Identification and evaluation of Escherichia coli strain ATCC 8739 as a surrogate for thermal inactivation of enterohemorrhagic Escherichia coli in fruit nectars: Impact of applied techniques on the decimal reduction time.

The objective of this study was to identify a suitable surrogate for E. coli O157:H7 strain 19685/91 and O113:H21 strain TS18/08, by assessing their thermal resistance at temperatures of 60 °C, 65 °C, and 72 °C in strawberry nectar. The influence of the matrix and the research methodology on the decimal reduction time (D-value) was investigated. Thermal kinetics and safety assessment demonstrated that E. coli ATCC 8739 is a suitable surrogate. The study demonstrated that the presence of fruit particles in the nectar increased thermal resistance of the tested strains. Variations in D-values were observed depending on the research method employed, with D-values in glass capillaries were up to 6.6 times lower compared to larger sample volumes. Encapsulation of E. coli ATCC 8739 exhibited high efficiency of 90.25 ± 0.26% and maintained stable viable counts after 26 days of storage in strawberry nectar at 4 °C. There were no significant differences in thermal resistance between surrogates directly inoculated into strawberry nectar and those encapsulated in alginate beads. Additionally, the encapsulated strains did not migrate outside the beads. Therefore, encapsulated E. coli ATCC 8739 in alginate beads can be effectively utilized in industrial settings to validate thermal treatments as a reliable and safe method.

Essential role of papillae flexibility in nectar capture by bees.

Many bees possess a tongue resembling a brush composed of a central rod (glossa) covered by elongated papillae, which is dipped periodically into nectar to collect this primary source of energy. In vivo measurements show that the amount of nectar collected per lap remains essentially constant for sugar concentrations lower than 50% but drops significantly for a concentration around 70%. To understand this variation of the ingestion rate with the sugar content of nectar, we investigate the dynamics of fluid capture by Bombus terrestris as a model system. During the dipping process, the papillae, which initially adhere to the glossa, unfold when immersed in the nectar. Combining in vivo investigations, macroscopic experiments with flexible rods, and an elastoviscous theoretical model, we show that the capture mechanism is governed by the relaxation dynamics of the bent papillae, driven by their elastic recoil slowed down through viscous dissipation. At low sugar concentrations, the papillae completely open before the tongue retracts out of nectar and thus, fully contribute to the fluid capture. In contrast, at larger concentrations corresponding to the drop of the ingestion rate, the viscous dissipation strongly hinders the papillae opening, reducing considerably the amount of nectar captured. This study shows the crucial role of flexible papillae, whose aspect ratio determines the optimal nectar concentration, to understand quantitatively the capture of nectar by bees and how physics can shed some light on the degree of adaptation of a specific morphological trait.

Sugar Concentration, Nitrogen Availability, and Phylogenetic Factors Determine the Ability of Acinetobacter spp. and Rosenbergiella spp. to Grow in Floral Nectar.

The floral nectar of angiosperms harbors a variety of microorganisms that depend predominantly on animal visitors for their dispersal. Although some members of the genus Acinetobacter and all currently known species of Rosenbergiella are thought to be adapted to thrive in nectar, there is limited information about the response of these bacteria to variation in the chemical characteristics of floral nectar. We investigated the growth performance of a diverse collection of Acinetobacter (n = 43) and Rosenbergiella (n = 45) isolates obtained from floral nectar and the digestive tract of flower-visiting bees in a set of 12 artificial nectars differing in sugar content (15% w/v or 50% w/v), nitrogen content (3.48/1.67 ppm or 348/167 ppm of total nitrogen/amino nitrogen), and sugar composition (only sucrose, 1/3 sucrose + 1/3 glucose + 1/3 fructose, or 1/2 glucose + 1/2 fructose). Growth was only observed in four of the 12 artificial nectars. Those containing elevated sugar concentration (50% w/v) and low nitrogen content (3.48/1.67 ppm) were limiting for bacterial growth. Furthermore, phylogenetic analyses revealed that the ability of the bacteria to grow in different types of nectar is highly conserved between closely related isolates and genotypes, but this conservatism rapidly vanishes deeper in phylogeny. Overall, these results demonstrate that the ability of Acinetobacter spp. and Rosenbergiella spp. to grow in floral nectar largely depends on nectar chemistry and bacterial phylogeny.

Global assessment of honeybee exposure to pesticides through guttation consumption: An indicator approach.

Guttation consumption is a potential pathway of pesticide residue exposure in honeybees. However, modeling tools for assessing honeybee exposure to pesticide residues in guttation drops are lacking. In this study, we propose an indicator-based approach for qualitatively or quantitatively analyzing the guttation-based exposure pathway, allowing us to conduct region-specific pesticide residue exposure assessments for honeybees. Exposure scores (the product of guttation production and residue level scores) were established to compare or rank honeybee exposure to pesticide residues via guttation intake across locations using three specified indicators (i.e., air temperature, relative humidity, and precipitation intensity). Warm, dry regions had high residue level scores (indicating high residue levels in guttation), whereas cold, wet regions had high guttation production scores (indicating high possibilities of guttation formation on leaf surfaces); their exposure scores were a combination of these two values. We evaluated and ranked honeybee exposure to imidacloprid residue across regions in Brazil, China, the United States, and selected European Union member states, revealing that pesticide application in many Brazilian federative units may raise honeybee risks due to high exposure scores. We also compared the guttation pathway to other common exposure pathways (nectar and pollen), suggesting that for some moderately lipophilic compounds, the guttation exposure pathway may not be ignored and should be further evaluated.

Residues of sulfoxaflor and its metabolites in floral and extrafloral nectar from Hibiscus rosa-sinensis L. (Malvaceae) with or without co-application of tebuconazole.

Systemic pesticide exposure through nectar is a growing global concern linked to loss of insect diversity, especially pollinators. The insecticide sulfoxaflor and the fungicide tebuconazole are currently widely used systemic pesticides which are toxic to certain pollinators. However, their metabolisms in floral or extrafloral nectar under different application methods have not yet been well studied. Hibiscus rosa-sinensis was exposed to sulfoxaflor and tebuconazole via soil drenching and foliar spraying. Sulfoxaflor, tebuconazole, and their main metabolites in floral and extrafloral nectar, soil, and leaves were identified and quantified using liquid chromatography coupled with triple quadrupole mass spectrometry (LC-QqQ MS). The chemical compositions of unexposed and contaminated H. rosa-sinensis floral nectar or extrafloral nectar were compared using regular biochemical methods. The activities of two pesticide detoxifying enzymes, glutathione-s-transferase and nitrile hydratase, in H. rosa-sinensis nectar were examined using LC-MS and spectrophotometry. The floral nectar proteome of H. rosa-sinensis was analysed using high-resolution orbitrap-based MS/MS analysis to screen for sulfoxaflor and tebuconazole detoxifying enzymes. H. rosa-sinensis can absorb sulfoxaflor and tebuconazole through its roots or leaf surfaces and secrete them into floral nectar and extrafloral nectar. Both sulfoxaflor and tebuconazole and their major metabolites were present at higher concentrations in extrafloral nectar than in floral nectar. X11719474 was the dominant metabolite of sulfoxaflor in the nectars we studied. Compared with soil application, more sulfoxaflor and tebuconazole remained in their original forms in floral nectar and extrafloral nectar after foliar application. Sulfoxaflor and tebuconazole exposure did not modify the chemical composition of floral or extrafloral nectar. No active components, including proteins in the nectar, were detected to be able to detoxify sulfoxaflor.

Floral Phytochemistry: Impact of Volatile Organic Compounds and Nectar Secondary Metabolites on Pollinator Behavior and Health.

Plants produce a plethora of phytochemicals including sugars, amino acids (AAs), volatile organic compounds (VOCs) and secondary metabolites (SMs) with different ecological functions. To attract pollinators and defenders and ensure reproductive success, plants mainly rely on VOCs, while to reward insects, plants synthesize nectar rich in sugars and AAs. Furthermore, plant SMs can play various roles. Some components are able to interact with the nervous system of insects by binding to neuron receptor proteins and thus manipulate pollinator behavior. Others, like alkaloids and phenolics, protect from nectar robbers and enhance memory and foraging efficiency, or, as in the case of flavonoids, exhibit high antioxidant activities supporting pollinator well-being. This review discusses the impact of VOCs and nectar SMs on insect behavior and pollinator health.

In Which Way Do the Flower Properties of the Specialist Orchid Goodyera repens Meet the Requirements of Its Generalist Pollinators?

This article is the next part of a series of studies documenting the influence of flower traits on the reproductive success (RS) of orchids. Knowledge of factors influencing RS helps to understand the mechanisms and processes crucial for shaping plant-pollinator interactions. The aim of the present study was to determine the role of flower structure and nectar composition in shaping the RS of the specialist orchid Goodyea repens, which is pollinated by generalist bumblebees. We found a high level of pollinaria removal (PR) and female reproductive success (fruiting, FRS) as well as a high level of variation between populations, although in certain populations pollination efficiency was low. Floral display traits, mainly inflorescence length, influenced FRS in certain populations. Among the flower traits, only the height of flowers was correlated with FRS in one population, suggesting that the flower structure of this orchid is well adapted to pollination by bumblebees. The nectar of G. repens is diluted and dominated by hexoses. Sugars were less important in shaping RS than amino acids. At the species level, twenty proteogenic and six non-proteogenic AAs were noted, along with their differentiated amounts and participation in particular populations. We found that distinct AAs or their groups mainly shaped PR, especially when correlations were considered at the species level. Our results suggest that both the individual nectar components and the ratios between them have an impact on G. repens RS. Because different nectar components influence the RS parameters in different ways (i.e., negatively or positively), we suggest that different Bombus species play the role of main pollinators in distinct populations.

Does Reproductive Success in Natural and Anthropogenic Populations of Generalist Epipactis helleborine Depend on Flower Morphology and Nectar Composition?

The purpose of our study was to determine the role of flower structure and nectar composition in shaping the reproductive success (RS) of the generalist orchid Epipactis helleborine in natural and anthropogenic populations. We supposed that the distinct character of two groups of habitats creates different conditions for plant-pollinator relationships, thus influencing reproductive success in E. helleborine populations. Both pollinaria removal (PR) and fruiting (FRS) were differentiated between the populations. On average, FRS was almost two times higher in the anthropogenic than in the natural populations. The difference between the two population groups in PR was smaller but still statistically significant. RS parameters were correlated with some floral display and flower traits. Floral display influenced RS only in three anthropogenic populations. Flower traits had a weak influence on RS (10 of the 192 cases analyzed). The more important trait in shaping RS was nectar chemistry. The nectar of E. helleborine is relatively diluted with a lower sugar concentration in the anthropogenic than in the natural populations. In the natural populations, domination of sucrose over hexoses was found, while in the anthropogenic populations, hexoses were more abundant and the participation of sugars was balanced. In some populations, sugars influenced RS. In E. helleborine nectar, 20 proteogenic and 7 non-proteogenic amino acids (AAs) were found with a clear domination of glutamic acid. We noted relationships between some AAs and RS, but distinct AAs shaped RS in different populations, and their impact was independent of their participation. Our results indicate that the flower structure and nectar composition of E. helleborine reflect its generalistic character and meet the requirements of a wide range of pollinators. Simultaneously, the differentiation of flower traits suggests a variation in pollinator assemblages in particular populations. Knowledge about the factors influencing RS in distinct habitats helps to understand the evolutionary potential of species and to understand mechanisms and processes crucial for shaping interactions between plants and pollinators.

The role of alanine synthesis and nitrate-induced nitric oxide production during hypoxia stress in Cucurbita pepo nectaries.

Floral nectar is a sugary solution produced by nectaries to attract and reward pollinators. Nectar metabolites, such as sugars, are synthesized within the nectary during secretion from both pre-stored and direct phloem-derived precursors. In addition to sugars, nectars contain nitrogenous compounds such as amino acids; however, little is known about the role(s) of nitrogen (N) compounds in nectary function. In this study, we investigated N metabolism in Cucurbita pepo (squash) floral nectaries in order to understand how various N-containing compounds are produced and determine the role of N metabolism in nectar secretion. The expression and activity of key enzymes involved in primary N assimilation, including nitrate reductase (NR) and alanine aminotransferase (AlaAT), were induced during secretion in C. pepo nectaries. Alanine (Ala) accumulated to about 35% of total amino acids in nectaries and nectar during peak secretion; however, alteration of vascular nitrate supply had no impact on Ala accumulation during secretion, suggesting that nectar(y) amino acids are produced by precursors other than nitrate. In addition, nitric oxide (NO) is produced from nitrate and nitrite, at least partially by NR, in nectaries and nectar. Hypoxia-related processes are induced in nectaries during secretion, including lactic acid and ethanolic fermentation. Finally, treatments that alter nitrate supply affect levels of hypoxic metabolites, nectar volume and nectar sugar composition. The induction of N metabolism in C. pepo nectaries thus plays an important role in the synthesis and secretion of nectar sugar.

Nectaries in ferns: their taxonomic distribution, structure, function, and sugar composition.

PREMISE: Extrafloral nectaries have mainly been studied in angiosperms, but have also been reported in 39 fern species. Here we provide a global review of nectaries in ferns and examined their structure, function, and nectar sugar composition in two genera. METHODS: We searched in the literature and living plant collections of botanical gardens for indications of fern nectaries, observed nectar-feeding animals, studied the morphoanatomy in the two genera Aglaomorpha and Campyloneurum, and analyzed the total sugar concentrations and ratios of 16 species. Diurnal nectar release was observed with time-lapse photography. RESULTS: We found evidence for nectaries in 101 species of ferns from 11 genera and 6 families. Most of the nectary-bearing species were tree ferns (Cyatheaceae) and epiphytic ferns of the family Polypodiaceae. Nectaries consisted of cytoplasm-rich parenchyma with large nuclei and an epidermis with or without stomata, were attached to amphiphloic vascular bundles, and released nectar on the lower leaf surface mainly on expanding leaves during the night. Sugar concentrations varied between species (3.8-15.3%) but not between genera, and were sucrose-dominant (3 spp.), sucrose-rich (7), or hexose-rich (3). In the greenhouse, introduced ants, scale insects, and snails fed on the nectar. CONCLUSIONS: The wide taxonomic distribution, variable morphology, locations, and sugar compositions point to multiple evolutionary origins of fern nectaries. Nectar release in young leaves might attract mutualistic ants to protect leaves against herbivores only during this most vulnerable developmental stage. Even ex-situ, fern nectar is a valuable food source because it attracted several opportunistic animal species.

Extranuptial nectaries in bromeliads: a new record for Pitcairnia burchellii and perspectives for Bromeliaceae.

Nectar plays important roles in the relationship between plants and other organisms, both within pollination systems and as a defense mechanism. In the latter case, extranuptial nectaries (ENNs) usually attract patrolling arthropods that reduce herbivory. ENNs have been frequently reported within the "xeric clade" of Bromeliaceae, but their occurrence in other groups of bromeliads is largely unexplored, especially considering their position, secretory activity and structure. After observing the presence of ants constantly patrolling the inflorescences of Pitcairnia burchellii Mez, we searched for the presence, secretory activity, and structure of ENNs in this species. We also provide a brief review of the occurrence ENNs in Bromeliaceae. The distribution of nectaries was assessed using ant-exclusion experiments, while structural analysis was performed using standard methods for light and scanning electron microscopy. The presence of sugars in the secretion was assessed by thin-layer chromatography and glucose strip tests. Nectaries in P. burchelli are non-structured glands on the adaxial surface of floral bracts and sepals. Bracts and sepals are distinct spatial units that act over time in the same strategy of floral bud protection. Literature data reveals that ENNs might be more common within Bromeliaceae than previously considered, comprising a homoplastic feature in the family. Future perspectives and evolutionary and taxonomic implications are discussed.

Modeling pesticide residues in nectar and pollen in support of pesticide exposure assessment for honeybees: A generic modeling approach.

Pesticide residues in nectar and pollen of plants can damage honeybees; however, few modeling approaches have simulated residue levels in nectar and pollen in support of exposure assessment for honeybees. This study introduced a generic modeling approach based on plant uptake models and simple partitioning rules that specifies soil incorporation and foliar spray application scenarios of pesticides and is flexible for conducting variability analysis for various environmental conditions, pesticide application patterns, chemical individuals, and plant varieties. The results indicated that, in general, systemic or moderate lipophilicity (log KOW of ~2.5) pesticides have relatively high simulated residue levels in nectar and pollen because of the enhanced residue uptake process from soil. For non-systemic or highly lipophilic pesticides, the residue uptake via leaf surface deposition pathway can be enhanced, and more residues will be bioaccumulated in pollen than nectar due to a relatively high lipid content of pollen (as compared to nectar), but the overall residue levels in nectar and pollen are lower than systemic or moderately lipophilic pesticides. The variability analysis showed that environmental conditions, pesticide application patterns, chemical properties, and plant varieties cause considerable variations in simulated residue levels in nectar and pollen, indicating that spatiotemporal, chemical, and plant-related factors must be considered in pesticide exposure assessment for honeybees. Moreover, the comparison between the simulated and measured data showed a high degree of consistency, indicating that the proposed model could be used to conduct a screening-level pesticide exposure assessment for honeybees.

Identification and quantitation of the novel insecticide sulfoxaflor and its metabolites in floral nectar from Salvia splendens Ker Gawl. (Lamiaceae).

Sulfoxaflor is a new systemic insecticide developed as a replacement for older neonicotinoids which are known to be toxic to pollinators. However, its metabolism in nectar and effect on nectar biosynthesis have not been investigated. After soil and foliar application, sulfoxaflor and its main metabolites in soil, leaf and Salvia splendens nectar, were measured by liquid chromatography coupled with triple quadrupole mass spectrometer (LC-MS/MS). The chemical composition between the clean and sulfoxaflor spiked nectar were also compared. The activities of two possible sulfoxaflor detoxifying enzymes in S. splendens nectar, nitrile hydratase and glutathione-s-transferase, were measured by LC-MS and spectrophotometry. S. splendens nectar proteome was investigated by high-resolution orbitrap-based MS/MS to screen for sulfoxaflor detoxifying relevant proteins. S. splendens could absorb sulfoxaflor through root or leaf surface and secrete a proportion of sulfoxaflor along with its metabolites into the nectar. After soil application, sulfoxaflor's low toxic metabolite X11719474 was dominant in the nectar and reached an average concentration of 8905 ppb. However, after foliar application, sulfoxaflor was dominant over its metabolites in the nectar. S. splendens nectar has no nitrile hydratase and glutathione-s-transferase activity and none of the 106 proteins identified in the nectar were predicted to function in detoxifying sulfoxaflor. Soil and foliar sulfoxaflor application can result in different profiles of sulfoxaflor and its metabolites presented in the nectar. However, sulfoxaflor had no effects on S. splendens nectar secretion and chemical composition and cannot be directly detoxified by components in the nectar.

A chemometric approach for the differentiation of 15 monofloral honeys based on physicochemical parameters.

BACKGROUND: Although the main method for authentication of monofloral honey is pollen analysis, other classification approaches have been also applied. However, the majority of the existing classification models so far have utilized a few honey types or a few honey samples of each honey type, which can lead to inaccurate results. Aiming at addressing this, the goal of the present study was to create a classification model by analysing in total 250 honey samples from 15 different monofloral honey types in ten physicochemical parameters and then, multivariate analysis [multivariate analysis of variance (MANOVA), principal component analysis (PCA) and multi-discriminant analysis (MDA)] was applied in an effort to distinguish and classify them. RESULTS: Electrical conductivity and colour were found to have the highest discriminative power, allowing the classification of monofloral honey types, such as oak, knotgrass and chestnut honey, as well as the differentiation between honeydew and nectar honeys. The classification model had a high predictive power, as the 84.4% of the group cases was correctly classified, while for the cases of chestnut, strawberry tree and sunflower honeys the respective prediction was correct by 91.3%, 95% and 100%, allowing further determination of unknown honey samples. CONCLUSION: It seems that the characterization of monofloral honeys based on their physicochemical parameters through the proposed model can be achieved and further applied on other honey types. The results could contribute to the development of methodologies for the determination of honey's botanical origin, based on simple techniques, so that these can be applied for routine analysis. © 2021 Society of Chemical Industry.

When a Tritrophic Interaction Goes Wrong to the Third Level: Xanthoxylin From Trees Causes the Honeybee Larval Mortality in Colonies Affected by the River Disease.

The "River Disease" (RD), a disorder impacting honeybee colonies located close to waterways with abundant riparian vegetation (including Sebastiania schottiana, Euphorbiaceae), kills newly hatched larvae. Forager bees from RD-affected colonies collect honeydew excretions from Epormenis cestri (Hemiptera: Flatidae), a planthopper feeding on trees of S. schottiana. First-instar honeybee larvae fed with this honeydew died. Thus, we postulated that the nectars of RD-affected colonies had a natural toxin coming from either E. cestri or S. schottiana. An untargeted metabolomics characterization of fresh nectars extracts from colonies with and without RD allowed to pinpoint xanthoxylin as one of the chemicals present in higher amounts in nectar from RD-affected colonies than in nectars from healthy colonies. Besides, xanthoxylin was also found in the aerial parts of S. schottiana and the honeydew excreted by E. cestri feeding on this tree. A larva feeding assay where xanthoxylin-enriched diets were offered to 1st instar larvae showed that larvae died in the same proportion as larvae did when offered enriched diets with nectars from RD-colonies. These findings demonstrate that a xenobiotic can mimic the RD syndrome in honeybee larvae and provide evidence of an interspecific flow of xanthoxylin among three trophic levels. Further, our results give information that can be considered when implementing measures to control this honeybee disease.

Seed coating with a neonicotinoid insecticide negatively affects wild bees.

Understanding the effects of neonicotinoid insecticides on bees is vital because of reported declines in bee diversity and distribution and the crucial role bees have as pollinators in ecosystems and agriculture. Neonicotinoids are suspected to pose an unacceptable risk to bees, partly because of their systemic uptake in plants, and the European Union has therefore introduced a moratorium on three neonicotinoids as seed coatings in flowering crops that attract bees. The moratorium has been criticized for being based on weak evidence, particularly because effects have mostly been measured on bees that have been artificially fed neonicotinoids. Thus, the key question is how neonicotinoids influence bees, and wild bees in particular, in real-world agricultural landscapes. Here we show that a commonly used insecticide seed coating in a flowering crop can have serious consequences for wild bees. In a study with replicated and matched landscapes, we found that seed coating with Elado, an insecticide containing a combination of the neonicotinoid clothianidin and the non-systemic pyrethroid ß-cyfluthrin, applied to oilseed rape seeds, reduced wild bee density, solitary bee nesting, and bumblebee colony growth and reproduction under field conditions. Hence, such insecticidal use can pose a substantial risk to wild bees in agricultural landscapes, and the contribution of pesticides to the global decline of wild bees may have been underestimated. The lack of a significant response in honeybee colonies suggests that reported pesticide effects on honeybees cannot always be extrapolated to wild bees.

Bees prefer foods containing neonicotinoid pesticides.

The impact of neonicotinoid insecticides on insect pollinators is highly controversial. Sublethal concentrations alter the behaviour of social bees and reduce survival of entire colonies. However, critics argue that the reported negative effects only arise from neonicotinoid concentrations that are greater than those found in the nectar and pollen of pesticide-treated plants. Furthermore, it has been suggested that bees could choose to forage on other available flowers and hence avoid or dilute exposure. Here, using a two-choice feeding assay, we show that the honeybee, Apis mellifera, and the buff-tailed bumblebee, Bombus terrestris, do not avoid nectar-relevant concentrations of three of the most commonly used neonicotinoids, imidacloprid (IMD), thiamethoxam (TMX), and clothianidin (CLO), in food. Moreover, bees of both species prefer to eat more of sucrose solutions laced with IMD or TMX than sucrose alone. Stimulation with IMD, TMX and CLO neither elicited spiking responses from gustatory neurons in the bees' mouthparts, nor inhibited the responses of sucrose-sensitive neurons. Our data indicate that bees cannot taste neonicotinoids and are not repelled by them. Instead, bees preferred solutions containing IMD or TMX, even though the consumption of these pesticides caused them to eat less food overall. This work shows that bees cannot control their exposure to neonicotinoids in food and implies that treating flowering crops with IMD and TMX presents a sizeable hazard to foraging bees.

Species coexistence through simultaneous fluctuation-dependent mechanisms.

Understanding the origins and maintenance of biodiversity remains one of biology's grand challenges. From theory and observational evidence, we know that variability in environmental conditions through time is likely critical to the coexistence of competing species. Nevertheless, experimental tests of fluctuation-driven coexistence are rare and have typically focused on just one of two potential mechanisms, the temporal storage effect, to the neglect of the theoretically equally plausible mechanism known as relative nonlinearity of competition. We combined experiments and simulations in a system of nectar yeasts to quantify the relative contribution of the two mechanisms to coexistence. Resource competition models parameterized from single-species assays predicted the outcomes of mixed-culture competition experiments with 83% accuracy. Model simulations revealed that both mechanisms have measurable effects on coexistence and that relative nonlinearity can be equal or greater in magnitude to the temporal storage effect. In addition, we show that their effect on coexistence can be both antagonistic and complementary. These results falsify the common assumption that relative nonlinearity is of negligible importance, and in doing so reveal the importance of testing coexistence mechanisms in combination.

How Are the Flower Structure and Nectar Composition of the Generalistic Orchid Neottia ovata Adapted to a Wide Range of Pollinators?

Plant-pollinator interactions significantly influence reproductive success (RS) and drive the evolution of pollination syndromes. In the context of RS, mainly the role of flower morphology is touched. The importance of nectar properties is less studied, despite its significance in pollination effectiveness. Therefore, the aim of this study was to test selection on flower morphology and nectar chemistry in the generalistic orchid Neottia ovata. In 2019-2020, we measured three floral displays and six flower traits, pollinaria removal (PR), female reproductive success (FRS), and determined the soil properties. The sugars and amino acids (AAs) were analyzed using the HPLC method. Data were analyzed using multiple statistical methods (boxplots, ternary plot, one-way ANOVA, Kruskal-Wallis test, and PCA). Variation of flower structure and nectar chemistry and their weak correlation with RS confirms the generalistic character of N. ovata. In particular populations, different traits were under selection. PR was high and similar in all populations in both years, while FRS was lower and varied among populations. Nectar was dominated by glucose, fructose, and included 28 AAs (Ala and Glu have the highest content). Sugars and AAs influenced mainly FRS. Among soil parameters, carbon and carbon:nitrogen ratio seems to be the most important in shaping flower structure and nectar chemistry.

Nectar Chemistry or Flower Morphology-What Is More Important for the Reproductive Success of Generalist Orchid Epipactis palustris in Natural and Anthropogenic Populations?

The aim of this study was to determine the level of reproductive success (RS) in natural and anthropogenic populations of generalist orchid Epipactis palustris and its dependence on flower structure and nectar composition, i.e., amino acids and sugars. We found that both pollinaria removal and female reproductive success were high and similar in all populations, despite differences in flower traits and nectar chemistry. Flower structures were weakly correlated with parameters of RS. Nectar traits were more important in shaping RS; although, we noted differentiated selection on nectar components in distinct populations. Individuals in natural populations produced nectar with a larger amount of sugars and amino acids. The sucrose to (fructose and glucose) ratio in natural populations was close to 1, while in anthropogenic ones, a clear domination of fructose and glucose was noted. Our results indicate that the flower traits and nectar composition of E. palustris reflect its generalist character and meet the requirements of a wide range of pollinators, differing according to body sizes, mouth apparatus, and dietary needs. Simultaneously, differentiation of nectar chemistry suggests a variation of pollinator assemblages in particular populations or domination of their some groups. To our knowledge, a comparison of nectar chemistry between natural and anthropogenic populations of orchids is reported for the first time in this paper.