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Abstract PremiseAs the sister clade to angiosperms, extant gymnosperms are crucial for reconstructing ancestral gene regulatory networks in seed plants. This highlights the need for model systems representing each of their distinct lineages. However, tools to quickly and effectively investigate gene function in gymnosperms are still limited due to the challenges of long life cycles and large genome sizes. Species within the xerophytic genusEphedra(Gnetales) have comparatively smaller genomes and shrubby growth habits with shorter life spans, making them better suited for greenhouse cultivation and laboratory experiments. MethodsWe implement virus‐induced gene silencing (VIGS) to manipulate gene expression inEphedra tweedieanaviaAgrobacterium‐mediated vacuum infiltration of tobacco rattle virus (TRV1 and TRV2) into seedlings. ResultsTreatment resulted in highly efficient gene silencing of theE. tweedieana PHYTOENE DESATURASE(PDS) orthologEtwPDS. The expected photobleaching phenotype was observed as early as two weeks, and lasted at least five months in stems, shoot tips, leaves, axillary meristems, and lateral branches of treated plants. DiscussionWe report on virus‐induced targeted gene silencing ofPDSin a Gnetales representative to further enable functional studies of the genetic mechanisms underpinning adaptations in gymnosperms, an important and underrepresented lineage of seed plants. 

Abstract BackgroundPlant dispersal units, or diaspores, allow the colonization of new environments expanding geographic range and promoting gene flow. Two broad categories of diaspores found in seed plants are dry and fleshy, associated with abiotic and biotic dispersal agents, respectively. Anatomy and developmental genetics of fleshy angiosperm fruits is advanced in contrast to the knowledge gap for analogous fleshy structures in gymnosperm diaspores. Improved understanding of the structural basis of modified accessory organs that aid in seed dispersal will enable future work on the underlying genetics, contributing to hypotheses on the origin of angiosperm fruits. To generate a structural framework for the development and evolution of gymnosperm fleshy diaspores, we studied the anatomy and histochemistry ofEphedra(Gnetales) seed cone bracts, the modified leaves surrounding the reproductive organs. We took an ontogenetic approach, comparing and contrasting the anatomy and histology of fleshy and papery-winged seed cone bracts, and their respective pollen cone bracts and leaves in four species from the South American clade. ResultsSeed bract fleshiness inEphedraderives from mucilage accumulated in chlorenchyma cells, also found in the reduced young leaves before they reach their mature, dry stage. Cellulosic fibers, an infrequent cell type in gymnosperms, were found inEphedra, where they presumably function as a source of supplementary apoplastic water in fleshy seed cone bracts. Papery-winged bract development more closely resembles that of leaves, with chlorenchyma mucilage cells turning into tanniniferous cells early on, and hyaline margins further extending into “wings”. ConclusionsWe propose an evolutionary developmental model whereby fleshy and papery-winged bracts develop from an early-stage anatomy shared with leaves that differs at the pollination stage. The ancestral fleshy bract state may represent a novel differentiation program built upon young leaf anatomy, while the derived dry, papery-winged state is likely built upon an existing differentiation pattern found in mature vegetative leaves. This model for the evolution of cone bract morphology in South AmericanEphedrahence involves a novel differentiation program repurposed from leaves combined with changes in the timing of leaf differentiation, or heterochrony, that can further be tested in other gymnosperms with fleshy diaspores. 

Abstract The Ranunculales are a hyperdiverse lineage in many aspects of their phenotype, including growth habit, floral and leaf morphology, reproductive mode, and specialized metabolism. Many Ranunculales species, such as opium poppy and goldenseal, have a high medicinal value. In addition, the order includes a large number of commercially important ornamental plants, such as columbines and larkspurs. The phylogenetic position of the order with respect to monocots and core eudicots and the diversity within this lineage make the Ranunculales an excellent group for studying evolutionary processes by comparative studies. Lately, the phylogeny of Ranunculales was revised, and genetic and genomic resources were developed for many species, allowing comparative analyses at the molecular scale. Here, we review the literature on the resources for genetic manipulation and genome sequencing, the recent phylogeny reconstruction of this order, and its fossil record. Further, we explain their habitat range and delve into the diversity in their floral morphology, focusing on perianth organ identity, floral symmetry, occurrences of spurs and nectaries, sexual and pollination systems, and fruit and dehiscence types. The Ranunculales order offers a wealth of opportunities for scientific exploration across various disciplines and scales, to gain novel insights into plant biology for researchers and plant enthusiasts alike. 

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

Abstract 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. 

Dioecious plants are obligate outcrossers with separate male and female individuals, which can result in decreased seed set with increasing distance between the sexes. Wind pollination is a common correlate of dioecy, yet combined wind and insect pollination (ambophily) could be advantageous in compensating for decreased pollen flow to isolated females. Dioecious, ambophilous gymnospermsEphedra(Gnetales) secrete pollination drops (PDs) in female cones that capture airborne pollen and attract ants that feed on them. Plant sugary secretions commonly reward ants in exchange for indirect plant defense against herbivores, and more rarely for pollination. We conducted field experiments to investigate whether ants are pollinators and/or plant defenders of South AmericanEphedra triandra, and whether their contribution to seed set and seed cone protection varies with distance between female and male plants. We quantified pollen flow in the wind and assessed the effectiveness of ants as pollinators by investigating their relative contribution to seed set, and their visitation rate in female plants at increasing distance from the nearest male. Ants accounted for most insect visits to female cones ofE. triandra, where they consumed PDs, and pollen load was larger on bigger ants without reduction in pollen viability. While wind pollination was the main contributor to seed set overall, the relative contribution of ants was distance dependent. Ant contribution to seed set was not significant at shorter distances, yet at the farthest distance from the nearest male (23 m), where 20 times less pollen reached females, ants enhanced seed set by 30% compared to plants depending solely on wind pollination. We found no evidence that ants contribute to plant defense by preventing seed cone damage. Our results suggest that, despite their short-range movements, ants can offset pollen limitation in isolated females of wind-pollinated plants with separate sexes. We propose that ants enhance plant reproductive success via targeted delivery of airborne pollen, through frequent contact with ovule tips while consuming PDs. Our study constitutes the first experimental quantification of distance-dependent contribution of ants to pollination and provides a working hypothesis for ambophily in other dioecious plants lacking pollinator reward in male plants.