Floral visitors of sesame (Sesamum indicum L.): Elucidating their nectar-robbing behaviour and impacts on the plant reproduction.

Nectar robbing is common in angiosperms, especially in long tubular flowers or flowers with spurs that keep nectar out of reach of visitors. However, the robbing behaviour of bees is less understood. Here, we studied the sesame visitors, their robbing behaviour, and the impacts of robbing on plant reproductive fitness. Diverse insect species (primarily members of Hymenoptera) visited sesame flowers. The most effective pollinators were Amegilla zonata, Apis cerana, Apis dorsata, Apis florea, Ceratina binghami, Halictus acrocephalus and Xylocopa amethystina. Almost all visitors with variable percentages revealed the nectar-robbing phenomenon. Robbing activity depended on a complex of multiple attributes, including the visitor's body size, the corolla tube length, the availability and accessibility of nectar, and the resource-collecting task allocation of bees. Robbing activity varied according to flower-visiting species, flowering period and daytime. Robbing was comparatively higher in the late flowering period at 10.00-14.00 h. In the case of robbing visits, flower handling time was lower, and the visitation rate remained higher than non-robbing visits. Robbing visits did not significantly affect fruit and seed sets of sesame. Therefore, we can interpret the nectar-robbing interactions on sesame as commensal, with pollinators benefitting without altering the plant's reproductive fitness.

Changes in landscape and climate in Mexico and Texas reveal small effects on migratory habitat of monarch butterflies (Danaus plexippus).

The decline of the iconic monarch butterfly (Danaus plexippus) in North America has motivated research on the impacts of land use and land cover (LULC) change and climate variability on monarch habitat and population dynamics. We investigated spring and fall trends in LULC, milkweed and nectar resources over a 20-year period, and ~ 30 years of climate variables in Mexico and Texas, U.S. This region supports spring breeding, and spring and fall migration during the annual life cycle of the monarch. We estimated a - 2.9% decline in milkweed in Texas, but little to no change in Mexico. Fall and spring nectar resources declined < 1% in both study extents. Vegetation greenness increased in the fall and spring in Mexico while the other climate variables did not change in both Mexico and Texas. Monarch habitat in Mexico and Texas appears relatively more intact than in the midwestern, agricultural landscapes of the U.S. Given the relatively modest observed changes in nectar and milkweed, the relatively stable climate conditions, and increased vegetation greenness in Mexico, it seems unlikely that habitat loss (quantity or quality) in Mexico and Texas has caused large declines in population size or survival during migration.

Nontarget impacts of neonicotinoids on nectar-inhabiting microbes.

Plant-systemic neonicotinoid (NN) insecticides can exert non-target impacts on organisms like beneficial insects and soil microbes. NNs can affect plant microbiomes, but we know little about their effects on microbial communities that mediate plant-insect interactions, including nectar-inhabiting microbes (NIMs). Here we employed two approaches to assess the impacts of NN exposure on several NIM taxa. First, we assayed the in vitro effects of six NN compounds on NIM growth using plate assays. Second, we inoculated a standardised NIM community into the nectar of NN-treated canola (Brassica napus) and assessed microbial survival and growth after 24 h. With few exceptions, in vitro NN exposure tended to decrease bacterial growth metrics. However, the magnitude of the decrease and the NN concentrations at which effects were observed varied substantially across bacteria. Yeasts showed no consistent in vitro response to NNs. In nectar, we saw no effects of NN treatment on NIM community metrics. Rather, NIM abundance and diversity responded to inherent plant qualities like nectar volume. In conclusion, we found no evidence that NIMs respond to field-relevant NN levels in nectar within 24 h, but our study suggests that context, specifically assay methods, time and plant traits, is important in assaying the effects of NNs on microbial communities.

Vertically stratified interactions of nectarivores and nectar-inhabiting bacteria in a liana flowering across forest strata.

PREMISE: Vertical stratification is a key feature of tropical forests and plant-frugivore interactions. However, it is unclear whether equally strong patterns of vertical stratification exist for plant-nectarivore interactions and, if so, which factors drive these patterns. Further, nectar-inhabiting bacteria, acting as "hidden players" in plant-nectarivore interactions, might be vertically stratified, either in response to differences among strata in microenvironmental conditions or to the nectarivore community serving as vectors. METHODS: We observed visitations by a diverse nectarivore community to the liana Marcgravia longifolia in a Peruvian rainforest and characterized diversity and community composition of nectar-inhabiting bacteria. Unlike most other plants, M. longifolia produces inflorescences across forest strata, enabling us to study effects of vertical stratification on plant-nectarivore interactions without confounding effects of plant species and stratum. RESULTS: A significantly higher number of visits were by nectarivorous bats and hummingbirds in the midstory than in the understory and canopy, and the visits were strongly correlated to flower availability and nectar quantity and quality. Trochiline hummingbirds foraged across all strata, whereas hermits remained in the lower strata. The Shannon diversity index for nectar-inhabiting bacterial communities was highest in the midstory. CONCLUSIONS: Our findings suggest that vertical niche differentiation in plant-nectarivore interactions seems to be partly driven by resource abundance, but other factors such as species-specific preferences of hummingbirds, likely caused by competition, play an important role. We conclude that vertical stratification is an important driver of a species' interaction niche highlighting its role for promoting biodiversity and ecosystem functioning.

Synergistic negative effects between a fungicide and high temperatures on homing behaviours in honeybees.

Interactions between environmental stressors may contribute to ongoing pollinator declines, but have not been extensively studied. Here, we examined the interaction between the agricultural fungicide Pristine (active ingredients: 25.2% boscalid, 12.8% pyraclostrobin) and high temperatures on critical honeybee behaviours. We have previously shown that consumption of field-realistic levels of this fungicide shortens worker lifespan in the field and impairs associative learning performance in a laboratory-based assay. We hypothesized that Pristine would also impair homing and foraging behaviours in the field, and that an interaction with hot weather would exacerbate this effect. Both field-relevant Pristine exposure and higher air temperatures reduced the probability of successful return on their own. Together, the two factors synergistically reduced the probability of return and increased the time required for bees to return to the hive. Pristine did not affect the masses of pollen or volumes of nectar or water brought back to the hive by foragers, and it did not affect the ratio of forager types in a colony. However, Pristine-fed bees brought more concentrated nectar back to the hive. As both agrochemical usage and heat waves increase, additive and synergistic negative effects may pose major threats to pollinators and sustainable agriculture.

Serial reversal learning in nectar-feeding bats.

We explored the behavioral flexibility of Commissaris's long-tongued bats through a spatial serial reversal foraging task. Bats kept in captivity for short periods were trained to obtain nectar rewards from two artificial flowers. At any given time, only one of the flowers provided rewards and these reward contingencies reversed in successive blocks of 50 flower visits. All bats detected and responded to reversals by making most of their visits to the currently active flower. As the bats experienced repeated reversals, their preference re-adjusted faster. Although the flower state reversals were theoretically predictable, we did not detect anticipatory behavior, that is, frequency of visits to the alternative flower did not increase within each block as the programmed reversal approached. The net balance of these changes was a progressive improvement in performance in terms of the total proportion of visits allocated to the active flower. The results are compatible with, but do not depend on, the bats displaying an ability to 'learn to learn' and show that the dynamics of allocation of effort between food sources can change flexibly according to circumstances.

Generation, degradation mechanism, and toxicity evaluation of pigmented compounds in Leucosceptrum canum nectar.

Leucosceptrum canum nectar (LCN) emerges as a novel food resource, distinguished by its unique dark brown hue. This study delves into the composition and toxicity assessment of novel pigments within LCN. Through liquid chromatography-tandem mass spectrometry (LC-MS/MS) and chemical synthesis, seventeen 2,5-di-(N-(-)-prolyl)-para-benzoquinone (DPBQ) analogs in LCN were identified. These compounds are synthesized in LCN via the Michael addition reaction, utilizing p-benzoquinone (BQ), derived from phenol metabolism, and amino acids as substrates in an alkaline environment (pH = 8.47 ± 0.06) facilitated by dissolved ammonia and the presence of alkaloids. Analytical techniques, including principal component analysis (PCA), orthogonal partial least squares discrimination analysis (OPLS-DA), and volcano plot analysis, were employed to investigate DPBQ analog degradation within the nectar and honey's unique environments. Toxicity assays revealed that DPBQ analogs exhibited no toxicity, displaying a significant difference in toxicity compared to the precursor compound BQ at concentrations exceeding 25 µM.

Imprints of indirect interactions on a resource-mediated ant-plant network across different levels of network organization.

Indirect interactions are pivotal in the evolution of interacting species and the assembly of populations and communities. Nevertheless, despite recently being investigated in plant-animal mutualism at the community level, indirect interactions have not been studied in resource-mediated mutualisms involving plant individuals that share different animal species as partners within a population (i.e., individual-based networks). Here, we analyzed an individual-based ant-plant network to evaluate how resource properties affect indirect interaction patterns and how changes in indirect links leave imprints in the network across multiple levels of network organization. Using complementary analytical approaches, we described the patterns of indirect interactions at the micro-, meso-, and macro-scale. We predicted that plants offering intermediate levels of nectar quantity and quality interact with more diverse ant assemblages. The increased number of ant species would cause a higher potential for indirect interactions in all scales evaluated. We found that nectar properties modified patterns of indirect interactions of plant individuals that share mutualistic partners, leaving imprints across different network scales. To our knowledge, this is the first study tracking indirect interactions in multiple scales within an individual-based network. We show that functional traits of interacting species, such as nectar properties, may lead to changes in indirect interactions, which could be tracked across different levels of the network organization evaluated.

Identification of nectar sources foraged by female mosquitoes in Canada.

For many mosquito species, the females must obtain vertebrate blood to complete a gonotrophic cycle. These blood meals are frequently supplemented by feeding on sugary plant nectar, which sustains energy reserves needed for flight, mating, and overall fitness. Our understanding of mosquito nectar foraging behaviors is mostly limited to laboratory experiments and direct field observations, with little research into natural mosquito-host plant relationships done in North America. In this study, we collected nectar-fed female mosquitoes over a 2-year period in Manitoba, Canada, and amplified a fragment of the chloroplast rbcL gene to identify the plant species fed upon. We found that mosquitoes foraged from diverse plant families (e.g., grasses, trees, ornamentals, and legumes), but preferred certain species, most notably soybean and Kentucky blue grass. Moreover, there appeared to be some associations between plant feeding preferences and mosquito species, date of collection, landscape, and geographical region. Overall, this study implemented DNA barcoding to identify nectar sources forage by mosquitoes in the Canadian Prairies.

Efficacy and safety of PD-1 blockade plus long-course chemoradiotherapy in locally advanced rectal cancer (NECTAR): a multi-center phase 2 study.

Adding PD-1 blockade in the neoadjuvant regimens for locally advanced rectal cancer (LARC) patients with microsatellite stable (MSS) / mismatch repair-proficient (pMMR) tumors is an attractive, but debatable strategy. This phase 2, multicenter, prospective, single-arm study enrolled patients from 6 centers from June 2021 to November 2022. Locally advanced rectal cancer (LARC, cT3-4aN0M0 and cT1-4aN1-2M0) patients aged ≥18 years with the distance from distal border of tumor to anal verge ≤10 cm (identified by Magnetic Resonance Imaging) were qualified for inclusion. The patients received long-course radiotherapy (50 Gy/25 fractions, 2 Gy/fraction, 5 days/week) and three 21-day cycles capecitabine (850-1000 mg/m2, bid, po, day1-14) and three 21-day cycles tislelizumab (200 mg, iv.gtt, day8) as neoadjuvant. Total mesorectal excision (TME) was 6-12 weeks after the end of radiotherapy to achieve radical resection. A total of 50 patients were enrolled in this study. The pathological complete response rate was 40.0% [20/50, 95% confidence interval (CI): 27.61-53.82%], while 15 (30.0%, 95% CI: 19.1-43.75%), 9 (18.0%, 95% CI: 9.77-30.8%), 2 (4.0%, 95% CI: 1.10-13.46%) patients respectively achieved grade 1, 2, and 3 tumor regression. Treatment-related adverse events (TRAEs) occurred in 28 (56.0%) LARC patients, including 26(52.0%) with grade I-II and 2 (4.0%) with grade III (1 with grade 3 immune-related colitis and 1 with grade 3 rash). PD-1 blockade plus long-course chemoradiotherapy (CRT) showed promising therapeutic effects according to pathological complete response rate and is well-tolerated in LARC patients. A larger randomized controlled study is desired to further validate the above findings.

Floral resources enhance fitness of the parasitoid Hadronotus pennsylvanicus (Hymenoptera: Scelionidae) but not biological control of its host Leptoglossus zonatus (Heteroptera: Coreidae).

The diet of adult parasitoid wasps is vital for their survival and reproduction. However, the availability of food resources, such as plant nectar, can vary widely in cropping systems, potentially affecting parasitoid fitness and thereby biological control of pests. The egg parasitoid Hadronotus pennsylvanicus (Ashmead) (Hymenoptera: Scelionidae) is a potential biological control agent of the pistachio pest Leptoglossus zonatus (Dallas) (Heteroptera: Coreidae). While H. pennsylvanicus is known to attack L. zonatus eggs in California, USA, parasitism rates in orchards are highly variable. Floral resource provisioning has the potential to enhance parasitoid longevity and thus improve parasitism rates, leading to reduced pest densities. Here, a combination of field and laboratory studies was used to assess the influence of flowering groundcovers on the reproductive fitness of H. pennsylvanicus and the abundance of L. zonatus. Evaluated groundcovers included oat (Avena sativa L.), cowpea (Vigna unguiculata L.), white mustard (Sinapis alba L.), and buckwheat (Fagopyrum esculentum Moench). Under laboratory conditions, buckwheat and mustard provided the greatest benefit to female H. pennsylvanicus longevity. However, females provided a buckwheat diet produced the greatest number of offspring over the course of their lifetime. In field trials, flowering groundcovers did not influence the abundance of H. pennsylvanicus nor parasitism rates on L. zonatus. While the availability of floral resources can improve the reproductive fitness of H. pennsylvanicus, the use of groundcovers in pistachio did not enhance biological control of L. zonatus.

Genome-Wide Identification and Expression Profile Analysis of Sugars Will Eventually Be Exported Transporter (SWEET) Genes in Zantedeschia elliottiana and Their Responsiveness to Pectobacterium carotovora subspecies Carotovora (Pcc) Infection.

SWEET, sugars will eventually be exported transporter, is a novel class of sugar transporter proteins that can transport sugars across membranes down a concentration gradient. It plays a key role in plant photosynthetic assimilates, phloem loading, nectar secretion from nectar glands, seed grouting, pollen development, pathogen interactions, and adversity regulation, and has received widespread attention in recent years. To date, systematic analysis of the SWEET family in Zantedeschia has not been documented, although the genome has been reported in Zantedeschia elliottiana. In this study, 19 ZeSWEET genes were genome-wide identified in Z. elliottiana, and unevenly located in 10 chromosomes. They were further clustered into four clades by a phylogenetic tree, and almost every clade has its own unique motifs. Synthetic analysis confirmed two pairs of segmental duplication events of ZeSWEET genes. Heatmaps of tissue-specific and Pectobacterium carotovora subsp. Carotovora (Pcc) infection showed that ZeSWEET genes had different expression patterns, so SWEETs may play widely varying roles in development and stress tolerance in Zantedeschia. Moreover, quantitative reverse transcription-PCR (qRT-PCR) analysis revealed that some of the ZeSWEETs responded to Pcc infection, among which eight genes were significantly upregulated and six genes were significantly downregulated, revealing their potential functions in response to Pcc infection. The promoter sequences of ZeSWEETs contained 51 different types of the 1380 cis-regulatory elements, and each ZeSWEET gene contained at least two phytohormone responsive elements and one stress response element. In addition, a subcellular localization study indicated that ZeSWEET07 and ZeSWEET18 were found to be localized to the plasma membrane. These findings provide insights into the characteristics of SWEET genes and contribute to future studies on the functional characteristics of ZeSWEET genes, and then improve Pcc infection tolerance in Zantedeschia through molecular breeding.

Protein/Lipid ratio of pollen biases the visitation of bumblebees (Bombus ignitus Smith) to male-fertile cultivars of the Japanese pear (Pyrus pyrifolia Nakai).

Bees have been known to visit the male-fertile cultivars of self-incompatible flowering plants more frequently than the male-sterile cultivars, but the origin of this preference is poorly understood. Here, we demonstrate that this preference is driven by the higher protein/lipid ratio of male-fertile pollen compared with male-sterile pollen by way of two caged-behavioral assays with six cultivars. In the first assay, flower-naïve bumblebees (Bombus ignitus Smith) showed a significantly higher flower-visitation rate to male-fertile cultivars (pollen germination rate > 55%; > 14 visits/10 min) of the Japanese pear (Pyrus pyrifolia Nakai) than male-sterile cultivars (pollen germination rate ≤ 20%; > 6 visits/10 min). In the second, bees still preferred the anthers of male-fertile cultivars (5-9 visits/10 min) more than those of male-sterile ones (less than 1 visit in 10 min) even in the absence of all other organs (i.e., petals, pistil, nectar), indicating that pollen is responsible for the preference. We then analyzed the macronutrient content of the pollen and its visual cues, and found that the bee preference was highly correlated with the protein/lipid ratio (0.3-1.6) but not color variables such as (a)chromatic contrast, intensity, and spectral purity. We conclude that the protein/lipid ratio influences the foraging behavior of the bumblebees likely by serving as (1) a chemotactile cue while antennating, (2) a gustatory cue after intake, and (3) an olfactory cue. In addition, the low bee visitation rate to poorly viable pollen could be due to its low protein/lipid ratio.

Pollution disrupts nighttime pollination by degrading scents.

Nitrate radicals, a common pollutant, break down the cues nocturnal insects follow to find nectar sources.

Illuminating the Mysteries of the Smallest Birds: Hummingbird Population Health, Disease Ecology, and Genomics.

Hummingbirds share biologically distinctive traits: sustained hovering flight, the smallest bird body size, and high metabolic rates fueled partially by nectar feeding that provides pollination to plant species. Being insectivorous and sometimes serving as prey to larger birds, they fulfill additional important ecological roles. Hummingbird species evolved and radiated into nearly every habitat in the Americas, with a core of species diversity in South America. Population declines of some of their species are increasing their risk of extinction. Threats to population health and genetic diversity are just beginning to be identified, including diseases and hazards caused by humans. We review the disciplines of population health, disease ecology, and genomics as they relate to hummingbirds. We appraise knowledge gaps, causes of morbidity and mortality including disease, and threats to population viability. Finally, we highlight areas of research need and provide ideas for future studies aimed at facilitating hummingbird conservation.

Histochemical, metabolic and ultrastructural changes in leaf patelliform nectaries explain extrafloral nectar synthesis and secretion in Clerodendrum chinense.

BACKGROUND AND AIMS: Extrafloral nectaries are nectar-secreting structures present on vegetative parts of plants which provide indirect defences against herbivore attack. Extrafloral nectaries in Clerodendrum chinense are patelliform-shaped specialized trichomatous structures. However, a complete understanding of patelliform extrafloral nectaries in general, and of C. chinense in particular, has not yet been established to provide fundamental insight into the cellular physiological machinery involved in nectar biosynthesis and secretory processes. METHODS: We studied temporal changes in the morphological, anatomical and ultrastructural features in the architectures of extrafloral nectaries. We also compared metabolite profiles of extrafloral nectar, nectary tissue, non-nectary tissue and phloem sap. Further, both in situ histolocalization and normal in vitro activities of enzymes related to sugar metabolism were examined. KEY RESULTS: Four distinct tissue regions in the nectar gland were revealed from histochemical characterization, among which the middle nectariferous tissue was found to be the metabolically active region, while the intermediate layer was found to be lipid-rich. Ultrastructural study showed the presence of a large number of mitochondria along with starch-bearing chloroplasts in the nectariferous region. However, starch depletion was noted with progressive maturation of nectaries. Metabolite analysis revealed compositional differences among nectar, phloem sap, nectary and non-nectary tissue. Invertase activity was higher in secretory stages and localized in nectariferous tissue and adjacent region. CONCLUSIONS: Our study suggests extrafloral nectar secretion in C. chinense to be both eccrine and merocrine in nature. A distinct intermediate lipid-rich layer that separates the epidermis from nectary parenchyma was revealed, which possibly acts as a barrier to water flow in nectar. This study also revealed a distinction between nectar and phloem sap, and starch could act as a nectar precursor, as evidenced from enzymatic and ultrastructural studies. Thus, our findings on changing architecture of extrafloral nectaries with temporal secretion revealed a cell physiological process involved in nectar biosynthesis and secretion.

A quantitative survey of the blueberry (Vaccinium spp.) culturable nectar microbiome: variation between cultivars, locations, and farm management approaches.

Microbes in floral nectar can impact both their host plants and floral visitors, yet little is known about the nectar microbiome of most pollinator-dependent crops. In this study, we examined the abundance and composition of the fungi and bacteria inhabiting Vaccinium spp. nectar, as well as nectar volume and sugar concentrations. We compared wild V. myrsinites with two field-grown V. corymbosum cultivars collected from two organic and two conventional farms. Differences in nectar traits and microbiomes were identified between V. corymbosum cultivars but not Vaccinium species. The microbiome of cultivated plants also varied greatly between farms, whereas management regime had only subtle effects, with higher fungal populations detected under organic management. Nectars were hexose-dominant, and high cell densities were correlated with reduced nectar sugar concentrations. Bacteria were more common than fungi in blueberry nectar, although both were frequently detected and co-occurred more often than would be predicted by chance. "Cosmopolitan" blueberry nectar microbes that were isolated in all plants, including Rosenbergiella sp. and Symmetrospora symmetrica, were identified. This study provides the first systematic report of the blueberry nectar microbiome, which may have important implications for pollinator and crop health.

Crop-emptying rate and nectar resource allocation in a nectivorous pollinator.

In nectivorous pollinators, timing and pattern of allocation of consumed nectar affects fitness traits and foraging behavior. Differences in male and female behaviors can influence these allocation strategies. These physiological patterns are not well studied in Lepidoptera, despite them being important pollinators. In this study we investigate crop-emptying rate and nectar allocation in Manduca sexta (Sphingidae), and how sex and flight influence these physiological patterns. After a single feeding event, moths were dissected at fixed time intervals to measure crop volume and analyze sugar allocation to flight muscle and fat body. Then we compared sedentary and flown moths to test how activity may alter these patterns. Sedentary males and females emptied their crops six hours after a feeding event. Both males and females preferentially allocated these consumed sugars to fat body over flight muscle. Moths began to allocate to the fat body during crop-emptying and retained these nutrients long-term (four and a half days after a feeding event). Males allocated consumed sugar to flight muscles sooner and retained these allocated nutrients in the flight muscle longer than did females. Flight initiated increased crop-emptying in females, but had no effect on males. Flight did not significantly affect allocation to flight muscle or fat body in either sex. This study showed that there are inherent differences in male and female nectar sugar allocation strategies, but that male and female differences in crop-emptying rate are context dependent on flight activity. These differences in physiology may be linked to distinct ways males and females maximize their own fitness.

Effect of the partial substitution of mango and ground chia on the antioxidant capacity in the elaboration of nectar based on sachatomate.

The consumption of products with high nutritional value and antioxidant capacity has increased notably in recent years. Due to health problems such as triglycerides and cardiovascular problems, its use is becoming reduced. So that, chia (Salvia hispánica) and sachatomate (Cyphomandra betacea) have gained interest as an alternative to develop nutraceutical products, compared to conventional products. The objective of the study was to determine the effect of the partial substitution of mango (Mangifera indica) and ground chia (Salvia hispánica) on the antioxidant capacity in the elaboration of nectar based on Sachatomate. The physicochemical characteristics were determined where sample 11 complies with the established parameters: 13.4° Brix, pH 4.323, 0.354 of C6-H8-O7 and viscosity 3967.3 mPas, according to the NTP 203.110 standard. Regarding the antioxidant capacity, sample 12 was the most optimal, according to the DPPH method, it has been determined 104.3 micromoles Trolox equivalents; according to the ABTS method, it was determined with an antioxidant content of 187.4 micromoles Trolox equivalents. Regarding the proximal chemical evaluation, sample 12 was determined to be the most suitable with a moisture percentage of 87.45%, ash 0.32%, crude fiber 0.09%, fat 0.10%, protein 0.45% and carbohydrates 11.59%. Concluding that substituting sachatomate and ground chia significantly influences the antioxidant capacity, increasing to 104.3 and 187.4 micromoles Trolox equivalents, determined by both methods, indicates that nectar consumption can be used to improve the health of consumers.

Lack of pollinators selects for increased selfing, restricted gene flow and resource allocation in the rare Mediterranean sage Salvia brachyodon.

Range contraction and habitat fragmentation can cause biodiversity loss by creating conditions that directly or indirectly affect the survival of plant populations. Fragmented habitats can alter pollinator guilds and impact their behavior, which may result in pollen/pollinator limitation and selection for increased selfing as a mechanism for reproductive assurance. We used Salvia brachyodon, a narrowly distributed and endangered sage from eastern Adriatic, to test the consequences of range contraction and habitat fragmentation. Molecular data indicate a severe and relatively recent species range reduction. While one population is reproductively almost completely isolated, moderate gene flow has been detected between the remaining two populations. The high pollen-to-ovule ratio and the results of controlled hand pollination indicate that S. brachyodon has a mixed mating system. Quantitative and qualitative differences in the community and behaviour of flower visitors resulted in limited pollination services in one population where no effective pollinator other than pollen and nectar robbers were observed. In this population, self-pollination predominated over cross-pollination. Various environmental factors, in which plant-pollinator interactions play a pivotal role, have likely created selection pressures that have led to genetic and phenotypic differentiation and different resource allocation strategies among populations.