Development and multiplexing of microsatellite markers in the polyploid perennial herb, Menyanthes trifoliata (Menyanthaceae)

• Premise of the study: We developed microsatellite primers to investigate genetic diversity and population genetic structure of the endangered herb Menyanthes trifoliata. • Methods and Results: Using the microsatellite-enriched library method, we identified 10 primer pairs in M. trifoliata. The primers amplified nine di- and one tri-nucleotide repeats with 4–13 alleles per locus in two Belgian populations. • Conclusions: The results indicate that these markers offer an appropriate amount of variation to investigate genetic diversity, pollen dispersal (through paternity inference), and other conservation issues.     

Repellence of nectar-thieving ants by a physical barrier: Adaptive role of petal hairs on Menyanthes trifoliata (Menyanthaceae)

Some plant species possess petal hairs, the defensive functions of which remain untested. I hypothesized that the petal hairs of an emergent plant species, Menyanthes trifoliata (Menyanthaceae), have a function in deterring nectar-thieving ants (Lasius japonicus). Lasius japonicus individuals were observed to stop moving forward after touching hairs and had difficulty in maintaining their balance when walking on hairs. In order to confirm the effectiveness of the hairs quantitatively, I trimmed hairs with a nose hair trimmer and then observed the behavior of flower-visiting L. japonicus. The success rate of L. japonicus in entering the floral tubes was significantly lower in the presence of intact hairs than when there were trimmed hairs. Moreover, it took L. japonicus twice as long to enter floral tubes with intact hairs, even when they succeeded. These results suggest that hairs on M. trifoliata petals are effective as a physical barrier against L. japonicus by restricting the movements of the ants.     

Crane flies and microlepidoptera also function as pollinators in Epidendrum (Orchidaceae: Laeliinae): the reproductive biology of E. avicula

Crane flies and microlepidoptera have been recorded as pollinators in unrelated orchid groups, but these insects have never been recorded in Epidendroideae, the most species‐rich orchid subfamily, which includes one of the most diverse genera among Orchidaceae, Epidendrum. Based on data on phenology, floral morpho‐anatomy, pollinators, pollination mechanisms and breeding system, the reproductive biology of E. avicula was studied in south‐eastern Brazil. Epidendrum avicula possess osmophores that produce a citric fragrance at night. The flowers attract Tipulidae flies and several families of microlepidoptera that drink the nectar produced in a tube formed by the adnation of the labellum and column. As is common in Epidendrum, after removing the pollinarium, both crane flies and micro‐moths get trapped by the proboscis, which frightens the insects and inhibits any possible intent to immediately visit another flower. The behavior of the pollinators on flowers, plus the retention of the anther cap by the pollinarium, results in a reduction in the occurrence of geitonogamy. Because E. avicula is self‐incompatible, the consequence of pollinator behavior and the floral mechanisms tend to reduce the pollen loss. As far as we know, this is the first study to report the reproductive biology of a species of Epidendroideae pollinated by crane flies and microlepidoptera. Based on more recent concepts of plant–pollinator interactions, although E. avicula is pollinated by several species belonging to two distinct orders, suggesting an unspecialized pollination system is involved, nectar‐seeking microlepidoptera and Tipulidae flies can be recognized as a single functional group.     

Population Structure and Reproductive Biology of Saururus cernuus L. (Saururaceae)

Abstract The population structure and reproductive biology of Saururus cernuus (Lizard's Tail; Saururaceae; Piperales), is documented in five sites in southern Louisiana (Mississippi Delta). The species is common throughout the southeastern United States in marshes, along streams, edges of lakes, and in the understory of moist forests. The clonal species exhibits sexual and vegetative reproduction. Wind and insects both play important roles in pollination. Pollen may be borne by insects directly. Alternatively, the pollen may be borne by wind after its release is triggered from pendulous floral spikes (the “Lizard's Tail”) by either wind or insect landings (insect-mediated wind pollination). The plants are self-incompatible and seed set results from a combination of pollination modes with wind pollination being the primary mode (rare in the Magnoliidae). Inflorescence and floral structure exhibit adaptive features that facilitate the various modes of pollen transfer, viz., numerous, small scented, protogynous flowers with no perianth, ultraviolet patterns produced by stamen filaments, small pollen grains, curved inflorescences, and exerted stamens, etc. Fruit production and seed germination are documented and plant growth and densities are compared in sunny versus forest sites.     

Reproductive biology of Nymphaea capensis Thunb. var. zanxibariensis (Casp.) Verdc. (Nymphaeaceae)

Anthesis in Nymphaea capensis var. zanzibariensis is diurnal with flowers opening and closing for three consecutive days. On the first day of anthesis, the stigmatic papillae secrete fluid and the outermost anthers are dehiscent. On the second day of anthesis the stamens form a cone above the dry stigmatic cup. The middle stamens open and turn outward. On the third day of flowering, all the stamens open and the dry stigmatic cup is exposed. The flowers are homogamous and not protogynous as the other Nymphaea. The gynoecium of the self-compatible N. capensis var. zanzibariensis , is characterized by a wet papillate stigma, a short hollow style, and secretory cells on the ventral surface of the ovary. The pollen is released on the receptive stigma. Following initial growth in intercellular spaces in the transmitting tract of the stigma, pollen tubes travel through the stylar canal and into the ovary.     

Heterostyly in Erythroxylum coca (Erythroxylaceae)

Erythroxylum coca is a distylous species with a strong self-incompatibility system linked with the floral dimorphism. The two sets of stamens in the flowers are usually unequal in length, but between individuals of both morphs there is considerable variation in the relative qengths of the two sets of stamens, which is unrelated to the incompatibility system. Pin flowers produce more pollen grains than thrum flowers, but thrum pollen is larger than pin pollen. Within each morph the two sets of stamens produce pollen grains of slightly different diameter. Erythroxylum novogranatewe is also distylous. Pin flowers of E. novogranatense var. novogranatense are partially self-compatible, while thrum flowers of E. novogranatense var. truxillense are self-incompatible. Reports of tristyly and of four different morphs in species of Erythroxylum are probably misinterpretations, resulting from limited sampling, of the continuous variation in the relative lengths of the two sets of stamens.     

A cladistic analysis of the genus Lopezia (Onagraceae)

Lopezia, a genus of 22 species largely restricted to Mexico, forms a monophyletic group defined by the possession of two stamens in tetramerous flowerS. A cladistic analysis of the genus was performed using 16 characters from morphology, anatomy and embryology. Twenty of the 22 species were considered the terminal taxa. Polarity of the characters was based on the outgroup comparison method, using three alternative outgroups: the genus Fuchsia, Onagraceae excluding Ludwigia, and Epilobieae-Onagreae. Using Fuchsia as outgroup, 29 equally parsimonious cladograms were produced, each with 24 steps and a consistency index of 0.75. A successive weighting procedure was applied, resulting in 15 cladograms with consistency index of 0.85. The strict consensus cladogram defines eight monophyletic groups and supports most of the current sectional classification of Lopezia, with the exception that no synapomorphy defines section Jehlia. The larger sections Lopezia and Pelozia, although well-defined as clades, are not fully resolved internally. All cladograms support section Riesenbachia as monophyletic. The two alternative hypotheses for outgroups produced similar results: 61 most parsimonious trees, reduced after the successive weighting procedure to 15, which are identical to those produced with Fuchsia as outgroup. These results are discussed in the context of data on cytology and pollination biology.     

Floral biology and histochemical analysis of Vanilla edwallii Hoehne (Orchidaceae: Vanilloideae): an orchid pollinated by Epicharis (Apidae: Centridini)

The genus Vanilla is the most diverse in Vanilloideae, with ca 90 species distributed among tropical regions. Despite their economic importance, studies on pollination of Vanilla are very scarce and data on pollinators of species endemic to Brazil are lacking. Based on fieldwork and laboratory investigations, the floral biology of V. edwallii was studied. The pollinators and pollination process were recorded at the Serra do Japi reserve, state of São Paulo, southeastern Brazil, and the presence of floral reward was also investigated. Vanilla edwallii blooms in summer. The lateral inflorescences produce up to four pale green flowers. The white labellum is united to the base of the column forming a mentum. In the studied population V. edwallii is pollinated by Epicharis (Hoplepicharis) affinis, where the males exhibit a territorial behavior, defending flowers from other possible flower visitors. The pollen is deposited on the scutellum of bees when they abandon the flower. The mentum region is dry, suggesting no nectar production. The only secretory structures are osmophores dispersed on the inner surface of the lip responsible for production of a sweet fragrance, which together with color and morphology of flowers is related to bee attraction. The labellum is rich in mucilaginous cells, while the mucilaginous substance is retained inside the cells. The histochemical analysis also detected the presence of phenolic compounds and starch concentrated mainly at the adaxial surface of the lip and around the vascular bundles.     

Breeding system and thrips (Thysanoptera) pollination in the endangered tree Ocotea porosa (Lauraceae): implications for conservation

Ocotea porosa has been extensively exploited over the past few decades because of the quality of its wood. Today, populations are reduced and the species is now included in the Red List of threatened species by the International Union for Nature Conservation. For conservation and management purposes, it is extremely important that we understand its reproductive ecology. Floral morphology was described based on field and scanning electron microscopic examination. The reproductive system was determined through experimentally controlled pollination along with observations of pollen tube growth. Pollinators were identified through field observations. Flowers of O. porosa are small, shallow, inconspicuous, asynchronous and grouped in inflorescences. This species presented self‐compatibility, but did not reproduce through apomixy and spontaneous self‐pollination was very rare (5%). Despite being monoclinal, flowers were protogynic, and the gynoecium was receptive after the first day of anthesis when the anthers were closed. Spontaneous self‐pollination was avoided by the extrorse position of the anthers of the internal stamens. Frankliniella gardeniae (Thysanoptera) was the only pollinating species observed and, after visiting several asynchronous flowers in the same inflorescence, favors geitonogamy. Together the small distance supposedly achieved by thrips in flight and the small population density of O. porosa can reduce the chances of cross‐pollination in this species. If so, conservation measures must include preservation of the current population and possibly planting to increase population density. This would reduce the distance between individuals and increase genetic variability. Thrips as pollinators must be included in conservation planning for O. porosa.     

Occurrence of Crassulacean acid metabolism in Cissus trifoliata L. (Vitaceae)

Summary Evidence for the operation of CAM in the deciduous climber, Cissus trifoliata L., was obtained in field and laboratory studies. Under natural conditions, diurnal oscillations of titratable acidity and colorimetric measurements of night CO₂ fixation, determined for a period of two and a half years, suggested that acid accumulation was related to plant water status, assessed through the daily courses of stomatal resistance and xylem water potential during dry and rainy seasons. These findings were confirmed by gas exchange studies under controlled conditions which showed that the plant fixed all its CO₂ during the day when it was well irrigated; as water stress increased, dark CO₂ uptake gradually replaced fixation during the day until the plant only performed dark fixation. In severe water stress, even the rate of the latter process decreased until leaves fell.Abbreviations CAM Crassulacean acid metabolism - FW leaf fresh weight - SWC relative soil water content - PAR photosynthetically active radiation - TR total radiation; r, leaf diffusive resistance - WSD water saturation deficit (leaf-air vapour concentration difference) - RWC relative water content of leaves     

Pollination biology and outcrossing rates in hermaphroditic Schiedea lydgatei (Caryophyllaceae)

The influence of outcrossing and pollination biology on the maintenance of hermaphroditism was studied for Schiedea lydgatei (Caryophyllaceae: Alsinoideae), a species endemic to Moloka`i in the Hawaiian Islands. Schiedea lydgatei is the only hermaphroditic species in an otherwise dimorphic clade and hermaphroditism is likely the result of a reversal from a gynodioecious ancestor. Both wind and native moths in the family Pyralidae are responsible for pollination in S. lydgatei. Outcrossing rates were generally high (0.80), especially in years when the greatest number of plants were flowering. The combination of high outcrossing rates and substantial inbreeding depression indicates that at present females would not be favored in the population. Pollination by both wind and insects is consistent with the hypothesis that hermaphroditism is the result of a relatively recent reversal, as the ancestor of S. lydgatei was probably wind pollinated and gynodioecious with few females in the populations. A shift from wind to predominately insect pollination on Moloka`i may have resulted in increased outcrossing rates and prevented the expression of high inbreeding depression among progeny of hermaphrodites, a condition that would select against females and favor a reversal to hermaphroditism. Because few females were likely to have been present in ancestral populations that colonized Moloka`i, founder effect is another potential explanation for loss of females. In either case, current high levels of outcrossing prevent re-establishment of females in populations of S. lydgatei.     

Observations on Gas Movement in the Rhizome of Menyanthes trifoliata L., with Comments on the Role of the Endodermis

Patterns of diffusion of oxygen in the rhizome of the aquaticMenyanthes have been investigated after the interruption ofeither stelar or cortical diffusion path-ways. Forty-eight hoursafter ringing, oxygen levels in cortex, stele, and root weresignificantly depressed, although never to danger point. Interruptionof the stele, involving also partial interruption of the cortex,was only significantly effective when made just above the samplingzone. The addition of M/1000 KCN to depress the metabolic uptakeof oxygen, resulted as expected in higher equilibrium levelsof oxygen in all zones. It was shown (a) that the velocity of diffusion of oxygen acrossthe endodermis essentially conformed to that of a process ofaqucous diffusion and (b) that oxygen diffused through the corticalair-space system at about 1/25 of its diffusion velocity inair. The diminished diffusion rate in the cortex is doubtlessdue to the frictional resistance imposed by the small poreswhich connect adjacent air cavities. An analysis has been made of the pertinent anatomical featuresof this rhizome, and the findings as a whole are discussed inrelation to their functional and ecological significance.     

Beyond the pollination syndrome: nectar ecology and the role of diurnal and nocturnal pollinators in the reproductive success of Inga sessilis (Fabaceae)

Inga species present brush‐type flower morphology allowing them to be visited by distinct groups of pollinators. Nectar features in relation to the main pollinators have seldom been studied in this genus. To test the hypothesis of floral adaptation to both diurnal and nocturnal pollinators, we studied the pollination ecology of Inga sessilis, with emphasis on the nectar secretion patterns, effects of sequential removals on nectar production, sugar composition and the role of diurnal and nocturnal pollinators in its reproductive success. Inga sessilis is self‐incompatible and pollinated by hummingbirds, hawkmoths and bats. Fruit set under natural conditions is very low despite the fact that most stigmas receive polyads with sufficient pollen to fertilise all ovules in a flower. Nectar secretion starts in the bud stage and flowers continually secreting nectar for a period of 8 h. Flowers actively reabsorbed the nectar a few hours before senescence. Sugar production increased after nectar removal, especially when flowers were drained during the night. Nectar sugar composition changed over flower life span, from sucrose‐dominant (just after flower opening, when hummingbirds were the main visitors) to hexose‐rich (throughout the night, when bats and hawkmoths were the main visitors). Diurnal pollinators contributed less than nocturnal ones to fruit production, but the former were more constant and reliable visitors through time. Our results indicate I. sessilis has floral adaptations, beyond the morphology, that encompass both diurnal and nocturnal pollinator requirements, suggesting a complementary and mixed pollination system.     

13: Lycoris sanguinea Maxim. (Amaryllidaceae)

The life-history characteristics of Lycoris sanguinea Maxim., a bulbous perennial of the Amaryllidaceae, are described here. Its geographical range is in the warm-temperate to cool-temperate zone of Honshu, Shikoku and Kyushu, Japan, extending further to the Korean Peninsula and China. Its growth form and seasonality are very unique, that is, the productive and reproductive phases are separate, which has been observed in other Lycoris species as well. In late March to mid-April for approximately 2 months, only radical leaves appear above ground; these leaves wither and disappear in late April to early May when the canopy of the deciduous trees casts heavy shade on the ground layer; then, in mid- to late-July when the forest floor becomes even darker, only scapes appear above ground, with two to six orange-red flowers borne on the umbels, occasionally forming large clumps here and there on the forest floor. Lycoris sanguinea is a typical insect-pollinated polycarpic perennial that is pollinated by small bees, such as Amegilla florae , and small butterflies, such as Thymelicus leoninus leoninus and Thymelicus sylvaticus sylvaticus . The seed output per plant is not very high, and 6–20 black spherical seeds, each approximately 6 mm in size, are produced.     

Reproductive Biology of the Distylous Manettia luteo-rubra (Rubiaceae)

The reproductive biology of Manettia luteo-rubra was studied in the coastal montane Atlantic rain forest of southeastern Brazil. This Rubiaceae is a perennial vine that flowers all the year round, but has a flowering peak during the dry season. It presents reciprocal herkogamy, thus the plants are morphologically distylous. The morphs occur in a 1:1 ratio, and pollen diameter and corolla length vary between short and long-styled flowers. Manettia luteo-rubra displays typical heterostylous self-incompatibility and sets almost no fruits from self- or intramorph pollinations. The flowers are tubular, red with yellow lobes, and odourless. Anthesis is asynchronous, and the flowers last about four days. Concentration of sugars in nectar is similar in both morphs, ca. 24%, this concentration being typical for hummingbird flowers. Three species of hummingbirds are the major pollinators of the flowers of M. luteo-rubra at the study site: the hermits Phaethornis eurynome and P. squalidus, and the trochiline Thalurania glaucopis. Three species of Heliconius butterflies act as minor pollinators. Both morphs of M. luteo-rubra exhibit natural fruit-set of about 80%, this reproductive output being maintained throughout the year by the pollinators' constancy to the flowers.     

Andromonoecy in Solanum lycocarpum A. St. -Hil. (Solanaceae): Floral attributes,visitors and variation in sexual expression over time

Sexual expression in andromonoecious species—those in which a single individual can bear both staminate and hermaphroditic flowers—may vary among reproductive events in the same plant, among individuals and across populations. This variation influences, in turn, the individual contribution of hermaphroditic plants via male and female fitness functions (i.e., Lloydʼs phenotypic gender). However, temporal variation in sexual expression in andromonoecious species and its relationship with seasonal changes in climatic conditions remain poorly understood. Here we analyze floral attributes, visitors and variation in sexual expression in three populations of Solanum lycocarpum A. St. -Hil. Seasonality in the production of floral types, the mating system and floral visitors were also investigated. Hermaphroditic flowers produced more pollen grains, but the pollen of staminate flowers had higher viability. Only hermaphroditic flowers produced fruits, and ovules in staminate flowers were sterile. Solanum lycocarpum is mainly pollinated by large bees with the ability to vibrate flowers. Phenotypic gender varied throughout the year, and the seasonal production of staminate flowers is associated with the local climate. We suggest that the higher and seasonally variable relative abundance of staminate flowers compared to the low and uniform production of hermaphroditic flowers may be explained by (a) the very high energetic costs incurred in producing large fruits, which in turn make hermaphroditic flower production very costly, and (b) the potentially lower energy expenditure of the smaller staminate flowers with reduced pistils and non-viable ovules that allow them to rapidly respond to climate variability.     

Seasonal changes in redox properties of peat, nutrition and phenology of Menyanthes trifoliata L. in a floating peat mat in Mizorogaike Pond, central Japan

Seasonal changes in leaf population per area, nutrient absorption rate and elemental concentration of Menyanthes trifoliata L. were studied in a floating peat mat in Mizorogaike Pond, central Japan, with reference to the peat redox potential. Leaf population of M. trifoliata showed a rapid increase in May, reached the maximum in June, and then decreased to 12% of the maximum density in July. The foliage density of M. trifoliata again increased and reached its second maximum in September, and then it decreased to zero in December. The decrease of the foliage density from June to July corresponded to the minimum of redox potential (Eh) of the surface peat. Low Eh in the rhizosphere of M. trifoliata relates to the temporary disappearance of the foliage of the plant from June to July. Nutrient concentration (K, Mg, Ca, Fe) in M. trifoliata roots showed minimum in June to August. The nutrient absorption rate of M. trifoliata evaluated from the rubidium absorption rate of excised root of M. trifoliata showed a minimum value in June and July. Peat redox properties would affect the nutrient absorption activity of the roots and the consequent foliage phenology of M. trifoliata in Mizorogaike Pond.     

Spatio‐temporal patterns in pollination of deceptive Aristolochia rotunda L. (Aristolochiaceae)

• Pollination success of highly specialised flowers is susceptible to fluctuations of the pollinator fauna. Mediterranean Aristolochia rotunda has deceptive trap flowers exhibiting a highly specialised pollination system. The sole pollinators are kleptoparasitic flies in search of food. This study investigates these pollinators on a spatio‐temporal scale and the impact of weather conditions on their availability. Two potential strategies of the plants to cope with pollinator limitation, i.e. autonomous selfing and an increased floral life span, were tested.
• A total of 6156 flowers were investigated for entrapped pollinators in 10 Croatian populations. Availability of the main pollinator was correlated to meteorological data. Artificial pollination experiments were conducted and the floral life span was recorded in two populations according to pollinator availability.
• Trachysiphonella ruficeps (Chloropidae) was identified as dominant pollinator, along with less abundant species of Chloropidae, Ceratopogonidae and Milichiidae. Pollinator compositions varied among populations. Weather conditions 15–30 days before pollination had a significant effect on availability of the main pollinator. Flowers were not autonomously selfing, and the floral life span exhibited considerable plasticity depending on pollinator availability.
• A. rotunda flowers rely on insect pollen vectors. Plants are specialised on a guild of kleptoparasitic flies, rather than on a single species. Pollinator variability may result in differing selection pressures among populations. The availability/abundance of pollinators depends on weather conditions during their larval development. Flowers show a prolonged trapping flower stage that likely increases outcrossing success during periods of pollinator limitation.

     

Inheritance and fitness effects analysis for a euchromatic supernumerary chromosome segment in Scilla autumnalis (Liliaceae)

A large euchromatic supernumerary segment (ss) occurs at a high frequency in a natural population of Scilla autumnalis from the west of the Iberian Peninsula. Mother-offspring analyses have shown random pollination of all bulbs lacking the extra segment but non-random pollination of some bulbs carrying it. With a single exception, ss transmission appeared to be Mendelian through the eggs of heterozygous bulbs, which was consistent with results of controlled crosses. An analysis of the effects of this ss on some female fitness components demonstrated significant differences between genotypes for mean seed weight. Heterozygous bulbs yielded significantly heavier seeds than standard homozygous bulbs, and slightly (but not significantly) heavier seeds than segment homozygotes. No trade-off was observed between seed number and seed size in this population. In heterozygotes there was a surprising positive correlation between fruit fertility (number of seeds per fruit) and seed weight. These results are discussed in relation to the maintenance of the segment in natural populations.