Floral trait variation across individual plants within a population enhances defense capability to nectar robbing.

Floral trait variation may help pollinators and nectar robbers identify their target plants and, thus, lead to differential selection pressure for defense capability against floral antagonists. However, the effect of floral trait variation among individuals within a population on multi-dimensional plant-animal interactions has been little explored. We investigated floral trait variation, pollination, and nectar robbing among individual plants in a population of the bumble bee-pollinated plant, Caryopteris divaricata, from which flowers are also robbed by bumble bees with varying intensity across individuals. We measured the variation in corolla tube length, nectar volume and sugar concentration among individual plants, and evaluated whether the variation were recognized by pollinators and robbers. We investigated the influence of nectar robbing on legitimate visitation and seed production per fruit. We found that the primary nectar robber (Bombus nobilis) preferred to forage on plants with long-tubed flowers, which produced less nectar and had lower sugar concentration compared to those with shorter corolla tubes. Individuals with shorter corolla tubes had comparatively lower nectar robbing intensity but higher visitation by legitimate visitors (mainly B. picipes) and higher seed production. Nectar robbing significantly reduced seed production because it decreased pollinator visits. However, neither pollination nor seed production differed between plants with long and short corolla tubes when nectar robbers were excluded. This finding suggests that floral trait variation might not be driven by pollinators. Such variation among individual plants thus allows legitimate visitors and nectar robbers to segregate niches and enhances population defense against nectar robbing in unpredictable conditions.

Correction: Identification of Hybrids in Potamogeton: Incongruence between Plastid and ITS Regions Solved by a Novel Barcoding Marker PHYB.

[This corrects the article DOI: 10.1371/journal.pone.0166177.].

Intergeneric Relationships within the Early-Diverging Angiosperm Family Nymphaeaceae Based on Chloroplast Phylogenomics.

The order Nymphaeales, consisting of three families with a record of eight genera, has gained significant interest from botanists, probably due to its position as a basal angiosperm. The phylogenetic relationships within the order have been well studied; however, a few controversial nodes still remain in the Nymphaeaceae. The position of the Nuphar genus and the monophyly of the Nymphaeaceae family remain uncertain. This study adds to the increasing number of the completely sequenced plastid genomes of the Nymphaeales and applies a large chloroplast gene data set in reconstructing the intergeneric relationships within the Nymphaeaceae. Five complete chloroplast genomes were newly generated, including a first for the monotypic Euryale genus. Using a set of 66 protein-coding genes from the chloroplast genomes of 17 taxa, the phylogenetic position of Nuphar was determined and a monophyletic Nymphaeaceae family was obtained with convincing statistical support from both partitioned and unpartitioned data schemes. Although genomic comparative analyses revealed a high degree of synteny among the chloroplast genomes of the ancient angiosperms, key minor variations were evident, particularly in the contraction/expansion of the inverted-repeat regions and in RNA-editing events. Genome structure, and gene content and arrangement were highly conserved among the chloroplast genomes. The intergeneric relationships defined in this study are congruent with those inferred using morphological data.

Investigating the molecular basis for heterophylly in the aquatic plant Potamogeton octandrus (Potamogetonaceae) with comparative transcriptomics.

Many plant species exhibit different leaf morphologies within a single plant, or heterophylly. The molecular mechanisms regulating this phenomenon, however, have remained elusive. In this study, the transcriptomes of submerged and floating leaves of an aquatic heterophyllous plant, Potamogeton octandrus Poir, at different stages of development, were sequenced using high-throughput sequencing (RNA-Seq), in order to aid gene discovery and functional studies of genes involved in heterophylly. A total of 81,103 unigenes were identified in submerged and floating leaves and 6,822 differentially expressed genes (DEGs) were identified by comparing samples at differing time points of development. KEGG pathway enrichment analysis categorized these unigenes into 128 pathways. A total of 24,025 differentially expressed genes were involved in carbon metabolic pathways, biosynthesis of amino acids, ribosomal processes, and plant-pathogen interactions. In particular, KEGG pathway enrichment analysis categorized a total of 70 DEGs into plant hormone signal transduction pathways. The high-throughput transcriptomic results presented here highlight the potential for understanding the molecular mechanisms underlying heterophylly, which is still poorly understood. Further, these data provide a framework to better understand heterophyllous leaf development in P. octandrus via targeted studies utilizing gene cloning and functional analyses.

Sequential stamen maturation and movement in a protandrous herb: mechanisms increasing pollination efficiency and reducing sexual interference.

Successive stamen movement directly controls pollen presentation schedules through sequential stamen maturation and changes the extent of herkogamy by altering the positions of sexual organs. However, the implications of such movements in terms of pollination are not well understood. Pollen presentation theory predicts that staggered pollen presentation should be favoured when plants are subject to diminishing returns on pollen transfer. Herkogamy on the other hand, has been interpreted as an adaptive trait that reduces sexual interference in hermaphrodite flowers. In this study, we conducted floral manipulations to determine the function of successive stamen movement in pollen transfer. By artificially manipulating the flowers to present two anthers simultaneously in the floral centre, we attempted to investigate whether changes in the anther presentation strategy affect pollen removal, deposition and the efficiency of pollinators. Compared with the natural treatment, the pollen transfer efficiency of halictid bees decreased significantly when the flowers were manipulated to present two anthers simultaneously. Although the presentation of two anthers simultaneously led to a similar pollen removal rate, there was a significant reduction in pollen deposition on neighbouring stigmas. To evaluate the effect of movement herkogamy on pollen export and deposition and seed set, the flowers were manipulated with or without the movement of stamen bending out from the floral centre. Pollen export decreased significantly when the central anther was moved away from the pistil, and pollen deposition and seed set declined significantly when the five spent anthers were retained on the pistil. Our study provides good support for the pollen presentation theory and provides direct experimental evidence that successive stamen movement could increase pollen transfer efficiency by sequential stamen maturation. Moreover, movement herkogamy promotes pollen export, deposition and seed set, and could therefore be regarded as an effective mechanism to reduce interference between male and female functions.

Two LEAFY homologs ILFY1 and ILFY2 control reproductive and vegetative developments in Isoetes L.

LEAFY (LFY) is a plant-specific transcription factor, which is found in algae and all land plants. LFY homologs exert ancestral roles in regulating cell division and obtain novel functions to control floral identity. Isoetes L. is an ancient genus of heterosporous lycophytes. However, characters about LFY homologs in lycophytes remain poorly investigated. In this study, two LFY homologs, ILFY1 and ILFY2, were cloned from five Isoetes species, including I. hypsophila, I. yunguiensis, I. sinensis, I. orientalis, and I. taiwanensis. The full length of ILFY1 was 1449-1456 bp with an open reading frame (ORF) of 927-936 bp. The full length of ILFY2 was 1768 bp with ORF of 726 bp. Phylogenetic tree revealed that ILFY1 and ILFY2 were separated into two clades, and I. hypsophila were separated with the others. Expression analysis demonstrated that IsLFY1 and IsLFY2 for I. sinensis did not show functional diversity. The two transcripts were similarly accumulated in both vegetative and reproductive tissues and highly expressed in juvenile tissues. In addition, the IsLFY1 and IsLFY2 transgenic Arabidopsis similarly did not promote precocious flowering, and they were inactive to rescue lfy mutants. The results facilitate general understandings about the characteristics of LFY in Isoetes and evolutionary process.

Identification of Hybrids in Potamogeton: Incongruence between Plastid and ITS Regions Solved by a Novel Barcoding Marker PHYB.

Potamogeton is one of the most difficult groups to clarify in aquatic plants, which has an extensive range of interspecific morphological and ecological diversity. Internal transcribed spacer (ITS) is prevalent for phylogenetic analysis in plants. However, most researches demonstrate that ITS has a high percentage of homoplasy in phylogenetic datasets. In this study, eighteen materials were collected in Potamogeton from China and incongruence was shown between the rbcL and ITS phylogenies. To solve the discrepancy, we employed a novel barcode PHYB to improve resolution and accuracy of the phylogenetic relationships. The PHYB phylogeny successfully resolved the incongruence between the rbcL and ITS phylogenies. In addition, six hybrids were confirmed using PHYB, including P. compressus × P. pusillus, P. octandrus × P. oxyphyllus, P. gramineus × P. lucens, P. distinctus × P. natans, P. distinctus × P. wrightii, and S. pectinata × S. amblyophylla. Whereas, only one hybrid was identified (P. compressus × P. pusillus) by ITS, indicating that ITS homoplasy was present in Potamogeton and ITS was completely homogenized to one parental lineage. Thus, ITS might have limited utility for phylogenetic relationships in Potamogeton. It is recommended that a three-locus combination of chloroplast DNA gene, ITS and PHYB is potential to effectively reveal more robust phylogenetic relationships and species identification.

Comparative analysis of the RTFL peptide family on the control of plant organogenesis.

Plant peptides play important roles in various aspects of plant growth and development. The RTFL/DVL family includes small peptides that are widely conserved among land plants. Overexpression of six RTFL genes in Arabidopsis was suggestive of their functions as negative regulators of cell proliferation and as positional cues along the longitudinal axis of the plant body . At this time, few reports are available on RTFL paralogs in other species and the evolutionary relationship of RTFL members among land plants remains unclear. In this study, we compared and analyzed whole amino acid sequences of 188 RTFL members from 22 species among land plants and identified 73 motifs. All RTFL members could be grouped into four clades, and each clade exhibited specific motif patterns, indicative of unique evolutionary traits in the RTFL family. In agreement with this hypothesis, we analyzed two RTFL members from Oryza sativa and Arabidopsis by overexpressing them in Arabidopsis, revealing similar phenotypes suggestive of a conserved function of the RTFL family between eudicots and monocots, as well as different phenotypes and unique functions.

Terrimonas arctica sp. nov., isolated from Arctic tundra soil.

A novel, Gram-stain-negative, aerobic, non-motile and rod-shaped bacterium, designated R9-86(T), was isolated from tundra soil collected near Ny-Ålesund, Svalbard Archipelago, Norway (78° N). Growth occurred at 4-28 °C (optimum, 22-25 °C) and at pH 6.0-9.0 (optimum, pH 7.0). Flexirubin-type pigments were absent. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain R9-86(T) belonged to the genus Terrimonas in the family Chitinophagaceae. 16S rRNA gene sequence similarities between strain R9-86(T) and the type strains of species of the genus Terrimonas with validly published names ranged from 93.7 to 95.0%. Strain R9-86(T) contained iso-C(15:1)-G (25.7%), iso-C(15:0) (24.5%), iso-C(17:0)-3OH (18.3%) and summed feature 3 (C(16:1)ω7c and/or C(16:1)ω6c, 8.7%) as its major cellular fatty acids; phosphatidylethanolamine and an unknown polar lipid as its main polar lipids, and MK-7 as its predominant respiratory quinone. The DNA G+C content was 48.4 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain R9-86(T) is considered to represent a novel species of the genus Terrimonas, for which the name Terrimonas arctica sp. nov. is proposed. The type strain is R9-86(T) ( =CCTCC AB 2011004(T) =NRRL B-59114(T)).

Desertibacter xinjiangensis sp. nov., isolated from the soil of a Euphrates poplar forest, and emended description of the genus Desertibacter.

A pale pink and strictly aerobic bacterium, designated strain M71(T), was isolated from the soil of a Euphrates poplar forest in Xingjiang, PR China. Cells of the strain were Gram-reaction-negative, rod-shaped and motile by means of a single polar flagellum. Growth occurred at 10-37 °C (optimum 30 °C), at pH 6.0-9.0 (optimum pH 7.0-8.0) and with 0-2.0% NaCl (w/v, optimum 0%). Phylogenetic analysis, based on 16S rRNA gene sequences, indicated that strain M71(T) belongs to the genus Desertibacter in the family Rhodospirillaceae. The 16S rRNA gene sequence of this strain showed 96.2% sequence similarity with the type strain of Desertibacter roseus 2262(T). The respiratory quinone was Q-10 and the predominant cellular fatty acids were C(18:1)ω7c (53.2%), C(16:1)ω5c (11.0%), summed feature 3 (C(16:1)ω7c and/or C(16:1)ω6c, 10.2%) and C(16:0) (8.5%). The DNA G+C content was 71.2 mol% (HPLC). The strain contained phosphatidylcholine and phosphatidylethanolamine as the predominant polar lipids. On the basis of the phenotypic, chemotaxonomic and phylogenetic data, strain M71(T) is considered to represent a novel species of the genus Desertibacter, for which the name Desertibacter xinjiangensis sp. nov. is proposed. The type strain is M71(T) ( =CCTCC AB 209291(T) =CIP 110127(T)).

Change of floral orientation within an inflorescence affects pollinator behavior and pollination efficiency in a bee-pollinated plant, Corydalis sheareri.

Vertical raceme or spike inflorescences that are bee-pollinated tend to present their flowers horizontally. Horizontal presentation of flowers is hypothesized to enhance pollinator recognition and pollination precision, and it may also ensure greater consistency of pollinator movement on inflorescences. We tested the hypotheses using bee-pollinated Corydalis sheareri which has erect inflorescences consisting of flowers with horizontal orientation. We altered the orientation of individual flowers and prepared three types of inflorescences: (i) unmanipulated inflorescences with horizontal-facing flowers, (ii) inflorescences with flowers turned upward, and (iii) inflorescences with flowers turned downward. We compared number of inflorescences approached and visited, number of successive probes within an inflorescence, the direction percentage of vertical movement on inflorescences, efficiency of pollen removal and seed production per inflorescence. Deviation from horizontal orientation decreased both approaches and visits by leafcutter bees and bumble bees to inflorescences. Changes in floral orientation increased the proportion of downward movements by leafcutter bees and decreased the consistency of pollinator movement on inflorescences. In addition, pollen removal per visit and seed production per inflorescence also declined with changes of floral orientation. In conclusion, floral orientation seems more or less optimal as regards bee behavior and pollen transfer for Corydalis sheareri. A horizontal orientation may be under selection of pollinators and co-adapt with other aspects of the inflorescence and floral traits.

Pollination in a patchily distributed lousewort is facilitated by presence of a co-flowering plant due to enhancement of quantity and quality of pollinator visits.

BACKGROUND AND AIMS: Plants surrounded by individuals of other co-flowering species may suffer a reproductive cost from interspecific pollen transfer (IPT). However, differences in floral architecture may reduce or eliminate IPT. METHODS: A study was made of Pedicularis densispica (lousewort) and its common co-flowering species, Astragalus pastorius, to compare reproductive and pollination success of lousewort plants from pure and mixed patches. Floral architecture and pollinator behaviour on flowers of the two plants were compared along with the composition of stigmatic pollen load of the louseworts. The extent of pollen limitation of plants from pure and mixed patches was also explored through supplemental pollination with self- and outcross pollen (PLs and PLx). KEY RESULTS: Mixed patches attracted many more nectar-searching individuals of Bombus richardsi. These bumble-bees moved frequently between flowers of the two species. However, they pollinated P. densispica with their dorsum and A. pastorius with their abdomen. This difference in handling almost completely eliminated IPT. Lousewort plants from mixed patches yielded more seeds, and seeds of higher mass and germinability, than those from pure patches. Moreover, louseworts from mixed patches had lower PLs and PLx compared with those from pure patches. CONCLUSIONS: Differences in floral architecture induced differences in pollinator behaviour that minimized IPT, such that co-flowering plants significantly enhanced quantity and quality of pollinator visits for the lousewort plants in patchy habitat. These findings add to our understanding of the mechanisms of pollination facilitation.

Mucilaginibacter soli sp. nov., isolated from Arctic tundra soil.

A novel pale-pink-coloured strain, designated R9-65(T), was isolated from a tundra soil near Ny-Ålesund, Svalbard Archipelago, Norway (78° N). The cells were facultatively anaerobic, Gram-staining-negative, non-motile and rod-shaped. Growth occurred at 4-32 °C (optimum, 25-28 °C), at pH 5.0-9.0 (optimum, pH 6.0-7.0) and with 0-1.0% (w/v) NaCl (optimum, no NaCl). Flexirubin-type pigments were absent. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain R9-65(T) belonged to the genus Mucilaginibacter in the family Sphingobacteriaceae. The 16S rRNA gene sequence similarity between strain R9-65(T) and type strains of related species ranged from 93.4 to 96.6%. Strain R9-65(T) contained summed feature 3 (C(16:1)ω7c and/or C(16:1)ω6c, 34.3%) and iso-C(15:0) (20.3%) as major cellular fatty acids, MK-7 as the major respiratory quinone, and phosphatidylethanolamine as the main polar lipid. The DNA G+C content of strain R9-65(T) was 47.2 mol%. On the basis of phylogenetic, physiological and chemotaxonomic data, strain R9-65(T) is considered to represent a novel species of the genus Mucilaginibacter, for which the name Mucilaginibacter soli sp. nov. is proposed. The type strain is R9-65(T) (=CCTCC AB 2010331(T)=NRRL B-59458(T)).

The presence of co-flowering species facilitates reproductive success of Pedicularis monbeigiana (Orobanchaceae) through variation in bumble-bee foraging behaviour.

Background and Aims The presence of co-flowering species can alter pollinator foraging behaviour and, in turn, positively or negatively affect the reproductive success of the focal species. Such interactions were investigated between a focal species, Pedicularis monbeigiana, and a co-flowering species, Vicia dichroantha, which was mediated by behaviour alteration of the shared bumble-bee pollinator. Methods Floral display size and floral colour change of P. monbeigiana were compared between pure (P. monbeigiana only) and mixed (P. monbeigiana and V. dichroantha) plots in two populations. Pollinator visitation rates, interspecific floral switching and successive within-plant pollinator visits were recorded. In addition, supplemental pollination at plant level was performed, and the fruit set and seed set were analysed in pure and mixed plots with different densities of P. monbeigiana. Key Results Pollinator visitation rates were dramatically higher in mixed plots than in pure plots. The higher pollinator visitation rates were recorded in both low- and high-density plots. In particular, successive flower visits within an individual plant were significantly lower in mixed plots. Supplemental pollination significantly increased fruit set and seed set of individuals in pure plots, while it only marginally increased seed set per fruit of plants in mixed plots. Conclusions The presence of V. dichroantha can facilitate pollination and increase female reproductive success of P. monbeigiana via both quantity (mitigating pollinator limitation) and quality (reducing geitonogamy) effects. This study suggests that successive pollinator movements among flowers within a plant, as well as pollinator visitation rates and interspecific flower switching, may be important determinants of the direction and mechanisms of interaction between species.

Function and evolution of sterile sex organs in cryptically dioecious Petasites tricholobus (Asteraceae).

BACKGROUND AND AIMS: Why are sterile anthers and carpels retained in some flowering plants, given their likely costs? To address this question, a cryptically dioecious species, Petasites tricholobus, in which male and female plants each have two floret types that appear pistillate and hermaphroditic, was studied. The aim was to understand the function of sterile hermaphroditic florets in females. In addition, the first examination of functions of sterile female structures in male plants was conducted in the hermaphroditic florets on males of this species. These female structures are exceptionally large in this species despite being sterile. METHODS: Differences in floret morphology between the sex morphs were documented and the possible functions of sterile sex organs investigated using manipulative experiments. Tests were carried out to find out if sterile female structures in male florets attract pollinators and if they aid in pollen dispersal, also to find out if the presence and quantity of sterile hermaphroditic florets in females increase pollinator attraction and reproductive success. To investigate what floret types provide nectar, all types of florets were examined under a scanning electron microscope to search for nectaries. KEY RESULTS: The sterile female structures in male florets did not increase pollinator visits but were essential to secondary pollen presentation, which significantly enhanced pollen dispersal. Sterile pistillate florets on male plants did not contribute to floral display and disappeared in nearly half of the male plants. The sterile hermaphroditic florets on female plants attracted pollinators by producing nectar and enhanced seed production. CONCLUSIONS: The presence of female structures in male florets and hermaphroditic florets on female plants is adaptive despite being sterile, and may be evolutionarily stable. However, the pistillate florets on male plants appear non-adaptive and are presumably in decline. Differential fates of the sterile sex organs in the species are determined by both the historical constraints and the ecological functions.

Differential effects of nectar robbing by the same bumble-bee species on three sympatric Corydalis species with varied mating systems.

BACKGROUND AND AIMS: Most research on the widespread phenomenon of nectar robbing has focused on the effect of the nectar robbers' behaviour on host-plant fitness. However, attention also needs be paid to the characteristics of host plants, which can potentially influence the consequences of nectar robbing as well. A system of three sympatric Corydalis species sharing the same nectar-robbing bumble-bee was therefore studied over 3 years in order to investigate the effect of nectar robbing on host reproductive fitness. METHODS: Three perennial species of Corydalis were studied in the Shennongjia Mountain area, central China. Observations were conducted on visitor behaviour and visitation frequencies of nectar-robbers and legitimate pollinators. KEY RESULTS: The results indicated that the effect of nectar robbing by Bombus pyrosoma varied among species, and the three species had different mating systems. Seed set was thus influenced differentially: there was no effect on seed set of the predominantly selfing C. tomentella; for the facultative outcrossing C. incisa, nectar robbing by B. pyrosoma had a positive effect; and nectar robbing had a significant negative effect on the seed set of outcrossing C. ternatifolia. CONCLUSIONS: A hypothesis is proposed that the type of host-plant mating system could influence the consequences of nectar robbing on host reproductive fitness.

Reproductive consequences of interactions between clonal growth and sexual reproduction in Nymphoides peltata: a distylous aquatic plant.

Distyly is a sexual polymorphism in which plant populations contain two floral morphs differing in morphology and physiology. The dimorphism serves to promote animal-mediated cross-pollination between the floral morphs. Clonal propagation can interfere with the functioning of distyly by compromising intermorph pollinations, resulting in reduced fertility. Here, we investigate the relations between clonal growth and sexual reproduction in the aquatic macrophyte Nymphoides peltata (Menyanthaceae). Surveys of morph representation in 30 populations from five regions of China revealed that most populations exhibited strongly biased morph ratios and 30% contained a single floral morph. Experimental pollinations indicate that N. peltata possesses a strong dimorphic incompatibility system preventing self and intramorph fertilizations. An experiment involving the manipulation of morph ratios in an experimental population and an investigation in a natural population with strong morph substructure both provided evidence that compatible pollen dilution limits fertility. Despite constraints on the functioning of distyly in N. peltata we found no evidence for evolutionary changes to the heterostylous syndrome, as reported in Nymphoides, including populations of N. peltata in other parts of its geographical range.

Temporal floral sex allocation in protogynous Aquilegia yabeana contrasts with protandrous species: support for the mating environment hypothesis.

We tested one of the predictions of Brunet and Charlesworth (1995) that relative floral sex allocation will vary temporally with the mating environment and that the form of dichogamy (protandry vs. protogyny) will select for the pattern of variation in male versus female resource allocation. In many hermaphroditic plant species, allocation to female function (ovule number) decreases from early to late flowers within inflorescences as a result of resource limitation or ontogenetic changes. This pattern may obscure the effects of the mating environment and dichogamy on selection for allocation patterns in protandrous species (male allocation increases regardless). By examining a protogynous species the alternative pattern of temporal variation in resource allocation is predicted, namely that allocation to male function should decrease (or female allocation increase) throughout the flowering sequence. This pattern was observed in protogynous Aquilegia yabeana (Ranunculaceae), in which ovule number per flower remained constant whereas pollen number decreased in sequentially blooming flowers. These observations support the temporal sex allocation hypothesis of Brunet and Charlesworth (1995).

Gender variation of sequential inflorescences in a monoecious plant Sagittaria trifolia (Alismataceae).

In protogynous plants, female flowers of early blooming plants are at a reproductive disadvantage because they cannot set fruit due to the lack of available pollen. To study this phenomenon, gender expression of the monoecious herb Sagittaria trifolia was investigated over the entire flowering season in two field and two cultivated populations in Hubei and Hunan Provinces, China. In racemes of S. trifolia, flowers open sequentially from bottom to top, with female flowers opening first followed by male flowers. This creates a temporal separation of sexes in the species. Under field conditions small plants are often male, with production of both male and female flowers increasing with plant size. Femaleness increased among sequential inflorescences since female flower production increased whereas male flower production did not. Seed production was greater in large inflorescences because they contain more female flowers, and the number of ovules increased in female flowers at basal positions within the raceme. A consistent pattern of high seed set was observed in flowers from both field and cultivated populations. About 1 % of unfertilized ovules resulted from no pollination and 2 % of the seeds produced were only partly developed due to resource limitation. In the first inflorescence of the six experimental populations, 6.7-40.0 % of individuals produced only male flowers, and female flowers of 1.9-6.5 % individuals were aborted. The occurrence of male flowers in early blooming inflorescences could be an adaptive strategy to conserve resources and enhance pollination of female flowers in protogynous S. trifolia.