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In recent years, our understanding of the complex chemistry of floral nectar and its ecological implications for plant-pollinator relationships has certainly increased. Nectar is no longer considered merely a reward for pollinators but rather a plant interface for complex interactions with insects and other organisms. A particular class of compounds, i.e., nectar secondary compounds (NSCs), has contributed to this new perspective, framing nectar in a more comprehensive ecological context. The aim of this review is to draft an overview of our current knowledge of NSCs, including emerging aspects such as non-protein amino acids and biogenic amines, whose presence in nectar was highlighted quite recently. After considering the implications of the different classes of NSCs in the pollination scenario, we discuss hypotheses regarding the evolution of such complex nectar profiles and provide cues for future research on plant-pollinator relationships.

International audience; Cuckoo bumblebees are a monophyletic group within the genus Bombus and social parasites of free-living bumblebees, upon which they rely to rear their offspring. Cuckoo bumblebees lack the worker caste and visit flowers primarily for their own sustenance and do not collect pollen. Although different flower-visiting behaviours can be expected between cuckoo and free-living bumblebees due to different biological constraints, no study has yet quantified such differences. Here, we provide the first empirical evidence of different flower-visiting behaviours between cuckoo and free-living bumblebees. We recorded the flower-visiting behaviour of 350 individual bumblebees over two years in a wild population of the entomophilous plant Gentiana lutea, of which they are among the main pollinators. In cuckoo bumblebees (28.9% of the total), we only found males, while we found both workers and males in free-living bumblebees. Cuckoo bumblebees visited significantly more flowers for longer time periods than both free-living bumblebee workers and males within whorls, while differences at the whole-plant level were less marked. Free-living bumblebee males visited more flowers and performed slightly longer flower visits than workers. Behavioural differences between cuckoo male bumblebees and free-living bumblebee workers are likely related to different foraging needs, while differences between cuckoo and free-living bumblebee males may be caused by differences in colony development and a delayed mating period of free-living bumblebees. The longer visits made by cuckoo male bumblebees will likely negatively affect plant reproductive success through increased within-plant pollen flow.

Bertoloni published Florula guatimalensis in 1840, which is commonly cited as the original publication. However, our findings show that previous publications of Alessandrini of 1838 and 1840 include the original descriptions by Bertoloni which predate Florula guatimalensis. Among the 60 new taxa authored by Bertoloni, 26 were published in 1838 and 34 in 1840 by Alessandrini. Additionally, original drawings were discovered inside an old copy of Bertoloni’s Florula guatimalensis. Also a comparison between the original material from Bertoloni’s herbarium and his original drawings is here reported for the first time. The collection of Guatemalan plants studied by Antonio Bertoloni preserved at BOLO was analysed and is here discussed. The new combinations Odontonema corymbulosum and Lamourouxia barbata are proposed. A neotype for Aristolochia podocarpa and an epitype for Philadelphus myrtoides are here designated.

Simple Summary Citizen science is the involvement of the public in scientific research, through the collection of data by volunteer members (“citizen scientists”), usually as part of a collaborative project with professional scientists. Beewatching is a citizen science project started in spring 2018. The project target is to acquire information on wild bee species in Italy and to promote knowledge among citizens on the diversity of bees and their importance as pollinators. Moreover, an attempt was made to follow the distribution of two alien bee species present in Italy. During the first two years of the project, 269 users contributed with 1086 reports. Overall, 38 Apoidea genera and 190 plant genera were reported. Furthermore, we received 22 reports of the alien species Megachile sculpturalis. The increasing number of reports and the higher percentage of correct ones throughout the first period of project implementation, but also the ecological data acquired, undoubtfully represent positive outputs, demonstrating the success of Beewatching project. Abstract Bees play a key role in natural and agro-ecosystems and their diversity is worldwide threatened by anthropogenic causes. Despite this, there is little awareness of the existence of the numerous species of wild bees, and the common name “bee” is very often exclusively associated with Apis mellifera. Our aim was to create a citizen science project in Italy with the following objectives: (a) raising awareness of the importance and diversity of bees, (b) obtaining data on the biology, ecology and distribution of Italian species, and (c) launching the monitoring of alien bees. The first step of the project was to create a website platform with a section containing informative datasheets of the wild bee families and of the most common bee genera present in Italy, a form to send reports of observed bees and an interactive map with all citizen’s reports. During the 2 years of the project 1086 reports were sent by 269 users, with 38 Apoidea genera reported on 190 plant genera; furthermore, 22 reports regarding the alien species Megachile sculpturalis arrived. The majority of bees (34 genera) were observed on spontaneous plants, including 115 genera native to Italy. Considering the increasing number of reports and data obtained in these first two years of the project, our objectives seem to be achieved. Future steps will be to outline the profile of beewatchers, to plan activities in a more targeted way, and also to start some sub-projects for conservation purposes.

Floral nectar is the primary reward directly consumed by floral visitors and its chemical composition affects their behaviour and fidelity. In turn, floral visitors are expected to alter floral nectar composition directly or indirectly through the introduction of external contaminants, such as pollen grains and microorganisms. To understand the effect of exogenous factors on nectar chemistry, we investigated the modifications of nectar sugar and amino acid composition in relation to floral visitors in the perennial herb Gentiana lutea. We quantified nectar contamination by floral visitors through visitor exclusion experiments in the field, estimating pollen and yeast concentration in visited flowers and in uncontaminated nectar at two different flower stages (flower bud and older open flowers). We then assessed changes in the composition and concentration of sugars and both protein and non-protein amino acid in the nectar. We found clear differences in the chemical composition of flower nectar in relation to flower stage and floral visitation. Nectar sugar and amino acid concentrations were significantly higher in older flowers than in flower buds. In addition, nectar sugar concentration was significantly higher in older flowers where visitors were excluded than in visited flowers. On the contrary, amino acid concentration was comparable between visited and non-visited older flowers. We also found that pollen contamination enriched the amino acid profile of the host nectar both quantitatively and qualitatively. Finally, nectar exposed to floral visitors showed a higher number of yeast cells than unvisited flowers although too low to cause the observed changes in the glucose:fructose ratio, suggesting the presence of fructose-related bacteria. Our results show that the chemical composition of floral nectar is altered by several endogenous and exogenous factors in a complex process of ecological relations, and suggest that the modulation of floral visitation through nectar is not solely related to the plant itself.

The decline of pollinators may alter the complex system of interactions that they establish with flowering plants, with potential negative consequences on both partners. Within this context, network analysis may be a useful tool to study ecological properties of plant-pollinator interactions and to evaluate the outcomes of conservation actions. Three conservation measures were implemented within the European LIFE + PP-ICON project to support the local pollinator community of a population of the rare plant Dictamnus albus in a protected area near Bologna, Italy. Artificial nesting sites were installed to support solitary bees, populations of native plants were reinforced to increase foraging resources for pollinators, and colonies of bumblebees reared from wild queens were released in the study area. In this work we evaluate the effects of these conservation actions on plant-pollinator networks over a period of four years, comparing a pre- (2011–2012) and a post-conservation (2013–2014) action period. The overall network generalisation increased after the implementation of conservation measures and interactions were more evenly distributed. Module composition significantly changed between the two periods, showing a marked rewiring of interactions. D. albus was a module hub both before and after conservation actions, thus emerging as an important node within its own module. In addition, some plant and pollinator species directly targeted by conservation measures became module connectors, highlighting their increased importance in linking different modules. Finally, the reinforcement of plant and pollinator populations led to increased flower visitation. These results indicate that conservation actions affected species both directly and indirectly and that the network of interactions has potentially increased its robustness and resilience towards possible species loss. This study highlights ways in which network analysis can be used to measure changes in plant-pollinator interactions in response to conservation actions.

Thisfile contains the main text of the article "Cuckoo bumblebees perform slower and longer flower visits than true bumblebees".

Even though plants represent an essential part of our lives offering exploitational, supporting and cultural services, we know very little about the biology of the rarest and most threatened plant species, and even less about their conservation status. Rapid changes in the environment and climate, today more pronounced than ever, affect their fitness and distribution causing rapid species declines, sometimes even before they had been discovered. Despite the high goals set by conservationists to protect native plants from further degradation and extinction, the initiatives for the conservation of threatened species in Europe are scattered and have not yielded the desired results. The main aim of this Action is to improve plant conservation in Europe through the establishment of a network of scientists and other stakeholders who deal with different aspects of plant conservation, from plant taxonomy, ecology, conservation genetics, conservation physiology and reproductive biology to protected area's managers, not forgetting social scientists, who are crucial when dealing with the general public., COST Action ConservePlants CA18201 "An integrated approach to conservation of threatened plants for the 21st Century", 1 SCIENTIFIC & TECHNOLOGICAL EXCELLENCE 1.1 SOUNDNESS OF THE CHALLENGE 1.1.1 DESCRIPTION OF THE STATE-OF-THE-ART 1.1.2 DESCRIPTION OF THE CHALLENGE (MAIN AIM) 1.2 PROGRESS BEYOND THE STATE-OF-THE-ART 1.2.1 APPROACH TO THE CHALLENGE AND PROGRESS BEYOND THE STATE-OF-THE-ART 1.2.2 OBJECTIVES 1.2.2.1 Research Coordination Objectives 1.2.2.2 Capacity-building Objectives 2 NETWORKING EXCELLENCE 2.1 ADDED VALUE OF NETWORKING IN S&T EXCELLENCE 2.1.1 ADDED VALUE IN RELATION TO EXISTING EFFORTS AT EUROPEAN AND/OR INTERNATIONAL LEVEL 2.2 ADDED VALUE OF NETWORKING IN IMPACT 2.2.1 SECURING THE CRITICAL MASS AND EXPERTISE 2.2.2 INVOLVEMENT OF STAKEHOLDERS 2.2.3 MUTUAL BENEFITS OF THE INVOLVEMENT OF SECONDARY PROPOSERS FROM NEAR NEIGHBOUR OR INTERNATIONAL PARTNER COUNTRIES OR INTERNATIONAL ORGANISATIONS 3 IMPACT 3.1 IMPACT TO SCIENCE, SOCIETY AND COMPETITIVENESS, AND POTENTIAL FOR INNOVATION/BREAKTHROUGHS 3.1.1 SCIENTIFIC, TECHNOLOGICAL, AND/OR SOCIOECONOMIC IMPACTS (INCLUDING POTENTIAL INNOVATIONS AND/OR BREAKTHROUGHS) 3.2 MEASURES TO MAXIMISE IMPACT 3.2.1 KNOWLEDGE CREATION, TRANSFER OF KNOWLEDGE AND CAREER DEVELOPMENT 3.2.2 PLAN FOR DISSEMINATION AND/OR EXPLOITATION AND DIALOGUE WITH THE GENERAL PUBLIC OR POLICY 4 IMPLEMENTATION 4.1 COHERENCE AND EFFECTIVENESS OF THE WORK PLAN 4.1.1 DESCRIPTION OF WORKING GROUPS, TASKS AND ACTIVITIES 4.1.2 DESCRIPTION OF DELIVERABLES AND TIME FRAME 4.1.3 RISK ANALYSIS AND CONTINGENCY PLANS Funding program References

There is increasing interest in evaluating biodiversity to preserve ecosystem services. Researchers can sustain policymakers by providing tools, such as indexes and indicators, that need constant implementation to become accepted standards. Implementation may vary from re-evaluation of existing indicators to introduction of new ones based on emerging threats to biodiversity. With the aim of contributing to the compelling need to estimate and counterbalance pollinator loss, we screened existing bioindicators. We first selected indexes/indicators applied to agricultural contexts and concurrently endorsed by a regulatory agency. We then extended our analysis to indexes/indicators based on arthropod taxa and formally recognized at least by national bodies. Our procedure identified a combination of surveys of various animal taxa and remote landscape analyses (e.g., using a GIS and other cartographic tools). When the animals are arthropods, most indexes/indicators can only address confined environments (e.g., grasslands, riversides). Indicator strength was improved by the simultaneous inclusion of biotic and abiotic components. Pollinator sensitivity to changes at micro-habitat level is widely appreciated and may help distinguish agricultural practices. A biodiversity index based on pollinators, including a wide monitoring scheme supplemented by citizen science, is currently fostered at the European level. The results obtained using such an index may finally enable focusing of strategic funding. Our analysis will help to reach this goal.

Floral nectar is a chemically complex aqueous solution within which several secondary metabolites have been identified that affect attractiveness for pollinators. Understanding preferences and aversions to nectar quality in flower visitors is crucial since this may influence the patterns of insect floral visitation with consequences on the plant fitness. We hypothesise that nectar chemical variation through different floral sexual phases may affect the number of insect visits that each phase receives. The study was realized on a population of Echium vulgare L. growing in a natural area close to Bologna. Nectar was collected from functionally male and female flowers to investigate its chemical composition through the HPLC technique. A total of 200min of behavioural observations on foraging insects was also carried out. Variation in nectar traits has been detected for the amino acid spectrum. The proportion of protein amino acids appeared to be significantly higher in male-phase flowers. This may explain the significantly higher number of visits on male flowers than expected observed for all bee taxa (except Hoplitis adunca females). Functionally male flowers presented higher concentrations of phenylalanine, whilst proline was highly represented in functionally female flowers. Since a recent study demonstrated that hymenopterans can oxidize proline at a high rate for ATP production, we can hypothesise that the quality of nectar offered by the two sexually distinct floral phases targets different insect behavioural traits and likely ensures an optimal pattern of visit among flower sexes, which are unequally distributed within and among individuals in the population.

To investigate the effects of two nectar nonprotein amino acids, β-alanine and γ-aminobutyric acid (GABA), on Osmia bicornis survival and locomotion, two groups of caged bees were fed with sugar syrup enriched with β-alanine and GABA, respectively. A further control group was fed with sugar syrup. Five behavioural categories were chosen according to the principle of parsimony and intrinsic unitary consistency from start to end, and recorded by scan sampling: two states (remaining under paper or in tubes) and three events (walking on net, feeding from flower and flying). We also analysed the amino acid content of haemolymph sampled from an additional 45 bees fed the same diets (15 per diet type). Bees fed with β-alanine had a significantly shorter survival time than those fed with the control and GABA diets. The GABA diet induced higher levels of locomotion than β-alanine. The former nonprotein amino acid was only detected in the haemolymph of bees fed GABA. The results suggest that insects consuming nonprotein amino-acid-rich diets absorb and transfer these substances to the haemolymph and that nonprotein amino acids affect survival and locomotion. Ecological consequences are discussed in the framework of plant reproductive biology.

Gentiana lutea L. (yellow gentian, Gentianaceae) is a protected orophyte of central and southern Europe, mainly threatened by the uncontrolled collection of its rhizome, used in traditional medicine and for liquor production. The species is self-compatible, but outcrossing mediated by pollinators is needed to obtain a viable progeny. In this study, we considered five natural populations belonging to the four subspecies of G. lutea. We performed controlled pollinations in the field (self- versus cross-pollination) followed by seed germination tests in laboratory conditions, adding a solution of gibberellic acid, in order to evaluate the seed performance. A cumulative index of inbreeding depression was calculated considering maternal reproductive output as well as seed performance traits. Seed weight and seed germination performance was similar between seeds resulting from naturally pollinated and pollen-augmented flowers and higher compared to selfed flowers, highlighting a disadvantage of selfing and the importance of cross-pollen transfer in natural conditions. However, in the small and isolated population of G. lutea subsp. symphyandra on Mt Grande we found a general reduction in seed germination rate, likely due to increased selfing or mating among close relatives as a consequence of a severe bottleneck. We discuss our results with regards to implications for conservation practices.

Background: Taxonomic analysis provides a basic understanding for taxon identification and contributes to preliminary information for several branches of applied biology, while studies on reproductive strategies and plant fitness are essential to interpret population status and dynamics. Aims: We tested the reliability of diagnostic characters for identification and to characterise sexual resource allocation, the breeding system and seed predation among subspecies of Gentiana lutea. Methods: We analysed morphological characters in 70 herbarium specimens. In five natural populations we counted pollen and ovule numbers, assessed reproductive output after pollination treatments and evaluated pre-dispersal predation. Results: Taxonomic traits previously indicated as diagnostic were not sufficient to discriminate among subspecies. The pollen number and pollen:ovule (P:O) ratio varied strongly among subspecies; self-pollinated flowers produced a significantly lower number of seeds than open-pollinated flowers. Retention of empty fruits and high levels of pre-dispersal seed predation were observed in every case. Conclusions: The variation of P:O ratios among subspecies suggests different efficiency in pollen transfer. The species is self-compatible, nevertheless all subspecies require pollen vectors to enhance cross pollination and viable seed production. Fruit retention may have evolved as a strategy to reduce predation, ensuring higher plant fitness.

Nectar mediates complex interactions between plants and animals. Recent research has focused on nectar secondary compounds that may play a role in regulating some of these interactions. These compounds may affect the behavior of nectar feeders by interacting with their neurobiology. Non-protein amino acids (NPAAs) can constitute a large portion of the amino acid content of floral nectar, but their ecological function has, to date, not been investigated. In this study, we tested the effects of diets with low and high concentrations of γ-amino butyric acid (GABA) and β-alanine on the survival and behavior of Bombus terrestris and Apis mellifera. The most apparent effect on longevity was observed for B. terrestris workers that fed on high concentration of GABA, with longevity increased. By contrast, neither of the two NPAAs (at either concentration) had an affect on A. mellifera longevity. At the low NPAA concentration, only B. terrestris workers showed a difference in consumption, consuming more β-alanine solution than the other two solutions. By contrast, at the high NPAA concentration, only A. mellifera workers showed a difference in consumption, consuming more β-alanine solution. The effects of the NPAAs on behavior differed between the two species, with B. terrestris appearing more sensitive to the NPAAs than A. mellifera. After consuming NPAAs, B. terrestris showed changes in three (walking, flying, stationary) of the four behaviors recorded, although the effects varied with concentration and compound. In contrast, honey bees only showed a change in feeding behavior, with consumption of both NPAAs (at low concentrations) resulting in a decrease. Thus, pollinator intake of NPAAs may have important behavioral/ecological implications.

Bees collect pollen as an important resource for offspring development while acting as pollen vectors for the plants visited. Foraging preferences of pollinators together with plant species availability shape the web of interactions at the local scale. In this study, we focused on the bee pollinator community of a population of the rare protected perennial herb Dictamnus albus, with the aim to characterise the pollen preferences and the foraging niche overlap among species through time. Bees were sampled during four consecutive years in a natural population of D. albus, throughout the blooming period of the plant species. We performed an analysis of insect pollen loads to investigate the interactions with the study species and the co-flowering plants in the area, and to evaluate the degree of foraging overlap among pollinators. Over the study years, all bee species showed a high fidelity to D. albus (60–80%), even if some taxa preferentially collected pollen from other flowering species. The foraging niche overlap in the pollinator community decreased together with an increased diversity of co-flowering plant species. The results obtained indicate that bees preferentially forage on D. albus in the studied area, but that co-flowering species contribute to complement their diet and likely reduce competition for foraging resources. It appears therefore important to maintain a high diversity of co-flowering plants to preserve the diversity in the studied pollinator community of D. albus.

Generalist flowers are visited by a broad variety of insects that function as pollinators, occasional visitors and as pollen and/or nectar robbers. Moreover, among legitimate pollinators the pollination efficiency can be different. Nectar greatly affects visitor behaviour and fidelity to a certain species, influencing plant reproductive effort. In this study we have investigated a generalist system (Gentiana lutea L.), examining the role of flower visitors and quantifying the contribution of each pollinating taxon in three natural populations. In order to verify the level of generalization, we introduce an index of Pollinator Performance (PoP), based on insect visitation rate and cross-pollen transport efficiency. Our results confirm the high degree of pollinator-generalization of the study species. Nevertheless, flower visitors show various degree of pollinating performance, mainly defined by their sedentary versus dynamic behaviour. Sedentary insects enhance geitonogamous pollen transfer, which results in reduced seed set and pollen limitation. In particular, an unusual sluggish behaviour was observed in bumblebees feeding on nectar. The hexose-rich abundant nectar offered by G. lutea flowers is remarkably rich in proline and β-alanine amino acids: this composition presumably influences feeding choice and insect dynamism, likely exerting a narcotic effect on pollinators. The consequences on plant fitness are discussed in an evolutionary perspective.

To investigate the effects of two nectar nonprotein amino acids, β-alanine and γ-aminobutyric acid (GABA), on Osmia bicornis survival and locomotion, two groups of caged bees were fed with sugar syrup enriched with β-alanine and GABA, respectively. A further control group was fed with sugar syrup. Five behavioural categories were chosen according to the principle of parsimony and intrinsic unitary consistency from start to end, and recorded by scan sampling: two states (remaining under paper or in tubes) and three events (walking on net, feeding from flower and flying). We also analysed the amino acid content of haemolymph sampled from an additional 45 bees fed the same diets (15 per diet type). Bees fed with β-alanine had a significantly shorter survival time than those fed with the control and GABA diets. The GABA diet induced higher levels of locomotion than β-alanine. The former nonprotein amino acid was only detected in the haemolymph of bees fed GABA. The results suggest that insects consuming nonprotein amino-acid-rich diets absorb and transfer these substances to the haemolymph and that nonprotein amino acids affect survival and locomotion. Ecological consequences are discussed in the framework of plant reproductive biology.

International audience; AbstractInbreeding is caused by the mating of closely related individuals and may produce a decrease in the fitness of offspring and have deleterious consequences for adults. In haplodiploid social Hymenoptera inbreeding has a further negative effect due to the production of unviable or sterile diploid males. As a consequence, mechanisms of inbreeding avoidance would be expected to evolve. In this study, we investigated the mating choice between related (inbred) or unrelated (outbred) gynes and males of Bombus terrestris reared in laboratory conditions by performing cage and tunnel experiments. Not only did we find no mating preference for related or unrelated partners (mating success 41.55 ± 3.7 and 39.69 ± 4.4%, respectively), but the mating latency was even shorter in inbred (6.97 ± 0.6 min) than in outbred matings (8.74 ± 0.8 min). We hypothesize that in wild populations of B. terrestris, the lack of incest avoidance could be compensated by tolerance of high levels of inbreeding.

International audience; Diapause control and colony initiation are among the major problems encountered in the rearing of bumble bee colonies in small-scale rearing. In this study, we used Bombus terrestris queens obtained from commercial colonies to investigate (1) the diapause survival in virgin and mated queens, (2) the diapause and colony initiation performance of mated bumble bee queens in relation to the pre-diapause weight and (3) the effect of pupae’s sex on colony initiation. We found that diapause survival is negatively affected by mating and by the low pre-diapause weight, but first egg deposition and development of the first adult worker were delayed in heavy queens. We found no significant differences in the egg-laying success in relation to pupae’s sex; however, queens stimulated with queen pupae laid more eggs per cell and developed a first brood larger than those stimulated with male pupae. Our results can be useful in small-scale rearing, including the rearing of wild queens for conservation purposes.

Disassortative mating in distylous self-incompatible species should result in the equilibrium of morph types in natural populations. Deviation from isoplethy may affect pollen transfer, and in isolated populations it could lead to Allee effect and genetic drift. Pollen limitation has been found to occur in several distylous species, for which mating opportunities are actually reduced to half population. In this study, we investigated the reproductive features and pollination ecology of the narrow endemic Primula apennina. We recorded equilibrium of morph frequencies in the studied population, reflecting the comparable fecundity found in the two morphs. Long-styled flowers produce more pollen grains of smaller size than short-styled ones: we hypothesize that in thrum flowers, pollen is more easily removed by the insect pollinator Macroglossum stellatarum, resulting in equal pollen amounts carried to both short styles and long styles. This lower pollen transfer efficiency from long-styled to short-styled flowers is also reflected in legitimate pollen-ovule ratio values. Despite results show no evidence of imminent threats to population persistence at study site, the strict dependence on one or very few pollinator species, and ecological traits, may increase extinction risks in the long-term period.

Plants with multiple flowers could be prone to autonomous self-pollination and insect-mediated geitonogamy, but physiological and ecological features have evolved preventing costs related to autogamy. We studied the rare perennial herb Dictamnus albus as a model plant, with the aim of describing the plant-pollinator system from both plant and pollinator perspectives and analysing features that promote outcrossing in an entomophilous species. The breeding system and reproductive success of D. albus were investigated in experimental and natural conditions, showing that it is potentially self-compatible, but only intra-inflorescence insect-mediated selfing is possible. Nectar analysis showed gender-biased production towards the female phase, which follows the male phase, and during flowering, full blooming is found in flowers at the bottom of the raceme. Among a wide spectrum of insect visitors, three genera (Bombus, Apis, Megachile) were found to be principal pollinators. A study of insect behaviour showed a tendency towards bottom-to-top flights for the most important pollinators Bombus spp. and Apis mellifera: upward movements on the racemes could be explained by foraging behaviour, from more to less rewarding flowers. In accordance with the 'declining reward hypothesis', bumblebees and honeybees leave the plant when gain of reward is low, after which few flowers are visited, reducing the chance of self-pollen transfer among flowers. Intra-flower self-pollination is prevented in D. albus by protandry and herkogamy, while the nectar-induced sequential pattern of pollinator visits avoids geitonogamy and tends to maximise pollen export, promoting outcrossing. All these features for preventing selfing benefit plant fitness and population genetic structure.

Knowledge about mixed mating systems can improve our understanding of the evolutionary dynamics of reproductive systems. Here we report a study of the floral and reproductive biology of Hypericum elodes, an Atlantic-European soft-water pools specialist which shows a floral architecture consistent with both self- and cross-pollination. Controlled pollination experiments were performed in a natural population during three consecutive years. Marked flowers were monitored until fruit production, and laboratory germination experiments were conducted with the seeds produced. Plants were self-compatible (SCI>0.75), however, compared with selfing, cross-pollination enhanced fruit-set, seed-set and seedling growth, but not seed germination. Inbreeding depression (δ) was mild in the pre-dispersal stages (δ = 0.22 for fruit set, 0.18 for seed set and 0.13 for seed mass), low for germination percentage (δ = 0.003) and mild for seedling growth (δ = 0.23). The breeding system of H. elodes promotes outcrossing and assures reproductive success by means of competitive autogamy. Our results suggest a mixed mating strategy for the studied population, characterized by mild inbreeding depression (cumulative δ = 0.57), highlighting the benefit of this reproductive mode in unpredictable habitat, as the typical shallow-water meadows where H. elodes grows.

Although several factors can limit female fertility in perennial plants, rarely have they been jointly studied in a single species over several years. In this study we experimentally manipulate seed production and simultaneously analyse the potential contribution of pollen limitation, costs of reproduction and plant size to variation in seed output over a 3-year period in the perennial herb Paeonia officinalis, in southern France. Since this rare species is threatened by forest closure in many sites we also examine the causes of female fertility variation in relation to habitat closure (open habitat vs. woodland). P. officinalis has a partial self-incompatibility system and only very low ability for autonomous self pollination in the absence of pollinators. However, supplementary pollination of individual plants in three consecutive years did not significantly increase seed production above natural levels. Forest closure was associated with a decline in ovule and seed production, which again was not due to pollen limitation since supplementary pollination had no significant effect on seed set in the woodland habitat. Comparison of the maternal fertility of plants which were previously excluded from reproduction with those which were hand pollinated to maximise seed set in two previous years produced no evidence that seed production in year three is limited by costs associated with prior reproduction. Likewise, flowering probability was not related to prior seed production but was however positively related to plant size. The absence of any influence of pollen limitation or prior reproduction on seed production suggests that sub-maximal seed production in long-lived perennial herbs may be part of a size-dependent strategy that maximises life-time seed production and fitness without compromising survival.

The interplay between insect and plant traits outlines the patterns of pollen transfer and the subsequent plant reproductive fitness. We studied the factors that affect the pollination efficiency of a pollinator community of Dictamnus albus L. by evaluating insect behaviour and morphological characteristics in relation to flowering phenology. In order to extrapolate the pollinator importance of single taxa and of the whole pollinator guild, we calculated an index distinguishing between potential (PPI) and realized (RPI) pollinator importance. Although the pollinator species spectrum appeared rather constant, we found high intra- and inter-annual variability of pollinator frequency and importance within the insect community. Flower visitation rate strictly depended on insect abundance and on the overlap between their flying period and flower blooming. All the pollinators visited flowers from the bottom to the top of the racemes, excluding intra-plant geitonogamous pollination, and most of them showed high pollen fidelity. Only medium large-sized bees could contact the upward bending stiles while feeding on nectar, highlighting a specialisation of the plant towards bigger pollinators. Moreover, we found evidence of functional specialisation, since all pollinators were restricted to a single taxonomic group (order: Hymenoptera; superfamily: Apoidea). Both the PPI and RPI indices indicate Habropoda tarsata as the most important pollinator of D. albus. Following hand cross-pollination experiments we revealed the presence of pollination limitation in 1 of the 3 years of field study. We discuss this result in relation to flowering abundance and to possible mismatches of phenological periods between plants and insects.

We studied the allocation of reproductive resources in 32 Mediterranean legume species. Correlations between main parameters were analysed at interspecific level. For each species we described the type of pollen presentation and examined its systematic significance. The high P/O values observed reflect low pollen transfer efficiency, typical of xenogamous species. Lowest P/Os were found in species with brush or explosive tripping mechanism, while highest ones were associated to primary pollen presentation. No trade-off was observed between P/O and the volume of a single pollen grain, nor between pollen number and pollen grain volume. Perennials showed higher P/Os compared to annuals, in accordance with the assumption that annual species are often self-compatible while most perennials are outcrossers. Fruit and seed set, evaluated in eight species under different experimental conditions, showed a close dependence on insects, and the existence of self-compatibility in three species (Colutea arborescens, Coronilla emerus, Hedysarum coronarium). In Colutea arborescens and Coronilla emerus pollen-pistil interaction and pre-zygotic barriers to selfing were investigated through stigma receptivity tests and in-vivo pollen germination trials. Results demonstrated the importance of the stigmatic cuticle in reducing levels of autogamy. Productivity tests confirmed the utility of P/O ratio as indirect measure of species' breeding system.

Papilionaceous flowers present functional traits commonly associated to the so-called “bee-syndrome”. Despite this evolutionary specialization, many legume species are visited by a large number of insect species. In a sample of seven perennial Mediterranean legumes we focus on the ecological aspects of plant-pollinator interactions. Our aim is to compare the degree of specialization of pollination systems among species showing similar floral traits associated with bee pollination. In particular we ask the following questions: (1) Do all flower visitors act as pollinators? (2) Are pollinators equally important? and (3) Is there any relationship between the degree of specialization of plant-pollinator system and plant fitness? We describe the spectrum of visitors and identify the potential pollinators by observing insects’ behaviour on flowers. The diversity of each population’s insect assemblage (flower visitors and potential pollinators) is calculated in order to estimate to which degree pollination systems are specialized. The importance value (PI) for main taxa of potential pollinators is computed considering both quality and quantity components of the pollination service: insect fidelity and relative abundance. Pollinator assemblages show different values of importance and dominance, revealing various degrees of plant-pollinator specificity. Visitors’ and pollinators’ diversity is negatively correlated with fruiting success, while there is a positive relation between maximum pollinator importance value and fruit set, suggesting the tendency towards a positive relationship between pollinator specificity and reproductive success.

Cytiseae have been reported to be mostly nectar-lacking, yet some taxa secrete nectar from extrastaminal nectaries. We studied the pollination biology of four shrubby species of Cytiseae (Cytisophyllum sessilifolium (L.) Lang, Spartium junceum L., Genista radiata (L.) Scop., Genista cilentina Valsecchi) which differ for ecology, distribution and population size. All species resulted obliged xenogamous, insect visits being necessary for successful pollination. Bumblebees and solitary bees are the principal pollinators, but also many beetles, some hover-flies, and few bugs visit flowers. Pollinator specificity is low, and this may be the reason of the scarce seed set compared to the number of ovules. Pollen is the main reward, but traces of glucose were detected in all species, at the base of vexillum or on the reproductive column. Nectar production is irregular in time, and apparently unpredictable. We suppose that nectar may play a role in attracting pollinators determining their right position for a successful pollination.

none 6 no In many species, inbreeding avoidance mechanisms prevent mating between close relatives, but these mechanisms are poorly studied in bumble bees. The probability of inbred matings within a colony in eusocial insects may depend on the timing of gyne and male emergence and on their sex ratio. In this study, we compared the development of 35 colonies of the bumble bee Bombus terrestris from founding to the emergence of the last gyne, and we investigated the probability of inbred mating in colonies with fertile newborn gynes and males. We calculated a novel colony inbreeding risk index (IRI), which considers the overlap period between fertile gynes and males, their numbers, and the colony sex ratio. We found that the IRI values were strictly correlated with the time elapsed between the gyne point and the switch point (i.e., from the moment of deposition of the first diploid egg that produces a gyne to the first haploid egg). We separated the colonies into two groups based on the mean value of the IRI: colonies with low IRI produced more gynes (93.8 ± 9.4), for a longer period (32.1 ± 1.8 days) and with a lower percentage of overlapped gynes (69 ± 5%) than colonies with high IRI (57.1 ± 21 gynes, 23.3 ± 2.9 days, and 100% overlapped gynes, respectively). A low IRI is connected to a reduced risk of inbred mating, while colonies with a high IRI may be advantaged in conditions of isolation, in case of the absence of non-related reproducers. Inbreeding risk index proved to be a good indicator of the colony reproductive strategy. none Gherardo Bogo; Natasha de Manincor; Alessandro Fisogni; Marta Galloni; Laura Zavatta; Laura Bortolotti Gherardo Bogo; Natasha de Manincor; Alessandro Fisogni; Marta Galloni; Laura Zavatta; Laura Bortolotti