Phylogenomic insights into the historical biogeography, character-state evolution, and species diversification rates of Cypripedioideae (Orchidaceae).

Cypripedioideae (slipper orchids; Orchidaceae) currently consist of âˆ¼200 herbaceous species with a strikingly disjunctive distribution in tropical and temperate regions of both hemispheres. In this study, an updated phylogeny with representatives from all five cypripedioid genera was presented based on maximum likelihood and Bayesian inference of plastome and low-copy nuclear genes. Phylogenomic analyses indicated that each genus is monophyletic, but some relationships (e.g., those among Cypripedium sects. Acaulia, Arietinum, Bifolia, Flabellinervia, Obtusipetala and Palangshanensia) conflict with those in previous studies based on Sanger data. Cypripedioideae appeared to have arisen in South America and/or the adjacent Qinghai-Tibet Plateau and Hengduan Mountains âˆ¼35 Mya. We inferred multiple dispersal events between East Asia and North America in Cypripedium, and between mainland Southeast Asia and the Malay Archipelago in Paphiopedilum. In the Americas, divergences among four genera (except Cypripedium) occurred around 31-20 Mya, long before the closure of the Isthmus of Panama, indicating the importance of long-distance dispersal. Evolutionary patterns between morphological and plastome character evolution suggested several traits, genome size and NDH genes, which are likely to have contributed to the success of slipper orchids in alpine floras and low-elevation forests. Species diversification rates were notably higher in epiphytic clades of Paphiopedilum than in other, terrestrial cypripedioids, paralleling similar accelerations associated with epiphytism in other groups. This study also suggested that sea-level fluctuations and mountain-building processes promoted the diversification of the largest genera, Paphiopedilum and Cypripedium.

Community-wide patterns in pollen and ovule production, their ratio (P/O), and other floral traits along an elevation gradient in southwestern China.

BACKGROUND: As the male and female gametophytes of flowering plants, pollen and ovules largely determine the upper and lower boundaries of plant reproductive success. It is commonly predicted that pollen and ovule number per flower should increase, and pollen-ovule ratio (P/O) per flower should decrease with increasing elevation in response to a more stochastic pollination environment. Here, we aimed to determine the response of pollen number, ovule number, and P/O to other floral traits and elevation gradients for 84 insect-pollinated herbaceous flowering plant species in five sub-alpine and alpine communities (2709 to 3896 m a.s.l.) on Yulong Snow Mountain, southwestern China. RESULTS: Six floral traits, including P/O, floral display area, flower number, tube depth, flower shape, and pollen presentation, were highly correlated with pollen and ovule number per flower. With increasing elevation, pollen number and P/O per flower increased marginally and significantly, respectively; ovule number per individual, flower number per individual, stigma stamen separation, and inflorescence height decreased significantly. However, ovule number per flower and other floral traits (i.e., floral display area, tube depth, stigma height, stamen height, and pollen and P/O per individual) did not change with elevation. We detected significant phylogenetic signals for pollen number, ovule number, and P/O, suggesting that these traits may be highly conserved and with limited response to changing environmental conditions. CONCLUSIONS: Results revealed patterns of plant reproductive character evolution along elevation gradients and the potential factors governing their spatial variation in high-elevation environments. Plant species at high elevations are more likely adapted to cross-pollination, indicated by increased P/O per flower at high elevations on Yulong Mountain. Combined effects of phylogenetic history and plant-pollinator interactions should determine plant trait evolution.

Floral nectar reabsorption and a sugar concentration gradient in two long-spurred Habenaria species (Orchidaceae).

BACKGROUND: Floral nectar is the most common reward flowers offered to pollinators. The quality and quantity of nectar produced by a plant species provide a key to understanding its interactions with pollinators and predicting rates of reproductive success. However, nectar secretion is a dynamic process with a production period accompanied or followed by reabsorption and reabsorption remains an understudied topic. In this study, we compared nectar volume and sugar concentration in the flowers of two long-spurred orchid species, Habenaria limprichtii and H. davidii (Orchidaceae). We also compared sugar concentration gradients within their spurs and rates of reabsorption of water and sugars. RESULTS: Both species produced diluted nectar with sugar concentrations from 17 to 24%. Analyses of nectar production dynamics showed that as flowers of both species wilted almost all sugar was reabsorbed while the original water was retained in their spurs. We established a nectar sugar concentration gradient for both species, with differences in sugar concentrations at their spur's terminus and at their spur's entrance (sinus). Sugar concentration gradient levels were 1.1% in H. limprichtii and 2.8% in H. davidii, both decreasing as flowers aged. CONCLUSION: We provided evidence for the reabsorption of sugars but not water occurred in wilted flowers of both Habenaria species. Their sugar concentration gradients vanished as flowers aged suggesting a slow process of sugar diffusion from the nectary at the spur's terminus where the nectar gland is located. The processes of nectar secretion/reabsorption in conjunction with the dilution and hydration of sugar rewards for moth pollinators warrant further study.

Observations on the pollination and breeding systems of two Corybas species (Diurideae; Orchidaceae) by fungus gnats (Mycetophilidae) in southwestern Yunnan, China.

Modes of floral presentation in some angiosperms attract flies that eat and/or oviposit on seasonal fruiting bodies of fungi. Mushroom mimesis by orchid flowers has been speculated in the geoflorous, Indo-Malaysian-Australasian, genus Corybas s.l. for decades but most studies remain fragmentary and are often inconclusive. Here we report the roles of fungus gnats as pollinators of Corybas geminigibbus and C. shanlinshiensis in southwestern Yunnan, China, combining results of field observations, lab analyses, and manipulative experiments. Hand pollination experiments suggested both species were self-compatible but incapable of mechanical self-pollination, thereby requiring pollinators for fruit production. A female of a Phthinia sp. (Mycetophilidae) carried a pollinarium of C. geminigibbus dorsally on its thorax. Two females and one male of Exechia sp. (Mycetophilidae) visiting flowers of C. shanlinshiensis carried dorsal depositions of pollinaria on their thoraces. Mycetophilid eggs were not found in the flowers of either species. The comparative fragrance analyses of these flowers and three co-fruiting mushroom species did not suggest that either orchid species was a brood-site mimic. This is the first confirmation of the dispersal of pollinaria of Corybas species by fungus gnats in subtropical-temperate Asia.

Morphological trait-matching in plant-Hymenoptera and plant-Diptera mutualisms across an elevational gradient.

Morphological trait-matching and species abundance are thought to be the main factors affecting the frequency and strength of mutualistic interactions. However, the relative importance of trait-matching and species abundance in shaping species interactions across environmental gradients remains poorly understood, especially for plant-insect mutualisms involving generalist species. Here, we characterised variation in species and trait composition and the relative importance of trait-matching and species abundance in shaping plant-Hymenoptera and plant-Diptera mutualisms in four meadows across an elevational gradient (2,725-3,910 m) in Yulong Snow Mountain, Southwest China. We also evaluated the effects of morphological traits of flower visitors and plant composition on their foraging specialisation (d' and normalised degree). There was a high degree of dissimilarity in the composition of Hymenoptera and Diptera visitors and their visited plants between communities. This variation was mainly driven by the spatial replacement of species. Both for plant-Hymenoptera and plant-Diptera networks, trait-matching between nectar tube depth and proboscis length was a stronger predictor of the interactions between temporally co-occurring plants and flower visitors than species abundance. Fourth-corner analyses revealed statistically significant trait-matching between nectar tube depth and proboscis length in plant-Hymenoptera networks at all sites, suggesting that Hymenoptera consistently foraged on plant species with nectar tube depths matching their proboscis lengths. By contrast, significant trait-matching in plant-Diptera networks was only observed at the two lower elevation sites. The species-level specialisation d' of flower visitors increased significantly as the proboscis length and the difference in nectar tube depth between the plant community and the plants visited by flower visitors increased. Our results highlight that the importance of trait-matching in shaping pairwise interactions and niche partitioning depends on the specific features (e.g. species composition and trait availability) of the plant-pollinator system. For specialised plant-Hymenoptera systems, trait-matching is an important determinant of species interactions, whereas for generalist plant-Diptera systems, trait-matching is relatively unimportant.

Changes in floral nectar are unlikely adaptive responses to pollinator flight sound.

Under noiseless experimental conditions, sugar concentration of secreted floral nectar may increase after flower exposure to nearby sounds of pollinator flight (Veits et al. 2019). However, we reject the argument that this represents adaptive plant behaviour, and consider that the appealing analogy between a flower and human ear is unjustified.

Specificity and seasonal prevalence of anther smut disease Microbotryum on sympatric Himalayan Silene species.

Host sympatry provides opportunities for cross-species disease transmission and compounded disease effects on host population and community structure. Using the Silene-Microbotryum interaction (the castrating anther smut disease), eleven Himalayan Silene species were assessed in regions of high host diversity to ascertain levels of pathogen specificity. We also investigated disease prevalence, seasonal dynamics of infection and flowering patterns in five co-blooming Silene species. We identified several new Microbotryum lineages with varying degrees of specialization that is likely influenced by degrees of host divergence and ecological similarities (i.e. shared pollinator guilds). Affected species had 15%-40% of plants infected by anther smut. Flowering was seasonally overlapping among host species (except for the species pair S. asclepiadea and S. atrocastanea), but diseased flowering onset was earlier than healthy plants, leading to dramatic seasonal shifts in observed disease prevalence. Overlapping distributions and flowering provides opportunities for floral pathogen movement between host species, but host specialization may be constrained by the plant phylogenetic relatedness, adaptation to micro-habitats and difference in pollinator/vector guilds.

Floral traits influence pollen vectors' choices in higher elevation communities in the Himalaya-Hengduan Mountains.

BACKGROUND: How floral traits and community composition influence plant specialization is poorly understood and the existing evidence is restricted to regions where plant diversity is low. Here, we assessed whether plant specialization varied among four species-rich subalpine/alpine communities on the Yulong Mountain, SW China (elevation from 2725 to 3910 m). We analyzed two factors (floral traits and pollen vector community composition: richness and density) to determine the degree of plant specialization across 101 plant species in all four communities. Floral visitors were collected and pollen load analyses were conducted to identify and define pollen vectors. Plant specialization of each species was described by using both pollen vector diversity (Shannon's diversity index) and plant selectiveness (d' index), which reflected how selective a given species was relative to available pollen vectors. RESULTS: Pollen vector diversity tended to be higher in communities at lower elevations, while plant selectiveness was significantly lower in a community with the highest proportion of unspecialized flowers (open flowers and clusters of flowers in open inflorescences). In particular, we found that plant species with large and unspecialized flowers attracted a greater diversity of pollen vectors and showed higher selectiveness in their use of pollen vectors. Plant species with large floral displays and high flower abundance were more selective in their exploitation of pollen vectors. Moreover, there was a negative relationship between plant selectiveness and pollen vector density. CONCLUSIONS: These findings suggest that flower shape and flower size can increase pollen vector diversity but they also increased plant selectiveness. This indicated that those floral traits that were more attractive to insects increased the diversity of pollen vectors to plants while decreasing overlap among co-blooming plant species for the same pollen vectors. Furthermore, floral traits had a more important impact on the diversity of pollen vectors than the composition of anthophilous insect communities. Plant selectiveness of pollen vectors was strongly influenced by both floral traits and insect community composition. These findings provide a basis for a better understanding of how floral traits and community context shape interactions between flowers and their pollen vectors in species-rich communities.

The evolution of floral deception in Epipactis veratrifolia (Orchidaceae): from indirect defense to pollination.

BACKGROUND: It is estimated that floral deception has evolved in at least 7500 species of angiosperms, of which two thirds are orchids. Epipactis veratrifolia (Orchidaceae) is a model system of aphid mimicry as aphidophagous hoverflies lay eggs on false brood sites on their flowers. To understand the evolutionary ecology of floral deception, we investigated the pollination biology of E. veratrifolia across 10 populations in the Eastern Himalayas. We reconstructed the phylogeny of Epipactis and mapped the known pollination systems of previously studied species onto the tree. RESULTS: Some inflorescences of E. veratrifolia were so infested with aphids while they were still in bud that the some larvae of hoverflies developed to the third instar while flower buds opened. This indicated that adult female hoverflies were partly rewarded for oviposition. Although flowers failed to secrete nectar, they mimicked both alarm pheromones and aphid coloring of to attract female hoverflies as their exclusive pollinators. Phylogenetic mapping indicate that pollination by aphidophagous hoverflies is likely an ancestral condition in the genus Epipactis. We suggest that the biological interaction of aphid (prey), orchid (primary producer) and hoverfly (predator) may represent an intermediate stage between mutualism and deception in the evolution of pollination-by-deceit in E. veratrifolia. CONCLUSIONS: Our analyses indicate that this intermediate stage may be used as a model system to interpret the origin of oviposition (brood site) mimicry in Epipactis. We propose the hypothesis that some deceptive pollination systems evolved directly from earlier (partly) mutualistic systems that maintained the fidelity of the original pollinator(s) even though rewards (nectar/ brood site) were lost.

Insect pollination and self-incompatibility in edible and/or medicinal crops in southwestern China, a global hotspot of biodiversity.

An increasing global demand for food, coupled with the widespread decline of pollinator diversity, remains an international concern in agriculture and genetic conservation. In particular, there are large gaps in the study of the pollination of economically important and traditionally grown species in China. Many plant species grown in China are both edible and used medicinally. The country retains extensive written records of agricultural and apicultural practices, facilitating contemporary studies of some important taxa. Here, we focus on Yunnan in southwestern China, a mega-biodiversity hotspot for medicinal/food plants. We used plant and insect taxa as model systems to understand the patterns and consequences of pollinator deficit to crops. We identified several gaps and limitations in research on the pollination ecology and breeding systems of domesticated taxa and their wild relatives in Yunnan and asked the following questions: (1) What is known about pollination systems of edible and medicinal plants in Yunnan? (2) What are the most important pollinators of Codonopsis subglobosa (Campanulaceae)? (3) How important are native pollinator species for maximizing yield in Chinese crops compared with the introduced Apis mellifera? We found that some crops that require cross-pollination now depend exclusively on hand pollination. Three domesticated crops are dependent primarily on the native but semidomesticated Apis cerana and the introduced A. mellifera. Other species of wild pollinators often play important roles for certain specialty crops (e.g., Vespa velutina pollinates Codonopsis subglobosa). We propose a more systematic and comprehensive approach to applied research in the future.

Flowers of Cypripedium fargesii (Orchidaceae) fool flat-footed flies (Platypezidae) by faking fungus-infected foliage.

Charles Darwin was fascinated by the orchid-pollinator interactions, but he did not realize that many orchid species are pollinated by deceit. Cypripedium, a model lineage of nonrewarding orchid flowers, is pollinated primarily by bees. Here we present both an example of floral mimesis of fungus-infected foliage in orchids and an example of flat-footed flies (Agathomyia sp.; Platypezidae) as pollen vectors for angiosperms. Cypripedium fargesii is a nectarless, terrestrial, endangered orchid from southwestern China that requires cross-pollination to produce the maximum number of viable embryos. All insects caught entering or leaving the labellum sac were Agathomyia sp. carrying conidia of Cladosporium sp. on their mouthparts and legs, suggesting mycophagy. Blackish hairy spots on the upper surface of foliage may imitate black mold spots, serving as short-term visual lures. Some odor molecules also associated with Cladosporium cultures were isolated in the floral scent. Mimesis of fungus-infected foliage probably represents an overlooked but important option in angiosperm diversification, because there are three to five more Cypripedium spp. in southwestern China with the same mode of floral presentation and black-spotted hairy leaves.

Flowering phenology differs among wet and dry sub-alpine meadows in southwestern China.

The effect of floral traits, floral rewards and plant water availability on plant-pollinator interactions are well-documented; however, empirical evidence of their impact on flowering phenology in high-elevation meadows remains scarce. In this study, we assessed three levels of flowering phenology, i.e. population-, individual- and flower-level (floral longevity), in two nearby but contrasting (wet versus dry) sub-alpine meadows on Yulong Snow Mountain, southwestern China. We also measured a series of floral traits (pollen number, ovule number, and the ratio of pollen to ovule number per flower, i.e. pollen:ovule ratio [P/O]) and floral rewards (nectar availability and pollen presentation) as plausible additional sources of variation for each phenological level. Floral longevity in the wet meadow was significantly longer than that for the dry meadow, whereas population- and individual-flowering duration were significantly shorter. Our results showed a significant positive relationship between flowering phenology with pollen number and P/O per flower; there was no relationship with ovule number per flower. Further, we found a significant effect of flowering phenology on nectar availability and pollen presentation. Our findings suggest that shorter floral longevity in dry habitats compared to wet might be due to water-dependent maintenance costs of flowers, where the population- and individual-level flowering phenology may be less affected by habitats. Our study shows how different levels of flowering phenology underscore the plausible effects of contrasting habitats on reproductive success.

The Functional Dilemma of Nectar Mimic Staminodes in Parnassia wightiana (Celastraceae): Attracting Pollinators and Florivorous Beetles.

While floral signaling plays a central role in the reproductive success of all animal-pollinated plants, it may also attract herbivores eager to feed on flowers. False nectaries with glossy surfaces reflecting incident light may produce signals that attract floral visitors guiding their movements to and within the flower. Whether false nectaries also attract herbivores that lower the reproductive success of natural populations requires attention. In this study, we focus on Parnassia wightiana, a subalpine species with a whorl of staminodes that act as false nectaries attracting bees, flies, and herbivorous beetles. We tested the functions of staminodes using controlled manipulative experiments under field and lab conditions. We found a significant decrease in pollinator visits, and subsequent seed set, in flowers in which we removed staminodes or staminode apices confirming the function of these organs. In our natural populations, we found that a beetle, Nonarthra variabilis (Alticinae; Chrysomelidae), chews first on staminode apices, then it eats the entire staminodes and other flower parts, but rarely feeds on ovaries. Additional experiments suggested these beetles preferred staminodes to ovaries. Our results suggest this is a case of selective florivory, in which staminodes play a dual role, attracting pollinators and herbivores at the same time causing the attractive dilemma. Although selective florivory by beetles did not directly damage fruits, it influenced plant-pollinator interactions, decreasing reproductive success in plant populations. Our study highlights the importance of plant-pollinator-herbivore interactions in selecting floral traits.

Evidence of introduced honeybees (Apis mellifera) as pollen wasters in orchid pollination.

Biological invasions threaten global biodiversity, altering landscapes, ecosystems, and mutualistic relationships like pollination. Orchids are one of the most threatened plant families, yet the impact of invasive bees on their reproduction remains poorly understood. We conduct a global literature survey on the incidence of invasive honeybees (Apis mellifera) on orchid pollination, followed by a study case on Australian orchids. Our literature survey shows that Apis mellifera is the primary alien bee visiting orchids worldwide. However, in most cases, introduced honeybees do not deposit orchid pollen. We also test the extent to which introduced honeybees affect orchid pollination using Diuris brumalis and D. magnifica. Diuris brumalis shows higher fruit set and pollination in habitats with both native and invasive bees compared to habitats with only introduced bees. Male and female reproductive success in D. magnifica increases with native bee abundance, while conversely pollinator efficiency decreases with honeybee abundance and rises with habitat size. Our results suggest that introduced honeybees are likely involved in pollen removal but do not effectively deposit orchid pollen, acting as pollen wasters. However, Apis mellifera may still contribute to pollination of Diuris where native bees no longer exist. Given the global occurrence of introduced honeybees, we warn that certain orchids may suffer from pollen depletion by these invaders, especially in altered habitats with compromised pollination communities.

Ethnobotanical study on medicinal plants used by the Yi people in Xiaoliangshan, Yunnan Province, SW China.

ETHNOPHARMACOLOGICAL RELEVANCE: The Yi people in the Xiaoliangshan region in southwest China have a unique practice of combining ritual treatment and traditional medicine to care for patients. Despite increasing urbanization in the area, they have managed to preserve their distinctive lifestyle and extensive knowledge of traditional medicinal plants, setting them apart from other regions. However, there is a lack of systematic documentation on the knowledge of traditional medicinal plants used by the Yi people in Xiaoliangshan. AIM OF THE STUDY: This research aims to achieve the following objectives: 1. Document the diversity of medicinal plants used by the Yi people and explore their therapeutic usages. 2. Evaluate and analyze the main types of diseases with a high incidence in the local area and identify the types of medicinal plants used to treat these diseases. 3. Explore the underlying geographical and human factors influencing both disease prevalence and medicinal plant usage. METHODS: Ethnobotanical research methods were used to record and analyze the medicinal plants used by the Yi in Xiaoliangshan. Experts identified all plant specimens collected during ethnobotanical field surveys. The types of diseases treated by medicinal plants were classified according to the International Classification of Primary Care -2nd. RESULTS: A total of 125 medicinal plants were recorded after interviewing 193 participants. Of the medicinal plants identified, those with over 100 use reports were Paris polyphylla (202 use reports), Taxillus sutchuenensis (183), Artemisia indica (149), and Papaver somniferum (113). A total of 14 disease categories were recorded, with those related to the following categories having higher Informant Consensus factor values (ICF ≥0.85): Pregnancy, Childbearing, Family Planning, General and Unspecified, Urological, Respiratory, Musculoskeletal, and Skin. The highest quantity of medicinal plants is utilized to improve specific diseases and health problems, namely those related to Digestion, Skin, and Musculoskeletal. Fewer plant species were utilized for diseases or health issues associated with Eyes, Psychological, or Pregnancy, Childbearing, and Family Planning. The use reports from the informants also revealed how some medicinal plants are used to treat a variety of diseases or health issues. For instance, Malva pusilla is used for inducing abortion, treating postpartum hemorrhage, and joint sprains; Artemisia indica is used for treating malaria; Argentina lineata is used to remedy tuberculosis and malaria. Taxillus sutchuenensis is used for dealing with cold, pneumonia, and other ailments. CONCLUSIONS: The Yi people in Xiaoliangshan have a rich knowledge of traditional medicinal plants. Decoction and wine brewing are the most common processing methods used for these plants, which are utilized to treat a wide range of diseases. The characteristics of the medicinal use of the Yi people reflects the alpine mountainous environment in which they live, and their medical practices are closely related to traditional healing culture. This study enhances our understanding of the Yi traditional medicine via documentation and offers a valuable reference for future research and the development of new drugs.

The adaptation of bumblebees to extremely high elevation associated with their gut microbiota.

Bumblebees are among the most abundant and important pollinators for sub-alpine and alpine flowering plant species in the Northern Hemisphere, but little is known about their adaptations to high elevations. In this article, we focused on two bumblebee species, Bombus friseanus and Bombus prshewalskyi, and their respective gut microbiota. The two species, distributed through the Hengduan Mountains of southwestern China, show species replacement at different elevations. We performed genome sequencing based on 20 worker bee samples of each species. Applying evolutionary population genetics and metagenomic approaches, we detected genes under selection and analyzed functional pathways between bumblebees and their gut microbes. We found clear genetic differentiation between the two host species and significant differences in their microbiota. Species replacement occurred in both hosts and their bacteria (Snodgrassella) with an increase in elevation. These extremely high-elevation bumblebees show evidence of positive selection related to diverse biological processes. Positively selected genes involved in host immune systems probably contributed to gut microbiota changes, while the butyrate generated by gut microbiota may influence both host energy metabolism and immune systems. This suggests a close association between the genomes of the host species and their microbiomes based on some degree of natural selection.IMPORTANCETwo closely related and dominant bumblebee species, distributed at different elevations through the Hengduan Mountains of southwestern China, showed a clear genomic signature of adaptation to elevation at the molecular level and significant differences in their respective microbiota. Species replacement occurred in both hosts and their bacteria (Snodgrassella) with an increase in elevation. Bumblebees' adaptations to higher elevations are closely associated with their gut microbiota through two biological processes: energy metabolism and immune response. Information allowing us to understand the adaptive mechanisms of species to extreme conditions is implicit if we are to conserve them as their environments change.

Floral nectar production: what cost to a plant?

Floral nectar production is central to plant pollination, and hence to human wellbeing. As floral nectar is essentially a solution in water of various sugars, it is likely a valuable plant resource, especially in terms of energy, with plants experiencing costs/trade-offs associated with its production or absorption and adopting mechanisms to regulate nectar in flowers. Possible costs of nectar production may also influence the evolution of nectar volume, concentration and composition, of pollination syndromes involving floral nectar, and the production of some crops. There has been frequent agreement that costs of floral nectar production are significant, but relevant evidence is scant and difficult to interpret. Convincing direct evidence comes from experimental studies that relate either enhanced nectar sugar production (through repeated nectar removal) to reduced ability to produce seeds, or increased sugar availability (through absorption of additional artificial nectar) to increased seed production. Proportions of available photosynthate allocated by plants to nectar production may also indicate nectar cost. However, such studies are rare, some do not include treatments of all (or almost all) flowers per plant, and all lack quantitative cost-benefit comparisons for nectar production. Additional circumstantial evidence of nectar cost is difficult to interpret and largely equivocal. Future research should repeat direct experimental approaches that relate reduced or enhanced nectar sugar availability for a plant with consequent ability to produce seeds. To avoid confounding effects of inter-flower resource transfer, each plant should experience a single treatment, with treatment of all or almost all flowers per plant. Resource allocation by plants, pathways used for resource transfer, and the locations of resource sources and sinks should also be investigated. Future research should also consider extension of nectar cost into other areas of biology. For example, evolutionary models of nectar production are rare but should be possible if plant fitness gains and costs associated with nectar production are expressed in the same currency, such as energy. It should then be possible to understand observed nectar production for different plant species and pollination syndromes involving floral nectar. In addition, potential economic benefits should be possible to assess if relationships between nectar production and crop value are evaluated.

Pollination crisis Down-Under: Has Australasia dodged the bullet?

Since mid-1990s, concerns have increased about a human-induced "pollination crisis." Threats have been identified to animals that act as plant pollinators, plants pollinated by these animals, and consequently human well-being. Threatening processes include loss of natural habitat, climate change, pesticide use, pathogen spread, and introduced species. However, concern has mostly been during last 10-15 years and from Europe and North America, with Australasia, known as Down-Under, receiving little attention. So perhaps Australasia has "dodged the bullet"? We systematically reviewed the published literature relating to the "pollination crisis" via Web of Science, focusing on issues amenable to this approach. Across these issues, we found a steep increase in publications over the last few decades and a major geographic bias towards Europe and North America, with relatively little attention in Australasia. While publications from Australasia are underrepresented, factors responsible elsewhere for causing the "pollination crisis" commonly occur in Australasia, so this lack of coverage probably reflects a lack of awareness rather than the absence of a problem. In other words, Australasia has not "dodged the bullet" and should take immediate action to address and mitigate its own "pollination crisis." Sensible steps would include increased taxonomic work on suspected plant pollinators, protection for pollinator populations threatened with extinction, establishing long-term monitoring of plant-pollinator relationships, incorporating pollination into sustainable agriculture, restricting the use of various pesticides, adopting an Integrated Pest and Pollinator Management approach, and developing partnerships with First Nations peoples for research, conservation and management of plants and their pollinators. Appropriate Government policy, funding and regulation could help.

Dark purple nectar as a foraging signal in a bird-pollinated Himalayan plant.

• Some plants secrete coloured nectar to attract pollinators, but little is known about the chemical origins of nectar colouration and its ecological function. Leucosceptrum canum stands out as the only plant with coloured nectar recorded in the Himalayas. Here, we focused on the compound associated with the dark colour of the nectar, as well as its secretion dynamics during the flowering season and its relationship to pollinators. • Fresh nectar was analysed by semi-preparative reversed-phase high-performance liquid chromatography (HPLC), LC-MS and HRESIMS (high resolution electronspray ionization mass spectroscopy) to determine which compound causes the nectar colouration. Behavioural experiments were conducted with birds and honeybees to elucidate the effect of the nectar colour and volume on pollinators. • We identified a purple anthocyanidin, 5-hydroxyflavylium, as a natural nectar product for the first time. Two short-billed birds were found to pollinate this plant, which employs two nectar-based mechanisms to direct bird pollinators to reproductively active flowers, controlling nectar palatability and presenting a foraging signal for birds by altering nectar volume and colour in a developmental stage-specific manner. • 5-Hydroxyflavylium was found to be the cause of the nectar colouration, the function of which is to act as a foraging signal to increase pollination efficiency through nectar visibility and palatability.

Did pollination exist before plants?

Research shows that seaweeds depend on crustaceans for fertilization.