Tangled webs and spider-flowers: Phylogenomics, biogeography, and seed morphology inform the evolutionary history of Cleomaceae.

PREMISE: Cleomaceae is an important model clade for studies of evolutionary processes including genome evolution, floral form diversification, and photosynthetic pathway evolution. Diversification and divergence patterns in Cleomaceae remain tangled as research has been restricted by its worldwide distribution, limited genetic sampling and species coverage, and a lack of definitive fossil calibration points. METHODS: We used target sequence capture and the Angiosperms353 probe set to perform a phylogenetic study of Cleomaceae. We estimated divergence times and biogeographic analyses to explore the origin and diversification of the family. Seed morphology across extant taxa was documented with multifocal image-stacking techniques and morphological characters were extracted, analyzed, and compared to fossil records. RESULTS: We recovered a well-supported and resolved phylogenetic tree of Cleomaceae generic relationships that includes 236 (~86%) species. We identified 11 principal clades and confidently placed Cleomella as sister to the rest of the family. Our analyses suggested that Cleomaceae and Brassicaceae diverged ~56 mya, and Cleomaceae began to diversify ~53 mya in the Palearctic and Africa. Multiple transatlantic disjunct distributions were identified. Seeds were imaged from 218 (~80%) species in the family and compared to all known fossil species. CONCLUSIONS: Our results represent the most comprehensive phylogenetic study of Cleomaceae to date. We identified transatlantic disjunctions and proposed explanations for these patterns, most likely either long-distance dispersals or contractions in latitudinal distributions caused by climate change over geological timescales. We found that seed morphology varied considerably but mostly mirrored generic relationships.

Developmental and genetic basis of the androgynophore in Gynandropsis gynandra.

PREMISE: Flowering plants have evolved a vast array of floral features involved in plant-pollinator interactions. A feature that seemingly increases the chance of pollen transfer is the androgynophore, a stalk-like structure that raises the reproductive organs of the flower. However, little is known about the developmental and genetic basis of this structure despite its presence in multiple, distantly related taxa. Here, we address this gap by investigating Gynandropsis gynandra (Cleomaceae), a species with a prominent androgynophore. METHODS: We combined morphological and anatomical analyses with a comparative transcriptomic study to provide a detailed description of the androgynophore throughout development, examine global gene expression patterns, and identify candidate genes putatively involved in androgynophore elongation. RESULTS: The radially symmetric androgynophore of G. gynandra rapidly lengthens primarily via cell elongation. Despite its structural uniformity, androgynophore development is characterized by complex gene expression patterns including differential expression of floral organ identity genes and genes associated with organ development and growth in Arabidopsis thaliana. CONCLUSIONS: Our morphological characterizations and high-quality transcriptome for G. gynandra suggest that the androgynophore is a novel structure formed via elaboration of both the receptacle and base of reproductive organs because it is structurally like an elongated internode but expresses the genetic repertoire typically associated with the reproductive organs. The drastic increase in cell length and uniform structure elevates the androgynophore as a potentially powerful model for cell elongation.

Phylogeny and multiple independent whole-genome duplication events in the Brassicales.

PREMISE: Whole-genome duplications (WGDs) are prevalent throughout the evolutionary history of plants. For example, dozens of WGDs have been phylogenetically localized across the order Brassicales, specifically, within the family Brassicaceae. A WGD event has also been identified in the Cleomaceae, the sister family to Brassicaceae, yet its placement, as well as that of WGDs in other families in the order, remains unclear. METHODS: Phylo-transcriptomic data were generated and used to infer a nuclear phylogeny for 74 Brassicales taxa. Genome survey sequencing was also performed on 66 of those taxa to infer a chloroplast phylogeny. These phylogenies were used to assess and confirm relationships among the major families of the Brassicales and within Brassicaceae. Multiple WGD inference methods were then used to assess the placement of WGDs on the nuclear phylogeny. RESULTS: Well-supported chloroplast and nuclear phylogenies for the Brassicales and the putative placement of the Cleomaceae-specific WGD event Th-ɑ are presented. This work also provides evidence for previously hypothesized WGDs, including a well-supported event shared by at least two members of the Resedaceae family, and a possible event within the Capparaceae. CONCLUSIONS: Phylogenetics and the placement of WGDs within highly polyploid lineages continues to be a major challenge. This study adds to the conversation on WGD inference difficulties by demonstrating that sampling is especially important for WGD identification and phylogenetic placement. Given its economic importance and genomic resources, the Brassicales continues to be an ideal group for assessing WGD inference methods.

Flowers up! The effect of floral height along the shoot axis on the fitness of bat-pollinated species.

BACKGROUND AND AIMS: Bat-pollination is an important system in terms of occurrence and distribution, although it remains little studied. Thus, the role of particular flower traits in this interaction remains uncertain. Flower height along the shoot axis, associated with flower exposure, has often been deemed a key trait in this system, but its effect on fitness has not previously been assessed. We aimed to test its role and propose that taller flowers attain higher fitness due to a higher degree of accessibility and conspicuity to foraging bats. METHODS: We assessed the effect of floral height on bat visiting rates to individual flowers of Crescentia cujete (Bignoniaceae), a cauliflorous model bat-pollinated species with a marked gradient in flower height along the shoot axis. Additionally, we tested the effect of this variable on seed/ovule ratio measurements from seven other species from different families along a herb-tree gradient. Hypotheses were tested through mixed-effect linear models. KEY RESULTS: Bat visiting rates varied positively as a function of flower height in C. cujete, but significance was found only for the subset of flowers located on the trunk, closer to the ground. Similarly, seed/ovule ratios were positively correlated with flower height only for the three species with the shortest statures along the height gradient and shortest average floral heights. These results suggest that proximity to the ground, associated with herbaceous or bushy surrounding vegetation, may be an obstacle to the foraging of nectar-feeding bats, which in turn should explain the morphological convergence of inflorescence length and exposure strategies of short-statured bat-pollinated plants. CONCLUSIONS: Flower height has a species-specific effect on plant fitness. This study provides a novel numerical perspective to the role of an unexplored trait in bat-pollination, and has elucidated some aspects of the adaptive importance of flower height based on limitations imposed by ecologically complex pollinators.

PISTILLATA paralogs in Tarenaya hassleriana have diverged in interaction specificity.

BACKGROUND: Floral organs are specified by MADS-domain transcription factors that act in a combinatorial manner, as summarized in the (A)BCE model. However, this evolutionarily conserved model is in contrast to a remarkable amount of morphological diversity in flowers. One of the mechanisms suggested to contribute to this diversity is duplication of floral MADS-domain transcription factors. Although gene duplication is often followed by loss of one of the copies, sometimes both copies are retained. If both copies are retained they will initially be redundant, providing freedom for one of the paralogs to change function. Here, we examine the evolutionary fate and functional consequences of a transposition event at the base of the Brassicales that resulted in the duplication of the floral regulator PISTILLATA (PI), using Tarenaya hassleriana (Cleomaceae) as a model system. RESULTS: The transposition of a genomic region containing a PI gene led to two paralogs which are located at different positions in the genome. The original PI copy is syntenic in position with most angiosperms, whereas the transposed copy is syntenic with the PI genes in Brassicaceae. The two PI paralogs of T. hassleriana have very similar expression patterns. However, they may have diverged in function, as only one of these PI proteins was able to act heterologously in the first whorl of A. thaliana flowers. We also observed differences in protein complex formation between the two paralogs, and the two paralogs exhibit subtle differences in DNA-binding specificity. Sequence analysis indicates that most of the protein sequence divergence between the two T. hassleriana paralogs emerged in a common ancestor of the Cleomaceae and the Brassicaceae. CONCLUSIONS: We found that the PI paralogs in T. hassleriana have similar expression patterns, but may have diverged at the level of protein function. Data suggest that most protein sequence divergence occurred rapidly, prior to the origin of the Brassicaceae and Cleomaceae. It is tempting to speculate that the interaction specificities of the Brassicaceae-specific PI proteins are different compared to the PI found in other angiosperms. This could lead to PI regulating partly different genes in the Brassicaceae, and ultimately might result in change floral in morphology.

Evolution of microRNA827 targeting in the plant kingdom.

Unlike most ancient microRNAs, which conservatively target homologous genes across species, microRNA827 (miR827) targets two different types of SPX (SYG1/PHO81/XPR1)-domain-containing genes, NITROGEN LIMITATION ADAPTATION (NLA) and PHOSPHATE TRANSPORTER 5 (PHT5), in Arabidopsis thaliana and Oryza sativa to regulate phosphate (Pi) transport and storage, respectively. However, how miR827 shifted its target preference and its evolutionary history are unknown. Based on target prediction analysis, we found that in most angiosperms, miR827 conservatively targets PHT5 homologs, but in Brassicaceae and Cleomaceae it preferentially targets NLA homologs, and we provide evidence for the transition of target preference during Brassicales evolution. Intriguingly, we found a lineage-specific loss of the miR827-regulatory module in legumes. Analysis of miR827-mediated cleavage efficiency and the expression of PHT5 in A. thaliana indicated that accumulation of mutations in the target site and the exclusion of the target site by alternative transcriptional initiation eliminated PHT5 targeting by miR827. Here, we identified a transition of miR827 target preference during plant evolution and revealed the uniqueness of miR827-mediated regulation among conserved plant miRNAs. Despite the change in its target preference, upregulation of miR827 by Pi starvation and its role in regulating cellular Pi homeostasis were retained.

Post-transcriptional regulation of photosynthetic genes is a key driver of C4 leaf ontogeny.

C4 photosynthesis allows highly efficient carbon fixation that originates from tightly regulated anatomical and biochemical modifications of leaf architecture. Recent studies showed that leaf transcriptome modifications during leaf ontogeny of closely related C3 (Tarenaya hassleriana) and C4 (Gynandropsis gynandra) species within the Cleomaceae family existed but they did not identify any dedicated transcriptional networks or factors specifically driving C4 leaf ontogeny. RNAseq analysis provides a steady-state quantification of whole-cell mRNAs but does not allow any discrimination between transcriptional and post-transcriptional processes that may occur simultaneously during leaf ontogeny. Here we use exon-intron split analysis (EISA) to determine the extent to which transcriptional and post-transcriptional processes are involved in the regulation of gene expression between young and expanded leaves in both species. C4-specific changes in post-transcriptional regulation were observed for genes involved in the Calvin-Benson cycle and some photosystem components but not for C4 core-cycle genes. Overall, this study provides an unbiased genome-wide insight into the post-transcriptional mechanisms that regulate gene expression through the control of mRNA levels and could be central to the onset of C4 photosynthesis. This mechanism is cytosolic which implies cell-specific modifications of mRNA stability. Understanding this mechanism may be crucial when aiming to transform C3 crops into C4 crops.

Biogeography and diversification of Brassicales: A 103million year tale.

Brassicales is a diverse order perhaps most famous because it houses Brassicaceae and, its premier member, Arabidopsis thaliana. This widely distributed and species-rich lineage has been overlooked as a promising system to investigate patterns of disjunct distributions and diversification rates. We analyzed plastid and mitochondrial sequence data from five gene regions (>8000bp) across 151 taxa to: (1) produce a chronogram for major lineages in Brassicales, including Brassicaceae and Arabidopsis, based on greater taxon sampling across the order and previously overlooked fossil evidence, (2) examine biogeographical ancestral range estimations and disjunct distributions in BioGeoBEARS, and (3) determine where shifts in species diversification occur using BAMM. The evolution and radiation of the Brassicales began 103Mya and was linked to a series of inter-continental vicariant, long-distance dispersal, and land bridge migration events. North America appears to be a significant area for early stem lineages in the order. Shifts to Australia then African are evident at nodes near the core Brassicales, which diverged 68.5Mya (HPD=75.6-62.0). This estimated age combined with fossil evidence, indicates that some New World clades embedded amongst Old World relatives (e.g., New World capparoids) are the result of different long distance dispersal events, whereas others may be best explained by land bridge migration (e.g., Forchhammeria). Based on these analyses, the Brassicaceae crown group diverged in Europe/Northern Africa in the Eocene, circa 43.4Mya (HPD=46.6-40.3) and Arabidopsis separated from close congeners circa 10.4Mya. These ages fall between divergent dates that were previously published, suggesting we are slowly converging on a robust age estimate for the family. Three significant shifts in species diversification are observed in the order: (1) 58Mya at the crown of Capparaceae, Cleomaceae and Brassicaceae, (2) 38Mya at the crown of Resedaceae+Stixis clade, and (3) 21Mya at the crown of the tribes Brassiceae and Sisymbrieae within Brassicaceae.

Flower power and the mustard bomb: Comparative analysis of gene and genome duplications in glucosinolate biosynthetic pathway evolution in Cleomaceae and Brassicaceae.

PREMISE OF THE STUDY: Glucosinolates (GS) are a class of plant secondary metabolites that provide defense against herbivores and may play an important role in pollinator attraction. Through coevolution with plant-interacting organisms, glucosinolates have diversified into a variety of chemotypes through gene sub- and neofunctionalization. Polyploidy has been of major importance in the evolutionary history of these gene families and the development of chemically separate GS types. Here we study the effects of polyploidy in Tarenaya hassleriana (Cleomaceae) on the genes underlying GS biosynthesis. METHODS: We established putative orthologs of all gene families involved in GS biosynthesis through sequence comparison and their duplication method through calculation of synonymous substitution ratios, phylogenetic gene trees, and synteny comparison. We drew expression data from previously published work of the identified genes and compared expression in several tissues. KEY RESULTS: We show that the majority of gene family expansion in T. hassleriana has taken place through the retention of polyploid duplicates, together with tandem and transpositional duplicates. We also show that the large majority (>75%) is actively expressed either globally or in specific tissues. We show that MAM and CYP83 gene families, which are crucial to GS diversification in Brassicaceae, are also recruited into specific tissue expression pathways in Cleomaceae. CONCLUSIONS: Many GS genes have expanded through polyploidy, gene transposition duplication, and tandem duplication in Cleomaceae. Duplicate retention through these mechanisms is similar to A. thaliana, but based on the expression of GS genes, Cleomaceae-specific diversification of GS genes has taken place.

Comparative anatomy, histochemistry and phytochemistry of three species of the genus Tarenaya Raf.

This study addresses the morphological similarities and taxonomic complexity of the genus Tarenaya Raf. of the family Cleomaceae Bercht. & J. Presl as well as the medicinal use of their species. The research compares potential anatomical diagnostic characters of the vegetative organs of species Tarenaya aculeata, Tarenaya diffusa, and Tarenaya spinosa to determine authenticity parameters. The study also carried out histochemical and phytochemical analyses of leaf blades to explore the medicinal use of these species. Semipermanent slides containing cross sections of the stems, petioles, and leaf blades, as well as paradermal sections of leaf blades, were prepared. The analyses were carried out in light and polarized optical microscopy. The histochemical analysis included different reagents depending on the target metabolite, which were analyzed by optical and fluorescence microscopy. Phytochemical tests of the methanolic extracts of the leaves were performed using thin layer chromatography. Anatomical characterization showed the characters of general occurrence in the family Cleomaceae and those that allow the differentiation of T. aculeata, T. diffusa, and T. spinosa. Histochemistry revealed the synthesis and/or storage sites of the metabolites and phytochemical tests; it was possible to observe the presence of different metabolites. The results bring comparative data on the anatomical and histochemical characterization of the species, thus increasing the taxonomic knowledge of the genus Tarenaya. RESEARCH HIGHLIGHTS: The comparative anatomy of three Tarenaya from Brazil was studied. Anatomical differences in different vegetative organs differ species. Differences in histochemistry and phytochemistry.

Phylogenetic relationships among the North American cleomoids (Cleomaceae): a test of Iltis's reduction series.

PREMISE OF STUDY: A monophyletic group composed of five genera of the Cleomaceae represents an intriguing lineage with outstanding taxonomic and evolutionary questions. Generic boundaries are poorly defined, and historical hypotheses regarding the evolution of fruit type and phylogenetic relationships provide testable questions. This is the first detailed phylogenetic investigation of all 22 species in this group. We use this phylogenetic framework to assess generic monophyly and test Iltis's evolutionary "reduction series" hypothesis regarding phylogeny and fruit type/seed number. • METHODS: Maximum likelihood and Bayesian analyses of four plastid intergenic spacer region sequences (rpl32-trnL, trnQ-rps16, ycf1-rps15, and psbA-trnH) and one nuclear (ITS) region were used to reconstruct phylogenetic relationships among the NA cleomoid species. Stochastic mapping and ancestral-state reconstruction were used to study the evolution of fruit type. • KEY RESULTS: Both analyses recovered nearly identical phylogenies. Three of the currently recognized genera (Wislizenia, Carsonia, and Oxystylis) are monophyletic while two (Cleomella and Peritoma) are para- or polyphyletic. There was a single origin of the two-seeded schizocarp in the ancestor of the Oxystylis-Wislizenia clade and a secondary derivation of elongated capsule-type fruits in Peritoma from a truncated capsule state in Cleomella. • CONCLUSIONS: Our well-resolved phylogeny supports most of the current species circumscriptions but not current generic circumscriptions. Additionally, our results are inconsistent with Iltis's hypothesis of species with elongated many-seed fruits giving rise to species with truncated few-seeded fruits. Instead, we find support for the reversion to elongated multiseeded fruits from a truncate few-seeded ancestor in Peritoma.

A comparison of stomatal conductance responses to blue and red light between C3 and C4 photosynthetic species in three phylogenetically-controlled experiments.

Introduction: C4 photosynthesis is an adaptation that has independently evolved at least 66 times in angiosperms. C4 plants, unlike their C3 ancestral, have a carbon concentrating mechanism which suppresses photorespiration, often resulting in faster photosynthetic rates, higher yields, and enhanced water use efficiency. Moreover, the presence of C4 photosynthesis greatly alters the relation between CO2 assimilation and stomatal conductance. Previous papers have suggested that the adjustment involves a decrease in stomatal density. Here, we tested if C4 species also have differing stomatal responses to environmental cues, to accommodate the modified CO2 assimilation patterns compared to C3 species. Methods: To test this hypothesis, stomatal responses to blue and red-light were analysed in three phylogenetically linked pairs of C3 and C4 species from the Cleomaceae (Gynandropsis and Tarenaya), Flaveria, and Alloteropsis, that use either C3 or C4 photosynthesis. Results: The results showed strongly decreased stomatal sensitivity to blue light in C4 dicots, compared to their C3 counterparts, which exhibited significant blue light responses. In contrast, in C3 and C4 subspecies of the monocot A. semialata, the blue light response was observed regardless of photosynthetic type. Further, the quantitative red-light response varied across species, but the presence or absence of a significant stomatal red-light response was not directly associated with differences in photosynthetic pathway. Interestingly, stomatal density and morphology patterns observed across the three comparisons were also not consistent with patterns commonly asserted for C3 and C4 species. Discussion: The strongly diminished blue-light sensitivity of stomatal responses in C4 species across two of the comparisons suggests a common C4 feature that may have functional implications. Altogether, the strong prevalence of species-specific effects clearly emphasizes the importance of phylogenetic controls in comparisons between C3 and C4 photosynthetic pathways.

Comparative Nectary Morphology across Cleomaceae (Brassicales).

Floral nectaries have evolved multiple times and rapidly diversified with the adaptive radiation of animal pollinators. As such, floral nectaries exhibit extraordinary variation in location, size, shape, and secretory mechanism. Despite the intricate ties to pollinator interactions, floral nectaries are often overlooked in morphological and developmental studies. As Cleomaceae exhibits substantial floral diversity, our objective was to describe and compare floral nectaries between and within genera. Floral nectary morphology was assessed through scanning electron microscopy and histology across three developmental stages of nine Cleomaceae species including representatives for seven genera. A modified fast green and safranin O staining protocol was used to yield vibrant sections without highly hazardous chemicals. Cleomaceae floral nectaries are most commonly receptacular, located between the perianth and stamens. The floral nectaries are supplied by vasculature, often contain nectary parenchyma, and have nectarostomata. Despite the shared location, components, and secretory mechanism, the floral nectaries display dramatic diversity in size and shape, ranging from adaxial protrusions or concavities to annular disks. Our data reveal substantive lability in form with both adaxial and annular floral nectaries interspersed across Cleomaceae. Floral nectaries contribute to the vast morphological diversity of Cleomaceae flowers and so are valuable for taxonomic descriptions. Though Cleomaceae floral nectaries are often derived from the receptacle and receptacular nectaries are common across flowering plants, the role of the receptacle in floral evolution and diversification is overlooked and warrants further exploration.

Nectary development in Cleome violacea.

Nectaries are a promising frontier for plant evo-devo research, and are particularly fascinating given their diversity in form, position, and secretion methods across angiosperms. Emerging model systems permit investigations of the molecular basis for nectary development and nectar secretion across a range of taxa, which addresses fundamental questions about underlying parallelisms and convergence. Herein, we explore nectary development and nectar secretion in the emerging model taxa, Cleome violacea (Cleomaceae), which exhibits a prominent adaxial nectary. First, we characterized nectary anatomy and quantified nectar secretion to establish a foundation for quantitative and functional gene experiments. Next, we leveraged RNA-seq to establish gene expression profiles of nectaries across three key stages of development: pre-anthesis, anthesis, and post-fertilization. We then performed functional studies on five genes that were putatively involved in nectary and nectar formation: CvCRABSCLAW (CvCRC), CvAGAMOUS (CvAG), CvSHATTERPROOF (CvSHP), CvSWEET9, and a highly expressed but uncharacterized transcript. These experiments revealed a high degree of functional convergence to homologues from other core Eudicots, especially Arabidopsis. CvCRC, redundantly with CvAG and CvSHP, are required for nectary initiation. Concordantly, CvSWEET9 is essential for nectar formation and secretion, which indicates that the process is eccrine based in C. violacea. While demonstration of conservation is informative to our understanding of nectary evolution, questions remain. For example, it is unknown which genes are downstream of the developmental initiators CvCRC, CvAG, and CvSHP, or what role the TCP gene family plays in nectary initiation in this family. Further to this, we have initiated a characterization of associations between nectaries, yeast, and bacteria, but more research is required beyond establishing their presence. Cleome violacea is an excellent model for continued research into nectary development because of its conspicuous nectaries, short generation time, and close taxonomic distance to Arabidopsis.

Complete chloroplast genome of the medicinal plant Cleome paradoxa R.Br. Ex DC: Comparative analysis, and phylogenetic relationships among the members of Cleomaceae.

The cleome species of the Cleomaceae family have several medical uses, including applications such as antioxidants and insecticides. In the present study, we sequenced the complete chloroplast genome (cp genome) of Cleome paradoxa. The chloroplast genome is 159,393 bp long, with a typical four-region structure: a large single copy (LSC) region of 88,191 bp, a small single copy (SSC) region of 18,620 bp, and inverted repeat regions (IRa and IRb) of 26,291 bp each. The proportion of GC content was 35.79 %. The chloroplast genome of C. paradoxa contains 133 genes, 81 of which encode proteins, 29 encode tRNA, and 4 encode rRNA. We noticed a divergence in the location and number of certain genes at the IR-LSC and IR-SSC boundaries. The phylogenetic tree constructed from the complete chloroplast genome data broadly supported the taxonomic situation of Cleome paradoxa as belonging to the Cleomaceae family and Cleome species. The cp genome of C. paradoxa was rich in single sequence repeats (SSRs), with a total of 314 SSRs. Additionally, several genes were under positive selection. These results could be useful for determining the genetic variations and resolving conflicting relationships among Cleomaceae species.

The complete chloroplast genome sequence of Gynandropsis gynandra (Cleomaceae).

Gynandropsis gynandra (L.) Briquet is a monoecious herb species in the family Cleomaceae. It is an important commercial crop with medicinal and nutritional values. Here, we sequenced, assembled, and analyzed the complete chloroplast genome of G. gynandra. The circular genome is 158,152 bp in size, consisting of two copies of inverted repeat (IR) regions of 26,181 bp, one large single-copy (LSC) region of 87,242 bp, and one small single-copy (SSC) region of 18,548 bp. The overall GC content was 35.81%. A total of 131 genes were annotated, including 37 tRNA genes, 87 protein-coding genes, and seven rRNA genes. Phylogenetic analysis based on 10 chloroplast genome sequences indicated that G. gynandra was more closely related to Tarenaya hassleriana.

New Insights Into the Evolution of C4 Photosynthesis Offered by the Tarenaya Cluster of Cleomaceae.

Cleomaceae is closely related to Brassicaceae and includes C3, C3-C4, and C4 species. Thus, this family represents an interesting system for studying the evolution of the carbon concentrating mechanism. However, inadequate genetic information on Cleomaceae limits their research applications. Here, we characterized 22 Cleomaceae accessions [3 genera (Cleoserrata, Gynandropsis, and Tarenaya) and 11 species] in terms of genome size; molecular phylogeny; as well as anatomical, biochemical, and photosynthetic traits. We clustered the species into seven groups based on genome size. Interestingly, despite clear differences in genome size (2C, ranging from 0.55 to 1.3 pg) in Tarenaya spp., this variation was not consistent with phylogenetic grouping based on the internal transcribed spacer (ITS) marker, suggesting the occurrence of multiple polyploidy events within this genus. Moreover, only G. gynandra, which possesses a large nuclear genome, exhibited the C4 metabolism. Among the C3-like species, we observed intra- and interspecific variation in nuclear genome size as well as in biochemical, physiological, and anatomical traits. Furthermore, the C3-like species had increased venation density and bundle sheath cell size, compared to C4 species, which likely predisposed the former lineages to C4 photosynthesis. Accordingly, our findings demonstrate the potential of Cleomaceae, mainly members of Tarenaya, in offering novel insights into the evolution of C4 photosynthesis.

Virus-induced gene silencing as a tool for functional studies in Cleome violacea.

PREMISE: Cleomaceae is emerging as a promising family to investigate a wide range of phenomena, such as C4 photosynthesis and floral diversity. However, functional techniques are lacking for elucidating this diversity. Herein, we establish virus-induced gene silencing (VIGS) as a method of generating functional data for Cleome violacea, bolstering Cleomaceae as a model system. METHODS: We leveraged the sister relationship of Cleomaceae and Brassicaceae by using constructs readily available for Arabidopsis thaliana to provide initial information about the feasibility of VIGS in C. violacea. We then developed endogenous constructs to optimize VIGS efficiency and viability for fruit development. RESULTS: PHYTOENE DESATURASE was successfully downregulated in C. violacea using both heterologous and endogenous constructs. The endogenous construct had the highest degree of downregulation, with many plants displaying strong photobleaching. FRUITFULL-treated plants were also successfully downregulated, with a high rate of survival but less effective silencing; only a small percentage of survivors showed a strong phenotype. DISCUSSION: Our optimized VIGS protocol in C. violacea enables functional gene analyses at different developmental stages. Additionally, C. violacea is amenable to heterologous knockdown, which suggests that a first pass using non-endogenous constructs is a possible route to test additional species of Cleomaceae.

Complete chloroplast genome sequences of Dipterygium glaucum and Cleome chrysantha and other Cleomaceae Species, comparative analysis and phylogenetic relationships.

This current study presents, for the first time, the complete chloroplast genome of two Cleomaceae species: Dipterygium glaucum and Cleome chrysantha in order to evaluate the evolutionary relationship. The cp genome is 158,576 bp in length with 35.74% GC content in D. glaucum and 158,111 bp with 35.96% GC in C. chrysantha. Inverted repeats IR 26,209 bp, 26,251 bp each, LSC of 87,738 bp, 87,184 bp and SSC of 18,420 bp, 18,425 bp respectively. There are 136 genes in the genome, which includes 80 protein coding genes, 31 tRNA genes and four rRNA genes were observed in both chloroplast genomes. 117 genes are unique while the remaining 19 genes are duplicated in IR regions. The analysis of repeats shows that the cp genome includes all types of repeats with more frequent occurrences of palindromic; Also, this analysis indicates that the total number of simple sequence repeats (SSR) were 323 in D. glaucum, and 313 in C. chrysantha, of which the majority of the SSRs in these plastid genomes were mononucleotide repeats A/T which are located in the intergenic spacer. Moreover, the comparative analysis of the four cp sequences revealed four hotspot genes (atpF, rpoC2, rps19, and ycf1), these variable regions could be used as molecular makers for the species authentication as well as resources for inferring phylogenetic relationships of the species. All the relationships in the phylogenetic tree are with high support, this indicate that the complete chloroplast genome is a useful data for inferring phylogenetic relationship within the Cleomaceae and other families. The simple sequence repeats identified will be useful for identification, genetic diversity, and other evolutionary studies of the species. This study reported the first cp genome of the genus Dipterygium and Cleome. The finding of this study will be beneficial for biological disciplines such as evolutionary and genetic diversity studies of the species within the core Cleomaceae.

A systematic revision of Capparaceae and Cleomaceae in Egypt: an evaluation of the generic delimitations of Capparis and Cleome using ecological and genetic diversity.

BACKGROUND: The Capparaceae family is commonly recognized as a caper, while Cleomaceae represents one of small flowering family within the order Brassicales. Earlier, Cleomaceae was included in the family Capparaceae; then, it was moved to a distinct family after DNA evidence. Variation in habits and a bewildering array of floral and fruit forms contributed to making Capparaceae a "trash-basket" family in which many unrelated plants were placed. Indeed, family Capparaceae and Cleomaceae are in clear need of more detailed systematic revision. RESULTS: Here, in the present study, the morphological characteristics and the ecological distribution as well as the genetic diversity analysis among the twelve species of both Capparaceae and Cleomaceae have been determined. The genetic analysis has been checked using 15 ISSR, 30 SRAP, and 18 ISTR to assess the systematic knots between the two families. In order to detect the molecular phylogeny, a comparative analysis of the three markers was performed based on the exposure of discriminating capacity, efficiency, and phylogenetic heatmap. Our results indicated that there is a morphological and ecological variation between the two families. Moreover, the molecular analysis confirmed that ISTR followed by SRAP markers has superior discriminating capacity for describing the genetic diversity and is able to simultaneously distinguish many polymorphic markers per reaction. Indeed, both the PCA and HCA data have drawn a successful annotation relationship in Capparaceae and Cleome species to evaluate whether the specific group sort individual or overlap groups. CONCLUSION: The outcomes of the morphological and ecological characterization along with the genetic diversity indicated an insight solution thorny interspecies in Cleome and Gynandropsis genera as a distinct family (Cleomaceae) and the other genera (Capparis, Cadaba, Boscia, and Maerua) as Capparaceae. Finally, we recommended further studies to elucidate the systematic position of Dipterygium glaucum.