Rhizome architecture, development and vascularization in the water lily Nymphaea alba.

BACKGROUND AND AIMS: Water lilies are of particular interest with regard to the evolution of angiosperms. They live in an aquatic environment and have been regarded as links to the monocots by some authors. Vascular bundles are sometimes described as scattered or atactostelar as in monocots. However, this view needs to be clarified as the morphology and vascularization of Nymphaea rhizomes remain to be understood. METHODS: The rhizome of Nymphaea alba was re-investigated morphologically and histologically. Developmental studies were conducted using scanning electron microscopy. Comprehensive histological analyses, including hand and microtome sections and a variety of specific staining procedures, were conducted to re-evaluate the composition of longitudinal and transverse tissue. KEY RESULTS: The rhizome is covered by parenchymatous nodal cushions each bearing a leaf and several adventitious roots. Internodes are extremely short. The apex is flat and early overtopped by developing leaf primordia and cushions. The phyllotaxis is spiral and passes alternately through vegetative and reproductive phases. Flowers appear in the leaf spiral, and lack a subtending bract and a cushion below the peduncle. The reproductive phase includes two or three flowers which alternate with a single leaf. The rhizome is histologically subdivided into a central core, an aerenchymatic cortex, and a parenchymatic exocortex formed to a great extent by the nodal cushions. The core contains strands of vascular bundles united to a complex vascular plexus. Vascular elements continuously anastomose and change shape and direction. Provascular strands originating from leaf primordia merge with the outer core vascular tissue whereas the flower strands run into the centre of the core. Roots originating from the parenchymatous cushions show the characteristic actinostelic pattern, which changes into a collateral pattern inside the rhizome. Several root traces merge and form one strand leading to the central core. Early cell divisions below the apical meristem dislocate leaf, flower and root primordia and their provascular strands outwards. Consequently, fully developed vascular strands insert horizontally into the vascular plexus at advanced rhizome stages. CONCLUSIONS: The absence of bracts and cushions below the flowers, the alternate leaf-flower sequence and the course of the peduncle strand suggest that the rhizome is sympodially instead of monopodially organized. The spiral phyllotaxis extends in this case over several shoot orders, masking the branching pattern. The vascular strands in the central plexus differ considerably from vascular bundles in monocots, confirming the unique vascularization in Nymphaea. Sclerenchymatic bundle sheaths are lacking, and vascular bundles continuously split and anastomose throughout the rhizome. Though vascular bundles in petioles and peduncles of N. alba show similarities with some Alismatales, the vascular system of N. alba in general has little in common with that of monocots.

Morphophysiological dormancy in the basal angiosperm order Nymphaeales.

Background and Aims: Substantial evidence supports the hypothesis that morphophysiological dormancy (MPD) is the basal kind of seed dormancy in the angiosperms. However, only physiological dormancy (PD) is reported in seeds of the ANA-grade genus Nymphaea. The primary aim of this study was to determine the kind of dormancy in seeds of six species of Nymphaea from the wet-dry tropics of Australia. Methods: The effects of temperature, light and germination stimulants on germination were tested on multiple collections of seeds of N. immutabilis, N. lukei, N. macrosperma, N. ondinea, N. pubescens and N. violacea. Embryo growth prior to hypocotyl emergence was monitored. Key Results: Germination was generally <10 % after 28 d in control treatments. Germination percentage was highest at 30 or 35 °C for seeds exposed to light and treated with ethylene or in anoxic conditions in sealed vials of water, and it differed significantly between collections of N. lukei, N. macrosperma and N. violacea. Seeds of N. pubescens did not germinate under any of the conditions. Embryo growth (8-37 % in length) occurred before hypocotyl emergence (germination) in seeds of the five species that germinated. Conclusions: Fresh seeds were dormant, and the amount of pregermination embryo growth in seeds of N. lukei and N. immutabilis was relatively small, while in seeds of N. macrosperma, N. ondinea and N. violacea it was relatively large. Thus, seeds of N. lukei and N. immutabilis had PD and those of N. macrosperma, N. ondinea and N. violacea had MPD. Overall, we found that seeds in the most phylogenetically derived clades within Nymphaea have MPD, suggesting that PD is the most likely basal trait within the Nymphaeales. This study also highlights the broad range of dormancy types and germination strategies in the ANA-grade angiosperms.

Abrupt out-of-plane edge folding of a circular thin plate: Implication for a mature Victoria regia leaf.

Inspired by the observation of the configurations of Victoria regia leaves, we establish a phenomenological buckling model for the abrupt out-of-plane edge folding of a circular thin sheet. A reduced model is first developed, and further refined by a more sophisticated growth strain field so that the resulting buckling morphology resembles that of a mature Victoria regia leaf. Parametric studies are carried out to investigate the effects of geometric, material, and strain field parameters on the buckling morphology. Several main characteristics discovered through numerical studies are verified by theoretical analysis of a simple geometry-based model. Besides, the roles of the thickness variation and cracks are examined. This work may not only shed some light on the morphogenesis of certain plants, but also provide some useful insights on three-dimensional fabrications using mechanical self-assembly.

Pollen structure and development in Nymphaeales: insights into character evolution in an ancient angiosperm lineage.

PREMISE OF THE STUDY: A knowledge of pollen characters in early-diverging angiosperm lineages is essential for understanding pollen evolution and the role of pollen in angiosperm diversification. In this paper, we report and synthesize data on mature pollen and pollen ontogeny from all genera of Nymphaeales within a comparative, phylogenetic context and consider pollen evolution in this early-diverging angiosperm lineage. We describe mature pollen characters for Euryale, Barclaya, and Nymphaea ondinea, taxa for which little to no structural data exist. METHODS: We studied mature pollen for all nymphaealean genera using light, scanning electron, and transmission electron microscopy. We reviewed published reports of nymphaealean pollen to provide a comprehensive discussion of pollen characters in water lilies. KEY RESULTS: Nymphaeales exhibit diversity in key pollen characters, including dispersal unit size, ornamentation, aperture morphology, and tapetum type. All Nymphaeales pollen are tectate-columellate, exhibiting one of two distinct patterns of infratectal ultrastructure-a thick infratectal space with robust columellae or a thin infratectal space with thin columellae. All genera have pollen with a lamellate endexine that becomes compressed in the proximal, but not distal wall. This endexine ultrastructure supports the operculate hypothesis for aperture origin. Nymphaeaceae pollen exhibit a membranous granular layer, which is a synapomorphy of the family. CONCLUSIONS: Variation in pollen characters indicates that significant potential for lability in pollen development was present in Nymphaeales at the time of its divergence from the rest of angiosperms. Structural and ontogenetic data are essential for interpreting pollen characters, such as infratectum and endexine ultrastructure in Nymphaeales.

Interrelationships of petiolar air canal architecture, water depth, and convective air flow in Nymphaea odorata (Nymphaeaceae).

PREMISE OF THE STUDY: Nymphaea odorata grows in water up to 2 m deep, producing fewer larger leaves in deeper water. This species has a convective flow system that moves gases from younger leaves through submerged parts to older leaves, aerating submerged parts. Petiolar air canals are the convective flow pathways. This study describes the structure of these canals, how this structure varies with water depth, and models how convective flow varies with depth. • METHODS: Nymphaea odorata plants were grown at water depths from 30 to 90 cm. Lamina area, petiolar cross-sectional area, and number and area of air canals were measured. Field-collected leaves and leaves from juvenile plants were analyzed similarly. Using these data and data from the literature, we modeled how convective flow changes with water depth. • KEY RESULTS: Petioles of N. odorata produce two central pairs of air canals; additional pairs are added peripherally, and succeeding pairs are smaller. The first three pairs account for 96% of air canal area. Air canals form 24% of petiolar cross-sectional area. Petiolar and air canal cross-sectional areas increase with water depth. Petiolar area scales with lamina area, but the slope of this relationship is lower in 90 cm water than at shallower depths. In our model, the rate of convective flow varied with depth and with the balance of influx to efflux leaves. • CONCLUSIONS: Air canals in N. odorata petioles increase in size and number in deeper water but at a decreasing amount in relation to lamina area. Convective flow also depends on the number of influx to efflux laminae.

A theoretical morphological model for quantitative description of the three-dimensional floral morphology in water lily (Nymphaea).

Water lilies (Nymphaea spp.) have diverse floral morphologies. Water lilies are not only commonly used as ornamental plants, but they are also important for understanding the diversification of basal angiosperms. Although the diversity in floral morphology of water lily provides useful information for evolutionary biology, horticulture, and horticultural science, it is difficult to describe and analyze the three-dimensional morphology of flowers. In this study, we propose a method to describe the floral morphology of water lily using a three-dimensional theoretical morphological model. The theoretical model was constructed based on three components, i.e., (1) the gradual change in size of floral organs, (2) spiral phyllotaxis, and (3) the interpolation of elevation angles, which were integrated into the model. We generated three-dimensional representation of water lily flowers and visualized theoretical morphospaces by varying each morphological parameter. The theoretical morphospace is a mathematical space of morphological spectrum generated by a theoretical morphological model. These morphospaces seems to display the large part of morphological variations of water lily. We measured morphological parameters of real flowers based on our theoretical model and display the occupation pattern of morphological parameters. We also surveyed the relation between morphological parameters and flower shape descriptions found in a catalog. In some parameters, we found breeders' description can link to our morphological model. In addition, the relationship between the global features of floral morphology and the parameters of the theoretical model was calculated with flower silhouettes simulated with a range of parameter values and the global features of the silhouette. We used two simple indices to assess the global morphological features, which were calculated with the convex hull. The results indicated that our method can effectively provide an objective and quantitative overview of the diversity in the floral morphology of water lily.

Environmental control of sepalness and petalness in perianth organs of waterlilies: a new Mosaic theory for the evolutionary origin of a differentiated perianth.

The conventional concept of an 'undifferentiated perianth', implying that all perianth organs of a flower are alike, obscures the fact that individual perianth organs are sometimes differentiated into sepaloid and petaloid regions, as in the early-divergent angiosperms Nuphar, Nymphaea, and Schisandra. In the waterlilies Nuphar and Nymphaea, sepaloid regions closely coincide with regions of the perianth that were exposed when the flower was in bud, whereas petaloid regions occur in covered regions, suggesting that their development is at least partly controlled by the environment of the developing tepal. Green and colourful areas differ from each other in trichome density and presence of papillae, features that often distinguish sepals and petals. Field experiments to test whether artificial exposure can induce sepalness in the inner tepals showed that development of sepaloid patches is initiated by exposure, at least in the waterlily species examined. Although light is an important environmental cue, other important factors include an absence of surface contact. Our interpretation contradicts the unspoken rule that 'sepal' and 'petal' must refer to whole organs. We propose a novel theory (the Mosaic theory), in which the distinction between sepalness and petalness evolved early in angiosperm history, but these features were not fixed to particular organs and were primarily environmentally controlled. At a later stage in angiosperm evolution, sepaloid and petaloid characteristics became fixed to whole organs in specific whorls, thus reducing or removing the need for environmental control in favour of fixed developmental control.

Comparative ovule and megagametophyte development in Hydatellaceae and water lilies reveal a mosaic of features among the earliest angiosperms.

BACKGROUND AND AIMS: The embryo sac, nucellus and integuments of the early-divergent angiosperms Hydatellaceae and other Nymphaeales are compared with those of other seed plants, in order to evaluate the evolutionary origin of these characters in the angiosperms. METHODS: Using light microscopy, ovule and embryo sac development are described in five (of 12) species of Trithuria, the sole genus of Hydatellaceae, and compared with those of Cabombaceae and Nymphaeaceae. KEY RESULTS: The ovule of Trithuria is bitegmic and tenuinucellate, rather than bitegmic and crassinucellate as in most other Nymphaeales. The seed is operculate and possesses a perisperm that develops precociously, which are both key features of Nymphaeales. However, in the Indian species T. konkanensis, perisperm is relatively poorly developed by the time of fertilization. Perisperm cells in Trithuria become multinucleate during development, a feature observed also in other Nymphaeales. The outer integument is semi-annular ('hood-shaped'), as in Cabombaceae and some Nymphaeaceae, in contrast to the annular ('cap-shaped') outer integument of some other Nymphaeaceae (e.g. Barclaya) and Amborella. The megagametophyte in Trithuria is monosporic and four-nucleate; at the two-nucleate stage both nuclei occur in the micropylar domain. Double megagametophytes were frequently observed, probably developed from different megaspores of the same tetrad. Indirect, but strong evidence is presented for apomictic embryo development in T. filamentosa. CONCLUSIONS: Most features of the ovule and embryo sac of Trithuria are consistent with a close relationship with other Nymphaeales, especially Cabombaceae. The frequent occurrence of double megagametophytes in the same ovule indicates a high degree of developmental flexibility, and could provide a clue to the evolutionary origin of the Polygonum-type of angiosperm embryo sac.

Disentangling historical signal and pollinator selection on the micromorphology of flowers: an example from the floral epidermis of the Nymphaeaceae.

The family Nymphaeaceae includes most of the diversity among the ANA-grade angiosperms. Among the species of this family, floral structures and pollination strategies vary. The genus Victoria, as well as subgenera Lotos and Hydrocallis in Nymphaea, present night-blooming, scented flowers pollinated by scarab beetles. Such similar pollination strategies have led to macromorphological similarities among the flowers of these species, which could be interpreted as homologies or convergences based on different phylogenetic hypotheses about the relationships of these groups. We employed scanning electron microscopy of floral epidermis for seven species of the Nymphaeaceae with contrasting pollination biology to identify the main characters of the floral organs and the potential homologous nature of the structures involved in pollinator attraction. Moreover, we used transmission electron microscopy to observe ultrastructure of papillate-conical epidermis in the stamen of Victoria cruziana. We then tested the phylogenetic or ecological distribution of these traits using both consensus network approaches and ancestral state reconstruction on fixed phylogenies. Our results show that the night-blooming flowers present different specialisations in their epidermis, with V. cruziana presenting the most elaborate floral anatomy. We also identify for the first time the presence of conical-papillate cells in the order Nymphaeales. The epidermal characters tend to reflect phylogenetic relationships more than convergence due to pollinator selection. These results point to an independent and parallel evolution of scarab pollination in Nymphaeaceae and demonstrate the promise of floral anatomy as a phylogenetic marker. Moreover, they indicate a degree of sophistication in the anatomical basis of cantharophilous flowers in the Nymphaeales that diverges from the most simplistic views of floral evolution in the angiosperms.

Permanently open stomata of aquatic angiosperms display modified cellulose crystallinity patterns.

Most floating aquatic plants have stomata on their upper leaf surfaces, and usually their stomata are permanently open. We previously identified 3 distinct crystallinity patterns in stomatal cell walls, with angiosperm kidney-shaped stomata having the highest crystallinity in the polar end walls as well as the adjacent polar regions of the guard cells. A numerical bio-mechanical model suggested that the high crystallinity areas are localized to regions where the highest stress is imposed. Here, stomatal cell wall crystallinity was examined in 4 floating plants from 2 different taxa: basal angiosperms from the ANITA grade and monocots. It appears that the non-functional stomata of floating plants display reduced crystallinity in the polar regions as compared with high crystallinity of the ventral (inner) walls. Thus their guard cells are both less flexible and less stress resistant. Our findings suggest that the pattern of cellulose crystallinity in stomata of floating plants from different families was altered as a consequence of similar evolutionary pressures.

Compared leaf anatomy of Nymphaea (Nymphaeaceae) species from Brazilian flood plain.

Nymphaea has seven species already catalogued in the flood prone areas of the Brazilian Pantanal. However, some species remain difficult to identify and descriptions of the anatomy of vegetative organs are an important tool for infrageneric separation to aid in group taxonomy. The species collected in the Pantanal and prepared according to the usual techniques for anatomical studies showed similar structural characteristics, and data on the arrangement of vascular bundles in the midrib and petiole, as well as the form and distribution of sclereids, were consistent. Nymphaea oxypetala stands out from the other evaluated species for having a greater number of differential characters, including angular collenchyma and the absence of bicollateral bundles in the petiole. Nymphaea lingulata stands out as the only species to feature bicollateral bundles in the leaf blade. The results, summarised in the dichotomous key, facilitate the identification of species that use the flower as the main differentiation, but are in a vegetative stage.

Seasonal variations and aeration effects on water quality improvements and physiological responses of Nymphaea tetragona Georgi.

Seasonal variations and aeration effects on water quality improvements and the physiological responses of Nymphaea tetragona Georgi were investigated with mesocosm experiments. Plants were hydroponically cultivated in six purifying tanks (aerated, non-aerated) and the characteristics of the plants were measured. Water quality improvements in purifying tanks were evaluated by comparing to the control tanks. The results showed that continuous aeration affected the plant morphology and physiology. The lengths of the roots, petioles and leaf limbs in aeration conditions were shorter than in non-aeration conditions. Chlorophyll and soluble protein contents of the leaf limbs in aerated tanks decreased, while peroxidase and catalase activities of roots tissues increased. In spring and summer, effects of aeration on the plants were less than in autumn. Total nitrogen (TN) and ammonia nitrogen (NH4(+)-N) in aerated tanks were lower than in non-aerated tanks, while total phosphorus (TP) and dissolved phosphorus (DP) increased in spring and summer. In autumn, effects of aeration on the plants became more significant. TN, NH4(+)-N, TP and DP became higher in aerated tanks than in non-aerated tanks in autumn. This work provided evidences for regulating aeration techniques based on seasonal variations of the plant physiology in restoring polluted stagnant water.

The AP2-like gene NsAP2 from water lily is involved in floral organogenesis and plant height.

APETALA2 (AP2) genes are ancient and widely distributed among the seed plants, and play an important role during the plant life cycle, acting as key regulators of many developmental processes. In this study, an AP2 homologue, NsAP2, was characterized from water lily (Nymphaea sp. cv. 'Yellow Prince') and is believed to be rather primitive in the evolution of the angiosperms. In situ RNA hybridization showed that NsAP2 transcript was present in all regions of the floral primordium, but had the highest level in the emerging floral organ primordium. After the differentiation of floral organs, NsAP2 was strongly expressed in sepals and petals, while low levels were found in stamens and carpels. The NsAP2 protein was suggested to be localized in the cell nucleus by onion transient expression experiment. Overexpression of NsAP2 in Arabidopsis led to more petal numbers, and Arabidopsis plants expressing NsAP2 exhibited higher plant height, which may be a result of down-regulated expression of GA2ox2 and GA2ox7. Our results indicated that the NsAP2 protein may function in flower organogenesis in water lily, and it is a promising gene for plant height improvement.

Compared leaf anatomy of Nymphaea (Nymphaeaceae) species from Brazilian flood plain / Anatomia comparativa da folha de espécies de Nymphaea (Nymphaeaceae) da planície de inundação brasileira

Nymphaea has seven species already catalogued in the flood prone areas of the Brazilian Pantanal. However, some species remain difficult to identify and descriptions of the anatomy of vegetative organs are an important tool for infrageneric separation to aid in group taxonomy. The species collected in the Pantanal and prepared according to the usual techniques for anatomical studies showed similar structural characteristics, and data on the arrangement of vascular bundles in the midrib and petiole, as well as the form and distribution of sclereids, were consistent. Nymphaea oxypetala stands out from the other evaluated species for having a greater number of differential characters, including angular collenchyma and the absence of bicollateral bundles in the petiole. Nymphaea lingulata stands out as the only species to feature bicollateral bundles in the leaf blade. The results, summarised in the dichotomous key, facilitate the identification of species that use the flower as the main differentiation, but are in a vegetative stage.

Nymphaea tem sete espécies catalogadas nas áreas inundáveis do Pantanal brasileiro. No entanto, algumas espécies são de difícil identificação e descrições da anatomia dos órgãos vegetativos são uma ferramenta importante para a separação infragenérica para auxiliar na taxonomia do grupo. As espécies coletadas no Pantanal e preparadas de acordo com as técnicas usuais para estudos anatômicos mostraram as mesmas características estruturais, e os dados de arranjo dos feixes vasculares na nervura central e pecíolo, bem como a forma e distribuição de esclereides, foram consistentes. Nymphaea oxypetala se destaca das outras espécies avaliadas por ter um maior número de caracteres diferenciais, incluindo colênquima angular e ausência de feixes bicolaterais no pecíolo. Nymphaea lingulata se destaca como a única espécie que apresenta feixes bicolaterais no limbo. Os resultados, resumidos em uma chave dicotômica, facilitam a identificação de espécies que utilizam a flor como principal diferenciação quando se encontram em estágio vegetativo.

Developmental morphology of branching flowers in Nymphaea prolifera.

Nymphaea and Nuphar (Nymphaeaceae) share an extra-axillary mode of floral inception in the shoot apical meristem (SAM). Some leaf sites along the ontogenetic spiral are occupied by floral primordia lacking a subtending bract. This pattern of flower initiation in leaf sites is repeated inside branching flowers of Nymphaea prolifera (Central and South America). Instead of fertile flowers this species usually produces sterile tuberiferous flowers that act as vegetative propagules. N. prolifera changes the meristem identity from reproductive to vegetative or vice versa repeatedly. Each branching flower first produces some perianth-like leaves, then it switches back to the vegetative meristem identity of the SAM with the formation of foliage leaves and another set of branching flowers. This process is repeated up to three times giving rise to more than 100 vegetative propagules. The developmental morphology of the branching flowers of N. prolifera is described using both microtome sections and scanning electron microscopy.