Floral mechanisms promote pollination success and reduce the incidence of self-pollination in a fly-pollinated self-incompatible orchid.

Among flowering plants, self-incompatibility is considered the most efficient system for avoiding self-fertilization. However, many self-incompatible plants have also evolved floral mechanisms to reduce sexual conflict. In China, some studies of Bulbophyllum have been reported to be self-incompatible and no fruit sets. However, we have observed relatively high fruit sets in Bulbophyllum funingense. Therefore, we speculated that if B. funingense is also self-incompatible, and it might present a floral mechanism to avoid sexual conflict. Natural fruit sets, pollinia removal and deposition rates were determined and breeding system was tested in a hand-pollination experiment. The pollination process and visiting frequency of pollinators and their behavior after escape from access were observed and recorded. Floral traits associated with pollination and pollinator size were measured. B. funingense was completely self-incompatible, the fruit sets of cross-pollination in 2 years were all more than 70%, and the natural fruit sets for 2 years were 1.70 ± 4.31% and 6.63 ± 5.29%, respectively. B. funingense did not produce strong odor or nectar, but produced a kind of secretions from its labellum that attracted flies. Calliphora vicina (Calliphoridae) was its only effective pollinator. When C. vicina licked the secretions, they were stuck in the access for a long time. Thus, when they escaped from access, they almost always flew quickly away from the inflorescence removing pollinia most of the times. In B. funingense, a floral mechanism improves pollinia transfer efficiency, reduces pollinia waste, promotes pollination success, reduces the incidence of self-pollination, and avoids sexual conflict to a certain extent.

Symbiosis between Dendrobium catenatum protocorms and Serendipita indica involves the plant hypoxia response pathway.

Mycorrhizae are ubiquitous symbioses established between fungi and plant roots. Orchids, in particular, require compatible mycorrhizal fungi for seed germination and protocorm development. Unlike arbuscular mycorrhizal fungi, which have wide host ranges, orchid mycorrhizal fungi are often highly specific to their host orchids. However, the molecular mechanism of orchid mycorrhizal symbiosis is largely unknown compared to that of arbuscular mycorrhizal and rhizobial symbiosis. Here, we report that an endophytic Sebacinales fungus, Serendipita indica, promotes seed germination and the development of protocorms into plantlets in several epiphytic Epidendroideae orchid species (6 species in 2 genera), including Dendrobium catenatum, a critically endangered orchid with high medicinal value. Although plant-pathogen interaction and high meristematic activity can induce the hypoxic response in plants, it has been unclear whether interactions with beneficial fungi, especially mycorrhizal ones, also involve the hypoxic response. By studying the symbiotic relationship between D. catenatum and S. indica, we determined that hypoxia-responsive genes, such as those encoding alcohol dehydrogenase (ADH), are highly induced in symbiotic D. catenatum protocorms. In situ hybridization assay indicated that the ADH gene is predominantly expressed in the basal mycorrhizal region of symbiotic protocorms. Additionally, the ADH inhibitors puerarin and 4-methylpyrazole both decreased S. indica colonization in D. catenatum protocorms. Thus, our study reveals that S. indica is widely compatible with orchids and that ADH and its related hypoxia-responsive pathway are involved in establishing successful symbiotic relationships in germinating orchids.

A novel method to produce massive seedlings via symbiotic seed germination in orchids.

Orchids produce large numbers of dust-like seeds that rely heavily on orchid mycorrhizal fungi (OMFs) for germination. Using OMFs to facilitate orchid proliferation is considered an effective method for orchid conservation but still presents challenges in practice. In this study, orchid seed-fungus complexes, in which orchid seeds and fungal mycelia were embedded together to form granules, were developed as platforms to facilitate seed germination and seedling production. Overall, seedlings were produced by seed-fungus complexes for five orchid species with large variations in the percentages of seedlings produced among species/treatments. For the different fungal treatments in Dendrobium officinale, Sebacinales LQ performed much better than the other fungal strains. At 90 days after sowing, 75.8±2.6% seedlings were produced in the LQ treatment, which was significantly higher than in the Tulasnella sp. JM (22.0±3.0%) and Tulasnella sp. TPYD-2 (5.3±1.0%) treatments, as well as in the LQ and TPYD-2 cocultured treatment (40.4±3.2%), while no seedlings were formed in the Tulasnella sp. SSCDO-5 or control treatments. For the other four orchid species, only one compatible fungus for each species was used, and the percentages of seedlings in epiphytic Dendrobium devonianum (67.2±2.9%) and D. nobile (38.9±2.8%) were much higher than those in terrestrial Paphiopedilum spicerianum (2.9±1.1%) and Arundina graminifolia (6.7±2.1%) at 90 days after sowing. Adding 1% polymer water-absorbent resin to the seed-fungus complexes of D. officinale seeds with fungal strain Sebacinales LQ significantly increased seedling formation, while other additional substances showed negative effects on seedling formation. For the storage of seed-fungus complexes, it is recommended to store the seed-fungus complexes in valve bags at room temperature for a short time and at a low temperature of 4°C for no more than 30 days. As a platform for symbiotic seed germination, the seed-fungus complex can facilitate seed germination, produce seedlings and support subsequent seedling growth, and its seedling productivity depends on seed germination characteristics, seed viability, and the efficiency of fungi. Seed-fungus complexes have great potential to be used as propagules in orchid conservation.

Using Ex Situ Seedling Baiting to Capture Seedling-Associated Mycorrhizal Fungi in Medicinal Orchid Dendrobium officinale.

Using orchid mycorrhizal fungi (OMFs) to facilitate orchid proliferation is considered an effective method of orchid conservation. Based on the success of using in situ seedling baiting to obtain plant growth-promoting fungi in our previous study, in this study, we developed the method of using ex situ seedling baiting to capture seedling-associated fungi from Dendrobium officinale. We collected substrates (e.g., litters, barks and mosses) from six original habitats of D. officinale in different geographical locations in China, and then, transplanted in vitro-produced seedlings of D. officinale into the substrates. After cultivation for 75 days, it was obvious that fungi colonized the seedling roots and formed large numbers of pelotons in all six groups. From these seedling roots, a total of 251 fungal strains, which were divided into 16 OMF and 11 non-OMF species, were successfully isolated. The 16 OMFs included 13 Tulasnella and 3 Serendipitaceae species. The fungal species isolated from the different groups (original habitat sources) were not identical, but the dominant OMFs with high isolation frequencies (more than 10 times) were commonly isolated from more than four original sources. Among the 11 non-OMFs, Fusarium oxysporum TP-18 and Muscodor sp. TP-26 were the dominant endophytes. Fusarium oxysporum is a common endophyte associated with many orchid species, including D. officinale. The results suggest that ex situ seedling baiting is an easy and efficient approach to obtaining seedling-associated fungi for this species and could be performed for other over-collected species, especially orchids for which wild plants have disappeared in the field but their original habitats are known. This approach has great potential for application in OMF studies in the future.

What role does the seed coat play during symbiotic seed germination in orchids: an experimental approach with Dendrobium officinale.

BACKGROUND: Orchids require specific mycorrhizal associations for seed germination. During symbiotic germination, the seed coat is the first point of fungal attachment, and whether the seed coat plays a role in the identification of compatible and incompatible fungi is unclear. Here, we compared the effects of compatible and incompatible fungi on seed germination, protocorm formation, seedling development, and colonization patterns in Dendrobium officinale; additionally, two experimental approaches, seeds pretreated with NaClO to change the permeability of the seed coat and fungi incubated with in vitro-produced protocorms, were used to assess the role of seed coat played during symbiotic seed germination. RESULTS: The two compatible fungi, Tulasnella sp. TPYD-2 and Serendipita indica PI could quickly promote D. officinale seed germination to the seedling stage. Sixty-two days after incubation, 67.8 ± 5.23% of seeds developed into seedlings with two leaves in the PI treatment, which was significantly higher than that in the TPYD-2 treatment (37.1 ± 3.55%), and massive pelotons formed inside the basal cells of the protocorm or seedlings in both compatible fungi treatments. In contrast, the incompatible fungus Tulasnella sp. FDd1 did not promote seed germination up to seedlings at 62 days after incubation, and only a few pelotons were occasionally observed inside the protocorms. NaClO seed pretreatment improved seed germination under all three fungal treatments but did not improve seed colonization or promote seedling formation by incompatible fungi. Without the seed coat barrier, the colonization of in vitro-produced protocorms by TPYD-2 and PI was slowed, postponing protocorm development and seedling formation compared to those in intact seeds incubated with the same fungi. Moreover, the incompatible fungus FDd1 was still unable to colonize in vitro-produced protocorms and promote seedling formation. CONCLUSIONS: Compatible fungi could quickly promote seed germination up to the seedling stage accompanied by hyphal colonization of seeds and formation of many pelotons inside cells, while incompatible fungi could not continuously colonize seeds and form enough protocorms to support D. officinale seedling development. The improvement of seed germination by seed pretreatment may result from improving the seed coat hydrophilicity and permeability, but seed pretreatment cannot change the compatibility of a fungus with an orchid. Without a seed coat, the incompatible fungus FDd1 still cannot colonize in vitro-produced protocorms or support seedling development. These results suggest that seed coats are not involved in symbiotic germination in D. officinale.

Compatible and Incompatible Mycorrhizal Fungi With Seeds of Dendrobium Species: The Colonization Process and Effects of Coculture on Germination and Seedling Development.

Orchids highly rely on mycorrhizal fungi for seed germination, and compatible fungi could effectively promote germination up to seedlings, while incompatible fungi may stimulate germination but do not support subsequent seedling development. In this study, we compared the fungal colonization process among two compatible and two incompatible fungi during seed germination of Dendrobium officinale. The two compatible fungi, i.e., Tulasnella SSCDO-5 and Sebacinales LQ, originally from different habitats, could persistently colonize seeds and form a large number of pelotons continuously in the basal cells, and both fungi promoted seed germination up to seedling with relative effectiveness. In contrast, the two incompatible fungi, i.e., Tulasnella FDd1 and Tulasnella AgP-1, could not persistently colonize seeds. No pelotons in the FDd1 treatment and only a few pelotons in the AgP-1 treatment were observed; moreover, no seedlings were developed at 120 days after incubation in either incompatible fungal treatment. The pattern of fungal hyphae colonizing seeds was well-matched with the morphological differentiation of seed germination and seedling development. In the fungal cocultural experiments, for both orchids of D. officinale and Dendrobium devonianum, cocultures had slightly negative effects on seed germination, protocorm formation, and seedling formation compared with the monocultures with compatible fungus. These results provide us with a better understanding of orchid mycorrhizal interactions; therefore, for orchid conservation based on symbiotic seed germination, it is recommended that a single, compatible, and ecological/habitat-specific fungus can be utilized for seed germination.

Transitions between the Terrestrial and Epiphytic Habit Drove the Evolution of Seed-Aerodynamic Traits in Orchids.

Orchids are globally distributed, a feature often attributed to their tiny dustlike seeds. They were ancestrally terrestrial but in the Eocene expanded into tree canopies, with some lineages later returning to the ground, providing an evolutionarily replicated system. Because seeds are released closer to the ground in terrestrial species than in epiphytic ones, seed traits in terrestrials may have been under selective pressure to increase seed dispersal efficiency. In this study, we test the expectations that seed airspace-a trait known to increase seed flotation time in the air-is (i) larger in terrestrial lineages and (ii) has increased following secondary returns to a terrestrial habit. We quantified and scored 20 seed traits in 121 species and carried out phylogenetically informed analyses. Results strongly support both expectations, suggesting that aerodynamic traits even in dust seeds are under selection to increase dispersal ability, following shifts in average release heights correlated with changes in habit.

Notes on the genus Gastrochilus (Orchidaceae) in Myanmar.

Myanmar is known for its high species richness of genus Gastrochilus; however, most of them lack proper information for taxonomic revision. During four years of field investigation in Myanmar, two new distributional records were encountered, namely, G. arunachalensis and G. corymbosus and one species, i.e. G. pechei was rediscovered after its original description. The three species were not easy to interpret from the available original descriptions and types due to severely shrunk or poorly preserved specimens. Therefore, we hereby present more detailed illustrations and updated descriptions for these species, based on freshly collected materials.

Are fungi from adult orchid roots the best symbionts at germination? A case study.

We studied mycobionts from advanced seedlings and adult mycorrhizal roots of the terrestrial orchid Arundina graminifolia. Fungi were isolated, identified by ITS sequencing, and tested for their impact on seed germination, protocorm formation, and development of advanced seedlings (emergence of first leaf) in vitro. Among the six fungal species isolated, four were not standard orchid mycorrhizal fungi (Fusarium solani, Cylindrocarpon sp., Acremonium sp., and Phlebiopsis flavidoalba) and did not support germination beyond imbibition and greening of the seeds during a span of 35 days. Over the same time, one Tulasnella species isolated from adult mycorrhiza allowed protocorm formation but not further development. However, another Tulasnella species isolated from advanced seedlings facilitated development to the advanced seedling stage. Our results support (i) the inability of occasional orchid root colonizers to support late seed germination, and (ii) the growing literature showing that fungal associates can change over orchid development. Functionally, we show that mycorrhizal taxa isolated from advanced seedlings can be more efficient than those from adults in supporting germination in some species, leading to recommendations for ex situ orchid conservation.

Host-specificity of symbiotic mycorrhizal fungi for enhancing seed germination, protocorm formation and seedling development of over-collected medicinal orchid, Dendrobium devonianum.

All orchids maintain an obligate relationship with mycorrhizal symbionts during seed germination. In most cases, germination-enhancing fungi have been isolated from roots of mature plants for conservation and cultivation purposes. To understand the germination biology of Dendrobium devonianum, an over-collected medicinal orchid, the seeds of D. devonianum were inoculated with a fungal strain (FDd1) isolated from naturally occurring protocorms of D. devonianum and two other germination-enhancing fungal strains (FDaI7 and FCb4) from D. aphyllum and Cymbidium mannii, respectively. The fungal strain was isolated from five protocorms of D. devonianum and identified as a species of the genus Epulorhiza. In germination trials, treatments with all of the three fungal strains showed a significant promoting effect on seed germination and protocorm formation, compared with the control treatment (no inoculation). However, FDd1 fungal strain showed the greatest effectiveness followed by FDaI7 and FCb4. For all inoculation and control treatments, seeds developed to protocorms regardless of the presence of illumination, whereas protocorms did not develop to seedlings unless illumination was provided. The results of our manipulative experiments confirmed the hypothesis that mycorrhizae associated with orchid seedlings are highly host-specific, and the degree of specificity may be life stagespecific under in vitro conditions. The specific mycorrhizal symbionts from protocorms can enhance restoration efforts and the conservation of orchids such as D. devonianum.

Using In Situ Symbiotic Seed Germination to Restore Over-collected Medicinal Orchids in Southwest China.

Due to increasing demand for medicinal and horticultural uses, the Orchidaceae is in urgent need of innovative and novel propagation techniques that address both market demand and conservation. Traditionally, restoration techniques have been centered on ex situ asymbiotic or symbiotic seed germination techniques that are not cost-effective, have limited genetic potential and often result in low survival rates in the field. Here, we propose a novel in situ advanced restoration-friendly program for the endangered epiphytic orchid species Dendrobium devonianum, in which a series of in situ symbiotic seed germination trials base on conspecific fungal isolates were conducted at two sites in Yunnan Province, China. We found that percentage germination varied among treatments and locations; control treatments (no inoculum) did not germinate at both sites. We found that the optimal treatment, having the highest in situ seed germination rate (0.94-1.44%) with no significant variation among sites, supported a warm, moist and fixed site that allowed for light penetration. When accounting for seed density, percentage germination was highest (2.78-2.35%) at low densities and did not vary among locations for the treatment that supported optimal conditions. Similarly for the same treatment, seed germination ranged from 0.24 to 5.87% among seasons but also did vary among sites. This study reports on the cultivation and restoration of an endangered epiphytic orchid species by in situ symbiotic seed germination and is likely to have broad application to the horticulture and conservation of the Orchidaceae.

[Effects of different endophytic fungi on seedling growth of Dendrobium devonianum].

To obtain seedling growth-promoting fungi is a key step in restoration-friendly cultivation of medicinal Dendrobium species, since there are a large number of functionally-unknown endophytic fungi in the roots of Dendrobium plants.In this study, six functionally-unknown endophytic fungal strains were isolated from roots of D.devonianum using single peleton isolation technology, and used in inoculation experiments to test their effectiveness for seedling growth in D.devonianum.After 90 days of inoculation, comparing with the control treatment, FDdS-1, FDdS-2 and FDdS-4 showed strong pathogenic or fatal effects on seedlings; while, FDdS-12, FDdS-9 and FDdS-5 had different effects on seedling growth.FDdS-5 had significant promoting effects on height, fresh and dry weight, stem diameter and root numbers, while FDdS-9 only had significant promoting effect on seedling height, and FDdS-12 had a negative effect on seedling growth.According to the anatomical features of the inoculated roots, FDdS-5 fungi could infect the velamina of seedlings and the existence of symbiosis pelotons in the cortex cells, suggesting that FDdS-5 is a mycorrhiza fungi of D.devonianum.FDdS-5 and FDdS-9 were identified as Sebacina vermifera and Sebacina sp.by molecular technologies.By using FDdS-5 in the restoration-friendly cultivation of D.devonianum, it could effectively promote seedling growth and shorten the seedling growth periods.The results will aid in reintroduction and cultivation of D.devonianum.

[Effects of different fungi on symbiotic seed germination of two Dendrobium species].

The epiphytic orchid, Dendrobium aphyllum and D. devonianum are used as traditional Chinese medicine, and became locally endangered in recent years because of over-collection. We test the effect of inoculations of endophytic fungi FDaI7 (Tulasnella sp.), FDd1 (Epulorhiza sp. ) and FCb4 (Epulorhiza sp.), which isolated from D. aphyllum, D. denonianum and Cymbidium mannii, respectively, on artificial substrate in these two Dendrobium species. In the symbiotic germination experiment, FDaI7 and FDd1 were effective for protocorm formation and seedling development of D. aphyllum and D. denonianum separately. After 60 days, 14.46% of the D. aphyllum seeds grown to protocorms and 12.07% developed to seedlings inoculated only with FDaI7, while contrasted with 0 when inoculated the other two isolates and non-inoculation treatment. However, in D. denonianum, seeds only grown to protocorms and developed to seedlings when inoculated with FDd1, the percentages were 44.36% and 42.91% distinguishingly. High specificity was shown in symbiotic germination on artificial substrate of Dendrobium. Protocorms could further develop to seedlings within or without light when inoculated the compatible fungi. However, light condition (12/12 h Light/Dark) produced the normal seedlings, while dark condition (0/24 h L/D) produced the abnormal seedlings. These may suggest that the development of young seedlings require light based on the effective symbiotic fungi. These findings will aid in seedling production of simulation-forestry ecology cultivation, conservation and reintroduction of Dendrobium.

In situ seed baiting to isolate germination-enhancing fungi for an epiphytic orchid, Dendrobium aphyllum (Orchidaceae).

Orchid conservation efforts, using seeds and species-specific fungi that support seed germination, require the isolation, identification, and germination enhancement testing of symbiotic fungi. However, few studies have focused on developing such techniques for the epiphytes that constitute the majority of orchids. In this study, conducted in Xishuangbanna Tropical Botanical Garden, Yunnan, China, we used seeds of Dendrobium aphyllum, a locally endangered and medicinally valuable epiphytic orchid, to attract germination promoting fungi. Of the two fungi isolated from seed baiting, Tulasnella spp. and Trichoderma spp., Tulasnella, enhanced seed germination by 13.6 %, protocorm formation by 85.7 %, and seedling development by 45.2 % (all P < 0.0001). Epulorhiza, another seed germination promoting fungi isolated from Cymbidium mannii, also enhanced seed germination (6.5 %; P < 0.05) and protocorm formation (20.3 %; P < 0.0001), but Trichoderma suppressed seed germination by 26.4 % (P < 0.0001). Tulasnella was the only treatment that produced seedlings. Light increased seed imbibition, protocorm formation, and two-leaved seed development of Tulasnella inoculated seeds (P < 0.0001). Because the germination stage success was not dependent on fungi, we recommend that Tulasnella be introduced for facilitating D. aphyllum seed germination at the protocorm formation stage and that light be provided for increasing germination as well as further seedling development. Our findings suggest that in situ seed baiting can be used to isolate seed germination-enhancing fungi for the development of seedling production for conservation and reintroduction efforts of epiphytic orchids such as D. aphyllum.

Rain pollination provides reproductive assurance in a deceptive orchid.

BACKGROUND AND AIMS: Abiotic pollination by wind or water is well established in flowering plants. In some species pollination by rain splashes, a condition known as ombrophily, has been proposed as a floral strategy. However, evidence for this type of abiotic pollination has remained controversial and many reported cases have subsequently been shown to be false. This study investigates ombrophily in the deceptive orchid Acampe rigida to determine the mechanism by which this species is able to maintain high fecundity, despite flowering during the rainy season in south-west China when pollinators are scarce. METHODS: The floral mechanisms promoting rain pollination in A. rigida were observed and described in detail. Controlled pollination experiments and observations of floral visitors were conducted. A field experiment using rain shelters at 14 sites in Guangxi, south-west China, evaluated the contribution of rain pollination to fruit-set. KEY RESULTS: During rainfall, raindrops physically flicked away the anther cap exposing the pollinarium. Raindrops then caused pollinia to be ejected upwards with the strap-like stipe pulling them back and causing them to fall into the stigmatic cavity, resulting in self-pollination. Neither flower nor pollen function were damaged by water. Although A. rigida is self-compatible, it is incapable of autonomous self-pollination without the assistance of rain splashes. The results of the rain-sheltering experiment indicated that rain pollination contributed substantially to increasing fruit-set, although there was variation among sites in the intensity of this effect. CONCLUSIONS: A. rigida flowers during the rainy season, when pollinators are scarce, and ombrophily functions to provide reproductive assurance without compromising opportunities for outcrossing.

Adaptive significance of flexistyly in Alpinia blepharocalyx (Zingiberaceae): a hand-pollination experiment.

BACKGROUND AND AIMS: Flexistyly is a sexual dimorphism where there are two morphs that differ in the temporal expression of sexual function and also involve reciprocal movement of the stigmatic surface through a vertical axis during the flowering period. The adaptive significance of flexistyly has been interpreted as a floral mechanism for outcrossing, but it may also function to reduce sexual interference in which styles and stigmas impede the pollen export. Here these two explanations of flexistyly were tested in Alpinia blepharocalyx through a hand-pollination experiment. METHODS: Hand-pollinations were performed in two temporal morphs and consisted of two sequential pollination treatments, namely self-pollination in the morning and inter-morph pollination in the afternoon (treatment 1) or conversely inter-morph pollination in the morning and self-pollination in the afternoon (treatment 2), and two simultaneous self- and inter-morph cross-pollination treatments either in the morning (treatment 3) or in the afternoon (treatment 4). Seed paternity was then determined to assess relative success of self- versus cross-pollen using allozyme markers. KEY RESULTS: In the sequential pollination treatments, whether the stigmas of recipients are receptive in the morning is crucial to the success of the pollen deposited. When the cataflexistylous (protandrous) morph served as pollen recipient, early-arriving pollen in the morning can sire only a very small proportion (<15%) of seeds because the stigmas were then unreceptive. However, when the anaflexistylous (protogynous) morph served as pollen recipient, early pollen did gain a large competitive advantage over the late pollen, particularly when cross-pollen arrived first. Simultaneous self- and inter-morph cross-pollination indicated that outcross-pollen is more competitive than self-pollen on receptive stigmas. CONCLUSIONS: Differential maturing of male and female organs in Alpinia blepharocalyx is sufficient for selfing avoidance, obviating the need for style movements. Instead, the upward style curvature of the cataflexistylous morph in the morning and the anaflexistylous morph in the afternoon most likely represents a means of reducing interference with pollen export.

The pollination ecology of Paraboea rufescens (Gesneriaceae): a buzz-pollinated tropical herb with mirror-image flowers.

BACKGROUND AND AIMS: Gesneriaceae is a pantropical plant family with over 3000 species. A great variety of pollination mechanisms have been reported for the neotropical members of the family, but the details of buzz-pollination and enantiostyly for the family have not been described. We investigated the floral biology and pollination ecology of Paraboea rufescens in Xishuangbanna, south-west China, considering three aspects: (1) the type of enantiostyly exhibited; (2) whether the species is self-compatible; and (3) whether pollinator behaviour could enhance the precision of pollen transfer between flowers of contrasting stylar orientation. METHODS: Flowering phenology was monitored once a month during vegetative growth, and once a week during flowering both in the field and under cultivation. Pollination manipulations and pollinator observation in the field were conducted. KEY RESULTS: Anthesis occurred early during the morning, and flowers remained open for 1-5 d, depending on weather conditions. Controlled pollinations revealed that P. rufescens is self-compatible, and exhibited inbreeding depression in seed set. Plants were pollinator limited in natural populations. The similar stylar deflection among flowers within a plant limits autonomous self-pollination as well as pollination between flowers. Two species of bumble bees (Bombus spp.), Amegila malaccensis and Nomia sp. effectively pollinated P. rufescens. These pollinators visited flowers in search of pollen with almost the same frequency. None of the pollinators appeared to discriminate between left- or right-handed flowers. CONCLUSIONS: Paraboea rufescens exhibits monomorphic enantiostylous flowers and a buzz-pollination syndrome. Floral morphology in P. rufescens and pollinator foraging behaviour seems likely to reduce self-pollination and pollinations between flowers of the same stylar deflection.

Predicting mating patterns from pollination syndromes: the case of "sapromyiophily" in Tacca chantrieri (Taccaceae).

Tacca, a genus of tropical herbs, possesses near black flowers, conspicuous involucral bracts and whisker-like filiform bracteoles. These features have been assumed to function as a "deceit syndrome" in which reproductive structures resemble decaying organic material attracting flies that facilitate cross-pollination (sapromyiophily). We investigated pollination and mating in Tacca chantrieri populations from SW China to evaluate this assumption. Contrary to this expectation, populations were highly selfing. Pollinator visitation was infrequent and bagged flowers set abundant seed. Pollen loads on stigmas indicated autonomous self-pollination, some of which occurred prior to flower opening. The seed set of inflorescences with bracts and bracteoles removed was not significantly different from unmanipulated inflorescences, suggesting that these structures play a limited role in pollinator attraction, at least at our study sites. Pollen : ovule ratios averaged 49, a value expected in a highly selfing species. Selfing rates estimated in four populations using allozyme markers averaged 0.86 (range 0.76-0.94), thus corroborating this inference. Our results indicate that despite considerable investment in extravagant display, populations of T. chantrieri are highly selfing. We propose several hypotheses to resolve this paradox and argue that future studies of pollination syndromes would benefit by investigation of both pollination and mating biology.

The floral biology of Curcumorpha longiflora (Zingiberaceae): a ginger with two-day flowers.

Curcumorpha longiflora is a perennial chasmophyte (rock fissure plant) ginger that usually grows in crevices of calcareous rocks and forms patches on the understory of limestone monsoon rainforests. The pollination ecology of C. longiflora was studied by monitoring phenology and flowering behavior, observing pollinator activity (frequency and behavior of visitors), and the quantity and quality of pollination services. We also investigated the germination of pollen grains and growth of pollen tubes after different pollination treatments to detect its breeding system. Based on the results: (1) for the first time in Zingiberaceae a new protandrous mechanism was found with a two-day flowering to avoid autogamy in this species; (2) under field conditions, all individuals of C. longiflora usually produced only one flower every other day to keep geitonogamy to a minimum; (3) germination of pollen grains and growth rates of pollen tubes under different pollination treatments were the same 4 h later after pollination, suggesting that C. longiflora is completely self-compatible; (4) among the limited visitors, Bombus sp. and Apis florae were effective pollinators, but they were active at different times and at different stages of the flower, probably receiving different rewards.