Variation in photosynthetic capacity of Salvia przewalskii along elevational gradients on the eastern Qinghai-Tibetan Plateau, China.

Elevational variation in plant growing environment drives diversification of photosynthetic capacity, however, the mechanism behind this reaction is poorly understood. We measured leaf gas exchange, chlorophyll fluorescence, anatomical characteristics, and biochemical traits of Salvia przewalskii at elevations ranging from 2400 m to 3400 m above sea level (a.s.l) on the eastern Qinghai-Tibetan Plateau, China. We found that photosynthetic capacity showed an initial increase and then a decrease with rising elevation, and the best state observed at 2800 m a.s.l. Environmental factors indirectly regulated photosynthetic capacity by affecting stomatal conductance (gs), mesophyll conductance (gm), maximum velocity of carboxylation (Vc max), and maximum capacity for photosynthetic electron transport (Jmax). The average temperature (T) and total precipitation (P) during the growing season had the highest contribution to the variation of photosynthetic capacity of S. przewalskii in subalpine areas, which were 25% and 24%, respectively. Photosynthetic capacity was mainly affected by diffusional limitations (71%-89%), and mesophyll limitation (lm) played a leading role. The variation of gm was attributed to the effects of environmental factors on the volume fraction of intercellular air space (fias), the thickness of cell wall (Tcw), the surface of mesophyll cells and chloroplasts exposed to intercellular airspace (Sm, Sc), and plasma membrane intrinsic protein (PIPs, PIP1, PIP2), independent of carbonic anhydrase (CA). Optimization of leaf tissue structure and adaptive physiological responses enabled plants to efficiently cope with variable climate conditions of high-elevation areas, and the while maintaining high levels of carbon assimilation.

Range of factors in the reduction of hyperhydricity associated with in vitro shoots of Salvia santolinifolia Bioss / Gama de fatores na redução da hiperidricidade associada a brotos in vitro de Salvia santolinifolia Bioss

Hyperhydricity is a serious physiological disorder and affects In vitro propagation of many plants and as well of Salvia santolinifolia. The donor material to initiate the in vitro culture was the callus taken from the in vitro shoots produced on Murashig and Skoogs (MS) medium at 4.0 mg/l BA. This callus formed numerous hyperhydric shoots on culturing upon the medium of the same composition. The aim was to systematically evaluate the effect of cytokinins (Benzyladnine (BA) and N6-(-2-isopentenyl) adenine (2iP), culture vessels magnitude, medium solidification, source of nitrogen and calcium chloride for the alleviation of hyperhydricity. In the tissue cultures of S. santolinifolia BA and 2iP induced severe hyperhydricity, when other factors i.e. culture vessels magnitude and a suitable concentration of agar, ammonium nitrate (NH4NO3), potassium nitrate (KNO3) & calcium chloride (CaCl2.2H2O) were not optimized. After 30 days' culture, we observed 83.82% hyperhydric shoots at increased level (1.5 mg/l 2iP) and 81.59% at decreased levels (1.0 mg/l 2iP). On the other hand, hyperhydricity percentage at decreased (0.4%) and at increased (0.8%) levels of agar were 72.37% and 39.08%, respectively. MS medium modification with NH4NO3 (412 mg/l), KNO3 (475 mg/l) and CaCl2.2H2O (880 mg/l) was found the best medium to reduced hyperhydricity (23.6%).

A hiperidricidade é um distúrbio fisiológico sério e afeta a propagação in vitro de muitas plantas e também da Salvia santolinifolia. O material doador para iniciar a cultura in vitro foi o calo retirado dos brotos in vitro produzidos em meio Murashig e Skoogs (MS) a 4,0 mg / l BA. Esse calo formou numerosos rebentos hiperídricos em cultura no meio da mesma composição. O objetivo foi avaliar sistematicamente o efeito das citocininas (Benziladnina (BA) e N6 - (- 2-isopentenil) adenina (2iP), magnitude dos vasos de cultura, solidificação do meio, fonte de nitrogênio e cloreto de cálcio para o alívio da hiperidricidade. culturas de tecidos de S. santolinifolia BA e 2iP induziram hiperidricidade severa, quando outros fatores, como magnitude dos vasos de cultura e uma concentração adequada de ágar, nitrato de amônio (NH4NO3), nitrato de potássio (KNO3) e cloreto de cálcio (CaCl2.2H2O), não foram otimizados. Após 30 dias de cultura, observamos 83,82% de brotos hiperídricos em níveis aumentados (1,5 mg / l 2iP) e 81,59% em níveis reduzidos (1,0 mg / l 2iP). Por outro lado, a porcentagem de hiperidricidade diminuiu (0,4%) e em níveis aumentados (0,8%) de ágar foram 72,37% e 39,08%, respectivamente. A modificação do meio MS com NH4NO3 (412 mg / l), KNO3 (475 mg / l) e CaCl2.2H2O (880 mg / l) foi encontrada melhor hiperidricidade média a reduzida (23,6%).

Chitosan nanoparticles improve physiological and biochemical responses of Salvia abrotanoides (Kar.) under drought stress.

BACKGROUND: The use of organic nanoparticles to improve drought resistance and water demand characteristics in plants seems to be a promising eco-friendly strategy for water resource management in arid and semi-arid areas. This study aimed to investigate the effect of chitosan nanoparticles (CNPs) (0, 30, 60 and 90 ppm) on some physiological, biochemical, and anatomical responses of Salvia abrotanoides under multiple irrigation regimes (30% (severe), 50% (medium) and 100% (control) field capacity). RESULTS: The results showed that drought stress decreases almost all biochemical parameters. However, foliar application of CNPs mitigated the effects caused by drought stress. This elicitor decreased electrolyte conductivity (35%), but improved relative water content (12.65%), total chlorophyll (63%), carotenoids (68%), phenol (23.1%), flavonoid (36.4%), soluble sugar (58%), proline (49%), protein (45.2%) in S. abrotanoides plants compared to the control (CNPs = 0). Furthermore, the activity of antioxidant enzymes superoxide dismutase (86%), polyphenol oxidase (72.8%), and guaiacol peroxidase (75.7%) were enhanced after CNPs treatment to reduce the effects of water deficit. Also, the CNPs led to an increase in stomatal density (5.2 and 6.6%) while decreasing stomatal aperture size (50 and 25%) and semi-closed stomata (26 and 53%) in leaves. CONCLUSION: The findings show that CNPs not only can considerably reduce water requirement of S. abrotanoides but also are able to enhance the drought tolerance ability of this plant particularly in drought-prone areas.

Herbaceous perennial ornamental plants can support complex pollinator communities.

Human-designed landscapes can host diverse pollinator communities, and the availability of floral resources is central to supporting insect biodiversity in highly modified environments. However, some urban landscapes have relatively few pollinator-attractive plant species and management in urban environments rarely considers the function of these plants in generating and supporting a stable ecological community. Evaluations of 25 cultivars within five commercially popular herbaceous perennial ornamental plant genera (Agastache, Echinacea, Nepeta, Rudbeckia, and Salvia) revealed variation in the total and proportional abundance of visitors attracted. These varieties supported multiple pollinator functional groups, however bees were the primary visitors to in this system. Cultivars were assessed according to their function within a plant-pollinator network. Comparisons of artificial networks created with the six most attractive and six least attractive cultivars demonstrated that a planting scheme using the most attractive cultivars would attract nearly four times as many bee species, including several specialists and rare species. Plant diversity in the landscape was correlated with abundance and diversity of pollinator visitors, demonstrating that community context shapes a plant's relative attractiveness to pollinators. We conclude that herbaceous perennial cultivars can support an abundance and diversity of pollinator visitors, however, planting schemes should take into consideration the effects of cultivar, landscape plant diversity, floral phenology, floral area, and contribution to a stable ecological community.

Cadmium toxicity in Salvia sclarea L.: An integrative response of element uptake, oxidative stress markers, leaf structure and photosynthesis.

The herbal plant Salvia sclarea L. (clary sage) is classified to cadmium (Cd) accumulators and considered as a potential plant for phytoremediation of heavy metal polluted soil. However, the effect of Cd only treatment on the function of the photosynthetic apparatus of S. sclarea, as well as the mechanisms involved in Cd tolerance have not yet been studied in detail. This study was conducted to examine the integrative responses of S. sclarea plants exposed to a high Cd supply (100 µM) for 3 and 8 days by investigating element nutrient uptake, oxidative stress markers, pigment composition, photosynthetic performance and leaf structure. Measurements of the functional activities of photosystem I (PSI, by P700 photooxidation), photosystem II (PSII, by chlorophyll fluorescence parameters), the oxygen-evolving complex (oxygen evolution by Joliot- and Clark-type electrodes), as well as the leaf pigment and phenolic contents, were used to evaluate the protective mechanisms of the photosynthetic apparatus under Cd stress. Data suggested that the molecular mechanisms included in the photosynthetic tolerance to Cd toxicity involve strongly increased phenolic and anthocyanin contents, as well as an increased non-photochemical quenching and accelerated cyclic electron transport around PSI up to 61%, which protect the function of the photosynthetic apparatus under stress. Furthermore, the tolerance of S. sclarea to Cd stress is also associated with increased accumulation of Fe in leaves by 25%. All the above, clearly suggest that S. sclarea plants employ several different mechanisms to protect the function of the photosynthetic apparatus against Cd stress, which are discussed here.

Multiple stressors in multiple species: Effects of different RDX soil concentrations and differential water-resourcing on RDX fate, plant health, and plant survival.

Response to simultaneous stressors is an important facet of plant ecology and land management. In a greenhouse trial, we studied how eight plant species responded to single and combined effects of three soil concentrations of the phytotoxic munitions constituent RDX and two levels of water-resourcing. In an outdoor trial, we studied the effects of high RDX soil concentration and two levels of water-resourcing in three plant species. Multiple endpoints related to RDX fate, plant health, and plant survival were evaluated in both trials. Starting RDX concentration was the most frequent factor influencing all endpoints. Water-resourcing also had significant impacts, but in fewer cases. For most endpoints, significant interaction effects between RDX concentration and water-resourcing were observed for some species and treatments. Main and interaction effects were typically variable (significant in one treatment, but not in another; associated with increasing endpoint values for one treatment and/or with decreasing endpoint values in another). This complexity has implications for understanding how RDX and water-availability combine to impact plants, as well as for applications like phytoremediation. As an additional product of these greenhouse and outdoor trials, three plants native or naturalized within the southeastern United States were identified as promising species for further study as in situ phytoremediation resources. Plumbago auriculata exhibited relatively strong and markedly consistent among-treatment mean proportional reductions in soil RDX concentrations (112% and 2.5% of the means of corresponding values observed within other species). Likewise, across all treatments, Salvia coccinea exhibited distinctively low variance in mean leaf chlorophyll content index levels (6.5% of the means of corresponding values observed within other species). Both species also exhibited mean wilting and chlorosis levels that were 66% and 35%, and 67% and 84%, of corresponding values observed in all other plants, respectively. Ruellia caroliniensis exhibited at least 43% higher mean survival across all treatments than any other test species in outdoor trials, despite exhibiting similar RDX uptake and bioconcentration levels.

Exogenous melatonin improves glutathione content, redox state and increases essential oil production in two Salvia species under drought stress.

This research was conducted to understand the influence of foliar applied melatonin (0, 50, 100, 150 and 200 µM) on two Salvia species (Salvia nemorosa L., and Salvia reuterana Boiss) under conditions of water stress. Water stress was applied using a reduced irrigation strategy based on re-watering at 80%, 60% and 40% of the field capacity (FC). Increasing water stress, while significantly enhancing malondialdehyde (MDA), H2O2, electrolyte leakage, oxidized glutathione (GSSG), and total glutathione (GT), reduced glutathione (GSH), catalase (CAT), peroxidase (POD), superoxide dismutase (SOD) and glutathione reductase (GR) activities, which led to a marked reduction in fluorescence (Fv/Fm). Foliar application of melatonin alleviated the oxidative stress by increasing GT, CAT, POD, SOD and GR activities and reducing GSSG. In particular, melatonin heightened GSH content as well as the ratio of GSH/GSSG when compared to non-sprayed water stressed plants. Melatonin-treated plants had significantly lower SOD and POD activities than control plants under drought stress, while the CAT activity was enhanced with the foliar treatment. Essential oil yield of both Salvia species increased with the decrease in irrigation from 80% to 60% FC but diminished with the more severe water deficit (40% FC). Essential oil components of Salvia nemorosa were ß- caryophyllene, germacrene- B, spathulenol, and cis- ß- farnesene, while (E) - ß- ocimene, α- gurjnnene, germacrene-D, hexyl acetate and aromadendrene was the major constituents of Salvia reuterana. When plants were subjected to water deficit, melatonin treatment increased the concentration and composition of the essential oil. In particular, melatonin treatments improved the primary oil components in both species when compared to non-melatonin treated plants. In conclusion, reduced irrigation regimes as well as melatonin treatments resulted in a significant improvement of essential oil production and composition in both Salvia species.

Sex-differential reproduction success and selection on floral traits in gynodioecious Salvia pratensis.

BACKGROUND: Gynodioecy, a sexual system with hermaphrodite and female individuals in a population, raises the question how the two sexual morphs are maintained. Salvia pratensis is a gynodioecious species featured by its modified stamens that act as a lever mechanism in pollination. Given sexual dimorphism in floral traits of the species, it is predictable that two sexual morphs differ in their interplay with pollinators and thus in their fitness. In this study, we investigated sex-specific reproduction success and floral adaptation in a population of S. pratensis. RESULTS: We found that two sexual morphs in S. pratensis distinctly differed in their floral proportions. Female flowers fitted better to the pollinators than hermaphrodites in terms of touching the stigmas when being probed, and hence were more efficient in pollen deposition. Floral traits overall underwent stronger selection in the population, with stigma position and corolla length subject to disruptive selection mediated by different body-sized bumble bees; some selections on floral traits were significantly different in the strength, even opposite in the direction between two morphs. Flower production tended to be under correlational selection with floral structural traits, implying that a large plant with many flowers did not show an advantage in fitness unless its flower construction mechanically matched the pollinators well. CONCLUSIONS: In conclusion, the pollinator-mediated selection likely played an important role in the evolution and maintenance of sexual dimorphism in the gynodioecious S. pratensis; and sex-divergent mechanical interaction with pollinators served as a critical mechanism by which female individuals were maintained in the population with a female advantage in pollen deposition efficiency (i.e. receiving pollen).

Evolutionary transition between bee pollination and hummingbird pollination in Salvia: Comparing means, variances and covariances of corolla traits.

Covariation among traits can modify the evolutionary trajectory of complex structures. This process is thought to operate at a microevolutionary scale, but its long-term effects remain controversial because trait covariation can itself evolve. Flower morphology, and particularly floral trait (co)variation, has been envisioned as the product of pollinator-mediated selection. Available evidence suggests that major changes in pollinator assemblages may affect the joint expression of floral traits and their phenotypic integration. We expect species within a monophyletic lineage sharing the same pollinator type will show not only similarity in trait means but also similar phenotypic variance-covariance structures. Here, we tested this expectation using eighteen Salvia species pollinated either by bees or by hummingbirds. Our findings indicated a nonsignificant multivariate phylogenetic signal and a decoupling between means and variance-covariance phenotypic matrices of floral traits during the evolution to hummingbird pollination. Mean trait value analyses revealed significant differences between bee- and hummingbird-pollinated Salvia species although fewer differences were detected in the covariance structure between groups. Variance-covariance matrices were much more similar among bee- than hummingbird-pollinated species. This pattern is consistent with the expectation that, unlike hummingbirds, bees physically manipulate the flower, presumably exerting stronger selection pressures favouring morphological convergence among species. Overall, we conclude that the evolution of hummingbird pollination proceeded through different independent transitions. Thus, although the evolution of hummingbird pollination led to a new phenotypic optimum, the process involved the diversification of the covariance structure.

Reproductive isolation between Salvia elegans and S. fulgens, two hummingbird-pollinated sympatric sages.

The integrity of species in sympatric contact sites is dependent on the existence of reproductive isolating mechanisms, which restrict gene flow between them. However, we know little about the mechanisms that enable the coexistence of species with similar floral morphologies. Here, we evaluated several reproductive isolation barriers between Salvia elegans and S. fulgens, two sympatric sages with a similar ornithophilous floral syndrome, offering nectar as the main reward. Over 3 years, we evaluated broad-scale geographic isolation, floral phenologies and floral visitors as pre-pollination barriers, and fruit set, seed number and seed germination as post-pollination barriers. We found considerable geographic isolation and significant altitudinal differences between the two sages. The flowering period of both sages always overlapped extensively during the 3 years of this study, but hummingbirds were highly specific, visiting one or the other Salvia species and showing aggressive territorial behaviour. Interspecific experimental crosses revealed that hybrid seeds might be formed although strong asymmetric barriers were found depending on the species acting as the maternal donor. Despite the low level of flowering asynchrony, reproductive isolation was remarkably high in the two sages. Geographic isolation and pollinator fidelity were the main factors responsible for maintaining species integrity. Despite an extensive review, we found very few studies quantifying the efficiency of isolation barriers in Neotropical plants or even the importance of hummingbirds as pollinators.

Salvia castanea Hairy Roots are More Tolerant to Phosphate Deficiency than Salvia miltiorrhiza Hairy Roots Based on the Secondary Metabolism and Antioxidant Defenses.

Salvia miltiorrhiza is a well-known traditional Chinese herb which is used to treat heart disease. Salvia castanea is a substitute product for S. miltiorrhiza in the medicinal field. Previous study has shown that phosphate (Pi) deficiency could promote the accumulation of secondary metabolism in herbs, and it has also developed a strategy for saving Pi resources and increasing the yield of active substances in herbs. In the present study, the hairy roots of S. miltiorrhiza and S. castanea were used to identify the Pi deficiency response mechanisms of these two Salvia species. The results showed that Pi deficiency increased the accumulation of specifically secondary metabolites, such as phenolic acids and tanshinones, which were caused by promoting the expression levels of key enzyme genes. In addition, Pi deficiency promoted the antioxidant activity in these two Salvia species. The data demonstrated that Pi deficiency increased the quality of the medicinal material in the plant. The hairy roots of S. castanea were more adaptive to Pi deficiency than those of S. miltiorrhiza in terms of biomass, secondary metabolism, and antioxidant activity. The results of this study provide insights into breeding herbs that are better adapted to Pi deficiency, which could increase the yield of active ingredients in herbs and save Pi resources.

Effect of environmental factors on the germination and emergence of Salvia verbenaca L. cultivars (verbenaca and vernalis): An invasive species in semi-arid and arid rangeland regions.

Salvia verbenaca (wild sage) is a commonly cultivated herbal medicine plant, which is native to the Mediterranean climate regions of Europe, Africa, Asia and the Middle East. However, it has become an invasive species in semi-arid and arid regions of southern Australia. Two varieties are present in this region, var. verbenaca and var. vernalis, each of which can be distinguished by differences in morphology and flowering period. Following trials to determine the optimum temperate regime for germination and response to light and dark, seeds of both varieties were tested for their response to variations in pH, moisture stress, salinity, and burial depth. The temperature and light trial was carried out using three different temperature regimes; 30/20°C, 25/15°C and 20/12°C, and two light regimes; 12 hours light/12 hours dark and 24 hours dark, with var. vernalis responding to relatively higher temperatures than var. verbenaca. The germination rate of neither species was significantly inhibited by complete darkness when compared to rates under periodic light exposure. Both varieties germinated at near optimum rates strongly to very strongly in all pH buffer solutions, from pH 5 to pH 10, but they responded most strongly at neutral pH. Var. vernalis showed slightly more tolerance to reduced moisture availability, moderate to strong salinity, and burial depth, compared to var. verbenaca. However, even a fairly shallow burial depth of 2 cm completely inhibited germination of both varieties. Thus, in circumstances where both varieties are present in a soil seedbank, var. vernalis could be expected to establish in more challenging conditions, where moisture is limited and salinity is 'moderate to high', implying that it is a more serious threat for invasive weed in conditions where crop plants are already challenged.

Salvia broussonetii Benth.: aroma profile and micromorphological analysis.

The volatile profiles (VOC) and the essential oil (EO) composition from the aerial parts of Salvia broussonetii were analysed. Sesquiterpene hydrocarbons dominate the VOCs from leaves (95.7%) and flowers (67.6%), followed by monoterpene hydrocarbons (2.6 and 29.7%, respectively). The main common compounds are germacrene D, ß-bourbonene, α-pinene, α-copaene and α-gurjunene, even if with divergent relative abundances. In the leaf EOs the sesquiterpenes prevail, even if not overwhelmingly (about 50.0%), followed by monoterpenes (23.0-35.0%) and by minor fractions of diterpene hydrocarbons and non-terpene derivates. The most abundant common compounds across the two sampling periods are α-pinene, ß-pinene, isobornyl acetate, α-gurjenene, germacrene D and bifloratriene. A morphological characterisation of the trichomes responsible for the productivity in terpenes was also performed. Four different morphotypes were observed on the above ground organs of S. brussonetii: peltates and capitates of type II and III resulted the only producers of volatile substances.

Nectar replenishment maintains the neutral effects of nectar robbing on female reproductive success of Salvia przewalskii (Lamiaceae), a plant pollinated and robbed by bumble bees.

Background and Aims: It has been suggested that the dynamics of nectar replenishment could differ for flowers after being nectar robbed or visited legitimately, but further experimental work is needed to investigate this hypothesis. This study aimed to assess the role of nectar replenishment in mediating the effects of nectar robbing on pollinator behaviour and plant reproduction. Methods: Plant-robber-pollinator interactions in an alpine plant, Salvia przewalskii , were studied. It is pollinated by long-tongued Bombus religiosus and short-tongued B. friseanus , but robbed by B. friseanus . Nectar production rates for flowers after they were either robbed or legitimately visited were compared, and three levels of nectar robbing were created to detect the effects of nectar robbing on pollinator behaviour and plant reproduction. Key Results: Nectar replenishment did not differ between flowers that had been robbed or legitimately visited. Neither fruit set nor seed set was significantly affected by nectar robbing. In addition, nectar robbing did not significantly affect visitation rate, flowers visited within a plant per foraging bout, or flower handling time of the legitimate pollinators. However, a tendency for a decrease in relative abundance of the pollinator B. religiosus with an increase of nectar robbing was found. Conclusions: Nectar robbing did not affect female reproductive success because nectar replenishment ensures that pollinators maintain their visiting activity to nectar-robbed flowers. Nectar replenishment might be a defence mechanism against nectar robbing to enhance reproductive fitness by maintaining attractiveness to pollinators. Further studies are needed to reveal the potential for interference competition among bumble bees foraging as robbers and legitimate visitors, and to investigate variation of nectar robbing in communities with different bumble bee species composition.

Environmental gradients shape the genetic structure of two medicinal Salvia species in Jordan.

Environmental gradients, and particularly climatic variables, exert a strong influence on plant distribution and, potentially, population genetic diversity and differentiation. Differences in water availability can cause among-population variation in ecological processes and can thus interrupt populations' connectivity and isolate them environmentally. The present study examines the effect of environmental heterogeneity on plant populations due to environmental isolation unrelated to geographic distance. Using AFLP markers, we analyzed genetic diversity and differentiation among 12 Salvia spinosa populations and 13 Salvia syriaca populations from three phytogeographical regions (Mediterranean, Irano-Turanian and Saharo-Arabian) representing the extent of the species' geographic range in Jordan. Differences in geographic location and climate were considered in the analyses. For both species, flowering phenology varied among populations and regions. Irano-Turanian and Saharo-Arabian populations had higher genetic diversity than Mediterranean populations, and genetic diversity increased significantly with increasing temperature. Genetic diversity in Salvia syriaca was affected by population size, while genetic diversity responded to drought in S. spinosa. For both species, high levels of genetic differentiation were found as well as two well-supported phytogeographical groups of populations, with Mediterranean populations clustering in one group and the Irano-Turanian and Saharo-Arabian populations in another. Genetic distance was significantly correlated to environmental distance, but not to geographic distance. Our data indicate that populations from moist vs. arid environments are environmentally isolated, where environmental gradients affect their flowering phenology, limit gene flow and shape their genetic structure. We conclude that environmental heterogeneity may act as driver for the observed variation in genetic diversity.

Spatio-temporal variation of nectar robbing in Salvia gesneriflora and its effects on nectar production and legitimate visitors.

Nectar robbing occurs when floral visitors remove floral nectar through floral damage and usually without providing pollination in return. Even though nectar robbing may have negative, neutral or even positive effects on plant fitness, few studies have investigated temporal and spatial variation in robbing rate and their consequences, particularly in the tropics. In this study, robbing levels were estimated during 3 years in four populations of Salvia gesneriflora, a hummingbird-pollinated shrub endemic to central Mexico that is mainly robbed by birds, carpenter bees and bumblebees. The effect of robbing on nectar availability, flower longevity and on visitation rate by floral visitors was also evaluated. Our results indicate great variation in robbing levels across years and populations and a positive relationship between robbing level and flower abundance per population. Moreover, our results show that nectar availability is about eight times higher in unrobbed flowers than in robbed flowers, and that nectar robbers prefer younger flowers, although lifespan of robbed and unrobbed flowers did not differ statistically. Primary and secondary nectar robbers showed a higher visitation rate compared to legitimate visitors, and neither legitimate nor illegitimate floral visitors seem to discriminate between robbed and unrobbed flowers. These results suggest that robbers may respond to food availability and that no floral visitors apparently could differentiate between robbed and unrobbed flowers. Finally, results show that nectar robbers prefer the youngest flowers, which suggests that strong competition for access to nectar between pollinators and robbers might occur, mainly at the first stages of the flowers.

Genetic variability and population structure of Salvia lachnostachys: implications for breeding and conservation programs.

The genetic diversity and population structure of Salvia lachnostachys Benth were assessed. Inter Simple Sequence Repeat (ISSR) molecular markers were used to investigate the restricted distribution of S. lachnostachys in Parana State, Brazil. Leaves of 73 individuals representing three populations were collected. DNA was extracted and submitted to PCR-ISSR amplification with nine tested primers. Genetic diversity parameters were evaluated. Our analysis indicated 95.6% polymorphic loci (stress value 0.02) with a 0.79 average Simpson's index. The Nei-Li distance dendrogram and principal component analysis largely recovered the geographical origin of each sample. Four major clusters were recognized representing each collected population. Nei's gene diversity and Shannon's information index were 0.25 and 0.40 respectively. As is typical for outcrossing herbs, the majority of genetic variation occurred at the population level (81.76%). A high gene flow (Nm = 2.48) was observed with a correspondingly low fixation index. These values were generally similar to previous studies on congeneric species. The results of principal coordinate analysis (PCA) and of arithmetic average (UPGMA) were consistent and all three populations appear distinct as in STRUCTURE analysis. In addition, this analysis indicated a majority intrapopulation genetic variation. Despite the human pressure on natural populations our study found high levels of genetic diversity for S. lachnostachys. This was the first molecular assessment for this endemic species with medicinal proprieties and the results can guide for subsequent bioprospection, breeding programs or conservation actions.

Are leaves 'freewheelin'? Testing for a wheeler-type effect in leaf xylem hydraulic decline.

A recent study found that cutting shoots under water while xylem was under tension (which has been the standard protocol for the past few decades) could produce artefactual embolisms inside the xylem, overestimating hydraulic vulnerability relative to shoots cut under water after relaxing xylem tension (Wheeler et al. 2013). That study also raised the possibility that such a 'Wheeler effect' might occur in studies of leaf hydraulic vulnerability. We tested for such an effect for four species by applying a modified vacuum pump method to leaves with minor veins severed, to construct leaf xylem hydraulic vulnerability curves. We tested for an impact on leaf xylem hydraulic conductance (Kx ) of cutting the petiole and minor veins under water for dehydrated leaves with xylem under tension compared with dehydrated leaves after previously relaxing xylem tension. Our results showed no significant 'cutting artefact' for leaf xylem. The lack of an effect for leaves could not be explained by narrower or shorter xylem conduits, and may be due to lesser mechanical stress imposed when cutting leaf petioles, and/or to rapid refilling of emboli in petioles. These findings provide the first validation of previous measurements of leaf hydraulic vulnerability against this potential artefact.

Flies as pollinators of melittophilous Salvia species (Lamiaceae).

UNLABELLED: • PREMISE OF THE STUDY: Floral adaptation to a functional pollinator group does not necessarily mean close specialization to a few pollinator species. For the more than 950 species of Salvia, only bee and bird pollinations are known. Restriction to these pollinators is mainly due to the specific flower construction (lever mechanism). Nevertheless, it has been repeatedly suggested that Salvia flowers might also be pollinated by flies. Are flies able to handle the lever mechanism? Are they functionally equivalent pollinators? In this study, we compared and quantified pollen transfer by bees and flies to test whether flies are true pollinators in Salvia.• METHODS: We identified pollinators using field observations and photos. Video documentation of the visitation rate and the site of pollen placement on the pollinator body, morphometric measurements, quantification of pollen placement, pollen load, handling time, and stigma contact ratio were analyzed.• KEY RESULTS: Field investigations revealed that 19 insect species pollinated S. virgata and four pollinated S. verticillata, including 16 bee species from seven genera of the Apidae and three fly species from three genera of the Nemestrinidae and Tabanidae.• CONCLUSIONS: Flies have been found to be pollinators in primarily bee-pollinated Salvia species. This result demonstrates the potential of a given "melittoid" flower construction to broaden the range of pollinators to guarantee successful pollination and seed production. Though bees, particularly Bombus terrestris, were more efficient than flies, the study shows that flies significantly contribute to pollen transfer in Salvia.

Why Africa matters: evolution of Old World Salvia (Lamiaceae) in Africa.

BACKGROUND AND AIMS: Salvia is the largest genus in Lamiaceae and it has recently been found to be non-monophyletic. Molecular data on Old World Salvia are largely lacking. In this study, we present data concerning Salvia in Africa. The focus is on the colonization of the continent, character evolution and the switch of pollination systems in the genus. METHODS: Maximum likelihood and Bayesian inference were used for phylogenetic reconstruction. Analyses were based on two nuclear markers [internal transcribed spacer (ITS) and external transcribed spacer (ETS)] and one plastid marker (rpl32-trnL). Sequence data were generated for 41 of the 62 African taxa (66 %). Mesquite was used to reconstruct ancestral character states for distribution, life form, calyx shape, stamen type and pollination syndrome. KEY RESULTS: Salvia in Africa is non-monophyletic. Each of the five major regions in Africa, except Madagascar, was colonized at least twice, and floristic links between North African, south-west Asian and European species are strongly supported. The large radiation in Sub-Saharan Africa (23 species) can be traced back to dispersal from North Africa via East Africa to the Cape Region. Adaptation to bird pollination in southern Africa and Madagascar reflects parallel evolution. CONCLUSIONS: The phenotypic diversity in African Salvia is associated with repeated introductions to the continent. Many important evolutionary processes, such as colonization, adaptation, parallelism and character transformation, are reflected in this comparatively small group. The data presented in this study can help to understand the evolution of Salvia sensu lato and other large genera.