Parallel evolution of apetalous lineages within the buttercup family (Ranunculaceae): outward expansion of AGAMOUS1, rather than disruption of APETALA3-3.

Complete loss of petals, or becoming apetalous, has occurred independently in many flowering plant lineages. However, the mechanisms underlying the parallel evolution of naturally occurring apetalous lineages remain largely unclear. Here, by sampling representatives of all nine apetalous genera/tribes of the family Ranunculaceae and conducting detailed morphological, expression, molecular evolutionary and functional studies, we investigate the mechanisms underlying parallel petal losses. We found that while non-expression/downregulation of the petal identity gene APETALA3-3 (AP3-3) is tightly associated with complete petal losses, disruptions of the AP3-3 orthologs were unlikely to be the real causes for the parallel evolution of apetalous lineages. We also found that, compared with their close petalous relatives, naturally occurring apetalous taxa usually bear slightly larger numbers of stamens, whereas the number of sepals remains largely unchanged, suggestive of petal-to-stamen rather than petal-to-sepal transformations. In addition, in the recently originated apetalous genus Enemion, the petal-to-stamen transformations have likely been caused by the mutations that led to the elevation and outward expansion of the expression of the C-function gene, AGAMOUS1 (AG1). Our results not only provide a general picture of parallel petal losses within the Ranunculaceae but also help understand the mechanisms underlying the independent originations of other apetalous lineages.

Using microscopic techniques for taxonomic implications of seed and fruits of Delphinium L. (sensu lato) (Ranunculaceae).

In present research, micromorphological characters of the genus Delphinium L. (sensu lato) Ranunculaceae seeds and fruits were studied using microscopic techniques. A total of 37 species were studied using light (LM) and scanning electron microscopy (SEM) to evaluate seeds and fruits micromorphological characteristics for the taxonomic identification and species relationship in the genus. For each taxon, 33 characters were observed and studied. All characters were analyzed using MVSP software and UPGMA method by using dissimilarity index (average taxonomic distance). Considerable differences were observed in trichomes shape of fruit and seed color features. The results revealed that all studied taxa are separated into three clades. The first clade consists of D. pallidiflorum and D. semibarbatum, which have black seed and two types of glandular and crispate trichomes in fruit. The second clade consists of D. speciosum, D. szowitsianum, D. dasystachyson, D. aquilegifolium, D. ursinum, C. trigonelloides, and C. camptocarpa, which had a brown color seeds, two types of glandular and villous trichomes on fruits also have been observed. The remaining species were classified as third clade having various colors including yellow, black, and brown color seeds, the trichome shapes included one of the following forms strigose, villous, trichomes less, glandular, hooked, and crispate trichomes in fruits. The aim of the present study is to use micromorphological characters of seeds and fruits for the taxonomic identification and species delimitation in the genus Delphinium.

Identification of green energy ranunculaceous flora of district Chitral, Northern Pakistan using pollen features through scanning electron microscopy.

Pollen micro-morphological features have proven to be helpful for the plant taxonomists in the identification and classification of plants. The utilization of this plantmayhelpfulin the areas of lignocellulosic conversion to biofuels and diversify application toward biomass. The current study was planned with the aim to evaluate the pollen features of complex Ranunculaceous flora of District Chitral, Northern Pakistan using both scanning electron microscopy (SEM) and Light Microscope (LM) for their taxonomic importance. Pollens of 18 Ranunculaceous species belonging to 6 genera were collected from different localities of the research area. SEM and LM were used to examine both qualitative and quantitative micro-morphological features. Sculptring of the sexine include; Scabrate, psilate, echinate, verrucate, perforate gemmate, and reticulate and so forth. Shape of the pollens was sub-spheroidal, spheroidal, prolate, subprolate and oblate and so forth. Type of pollen was ranged from mono to tricolpate and tricolporate. Quantitative characters include length/width of the pollen, colpus, exine thickness, and P/E ratio. Based on these micro-morphological features a taxonomic key was prepared for the fast and correct identification. RESEARCH HIGHLIGHT: Study of the pollen micro-morphological features of Ranunculaceous species by SEM and LM. Analysing both qualitative and quantitative characters of the pollens. Preparation of taxonomic key based on micro-morphological features for the correct and fast identification.

Relationship between the species-representative phenotype and intraspecific variation in Ranunculaceae floral organ and Asteraceae flower numbers.

BACKGROUND AND AIMS: Phenotypic variation in floral morphologies contributes to speciation by testing various morphologies that might have higher adaptivity, leading eventually to phylogenetic diversity. Species diversity has been recognized, however, by modal morphologies where the variation is averaged out, so little is known about the relationship between the variation and the diversity. METHODS: We analysed quantitatively the intraspecific variation of the organ numbers within flowers of Ranunculaceae, a family which branched near the monocot-eudicot separation, and the numbers of flowers within the capitula of Asteraceae, one of the most diverse families of eudicots. We used four elementary statistical quantities: mean, standard deviation (s.d.), degree of symmetry (skewness) and steepness (kurtosis). KEY RESULTS: While these four quantities vary among populations, we found a common relationship between s.d. and the mean number of petals and sepals in Ranunculaceae and number of flowers per capitulum in Asteraceae. The s.d. is equal to the square root of the difference between the mean and specific number, showing robustness: for example, 3 in Ficaria sepals, 5 in Ranunculus petals and Anemone tepals, and 13 in Farfugium ray florets. This square-root relationship was not applicable to Eranthis petals which show little correlation between the s.d. and mean, and the stamens and carpels of Ranunculaceae whose s.d. is proportional to the mean. The specific values found in the square-root relationship provide a novel way to find the species-representative phenotype among varied morphologies. CONCLUSIONS: The representative phenotype is, in most cases, unique to the species or genus level, despite intraspecific differences of average phenotype among populations. The type of variation shown by the statistical quantities indicates not only the robustness of the morphologies but also how flowering plants changed during evolution among representative phenotypes that eventually led to phylogenetic diversification.

Selection by pollinators on floral traits in generalized Trollius ranunculoides (Ranunculaceae) along altitudinal gradients.

Abundance and visitation of pollinator assemblages tend to decrease with altitude, leading to an increase in pollen limitation. Thus increased competition for pollinators may generate stronger selection on attractive traits of flowers at high elevations and cause floral adaptive evolution. Few studies have related geographically variable selection from pollinators and intraspecific floral differentiation. We investigated the variation of Trollius ranunculoides flowers and its pollinators along an altitudinal gradient on the eastern Qinghai-Tibet Plateau, and measured phenotypic selection by pollinators on floral traits across populations. The results showed significant decline of visitation rate of bees along altitudinal gradients, while flies was unchanged. When fitness is estimated by the visitation rate rather than the seed number per plant, phenotypic selection on the sepal length and width shows a significant correlation between the selection strength and the altitude, with stronger selection at higher altitudes. However, significant decreases in the sepal length and width of T. ranunculoides along the altitudinal gradient did not correspond to stronger selection of pollinators. In contrast to the pollinator visitation, mean annual precipitation negatively affected the sepal length and width, and contributed more to geographical variation in measured floral traits than the visitation rate of pollinators. Therefore, the sepal size may have been influenced by conflicting selection pressures from biotic and abiotic selective agents. This study supports the hypothesis that lower pollinator availability at high altitude can intensify selection on flower attractive traits, but abiotic selection is preventing a response to selection from pollinators.

Petal-specific subfunctionalization of an APETALA3 paralog in the Ranunculales and its implications for petal evolution.

• The petals of the lower eudicot family Ranunculaceae are thought to have been derived many times independently from stamens. However, investigation of the genetic basis of their identity has suggested an alternative hypothesis: that they share a commonly inherited petal identity program. This theory is based on the fact that an ancient paralogous lineage of APETALA3 (AP3) in the Ranunculaceae appears to have a conserved, petal-specific expression pattern. • Here, we have used a combination of approaches, including RNAi, comparative gene expression and molecular evolutionary studies, to understand the function of this petal-specific AP3 lineage. • Functional analysis of the Aquilegia locus AqAP3-3 has demonstrated that the paralog is required for petal identity with little contribution to the identity of the other floral organs. Expanded expression studies and analyses of molecular evolutionary patterns provide further evidence that orthologs of AqAP3-3 are primarily expressed in petals and are under higher purifying selection across the family than the other AP3 paralogs. • Taken together, these findings suggest that the AqAP3-3 lineage underwent progressive subfunctionalization within the order Ranunculales, ultimately yielding a specific role in petal identity that has probably been conserved, in stark contrast with the multiple independent origins predicted by botanical theories.

Optimal pollinator attraction strategies in Trollius ranunculoides Hemsl. (Ranunculaceae) at different altitudes: increased floral display or promotion of nectar output?

For alpine plant species, patterns of resource allocation to functional floral traits for pollinator attraction can be highly significant in adaptation to low pollinator abundance and consequent pollen limitation. Increased pollination can be achieved either through a larger floral display or production of more pollen rewards. In this study, variation in resource allocation to different components for pollinator attraction was studied along an altitudinal gradient in Trollius ranunculoides, an obligate self-incompatible out-crosser of the Qinghai-Tibet Plateau. We compared resource allocation to conspicuous yellow sepals (which mainly provide visual attraction) and degenerate petals (which provide the major nectar reward) between populations at four altitudes. Furthermore, we investigated the contribution of sepals and petals to pollinator attraction and female reproductive success in an experiment with sepal or petal removal at sites at different altitudes. At the level of single flowers, resource allocation increased to sepals but decreased to petals with increasing altitude. Consistent with these results, sepals contributed much more to visitation rate and seed set than petals, as confirmed in the sepal or petal removal experiment. Sepals and petals contributed to female reproductive success by ensuring visitation rate rather than visitation duration. To alleviate increasing pollen limitation with increasing altitude, resource allocation patterns of T. ranunculoides altered to favour development of sepals rather than petals. This strategy may improve pollination and reproductive success through visual attraction (sepal) rather than nectar reward (petal) over a gradient of decreasing pollinator abundance.

The role of volatile organic compounds, morphology and pigments of globeflowers in the attraction of their specific pollinating flies.

• Floral scents and visual cues of the globeflower Trollius europaeus may play a key role in the attraction of Chiastocheta flies, involved in a highly specific nursery pollination mutualism. • Here, headspace collection and GC-MS were used to identify and quantify the volatile organic compounds emitted by the globeflower. • Scents are produced in three different floral parts by four structures: secretory glands and flat epidermis cells in the abaxial sepal epidermis, conical cells in the adaxial sepal epidermis, and pollen. The blend is made up of 16 compounds commonly found in floral scents. Geographical variation among populations is low compared with variation amongst individuals within populations. Electroantenno-graphic analyses revealed that six compounds emitted by both anthers and sepals are detected by Chiastocheta flies. Removing the anthers hidden inside the globe from flowers in the field decreased the number of fly visits to globeflowers. • A multivariate analysis of the effect of several floral traits on pollinator visitation rate conducted in the field showed that both floral scents and visual flower cues play a role in pollinator attraction. However, their relative roles and the intensity of the selective pressures exerted on floral traits by pollinators appear to vary in time and space.

Early reproductive developmental anatomy in Decaisnea (Lardizabalaceae) and its systematic implications.

BACKGROUND AND AIMS: Decaisnea insignis, known as 'dead man's fingers' (Lardizabalaceae), is widely distributed in China and the Himalayan foothill countries. This economically important plant, which is the only species in the genus, has not been the subject of any embryological studies aside from one brief, older paper that lacks micrographs. Data on Decaisnea are also important because its systematic position has been unstable since the genus was established in 1855. Therefore, the objectives of this study were: (a) to use modern microscopy to document early reproductive anatomical development in Decaisnea; and (b) to compare qualitatively these early embryological characters with allied taxa in a systematic context. METHODS: Decaisnea insignis floral buds and inflorescences were regularly collected from Shaanxi Province, China and prepared for light microscopy. The embryological characters studied were qualitatively compared with those of allied taxa via a thorough examination of the existing literature. KEY RESULTS: Early reproductive anatomy in Decaisnea was documented and novel revelations made. It was discovered that the pollen is shed when three-celled (not two-celled, as previously reported), and that endosperm formation is nuclear (not cellular or helobial, as previously reported). These two newly revealed embryological characters are not found in any other members of Lardizabalaceae. Furthermore, neither are persistent antipodal cells, which we confirmed to be present in Decaisnea. CONCLUSIONS: Decaisnea and other Lardizabalaceae characteristically have tetrasporangiate anthers, a secretory tapetum, simultaneous microsporocyte cytokinesis, primarily bitegmic, crassinucellate ovules, and a Polygonum type embryo sac. However, in the family, persistent antipodals, nuclear endosperm, and pollen shed at the three-celled stage are only found in Decaisnea. These embryological data prompted the suggestion that Decaisnea needs elevation above the level of genus.

Stability of floral specialization in Trollius europaeus in contrasting ecological environments.

Specialization of some plants on seed-eating pollinators is intriguing, especially when co-pollinators exclusively feeding on nectar are also present. We examined the stability of the morphological specialization of Trollius europaeus (L.) globeflowers with respect to Chiastocheta (Pokorny) flies by artificially opening the flowers. In the montane and subalpine environments studied, other visitors contributed 2% and 28% of all the visits, respectively, and visited open flowers nearly eight times more often than closed flowers, but in both environments their contribution to pollination did not compensate for Chiastocheta aversion against open phenotypes. Net seed set (female success) was slightly higher (+4%) and pollen export (male success) was much higher (+85%) for closed than open flowers. Selection in favour of the closed phenotype was even more intense in patches where open phenotypes were most common, precluding the evolution of open flowers in the study populations.

Chromones from the tubers of Eranthis cilicica and their antioxidant activity.

Chemical studies on the constituents of Eranthis cilicica led to isolation of ten chromone derivatives, two of which were previously known. Comprehensive spectroscopic analysis, including extensive 1D and 2D NMR data, and the results of enzymatic hydrolysis allowed the chemical structures of the compounds to be assigned as 8,11-dihydro-5-hydroxy-2,9-dihydroxymethyl-4H-pyrano[2,3-g][1]benzoxepin-4-one, 5,7-dihydroxy-8-[(2E)-4-hydroxy-3-methylbut-2-enyl]-2-methyl-4H-1-benzopyran-4-one, 5,7-dihydroxy-2-hydroxymethyl-8-[(2E)-4-hydroxy-3-methylbut-2-enyl]-4H-1-benzopyran-4-one, 7-[(beta-d-glucopyranosyl)oxy]-5-hydroxy-8-[(2E)-4-hydroxy-3-methylbut-2-enyl]-2-methyl-4H-1-benzopyran-4-one, 7-[(beta-d-glucopyranosyl)oxy]-5-hydroxy-2-hydroxymethyl-8-[(2E)-4-hydroxy-3-methylbut-2-enyl]-4H-1-benzopyran-4-one, 9-[(O-beta-d-glucopyranosyl-(1-->6)-beta-d-glucopyranosyl)oxy]methyl-8,11-dihydro-5,9-dihydroxy-2-methyl-4H-pyrano[2,3-g][1]benzoxepin-4-one, 8,11-dihydro-5,9-dihydroxy-9-hydroxymethyl-2-methyl-4H-pyrano[2,3-g][1]benzoxepin-4-one, and 7-[(O-beta-d-glucopyranosyl-(1-->6)-beta-d-glucopyranosyl)oxy]methyl-4-hydroxy-5H-furo[3,2-g][1]benzopyran-5-one, respectively. The isolated compounds were evaluated for their antioxidant activity.

[Pharmacognostical study of tall monkshood root].

OBJECTIVE: To provide the scientific basis for gruffs identification and utilization of tall monkshood root. METHODS: We studied tall monkshood root through the macroscopic, microscopical and Physicochemical identification. RESULTS: In the cross section of tall monkshood root, there were cork cortex cells, a cricoid range hetero-vascular bundle, radial-cell including radial little brown cell, a great deal of starch grain, cortex organize fragment was brown, cell squareness and polygon. CONCLUSION: These character can provide scientific basis for its quality standard and development.

Evolution of perianth and stamen characteristics with respect to floral symmetry in Ranunculales.

BACKGROUND AND AIMS: Floral symmetry presents two main states in angiosperms, namely polysymmetry and monosymmetry. Monosymmetry is thought to have evolved several times independently from polysymmetry, possibly in co-adaptation with specialized pollinators. Monosymmetry commonly refers to the perianth, even though associated androecium modifications have been reported. The evolution of perianth symmetry is examined with respect to traits of flower architecture in the Ranunculales, the sister group to all other eudicots, which present a large diversity of floral forms. METHODS: Characters considered were perianth merism, calyx, corolla and androecium symmetry, number of stamens and spurs. Character evolution was optimized on a composite phylogenetic tree of Ranunculales using maximum parsimony. KEY RESULTS: The ancestral state for merism could not be inferred because the basalmost Eupteleaceae lack a perianth and have a variable number of stamens. The Papaveraceae are dimerous, and the five other families share a common trimerous ancestor. Shifts from trimery to dimery (or reverse) are observed. Pentamery evolved in Ranunculaceae. Ranunculales except Eupteleaceae, present a polysymmetric ancestral state. Monosymmetry evolved once within Papaveraceae, Ranunculaceae and Menispermaceae (female flowers only). Oligandry is the ancestral state for all Ranunculales, and polyandry evolved several times independently, in Papaveraceae, Menispermaceae, Berberidaceae and Ranunculaceae, with two reversions to oligandry in the latter. The ancestral state for androecium symmetry is ambiguous for the Ranunculales, while polysymmetry evolved immediately after the divergence of Eupteleaceae. A disymmetric androecium evolved in Papaveraceae. The ancestral state for spurs is none. Multiple spurs evolved in Papaveraceae, Berberidaceae and Ranunculaceae, and single spurs occur in Papaveraceae and Ranunculaceae. CONCLUSIONS: The evolution of symmetry appears disconnected from changes in merism and stamen number, although monosymmetry never evolved in the context of an open ground plan. In bisexual species, monosymmetry evolved coincidently with single spurs, allowing us to propose an evolutionary scenario for Papaveraceae.

Design and construction of a new temperature-controlled chamber for light and confocal microscopy under monitored conditions: biological application for plant samples.

A new light microscope-temperature-controlled chamber (LM-TCC) has been constructed. The special feature of the light microscope-temperature-controlled chamber is the Peltier-element temperature control of a specimen holder for biological samples, with a volume capacity of 1 mL. This system has marked advantages when compared to other approaches for temperature-controlled microscopy. It works in a temperature range of -10 degrees C to +95 degrees C with an accuracy of +/-0.1 degrees C in the stationary phase. The light microscope-temperature-controlled chamber allows rapid temperature shift rates. A maximum heating rate of 12.9 degrees C min(-1) and a maximum cooling rate of 6.0 degrees C min(-1) are achieved with minimized overshoots (

Geographic and within-population variation in the globeflower-globeflower fly interaction: the costs and benefits of rearing pollinators' larvae.

Interspecific interactions can vary within and among populations and geographic locations, and this variation can influence the nature of the interaction (e.g. mutualistic vs. antagonistic) and its evolutionary stability. Globeflowers are exclusively pollinated by flies, whose larvae feed only on their seeds. Here we document geographic variability in costs and benefits in globeflowers in sustaining their pollinating flies throughout the range of this arctic-alpine European plant over several years. A total of 1,710 flower heads from 38 populations were analysed for their carpel, egg and seed contents. Individual and population analyses control for the confounding influences of variation in both: (1) population traits, such as fly density and egg distribution among flower heads; and (2) individuals traits, such as carpel and egg numbers per flower head. Despite considerable variation in ecological conditions and pollinator densities across populations, large proportions (range 33-58%) of seeds were released after predation, with a benefit-to-cost ratio of 3, indicating that the mutualism is stable over the whole globeflower geographical range. The stability of the mutualistic interaction relies on density-dependent competition among larvae co-developing in a flower head. This competition is revealed by a sharp decrease in the number of seeds eaten per larva with increasing larval number, and is intensified by non-uniform egg distribution among globeflowers within a population. Carpel number is highly variable across globeflowers (range 10-69), and flies lay more eggs in large flowers. Most plants within a population contribute to the rearing of pollinators, but the costs are greater for some than for others. Large globeflowers lose more seed to pollinator larvae, but also release more seed than smaller plants. The apparent alignment of interests between fly and plants (positive relationship between numbers of seed released and destroyed) is shown to hide a conflict of interest found when flower size is controlled for.

[Pharmaphylogenetic study on isopyroideae (Ranunculaceae)].

OBJECTIVE: To study the pharmaphylogenetic of medicinal plants of Isopyroideae (Ranunculaceae). METHOD: Comprehensively analyze the correlation between phylogeny, chemical constituents and pharmaceutical aspects of Isopyroideae plants, based on chemical, pharmaceutical (both ethnopharmacologic and pharmacological) information, linking with different plant systems of Ranunculaceae. RESULT: Plants from Aquilegia mainly contain flavonoids constituents while the major chemical constituents of Isopyrum are bisbenzylisoquinoline alkaloids. Chemical characteristics also support that this taxon should be separated from Thalictrodeae, and constituted an independent subfamily, namely, Isopyroideae.

Recruitment of CRABS CLAW to promote nectary development within the eudicot clade.

Nectaries are secretory organs that are widely present in flowering plants that function to attract floral pollinators. Owing to diversity in nectary positions and structures, they are thought to have originated multiple times during angiosperm evolution, with their potential contribution to the diversification of flowering plants and pollinating animals being considerable. We investigated the genetic basis of diverse nectary forms in eudicot angiosperm species using CRABS CLAW (CRC), a gene required for nectaries in Arabidopsis. CRC expression is conserved in morphologically different nectaries from several core eudicot species and is required for nectary development in both rosids and asterids, two major phylogenetic lineages of eudicots. However, in a basal eudicot species, no evidence of CRC expression in nectaries was found. Considering the phylogenetic distribution of nectary positions and CRC expression analyses in eudicots, we propose that diverse nectaries in core eudicots share conserved CRC gene regulation, and that derived nectary positions in eudicots have altered regulation of CRC. As the ancestral function of CRC lies in the regulation of carpel development, it may have been co-opted as a regulator of nectary development within the eudicots, concomitant with the association of nectaries with reproductive organs in derived lineages.

[Microscopy identification of Caltha palustris L. var. membranacea Turcz].

OBJECTIVE: To identify Caltha palustris L. var. membranacea Turcz. METHODS: Morphological and histological identification. RESULTS: There are crystals in root and mesophyll from Caltha palustris L. var. membrancea Turcz.. Anomocytic type stomata are visible. CONCLUSION: Above mentioned characters can be used for the identification of Caltha palustris L. var. membranacea Turcz.

Sex and pollen: the role of males in stabilising a plant-seed eater pollinating mutualism.

Some plants are exclusively pollinated by an insect whose larvae feed on their seeds. The net outcome of a single visit for the plant depends on the number of ovules fertilised by the visitor, the number of eggs laid, and the number of seeds eaten by each larva. Unlike other known plant-seed eater pollinating mutualisms, the globeflower-globeflower fly mutualism (Trollius europaeus-Chiastocheta spp.) is unique in that not only females but also males visit flowers, and both sexes are potential pollinators. I analysed the relative efficiency of Chiastocheta males versus females in transporting pollen and fertilising globeflower ovules. I show that there is no sex-specific morphological adaptation or behaviour to enhance pollen collection and transportation in Chiastocheta flies, and that males contribute to pollination. However, because of their smaller body size, males transport significantly less pollen than females. Less seeds are produced after a visit from a male than after a visit from a female. A single female visit contributes to about 12% of total seed production, and a single male visit to only 5.4%. Females tend to spend more time inside the flower than males, and the number of ovules fertilised is significantly correlated with the time insects spent inside the closed corolla. The lower efficiency of ovule fertilisation by a male's single visit is compensated for by the higher rate of flower visitation by males: a flower receives about twice as many visits from males as from females during a time unit. The contribution of males to pollination is of major importance with respect to understanding the evolutionary stability of the globeflower-globeflower fly mutualism, as males satiate pollen requirement of flowers, masking the antagonistic effect of ovipositing females.