polyploidy in meadow-rues (thalictrum, ranunculaceae).

The discovery of ancient whole-genome duplications in eukaryotic lineages has renewed the interest in polyploidy and its effects on the diversification of organisms. Polyploidy has large-scale effects on both genotype and phenotype and has been linked to the evolution of genome size, dioecy, and changes in ecological interactions, such as pollinator visitation. Here, we take a molecular systematics approach to examine the evolution of polyploidy in the plant genus Thalictrum (Ranunculaceae) and test its correlation to changes in genome size, sexual system, and pollination mode. Thalictrum is an ideal study system due to its extensive ploidy range and floral diversity. Phylogenetic analyses were used for character reconstructions, correlation tests, and dating estimates. Our results suggest that polyploidization occurred frequently and recently in the evolution of Thalictrum, mostly within the last 10.6-5.8 My, coinciding with the diversification of particular clades. In spite of an overall trend of genomic downsizing accompanying polyploidy in angiosperms and proportional increases observed at finer scales, our genome size estimates for Thalictrum show no correlation with chromosome number. Instead, we observe genomic expansion in diploids and genomic contraction in polyploids with increased age. Additionally, polyploidy is not correlated with dioecy in Thalictrum; therefore, other factors must have influenced the evolution of separate sexes in this group. A novel finding from our study is the association of polyploidy with shifts to wind pollination, in particular, during a time period of global cooling and mountain uplift in the Americas.

Phylogenetic insights into the correlates of dioecy in meadow-rues (Thalictrum, Ranunculaceae).

Numerous studies have examined the evolution of sexual systems in angiosperms, but few explore the interaction between these and the evolution of pollination mode. Wind pollination is often associated with unisexual flowers, but which evolved first and played a causative role in the evolution of the other is unclear. Thalictrum, meadow-rues (Ranunculaceae), provides a unique opportunity to study the evolution of these traits because it contains insect and wind pollination and four sexual systems. We used a phylogenetic approach to reconstruct ancestral states for sexual system, pollination mode, and geographic distribution in Thalictrum, and tested for correlations to uncover the factors involved in the evolution of unisexuality and wind pollination. Our results show that dioecy, andro- and gynomonoecy evolved at least twice from hermaphroditism. Wind pollination, unisexual flowers, and New World distribution were all significantly correlated. Wind pollination may have evolved early in the genus, followed by multiple losses and gains, and likely preceded the origin of unisexual flowers in several cases; we found no evidence for unisexual flowers evolving prior to wind pollination. Given a broad scale study showing the evolution of dioecy before wind pollination, our results from a finer scale analysis highlight that different evolutionary pathways are likely to occur throughout angiosperms.

Evolution of breeding systems and fruits in New World Galium and relatives (Rubiaceae).

PREMISE OF THE STUDY: Dioecy occurs in only about 6% of angiosperms, yet it has evolved many times from hermaphroditism. Polygamy is an even more uncommon condition within angiosperms, in which both unisexual and bisexual flowers occur within a species. Polygamy, dioecy, and hermaphroditism all occur within a New World clade of Galium (Rubiaceae), in which dioecy is hypothesized to have evolved from hermaphroditism via polygamy. At least five sections of Galium as traditionally defined by fruit morphology occur within this group. We tested the monophyly of sections defined by fruit morphology and sought to determine origins and pathways of breeding systems within this group. • METHODS: We obtained chloroplast (rpoB-trnC, trnC-psbM, trnL-ndhJ) and nuclear ribosomal (external transcribed spacer) DNA sequences for 89 taxa from the Cruciata-Galium-Valantia (CGV) clade to estimate the phylogeny. Ancestral states for breeding systems, fruit types, and fruit hairs were reconstructed using parsimony and likelihood analyses. • KEY RESULTS: We identified nine well-supported lineages of New World Galium taxa. However, none of the sections traditionally defined by fruit morphology are monophyletic. Dioecy is inferred to have arisen at least three times from hermaphroditism; polygamy is inferred to have arisen at least twice from dioecy and at least six times from hermaphroditism. • CONCLUSIONS: Polygamy appears to be a terminal condition in the CGV clade and not a pathway to dioecy. Fruit characters traditionally used in the taxonomy of this group have arisen multiple times within this clade of Galium and are not reliable indicators of shared evolutionary history.