Whole-genome sequence and annotation of Penstemon davidsonii.

Penstemon is the most speciose flowering plant genus endemic to North America. Penstemon species' diverse morphology and adaptation to various environments have made them a valuable model system for studying evolution. Here, we report the first full reference genome assembly and annotation for Penstemon davidsonii. Using PacBio long-read sequencing and Hi-C scaffolding technology, we constructed a de novo reference genome of 437,568,744 bases, with a contig N50 of 40 Mb and L50 of 5. The annotation includes 18,199 gene models, and both the genome and transcriptome assembly contain over 95% complete eudicot BUSCOs. This genome assembly will serve as a valuable reference for studying the evolutionary history and genetic diversity of the Penstemon genus.

A few essential genetic loci distinguish Penstemon species with flowers adapted to pollination by bees or hummingbirds.

In the formation of species, adaptation by natural selection generates distinct combinations of traits that function well together. The maintenance of adaptive trait combinations in the face of gene flow depends on the strength and nature of selection acting on the underlying genetic loci. Floral pollination syndromes exemplify the evolution of trait combinations adaptive for particular pollinators. The North American wildflower genus Penstemon displays remarkable floral syndrome convergence, with at least 20 separate lineages that have evolved from ancestral bee pollination syndrome (wide blue-purple flowers that present a landing platform for bees and small amounts of nectar) to hummingbird pollination syndrome (bright red narrowly tubular flowers offering copious nectar). Related taxa that differ in floral syndrome offer an attractive opportunity to examine the genomic basis of complex trait divergence. In this study, we characterized genomic divergence among 229 individuals from a Penstemon species complex that includes both bee and hummingbird floral syndromes. Field plants are easily classified into species based on phenotypic differences and hybrids displaying intermediate floral syndromes are rare. Despite unambiguous phenotypic differences, genome-wide differentiation between species is minimal. Hummingbird-adapted populations are more genetically similar to nearby bee-adapted populations than to geographically distant hummingbird-adapted populations, in terms of genome-wide dXY. However, a small number of genetic loci are strongly differentiated between species. These approximately 20 "species-diagnostic loci," which appear to have nearly fixed differences between pollination syndromes, are sprinkled throughout the genome in high recombination regions. Several map closely to previously established floral trait quantitative trait loci (QTLs). The striking difference between the diagnostic loci and the genome as whole suggests strong selection to maintain distinct combinations of traits, but with sufficient gene flow to homogenize the genomic background. A surprisingly small number of alleles confer phenotypic differences that form the basis of species identity in this species complex.

A few genetic variants go a long way in differentiating Penstemon species.

The integrity of hybridizing species is usually maintained by genome-wide selection or by selection on a few genomic regions. A study published in PLOS Biology finds a different pattern-60 SNPs spread across the genome differentiate a Penstemon species pair.

Predictive Links between Petal Color and Pigment Quantities in Natural Penstemon Hybrids.

Flowers have evolved remarkable diversity in petal color, in large part due to pollinator-mediated selection. This diversity arises from specialized metabolic pathways that generate conspicuous pigments. Despite the clear link between flower color and floral pigment production, quantitative models inferring predictive relationships between pigmentation and reflectance spectra have not been reported. In this study, we analyze a dataset consisting of hundreds of natural Penstemon hybrids that exhibit variation in flower color, including blue, purple, pink, and red. For each individual hybrid, we measured anthocyanin pigment content and petal spectral reflectance. We found that floral pigment quantities are correlated with hue, chroma, and brightness as calculated from petal spectral reflectance data: hue is related to the relative amounts of delphinidin vs. pelargonidin pigmentation, whereas brightness and chroma are correlated with the total anthocyanin pigmentation. We used a partial least squares regression approach to identify predictive relationships between pigment production and petal reflectance. We find that pigment quantity data provide robust predictions of petal reflectance, confirming a pervasive assumption that differences in pigmentation should predictably influence flower color. Moreover, we find that reflectance data enables accurate inferences of pigment quantities, where the full reflectance spectra provide much more accurate inference of pigment quantities than spectral attributes (brightness, chroma, and hue). Our predictive framework provides readily interpretable model coefficients relating spectral attributes of petal reflectance to underlying pigment quantities. These relationships represent key links between genetic changes affecting anthocyanin production and the ecological functions of petal coloration.

Identification of a highly promiscuous glucosyltransferase from Penstemon barbatus for natural product glycodiversification.

Glycosylation reactions mediated by UDP-glycosyltransferases (UGTs) are common post-modifications involved in plant secondary metabolism and significantly improve the solubility and bioactivity of aglycones. Penstemon barbatus is rich in phenylethanoid glycosides (PhGs), such as echinacoside and verbascoside. In this study, a promiscuous glycosyltransferase UGT84A95 was identified from P. barbatus. In vitro enzyme assays showed that UGT84A95 catalyzed the glucosylation of the phenol hydroxyl group of PhGs efficiently as well as other structurally diverse phenolic glycosides, including flavonoids, terpenoids, stilbene glycosides, coumarins, and simple polyphenols. By using UGT84A95, 12 glycosylated products were prepared and structurally identified by NMR spectroscopy, among which 7 are new compounds. These findings suggest that UGT84A95 could be a potential biocatalyst to synthesize multi-glycosylated glycosides.

Testing hypotheses of hybrid taxon formation in the shrubby beardtongues (Penstemon subgenus Dasanthera).

PREMISE: Hybridization is increasingly being identified in the genomes of species across the tree of life, leading to a general recognition that hybridization plays an important role in the generation of species diversity. While hybridization may increase species diversity directly via the formation of new taxa through hybrid speciation, it may also act indirectly via the exchange of phenotypic and genetic variance between species, which may in turn stimulate future speciation events. METHODS: Using high-throughput sequence data, we resolved phylogenetic relationships and investigated the role of hybridization as a diversification mechanism in the shrubby beardtongues (Penstemon subgenus Dasanthera), a group of North American wildflowers that has undergone a recent and rapid adaptive radiation. Specifically, we tested four hypotheses of hybrid taxon formation resulting from hybridization between P. davidsonii and P. fruticosus. RESULTS: Species tree inference supports the monophyly of subgenus Dasanthera and elucidates relationships between taxa distributed in the Cascades and Sierra Nevada Mountains. Results also provide evidence of gene flow between P. davidsonii and P. fruticosus and support at least one hybrid origin hypothesis (P. davidsonii var. menziesii) in a region of contemporary distributional overlap. Hybridization may have also been facilitated by historical overlap in geographic distribution caused by species' responses to climatic changes during the Pleistocene. CONCLUSIONS: Our results support a history of hybridization between focal taxa in a rapidly radiating clade of plants and more broadly contribute to our growing understanding of the role of hybridization as a diversification mechanism in plants.

Adaptation to lower latitudes and lower elevations precedes the evolution of hummingbird pollination in western North American Penstemon.

PREMISE: A switch in pollinator can occur when a plant lineage enters a new habitat where the ancestral pollinator is less common, and a novel pollinator is more common. Because pollinator communities vary according to environmental tolerances and availability of resources, there may be consistent associations between pollination mode and specific regions and habitats. Such associations can be studied in lineages that have experienced multiple pollinator transitions, representing evolutionary replicates. METHODS: Our study focused on a large clade of Penstemon wildflower species in western North America, which has repeatedly evolved hummingbird-adapted flowers from ancestral bee-adapted flowers. For each species, we estimated geographic ranges from occurrence data and inferred environmental niches from climate, topographical, and soil data. Using a phylogenetic comparative approach, we investigated whether hummingbird-adapted species occupy distinct geographic regions or habitats relative to bee-adapted species. RESULTS: Hummingbird-adapted species occur at lower latitudes and lower elevations than bee-adapted species, resulting in a difference in their environmental niche. Bee-adapted species sister to hummingbird-adapted species are also found in relatively low elevations and latitudes, similar to their hummingbird-adapted sister species, suggesting ecogeographic shifts precede pollinator divergence. Sister species pairs-regardless of whether they differ in pollinator-show relatively little geographic range overlap. CONCLUSIONS: Adaptation to a novel pollinator may often occur in geographic and ecological isolation from ancestral populations. The ability of a given lineage to adapt to novel pollinators may critically depend on its ability to colonize regions and habitats associated with novel pollinator communities.

Improving phylogenetic resolution of the Lamiales using the complete plastome sequences of six Penstemon species.

The North American endemic genus Penstemon (Mitchell) has a recent geologic origin of ca. 3.6 million years ago (MYA) during the Pliocene/Pleistocene transition and has undergone a rapid adaptive evolutionary radiation with ca. 285 species of perennial forbs and sub-shrubs. Penstemon is divided into six subgenera occupying all North American habitats including the Arctic tundra, Central American tropical forests, alpine meadows, arid deserts, and temperate grasslands. Due to the rapid rate of diversification and speciation, previous phylogenetic studies using individual and concatenated chloroplast sequences have failed to resolve many polytomic clades. We investigated the efficacy of utilizing the plastid genomes (plastomes) of 29 species in the Lamiales order, including five newly sequenced Penstemon plastomes, for analyzing phylogenetic relationships and resolving problematic clades. We compared whole-plastome based phylogenies to phylogenies based on individual gene sequences (matK, ndhF, psaA, psbA, rbcL, rpoC2, and rps2) and concatenated sequences. We also We found that our whole-plastome based phylogeny had higher nodal support than all other phylogenies, which suggests that it provides greater accuracy in describing the hierarchal relationships among taxa as compared to other methods. We found that the genus Penstemon forms a monophyletic clade sister to, but separate from, the Old World taxa of the Plantaginaceae family included in our study. Our whole-plastome based phylogeny also supports the rearrangement of the Scrophulariaceae family and improves resolution of major clades and genera of the Lamiales.

Pollinator divergence and pollination isolation between hybrids with different floral color and morphology in two sympatric Penstemon species.

Differential visitation of pollinators due to divergent floral traits can lead to reproductive isolation via assortative pollen flow, which may ultimately be a driving force in plant speciation, particularly in areas of overlap. We evaluate the effects of pollinator behavioral responses to variation of intraspecific floral color and nectar rewards, on reproductive isolation between two hybrid flower color morphs (fuchsia and blue) and their parental species Penstemon roseus and P. gentianoides with a mixed-pollination system. We show that pollinators (bumblebees and hummingbirds) exhibit different behavioral responses to fuchsia and blue morphs, which could result from differential attraction or deterrence. In addition to differences in color (spectral reflectance), we found that plants with fuchsia flowers produced more and larger flowers, produced more nectar and were more visited by pollinators than those with blue flowers. These differences influenced the foraging behavior and effectiveness as pollinators of both bumblebees and hummingbirds, which contributed to reproductive isolation between the two hybrid flower color morphs and parental species. This study demonstrates how differentiation of pollination traits promotes the formation of hybrid zones leading to pollinator shifts and reproductive isolation. While phenotypic traits of fuchsia and red flowers might encourage more efficient hummingbird pollination in a mixed-pollination system, the costs of bumblebee pollination on plant reproduction could be the drivers for the repeated shifts from bumblebee- to hummingbird-mediated pollination.

Nectary size is a pollination syndrome trait in Penstemon.

Evolution of complex phenotypes depends on the adaptive importance of individual traits, and the developmental changes required to modify traits. Floral syndromes are complex adaptations to pollinators that include color, nectar, and shape variation. Hummingbird-adapted flowers have evolved a remarkable number of times from bee-adapted ancestors in Penstemon, and previous work demonstrates that color over shape better distinguishes bee from hummingbird syndromes. Here, we examined the relative importance of nectar volume and nectary development in defining Penstemon pollination syndromes. We tested the evolutionary association of nectar volume and nectary area with pollination syndrome across 19 Penstemon species. In selected species, we assessed cellular-level processes shaping nectary size. Within a segregating population from an intersyndrome cross, we assessed trait correlations between nectar volume, nectary area, and the size of stamens on which nectaries develop. Nectar volume and nectary area displayed an evolutionary association with pollination syndrome. These traits were correlated within a genetic cross, suggesting a mechanistic link. Nectary area evolution involves parallel processes of cell expansion and proliferation. Our results demonstrate that changes to nectary patterning are an important contributor to pollination syndrome diversity and provide further evidence that repeated origins of hummingbird adaptation involve parallel developmental processes in Penstemon.

"Pro-bird" floral traits discourage bumblebee visits to Penstemon gentianoides (Plantaginaceae), a mixed-pollinated herb.

The pollination syndrome concept implies that flowers evolved with particular sets of characteristics, such as colors, shapes, orientations, and rewards, as a means of attracting pollinators. However, these traits may have also evolved to deter unwanted visitors. The North American genus Penstemon exhibits a great floral diversity that is mainly associated with bumblebee and hummingbird pollination. Evolutionary shifts from insect pollination to hummingbird pollination have occurred in Penstemon repeatedly, but some species maintain mixed-pollination systems and intermediate floral traits between bee- and hummingbird-pollination modes. The apparently intermediate floral traits of species with mixed-pollination systems might be potentially acting to deter bumblebee foragers. Then, bird-flower traits might be selected with increased hummingbird visitation over evolutionary time might, resulting in specialization to and the evolution of floral traits present in hummingbird-pollinated species. Here, we modified bee-pollination floral traits in Penstemon gentianoides with a mixed pollination system, to resemble hummingbird-pollination traits, and measured the effects of trait modification on bumblebee foraging behavior and plant female reproductive fitness. Our results showed that reduction in the width of the corolla tube and the absence of the corolla lip negatively affects bumblebee visitation and their efficiency as pollinators, and that the synergistic interaction of both traits enhanced the "anti-bee" effect. We conclude that acquisition of floral traits that resemble those of hummingbird-pollination enables Penstemon plant species to deter bumblebee visits.

SNP-skimming: A fast approach to map loci generating quantitative variation in natural populations.

Genome-wide association mapping (GWAS) is a method to estimate the contribution of segregating genetic loci to trait variation. A major challenge for applying GWAS to nonmodel species has been generating dense genome-wide markers that satisfy the key requirement that marker data are error-free. Here, we present an approach to map loci within natural populations using inexpensive shallow genome sequencing. This "SNP-skimming" approach involves two steps: an initial genome-wide scan to identify putative targets followed by deep sequencing for confirmation of targeted loci. We apply our method to a test data set of floral dimension variation in the plant Penstemon virgatus, a member of a genus that has experienced dynamic floral adaptation that reflects repeated transitions in primary pollinator. The ability to detect SNPs that generate phenotypic variation depends on population genetic factors such as population allele frequency, effect size and epistasis, as well as sampling effects contingent on missing data and genotype uncertainty. However, both simulations and the Penstemon data suggest that the most significant tests from the initial SNP skim are likely to be true positives-loci with subtle but significant quantitative effects on phenotype. We discuss the promise and limitations of this method and consider optimal experimental design for a given sequencing effort. Simulations demonstrate that sampling a larger number of individual at the expense of average read depth per individual maximizes the power to detect loci.

The Perennial Penstemon: Variation in Defensive Chemistry Across Years, Populations, and Tissues.

Plants produce a variety of secondary metabolites that function as a defense against their natural enemies. Production of these secondary metabolites is genetically controlled, but is also phenotypically plastic and varies in response to both biotic and abiotic factors. Therefore, plant species may vary widely in their chemical defenses and such variation can be evident at temporal, spatial and tissue levels. Focusing on the chemical defenses of a native Colorado wildflower, Penstemon virgatus, we assessed the variation in iridoid glycoside (IG) content across two non-consecutive growing seasons, six natural populations and three tissue types: leaves, stems and flowers. Our results indicate that P. virgatus plants contain high concentrations of IGs (mean = 23.36% dry weight of leaves) and that IGs were differentially allocated among tissue types. Leaves contained the highest concentration of IGs, which varied quantitatively between sampling years, among plant populations, and plant parts. We also quantified leaf herbivore damage at all six populations but we found very little herbivore damage. Our study indicates that the IG concentrations of P. virgatus plants are both spatially and temporally variable. Furthermore, the high concentrations of secondary metabolites combined with the low levels of damage suggest that these plants are well defended against generalist herbivores.

Multiplexed shotgun genotyping resolves species relationships within the North American genus Penstemon.

PREMISE OF THE STUDY: Evolutionary radiations provide opportunities to examine large-scale patterns in diversification and character evolution, yet are often recalcitrant to phylogenetic resolution due to rapid speciation events. The plant genus Penstemon has been difficult to resolve using Sanger sequence-based markers, leading to the hypothesis that it represents a recent North American radiation. The current study demonstrates the utility of multiplexed shotgun genotyping (MSG), a style of restriction site-associated DNA sequencing (RADseq), to infer phylogenetic relationships within a subset of species in this genus and provide insight into evolutionary patterns. METHODS: We sampled genomic DNA, primarily from herbarium material, and subjected it to MSG library preparation and Illumina sequencing. The resultant sequencing reads were clustered into homologous loci, aligned, and concatenated into data matrices that differed according to clustering similarity and amount of missing data. We performed phylogenetic analyses on these matrices using maximum likelihood (RAxML) and a species tree approach (SVDquartets). KEY RESULTS: MSG data provide a highly resolved estimate of species relationships within Penstemon. While most species relationships were highly supported, the position of certain taxa remains ambiguous, suggesting that increased taxonomic sampling or additional methodologies may be required. The data confirm that evolutionary shifts from hymenopteran- to hummingbird-adapted flowers have occurred independently many times. CONCLUSIONS: This study demonstrates that phylogenomic approaches yielding thousands of variable sites can greatly improve species-level resolution of recent and rapid radiations. Similar to other studies, we found that less conservative similarity and missing data thresholds resulted in more highly supported topologies.

Predicting the genetic consequences of future climate change: The power of coupling spatial demography, the coalescent, and historical landscape changes.

PREMISE OF THE STUDY: Climate change is a widely accepted threat to biodiversity. Species distribution models (SDMs) are used to forecast whether and how species distributions may track these changes. Yet, SDMs generally fail to account for genetic and demographic processes, limiting population-level inferences. We still do not understand how predicted environmental shifts will impact the spatial distribution of genetic diversity within taxa. METHODS: We propose a novel method that predicts spatially explicit genetic and demographic landscapes of populations under future climatic conditions. We use carefully parameterized SDMs as estimates of the spatial distribution of suitable habitats and landscape dispersal permeability under present-day, past, and future conditions. We use empirical genetic data and approximate Bayesian computation to estimate unknown demographic parameters. Finally, we employ these parameters to simulate realistic and complex models of responses to future environmental shifts. We contrast parameterized models under current and future landscapes to quantify the expected magnitude of change. KEY RESULTS: We implement this framework on neutral genetic data available from Penstemon deustus. Our results predict that future climate change will result in geographically widespread declines in genetic diversity in this species. The extent of reduction will heavily depend on the continuity of population networks and deme sizes. CONCLUSIONS: To our knowledge, this is the first study to provide spatially explicit predictions of within-species genetic diversity using climatic, demographic, and genetic data. Our approach accounts for climatic, geographic, and biological complexity. This framework is promising for understanding evolutionary consequences of climate change, and guiding conservation planning.

In vitro COX-1 and COX-2 enzyme inhibitory activities of iridoids from Penstemon barbatus, Castilleja tenuiflora, Cresentia alata and Vitex mollis.

A group of sixteen iridoids isolated from plants used as anti-inflammatory remedies in Mexican folk medicine were evaluated for their potential to inhibit cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) enzymes. From these assays, loganic acid (10) was identified as the most promising compound with both COX-1 (36.0 ± 0.6%) and COX-2 (80.8 ± 4.0%) inhibition at 10 µM. Compound 10 shows a better inhibition against the COX-2 enzyme. Other iridoids tested in the present study showed weak or no inhibition against these enzymes. Furthermore, herein are presented key interactions of iridoid 10 with COX-1 and COX-2 enzymes through molecular docking studies. These studies suggest that 10 exhibits anti-inflammatory activity due to COX inhibition.

Spatiotemporal Floral Scent Variation of Penstemon digitalis.

Variability in floral volatile emissions can occur temporally through floral development, during diel cycles, as well as spatially within a flower. These spatiotemporal patterns are hypothesized to provide additional information to floral visitors, but they are rarely measured, and their attendant hypotheses are even more rarely tested. In Penstemon digitalis, a plant whose floral scent has been shown to be under strong phenotypic selection for seed fitness, we investigated spatiotemporal variation in floral scent by using dynamic headspace collection, respectively solid-phase microextraction, and analyzed the volatile samples by combined gas chromatography-mass spectrometry. Total volatile emission was greatest during flowering and peak pollinator activity hours, suggesting its importance in mediating ecological interactions. We also detected tissue and reward-specific compounds, consistent with the hypothesis that complexity in floral scent composition reflects several ecological functions. In particular, we found tissue-specific scents for the stigma, stamens, and staminode (a modified sterile stamen common to all Penstemons). Our findings emphasize the dynamic nature of floral scents and highlight a need for greater understanding of ecological and physiological mechanisms driving spatiotemporal patterns in scent production.

Ecological transition predictably associated with gene degeneration.

Gene degeneration or loss can significantly contribute to phenotypic diversification, but may generate genetic constraints on future evolutionary trajectories, potentially restricting phenotypic reversal. Such constraints may manifest as directional evolutionary trends when parallel phenotypic shifts consistently involve gene degeneration or loss. Here, we demonstrate that widespread parallel evolution in Penstemon from blue to red flowers predictably involves the functional inactivation and degeneration of the enzyme flavonoid 3',5'-hydroxylase (F3'5'H), an anthocyanin pathway enzyme required for the production of blue floral pigments. Other types of genetic mutations do not consistently accompany this phenotypic shift. This pattern may be driven by the relatively large mutational target size of degenerative mutations to this locus and the apparent lack of associated pleiotropic effects. The consistent degeneration of F3'5'H may provide a mechanistic explanation for the observed asymmetry in the direction of flower color evolution in Penstemon: Blue to red transitions are common, but reverse transitions have not been observed. Although phenotypic shifts in this system are likely driven by natural selection, internal constraints may generate predictable genetic outcomes and may restrict future evolutionary trajectories.

Identification of major quantitative trait loci underlying floral pollination syndrome divergence in Penstemon.

Distinct floral pollination syndromes have emerged multiple times during the diversification of flowering plants. For example, in western North America, a hummingbird pollination syndrome has evolved more than 100 times, generally from within insect-pollinated lineages. The hummingbird syndrome is characterized by a suite of floral traits that attracts and facilitates pollen movement by hummingbirds, while at the same time discourages bee visitation. These floral traits generally include large nectar volume, red flower colour, elongated and narrow corolla tubes and reproductive organs that are exerted from the corolla. A handful of studies have examined the genetic architecture of hummingbird pollination syndrome evolution. These studies find that mutations of relatively large effect often explain increased nectar volume and transition to red flower colour. In addition, they suggest that adaptive suites of floral traits may often exhibit a high degree of genetic linkage, which could facilitate their fixation during pollination syndrome evolution. Here, we explore these emerging generalities by investigating the genetic basis of floral pollination syndrome divergence between two related Penstemon species with different pollination syndromes--bee-pollinated P. neomexicanus and closely related hummingbird-pollinated P. barbatus. In an F2 mapping population derived from a cross between these two species, we characterized the effect size of genetic loci underlying floral trait divergence associated with the transition to bird pollination, as well as correlation structure of floral trait variation. We find the effect sizes of quantitative trait loci for adaptive floral traits are in line with patterns observed in previous studies, and find strong evidence that suites of floral traits are genetically linked. This linkage may be due to genetic proximity or pleiotropic effects of single causative loci. Interestingly, our data suggest that the evolution of floral traits critical for hummingbird pollination was not constrained by negative pleiotropy at loci that show co-localization for multiple traits.

Predictability and irreversibility of genetic changes associated with flower color evolution in Penstemon barbatus.

Two outstanding questions in evolutionary biology are whether, and how often, the genetic basis of phenotypic evolution is predictable; and whether genetic change constrains evolutionary reversibility. We address these questions by studying the genetic basis of red flower color in Penstemon barbatus. The production of red flowers often involves the inactivation of one or both of two anthocyanin pathway genes, Flavonoid 3',5'-hydroxylase (F3'5'h) and Flavonoid 3'-hydroxylase (F3'h). We used gene expression and enzyme function assays to determine that redundant inactivating mutations to F3'5'h underlie the evolution of red flowers in P. barbatus. Comparison of our results to previously characterized shifts from blue to red flowers suggests that the genetic change associated with the evolution of red flowers is predictable: when it involves elimination of F3'5'H activity, functional inactivation or deletion of this gene tends to occur; however, when it involves elimination of F3'H activity, tissue-specific regulatory substitutions occur and the gene is not functionally inactivated. This pattern is consistent with emerging data from physiological experiments indicating that F3'h may have pleiotropic effects and is thus subject to purifying selection. The multiple, redundant inactivating mutations to F3'5'h suggest that reversal to blue-purple flowers in this group would be unlikely.