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Multiple transitions from insect to wind pollination are associated with polyploidy and unisexual flowers inThalictrum(Ranunculaceae), yet the underlying genetics remains unknown. We generated a draft genome ofThalictrum thalictroides, a representative of a clade with ancestral floral traits (diploid, hermaphrodite, and insect pollinated) and a model for functional studies. Floral transcriptomes ofT. thalictroidesand of wind‐pollinated, andromonoeciousT. hernandeziiare presented as a resource to facilitate candidate gene discovery in flowers with different sexual and pollination systems.

A draft genome ofT. thalictroidesand two floral transcriptomes ofT. thalictroidesandT. hernandeziiwere obtained from HiSeq 2000 Illumina sequencing and de novo assembly.

TheT. thalictroidesde novo draft genome assembly consisted of 44,860 contigs (N50 = 12,761 bp, 243 Mbp total length) and contained 84.5% conserved embryophyte single‐copy genes. Floral transcriptomes contained representatives of most eukaryotic core genes, and most of their genes formed orthogroups.

To validate the utility of these resources, potential candidate genes were identified for the different floral morphologies using stepwise data set comparisons. Single‐copy gene analysis and simple sequence repeat markers were also generated as a resource for population‐level and phylogenetic studies.

 

The regulation of floral organ identity was investigated using a forward genetic approach in five floral homeotic mutants ofThalictrum, a noncore eudicot. We hypothesized that these mutants carry defects in the floral patterning genes. Mutant characterization comprised comparative floral morphology and organ identity gene expression at early and late developmental stages, followed by sequence analysis of coding and intronic regions to identify transcription factor binding sites and protein–protein interaction (PPI) motifs. Mutants exhibited altered expression of floral MADS‐box genes, which further informed the function of paralogs arising from gene duplications not found in reference model systems. The ensuing modified BCE models for the mutants supported instances of neofunctionalization (e.g., B‐class genes expressed ectopically in sepals), partial redundancy (E‐class), or subfunctionalization (C‐class) of paralogs. A lack of deleterious mutations in the coding regions of candidate floral MADS‐box genes suggested thatcis‐regulatory ortrans‐acting mutations are at play. Consistent with this hypothesis, double‐flower mutants had transposon insertions or showed signs of transposon activity in the regulatory intron ofAGAMOUS(AG) orthologs. Single amino acid substitutions were also found, yet they did not fall on any of the identified DNA binding or PPI motifs. In conclusion, we present evidence suggesting that transposon activity and regulatory mutations in floral homeotic genes likely underlie the striking phenotypes of theseThalictrumfloral homeotic mutants.

 

TheMIXTAfamily of MYB transcription factors modulate the development of diverse epidermal features in land plants. This study investigates the evolutionary history and function of theMIXTAgene family in the early-diverging eudicot model lineageThalictrum(Ranunculaceae), with R2R3 SBG9-A MYB transcription factors representative of the pre-core eudicot duplication and thus hereby referred to as “paleoMIXTA” (PMX). Cloning and phylogenetic analysis ofThalictrum paleoMIXTA(ThPMX) orthologs across 23 species reveal a genus-wide duplication coincident with a whole-genome duplication. Expression analysis by qPCR confirmed that the highest expression is found in carpels, while newly revealing high expression in leaves and nuanced differences between paralogs in representative polyploid species. The single-copy ortholog from the diploid speciesT. thalictroides(TthPMX, previouslyTtMYBML2), which has petaloid sepals with conical–papillate cells and trichomes on leaves, was functionally characterized by virus-induced gene silencing (VIGS), and its role in leaves was also assessed from heterologous overexpression in tobacco. Another ortholog from a species with conical–papillate cells on stamen filaments,TclPMX, was also targeted for silencing. Overexpression assays in tobacco provide further evidence that thepaleoMIXTAlineage has the potential for leaf trichome function in a core eudicot. Transcriptome analysis by RNA-Seq on leaves of VIGS-treated plants suggests thatTthPMXmodulates leaf trichome development and morphogenesis through microtubule-associated mechanisms and that this may be a conserved pathway for eudicots. These experiments provide evidence for a combined role forpaleoMIXTAorthologs in (leaf) trichomes and (floral) conical–papillate cells that, together with data from other systems, makes the functional reconstruction of a eudicot ancestor most likely as also having a combined function.

 

Abstract 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 additionally allows to test 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 while longer structures tracked wind-pollinated ones, consistent with selective pressures exerted by biotic vs. abiotic pollination vectors, respectively. Conclusions While detectable suites of integrated floral traits across Thalictrum 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 pollination mode evolution in Thalictrum, presumably via different paths from an ancestral mixed pollination state. 

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.