The Conserved and Particular Roles of the R2R3-MYB Regulator FhPAP1 from Freesia hybrida in Flower Anthocyanin Biosynthesis.

Anthocyanin biosynthesis is mainly controlled by MYB-bHLH-WD40 (MBW) complexes that modulate the expression of anthocyanin biosynthetic genes (ABGs). The MYB regulators involved in anthocyanin biosynthesis arose early during plant evolution and thus might function divergently in different evolutionary lineages. Although the anthocyanin-promoting R2R3-MYB regulators in eudicots have been comprehensively explored, little consensus has been reached about functional discrepancies versus conservation among MYB regulators from different plant lineages. Here, we integrated transcriptome analysis, gene expression profiles, gain-of-function experiments and transient protoplast transfection assays to functionally characterize the monocot Freesia hybrida anthocyanin MYB regulator gene FhPAP1, which showed correlations with late ABGs. FhPAP1 could activate ABGs as well as TT8-clade genes FhTT8L, AtTT8 and NtAN1 when overexpressed in Freesia, Arabidopsis and tobacco, respectively. Consistently, FhPAP1 could interact with FhTT8L and FhTTG1 to form the conserved MBW complex and shared similar target genes with its orthologs from Arabidopsis. Most prominently, FhPAP1 displayed higher transactivation capacity than its homologs in Arabidopsis and tobacco, which was instantiated in its powerful regulation on ABGs. Moreover, we found that FhPAP1 might be the selected gene during the domestication and rapid evolution of the wild Freesia species to generate intensive flower pigmentation. These results showed that while the MBW complex was highly evolutionarily conserved between tested monocot and core eudicot plants, participating MYB regulators showed functional differences in transactivation capacity according to their activation domain and played important roles in the flower coloration domestication and evolution of angiosperms.

GhTCP19 Transcription Factor Regulates Corm Dormancy Release by Repressing GhNCED Expression in Gladiolus.

Dormancy is one of the least understood phenomena in plant biology; however, bud/corm dormancy is an important economic trait in agricultural/horticultural breeding. In this study, we isolated an ABA biosynthesis gene, GhNCED, from the transcriptome database of corm dormancy release (CDR), and characterized its negative role in regulating CDR. To understand transcriptional regulation of GhNCED, yeast one-hybrid screening was conducted and GhTCP19 was identified and shown to regulate GhNCED expression directly. An in planta assay showed that GhTCP19 negatively regulates GhNCED expression. GhTCP19 is dramatically induced by exogenous cytokinins (CKs) and is induced during CDR. Silencing of GhTCP19 in dormant cormels delayed CDR, resulting in higher expression of GhNCED and ABA levels. Meanwhile, endogenous CK biosynthesis and signaling were inhibited in GhTCP19-silenced cormels. Taken together, our results reveal that GhTCP19 is a positive regulator of the CDR process by repressing expression of an ABA biosynthesis gene (GhNCED), promoting CK biosynthesis (GhIPT) and signal transduction (GhARR) as well as inducing cyclin genes. This study expands our knowledge on CDR which is mediated by TCP family members.

Intraspecific divergence in floral-tube length promotes asymmetric pollen movement and reproductive isolation.

The causative link between phenotypic divergence and reproductive isolation is an important but poorly understood part of ecological speciation. We studied the effects of floral-tube length variation on pollen placement/receipt positions and reproductive isolation. In a population of Lapeirousia anceps (Iridaceae) with bimodal floral-tube lengths, we labelled pollen of short- and long-tubed flowers with different colour fluorescent nanoparticles (quantum dots). This enabled us to map pollen placement by long- and short-tubed flowers on the only floral visitor, a long-proboscid fly. Furthermore, it allowed us to quantify pollen movement within and between short- and long-tubed flowers. Short- and long-tubed flowers placed pollen on different parts of the pollinator, and long-tubed flowers placed more pollen per visit than short-tubed flowers. This resulted in assortative pollen receipt (most pollen received comes from the same phenotype) and strong but asymmetric reproductive isolation, where short-tubed plants are more reproductively isolated than long-tubed plants. These results suggest that floral-tube length divergence can promote mechanical isolation in plants through divergence in pollen placement sites on pollinators. Consequently, in concert with other reproductive isolation mechanisms, selection for differences in floral-tube length can play an important role in ecological speciation of plants.

GhNAC83 inhibits corm dormancy release by regulating ABA signaling and cytokinin biosynthesis in Gladiolus hybridus.

Corm dormancy is an important trait for breeding in many bulb flowers, including the most cultivated Gladiolus hybridus. Gladiolus corms are modified underground stems that function as storage organs and remain dormant to survive adverse environmental conditions. Unlike seed dormancy, not much is known about corm dormancy. Here, we characterize the mechanism of corm dormancy release (CDR) in Gladiolus. We identified an important ABA (abscisic acid) signaling regulator, GhPP2C1 (protein phosphatase 2C1), by transcriptome analysis of CDR. GhPP2C1 expression increased during CDR, and silencing of GhPP2C1 expression in dormant cormels delayed CDR. Furthermore, we show that GhPP2C1 expression is directly regulated by GhNAC83, which was identified by yeast one-hybrid library screening. In planta assays show that GhNAC83 is a negative regulator of GhPP2C1, and silencing of GhNAC83 promoted CDR. As expected, silencing of GhNAC83 decreased the ABA level, but also dramatically increased cytokinin (CK; zeatin) content in cormels. Binding assays demonstrate that GhNAC83 associates with the GhIPT (ISOPENTENYLTRANSFERASE) promoter and negatively regulates zeatin biosynthesis. Taken together, our results reveal that GhNAC83 promotes ABA signaling and synthesis, and inhibits CK biosynthesis pathways, thereby inhibiting CDR. These findings demonstrate that GhNAC83 regulates the ABA and CK pathways, and therefore controls corm dormancy.

Cloning and characterization of a novel Gladiolus hybridus AFP family gene (GhAFP-like) related to corm dormancy.

Abscisic acid (ABA) is an important phytohormone controlling seed dormancy. AFPs (ABA INSENSITIVE FIVE BINDING PROTEINS) are reported to be negative regulators of the ABA signaling pathway. The involvement of AFPs in dormant vegetative organs remains poorly understood. Here, we isolated and characterized a novel AFP family member from Gladiolus dormant cormels, GhAFP-like, containing three conserved domains of the AFP family. Quantitative PCR analysis revealed that GhAFP-like was expressed in dormant organs and its expression was down-regulated along with corm storage. GhAFP-like was verified to be a nuclear-localized protein. Overexpressing GhAFP-like in Arabidopsis thaliana not only showed weaker seed dormancy with insensitivity to ABA, but also changed the expression of some ABA related genes. In addition, a primary root elongation assay showed GhAFP-like may involve in auxin signaling response. The results in this study indicate that GhAFP-like acts as a negative regulator in ABA signaling and is related to dormancy.

Pollination, mating and reproductive fitness in a plant population with bimodal floral-tube length.

Mating patterns and natural selection play important roles in determining whether genetic polymorphisms are maintained or lost. Here, we document an atypical population of Lapeirousia anceps (Iridaceae) with a bimodal distribution of floral-tube length and investigate the reproductive mechanisms associated with this pattern of variation. Flowers were visited exclusively by the long-proboscid fly Moegistorhynchus longirostris (Nemestrinidae), which exhibited a unimodal distribution of proboscis length and displayed a preference for long-tubed phenotypes. Despite being visited by a single pollinator species, allozyme markers revealed significant genetic differentiation between open-pollinated progeny of long- and short-tubed phenotypes suggesting mating barriers between them. We obtained direct evidence for mating barriers between the floral-tube phenotypes through observations of pollinator foraging, controlled hand pollinations and measurements of pollen competition and seed set. Intermediate tube-length phenotypes produced fewer seeds in the field than either long- or short-tubed phenotypes. Although floral-tube length bimodality may not be a stable state over long timescales, reproductive barriers to mating and low 'hybrid' fitness have the potential to contribute to the maintenance of this state in the short term.

The NPR1 homolog GhNPR1 plays an important role in the defense response of Gladiolus hybridus.

KEY MESSAGE: GhNPR1 shares similar functions as Arabidopsis NPR1 . Silencing of GhNPR1 in Gladiolus results in an enhanced susceptibility to Curvularia gladioli. We propose that GhNPR1 plays important roles in plant immunity. Gladiolus plants and corms are susceptible to various types of pathogens including fungi, bacteria and viruses. Understanding the innate defense mechanism in Gladiolus is a prerequisite for the development of new protection strategies. The non-expressor of pathogenesis-related gene 1 (NPR1) and bzip transcription factor TGA2 play a key role in regulating salicylic acid (SA)-mediated systemic acquired resistance (SAR). In this study, the homologous genes, GhNPR1 and GhTGA2, were isolated from Gladiolus and functionally characterized. Expression of GhNPR1 exhibited a 3.8-fold increase in Gladiolus leaves following salicylic acid treatment. A 1332 bp fragment of the GhNPR1 promoter from Gladiolus hybridus was identified. Inducibility of the GhNPR1 promoter by SA was demonstrated using transient expression assays in the leaves of Nicotiana benthamiana. The GhNPR1 protein is located in the nucleus and cytomembrane. GhNPR1 interacts with GhTGA2, as observed using the bimolecular fluorescence complementation system. Overexpression of GhNPR1 in an Arabidopsis npr1 mutant can restore its basal resistance to Pseudomonas syringae pv. tomato DC3000. Silencing of GhNPR1, using a tobacco rattle virus-based silencing vector, resulted in an enhanced susceptibility to Curvularia gladioli. In conclusion, these results suggest that GhNPR1 plays a pivotal role in the SA-dependent systemic acquired resistance in Gladiolus.

Intraspecific divergence and convergence of floral tube length in specialized pollination interactions.

Floral tubes are often thought to be a consequence of adaptive specialization towards pollinator morphology. We explore floral tube length evolution within Tritoniopsis revoluta (Iridaceae), a species with considerable geographical tube length variation. We ask whether tube lengths of T. revoluta populations are associated with pollinator proboscis lengths, whether floral divergence occurs in the presence of different pollinators and whether floral convergence occurs between distantly related populations pollinated by the same pollinator. Finally, we ask whether tube length evolution is directional. Shifts between morphologically different pollinators were always associated with shifts in floral morphology, even when populations were very closely related. Distantly related populations had similar tube lengths when they were pollinated by the same pollinator. Shifts in tube length tended to be from short to long, although reversals were not infrequent. After correcting for the population-level phylogeny, there was a strong positive, linear relationship between floral tube length and pollinator proboscis length, suggesting that plants are functionally specialized on different pollinators at different sites. However, because tube length evolution in this system can be a bidirectional process, specialization to the local pollinator fauna is unlikely to result in evolutionary or ecological dead-ends such as canalization or range limitation.

Pollinator shifts as triggers of speciation in painted petal irises (Lapeirousia: Iridaceae).

BACKGROUND AND AIMS: Adaptation to different pollinators has been hypothesized as one of the main factors promoting the formation of new species in the Cape region of South Africa. Other researchers favour alternative causes such as shifts in edaphic preferences. Using a phylogenetic framework and taking into consideration the biogeographical scenario explaining the distribution of the group as well as the distribution of pollinators, this study compares pollination strategies with substrate adaptations to develop hypotheses of the primary factors leading to speciation in Lapeirousia (Iridaceae), a genus of corm-bearing geophytes well represented in the Cape and presenting an important diversity of pollination syndromes and edaphic preferences. METHODS: Phylogenetic relationships are reconstructed within Lapeirousia using nuclear and plastid DNA sequence data. State-of-the-art methods in biogeography, divergence time estimation, character optimization and diversification rate assessments are used to examine the evolution of pollination syndromes and substrate shifts in the history of the group. Based on the phylogenetic results, ecological factors are compared for nine sister species pairs in Lapeirousia. KEY RESULTS: Seventeen pollinator shifts and ten changes in substrate types were inferred during the evolution of the genus Lapeirousia. Of the nine species pairs examined, all show divergence in pollination syndromes, while only four pairs present different substrate types. CONCLUSIONS: The available evidence points to a predominant influence of pollinator shifts over substrate types on the speciation process within Lapeirousia, contrary to previous studies that favoured a more important role for edaphic factors in these processes. This work also highlights the importance of biogeographical patterns in the study of pollination syndromes.

Phytotoxicity of atrazine to emergent hydrophyte, Iris pseudacorus L.

The emergent hydrophyte Iris pseudacorus was constantly exposed over a 35-day period to atrazine in the laboratory. It could survive at an atrazine level up to 32 mg/L. Its relative growth rates were inhibited significantly when exposure dosage reached at or exceeded 2 mg/L (p < 0.05). No observed effect concentration and lowest observed effect concentration for growth were 1 and 2 mg/L, respectively. Chlorophyll a and b contents of the plant in all treatment groups were affected significantly, and chlorophyll a/b ratios of all atrazine treatment levels were pronouncedly higher than those of the control within 5 days of exposure (p < 0.05), but thereafter recovered to the level of the control. Differences of photosynthetic efficiency were significant between all atrazine treatments and the control; except for 1 mg/L on day 1 and 5, and 2 mg/L on day 1. I. pseudacorus did not show phytotoxicity symptoms after 35 days exposure to atrazine below 2 mg/L level, but photosynthetic efficiency had begun to decline.

[Study on relieving effects of exogenous SNP, Spd on Belamcanda chinensis under salt-alkalline stress].

The study is aimed to provide the theoretical basis for exploiting and utilization of salt-alkaline soil and cultivating Belamcanda chinensis. In this study, we exerted exogenous substances SNP, Spd to relieve the damage of the mixing salt-alkaline stress on B. chinensis seedling which is NaCl, Na2SO4, NaHCO3 and Na2CO3 four kinds of salt molar ratio of 9: 1: 9: 1, salt concentration of 100 mmol x L(-1). The result illustrated that high pH stress is a major factor caused the salt-alkaline stress, the interaction between time and the concentration of each, treatment was observed, what is more, there are synergies between the salt and alkali stress. The content of B. chinensis seedling leaves' membrane peroxidation index (MDA, O2-*) and metabolites (soluble protein, soluble sugars, organic acids) are showing an upward trend in varying degrees under 100 mmol x L(-1) salt-alkaline stress. It is effective to reduce the content of MDA and O2-*. and improve the levels of metabolites, in which the SNP (0.05 mmol x L(-1)) and Spd (0.5 mmol x L(-1)) to alleviate damage effects is the best. Therefore we can hold the conclusion that SNP and Spd can effectively mitigate the damage of B. chinensis seedling on salt-alkaline stress, improve the resistance ability of B. chinensis seedling which can provide the scientific basis for the utilization of salt-alkaline soil, and the cultivation of B. chinensis.

Did pollination shifts drive diversification in southern African Gladiolus? Evaluating the model of pollinator-driven speciation.

The pollinator-driven ecological speciation model has frequently been invoked to explain plant richness in biodiversity hotspots. Here, by focusing on Gladiolus (260 species), a flagship example of a clade with diverse pollination biology, we test the hypothesis that high species diversity in southern Africa, one of the world's most floristically rich regions, has primarily been driven by ecological shifts in pollination systems. We use phylogenetic methods to estimate rates of transition between the seven highly specialized pollination strategies in Gladiolus. We find that pollination systems have evolved multiple times and that some pollination strategies arose by a variety of evolutionary pathways. Pollination shifts account for up to one-third of all lineage splitting events in the genus, providing partial support for the pollinator-driven speciation model. Transitions from the ancestral pollination mode to derived systems have also resulted in increased rates of diversification, suggesting that certain pollination systems may speed up speciation processes, independently of pollination shifts per se. This study suggests that frequent pollination shifts have played a role in driving high phenotypic and species diversity but indicates that additional factors need to be invoked to account for the spectacular diversification in southern African Gladiolus.

The natural history of pollination and mating in bird-pollinated Babiana (Iridaceae).

BACKGROUND AND AIMS: Floral variation, pollination biology and mating patterns were investigated in sunbird-pollinated Babiana (Iridaceae) species endemic to the Western Cape of South Africa. The group includes several taxa with specialized bird perches and it has been proposed that these function to promote cross-pollination. METHODS: Pollinator observations were conducted in 12 populations of five taxa (B. ringens subspp. ringens, australis, B. hirsuta, B. avicularis, B. carminea) and geographic variation in morphological traits investigated in the widespread B. ringens. Experimental pollinations were used to determine the compatibility status, facility for autonomous self-pollination and intensity of pollen limitation in six populations of four taxa. Allozyme markers were employed to investigate mating patterns in four populations of three species. KEY RESULTS: Sunbirds were the primary pollinators of the five Babiana taxa investigated. Correlated geographical variation in perch size, flower size and stigma-anther separation was evident among B. ringens populations. Experimental pollinations demonstrated that B. ringens and B. avicularis were self-compatible with variation in levels of autonomous self-pollination and weak or no pollen limitation of seed set. In contrast, B. hirsuta was self-incompatible and chronically pollen limited. Estimates of outcrossing rate indicated mixed mating with substantial self-fertilization in all species investigated. CONCLUSIONS: Despite the possession of specialized bird perches in B. ringens and B. avicularis, these structures do not prevent considerable selfing from occurring, probably as a result of autonomous self-pollination. In eastern populations of B. ringens, smaller flowers and reduced herkogamy appear to be associated with a shift to predominant selfing. Relaxed selection on perch function due to increased selfing may explain the increased incidence of apical flowers in some populations.

The effect of mammalian herbivory on inflorescence architecture in ornithophilous Babiana (Iridaceae): implications for the evolution of a bird perch.

PREMISE OF THE STUDY: The showiness of floral displays is usually explained as an adaptation to attract pollinators. However, selection for less attractive displays imposed by non-pollinating agents, particularly herbivores, may balance pollinator-driven selection for highly visible inflorescences. We investigated whether inflorescence architecture, particularly the unusual ground-level flowering associated with a specialized bird perch in Babiana ringens may have originated, in part, as an adaptive response to mammalian herbivory. METHODS: We measured levels of herbivory by antelope in populations of B. hirsuta, the putative sister species of B. ringens, which possesses the likely ancestral form of inflorescence architecture. To test for position-dependent effects of herbivory on flowers, we compared the herbivory rates and seed production of manipulated inflorescences in a field experiment. We predicted that flowers at the base of inflorescences would suffer less herbivory than those in apical positions. RESULTS: We found herbivore damage to flowers in 50% of naturally occurring B. hirsuta plants. Manipulated inflorescences with only basal flowers, and consequently similar inflorescence architecture to B. ringens, experienced significantly lower herbivory and higher seed set than inflorescences manipulated to have only apical flowers. CONCLUSIONS: Our results are consistent with the hypothesis that position-dependent herbivory on inflorescences could have played a role in the evolution of inflorescence design. More specifically, position-dependent herbivory may have selected for the loss of apical flowers. Position-dependent herbivory may have contributed toward the evolution of a naked inflorescence axis, a structure that characterizes B. ringens and functions as a bird perch facilitating cross-pollination by sunbirds.

Botany: specialized bird perch aids cross-pollination.

Birds may hover over or perch on flowers when feeding on nectar, and this assists cross-pollination if they then visit other plants. Here we investigate the curious sterile inflorescence axis of the South African Cape endemic 'rat's tail' plant (Babiana ringens, Iridaceae), whose function--unlike in other bird-pollinated plants--is exclusively to provide a perch for foraging birds. We find that this structure promotes the plant's mating success by causing the malachite sunbird (Nectarinia famosa), its main pollinator, to adopt a position ideal for the cross-pollination of its unusual ground-level flowers.

cDNA cloning and characterization of UDP-glucose: anthocyanidin 3-O-glucosyltransferase in Freesia hybrida.

The enzyme that catalyzes the formation of the first stable anthocyanin in the biosynthesis of natural compounds is UDP-glucose: anthocyanidin 3-O-glucosyltransferase (UF3GT). A cDNA clone (Fh3GT1) encoding UF3GT was isolated from Freesia hybrida. Phylogenetic tree analysis indicated that Fh3GT1 was a novel member of glycosyltransferase, which was classified into monocot subgroups. Semi-quantitative RT-PCR analysis detected transcripts of Fh3GT1 in different organs of F. hybrida and in petals of Freesia cultivars of different colors, and the expression level reached the maximum at the fully opened stage of petals. Characterization of the enzymatic assays indicated that Fh3GT1 had a role in anthocyanin glycoside biosyntheses in vitro. To elucidate the function of Fh3GT1, RNA interference vector (pART-Fh3GT1i) was constructed, and introduced into Petunia grandiflora by Agrobacterium-mediated transformation. Integration of the Fh3GT1 in petunia genome was confirmed by PCR and Southern blotting. SqRT-PCR revealed that the endogenous Ph3GT1 mRNA expression levels decreased in transgenic lines compared with the wild-type. The content of total anthocyanin pigments also decreased with the reduction of mRNA transcript levels, and the transgenic petunia plants had significant changes on their flower colors. In summary, this work identified a UF3GT gene from Freesia hybrida and demonstrated a method to modify plant flower color by redirecting the anthocyanin biosynthesis.

Modulation of salt-induced stress impact in Gladiolus grandiflorus L. by exogenous application of salicylic acid.

Salinity is challenging threats to the agricultural system and leading cause of crop loss. Salicylic acid (SA) is an important endogenous signal molecule, which by regulating growth and physiological processes improves the plant ability to tolerate salt stress. Considering the prime importance of Gladiolus grandiflorus (L.) in the world's cut-flower market, the research work was undertaken to elucidate salinity tolerance in G. grandiflorus by exogenous application of SA irrigated with saline water. Results revealed that increasing salinity (EC: 2, 4 and 6 dS m-1) considerably altered morpho-growth indices (corm morphology and plant biomass) in plants through increasing key antioxidants including proline content and enzymes activity (superoxide dismutase, catalase and peroxidase), while negatively affected the total phenolic along with activity of defense-related enzymes (phenylalanine ammonia lyase, and polyphenol oxidase activity). SA application (50-200 ppm) in non-saline control or saline conditions improved morpho-physiological traits in concentration-dependent manners. In saline conditions, SA minimized salt-stress by enhancing chlorophyll content, accumulating organic osmolytes (glycine betaine and proline content), total phenolic, and boosting activity of antioxidant and defense-related enzymes. Principle component analysis based on all 16 morphological and physiological variables generated useful information regarding the classification of salt tolerant treatment according to their response to SA. These results suggest SA (100 or 150 ppm) could be used as an effective, economic, easily available and safe phenolic agent against salinity stress in G. grandiflorus.

Phylogenetically independent associations between autonomous self-fertilization and plant invasiveness.

Many plant species have been introduced from their native ranges to new continents, but few have become naturalized or, ultimately, invasive. It has been predicted that species that do not require the presence of compatible mates and the services of pollinators for reproduction will be favored in establishment after long-distance dispersal. We tested whether this hypothesis, generally referred to as Baker's law, holds for South African species of Iridaceae (iris family) that have been introduced in other regions for horticultural purposes. Fruit and seed production of flowers from which pollinators had been experimentally excluded was assessed for 10 pairs of species from nine different genera or subgenera. Each species pair comprised one naturalized and one nonnaturalized species, all of which are used in international horticulture. On average, species of Iridaceae that have become naturalized outside their native ranges showed a higher capacity for autonomous fruit and seed production than congeneric species that have not become naturalized. This was especially true for the naturalized species that are considered to be invasive weeds. These results provide strong evidence for the role of autonomous seed production in increasing potential invasiveness in plants.

Comparison of Iris pseudacorus wetland systems with unplanted systems on pollutant removal and microbial community under nanosilver exposure.

Rapidly developing industry raises concerns about the environmental risks of silver nanoparticles (AgNPs), but the effects of AgNPs on the performance and microbial community in the constructed wetlands remain unclear. In this study, long-term exposure of AgNPs in two VFCWs was conducted to determine the effects of AgNPs on the pollutant removal and microbial community structure. Before exposing AgNPs, the water quality of effluent was better in planted wetland (CW2), compared with unplanted wetland (CW1). After continuous exposure of 100µg/L AgNPs, the COD (chemical oxygen demand) removal of two CWs had no difference. However, addition of AgNPs reduced the nitrogen and phosphorus removal in two CWs, with decreasing average removal efficiencies of ammonia nitrogen from 46.31% to 32.09% and 59.66% to 51.06%, total nitrogen from 57.76% to 43.78% and 67.35 to 60.58%, total phosphorus from 71.29% to 59.31% and 67.35% to 60.58%, respectively. The vegetable wetlands showed higher resistances to AgNPs loading than unplanted wetlands. In addition, AgNPs accumulated in the wetland substrate, especially in the soil layer with the silver concentration of approximately 4.32µg/g. The small portion of silver was found in plant tissues, and plants played a minor role to remove the AgNPs from wastewater. Moreover, the constructed wetlands could effectively remove the AgNPs from the synthetic wastewater. The illumine high-throughput sequencing results demonstrated the variations of the bacterial community structure at the exposure of AgNPs. The results showed that the dominant phyla were Proteobacteria, Acidobacteria and Bacteroidetes. Compared with unplanted wetlands, the contents of several nitrifying bacteria such as Candidatus Nitrososphaera (AOA) and Nitrospira (NOB) at genus level increased, leading to the higher nitrogen removal in the planted wetlands.

Pollinators shift to nectar robbers when florivory occurs, with effects on reproductive success in Iris bulleyana (Iridaceae).

Studies have indicated that florivory and nectar robbing may reduce reproductive success of host plants. However, whether and how these effects might interact when plants are simultaneously attacked by both florivores and nectar robbers still needs further investigation. We used Iris bulleyana to detect the interactions among florivory, nectar robbing and pollination, and moreover, their effects on plant reproductive success. Field investigations and hand-pollination treatments were conducted on two experimental plots from a natural population, in which Experimental plot was protected from florivores and Control plot was not manipulated. The flower calyx was bitten by sawflies to consume the nectary, and three bumblebee species were pollinators. In addition, the short-tongued pollinator, Bombus friseanus, was the only robber when there was a hole made by a sawfly. The bumblebee had significantly shortened flower handling time when robbing, as compared to legitimate visits. Pollinator visitation and seed production decreased significantly in damaged flowers. However, seed production per flower after supplementary hand-pollination did not differ significantly between damaged and undamaged flowers. Compared to the Experimental plot, bumblebees visited fewer flowers per plant in a foraging bout in the Control plot. The flowers damaged by florivory allowed B. friseanus to shift to a nectar robber. Florivory and nectar robbing collectively decreased plant reproductive success by consuming nectar resources, which may reduce attractiveness to pollinators of the damaged flowers. However, the changes in pollinator behaviour might be beneficial to the plant by reducing the risk of geitonogamous mating.