Staminal hairs enhance fecundity in the pollen‐rewarding self‐incompatible lily Bulbine abyssinica

Flowering plants typically use floral rewards to attract animal pollinators. Unlike nectar, pollen rewards are usually visible and may thus function as a signal that influences landing decisions by pollen‐seeking insects. Here we artificially manipulate the presence of both pollen and staminal hairs (a putative false signal of pollen reward availability) in the hermaphroditic lily Bulbine abyssinica (Xanthorrhoeaceae) to investigate their effects on bee visitation and fecundity, and also test for trade‐offs between pollen production and seed production. Honeybees, the primary floral visitors, are probably not able to distinguish between colours of petals, staminal hairs and pollen of B. abyssinica, according to analysis of reflectance spectra in a bee vision model. Flowers with both pollen and hairs removed had the lowest levels of bee visitation, seed set and seed abortions. Flowers containing hairs had an ～50% increase in visitation rate and seed set compared with emasculated flowers, while intact controls had the highest seed abortion rate. Ovule discounting in intact flowers is probably due to ovarian self‐incompatibility (or strong early inbreeding depression) as ovules penetrated by tubes from self‐pollen uniformly failed to develop into seeds. These results show that staminal hairs can enhance plant fecundity by increasing attraction of pollen‐seeking insects to flowers without increasing the risk of ovule discounting through pollinator‐mediated self‐pollination. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 177 , 481–490.     

Breeding systems in Cyrtanthus (Amaryllidaceae): variation in self‐sterility and potential for ovule discounting

• Breeding systems of plants determine their reliance on pollinators and ability to produce seeds following self‐pollination. Self‐sterility, where ovules that are penetrated by self‐pollen tubes that do not develop into seeds, is usually considered to represent either a system of late‐acting self‐incompatibility or strong early inbreeding depression. Importantly, it can lead to impaired female function through ovule or seed discounting when stigmas receive mixtures of self and cross pollen, unless cross pollen is able to reach the ovary ahead of self pollen (‘prepotency’). Self‐sterility associated with ovule penetration by self‐pollen tubes appears to be widespread among the Amaryllidaceae.
• We tested for self‐sterility in three Cyrtanthus species – C. contractus, C. ventricosus and C. mackenii – by means of controlled hand‐pollination experiments. To determine the growth rates and frequency of ovule penetration by self‐ versus cross‐pollen tubes, we used fluorescence microscopy to examine flowers of C. contractus harvested 24, 48 and 72 h after pollination, in conjunction with a novel method of processing these images digitally. To test the potential for ovule discounting (loss of cross‐fertilisation opportunities when ovules are disabled by self‐pollination), we pollinated flowers of C. contractus and C. mackenii with mixtures of self‐ and cross pollen.
• We recorded full self‐sterility for C. contractus and C. ventricosus, and partial self‐sterility for C. mackenii. In C. contractus, we found no differences in the growth rates of self‐ and cross‐pollen tubes, nor in the proportions of ovules penetrated by self‐ and cross‐pollen tubes. In this species, seed set was depressed (relative to cross‐pollinated controls) when flowers received a mixture of self and cross pollen, but this was not the case for C. mackenii.
• These results reveal variation in breeding systems among Cyrtanthus species and highlight the potential for gender conflict in self‐sterile species in which ovules are penetrated and disabled by pollen tubes from self pollen.

     

Breeding systems in Clivia (Amaryllidaceae): late‐acting self‐incompatibility and its functional consequences

Late‐acting (ovarian) self‐incompatibility, characterized by minimal or zero seed production following self‐pollen tube growth to the ovules, is expected to show phylogenetic clustering, but can otherwise be difficult to distinguish from early‐acting inbreeding depression. In Amaryllidaceae, late‐acting self‐incompatibility has been proposed for Narcissus (Narcisseae) and Cyrtanthus (Cyrtantheae). Here, we investigate whether it occurs in the horticulturally important genus Clivia (Haemantheae) and test whether species in this genus experience ovule discounting in wild populations. Seed‐set results following controlled hand pollinations revealed that Clivia miniata and C. gardenii are largely self‐sterile. Self‐ and cross‐pollinated flowers of both species had similar proportions of pollen tubes entering the ovary, and those of C. gardenii also did not differ in the proportions of pollen tubes that penetrated ovules, thus ruling out classical gametophytic self‐incompatibility acting in the style, but not early inbreeding depression. Flowers that received equal mixtures of self‐ and cross‐pollen set fewer seeds than those that received cross‐pollen only, but it was unclear whether this effect was a result of ovule discounting or interactions on the stigma. The prevention of self‐pollination by the emasculation of either single flowers or whole inflorescences in wild populations did not affect seed set, suggesting that ovule discounting is not a major natural limitation on seed production. Flowers typically produce one to three large fleshy seeds from approximately 16 available ovules, even when supplementally hand pollinated, suggesting that fecundity is mostly resource limited. The results of this study suggest that Clivia spp. are largely self‐sterile as a result of either a late‐acting self‐incompatibility system or severe early inbreeding depression, but ovule discounting caused by self‐pollination is not a major constraint on fecundity. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 175 , 155–168.     

S-allele diversity suggests no mate limitation in small populations of a self-incompatible plant

Small populations of self-incompatible plants are assumed to be threatened by a limitation of compatible mating partners due to low genetic diversity at the self-incompatibility (S) locus. In contrast, we show by using a PCR-RFLP approach for S-genotype identification that 15 small populations (N = 8-88) of the rare wild pear (Pyrus pyraster) displayed no mate limitation. S-allele diversity within populations was high (N = 9-21) as was mate availability (92.9-100%). Although population size and S-allele diversity were strongly related, no relationship was found between population size and mate availability, gene diversity (He), or fixation index (F(IS)), based on five neutral microsatellite loci. As we determined the principal mate availability within populations based on the S-genotypes observed, the realized mate availability under natural conditions may differ from our estimates, for example, due to spatially limited pollen dispersal. We therefore urge studies on self-incompatible plants to proceed from the simple assessment of principal mate availability to the determination of realized mate availability in natural populations.     

Effects of landscape structure and climate seasonality on pollen intake by honeybees in Neotropical highland agroecosystems

1. Pollen is the main protein source for honeybee brood and so colony development relies heavily on the availability of pollen in the environment. Intensification of agriculture and climate seasonality are known to alter honeybee pollen intake in temperate regions through changes in resource availability; however, little is known about how honeybees respond to such environmental factors in tropical regions.
2. Pollen collected by honeybees was sampled from apiaries in a Neotropical highland region of Colombia. Pollen species were identified and the effects of landscape diversity, forest area and mean monthly precipitation on the pollen intake by honeybees were evaluated for all pollen species together and pollen species segregated according to forest and anthropic areas.
3. Honeybees were found to be much more associated with anthropic than forest pollen species regardless of landscape structure or precipitation. However, pollen intake from all species and forest species responded positively to landscape diversity and forest area. Precipitation was found not to be related to the overall amount and overall richness of pollen collected by honeybees. Nonetheless, overall pollen diversity was negatively affected by precipitation in less diverse landscapes, whereas anthropic pollen diversity was negatively affected by precipitation in more forested landscapes.

     

蜜蜂人工代用花粉中适宜钙磷水平的初步研究

钙磷是蜜蜂饲粮中必需的常量元素。为了探讨蜜蜂人工代用花粉中适宜的钙磷水平,本试验选取蜂王、群势基本一致的意大利蜜蜂Apis mellifera ligustica Spinola 70群,随机分为14个组,每组5群蜂,前12组饲喂采用均匀设计法配制的不同钙磷水平的人工代用花粉。第13组饲喂不加钙磷的人工代用花粉为负对照,第14组饲喂纯油菜花粉为正对照。试验从春繁开始,到刺槐流蜜期结束为止。试验期间测定蜂群的采食量、蜂群群势、幼蜂初生重,成蜂蜂体组织内钙磷含量。结果表明:当人工代用花粉中钙磷水平分别为0,0.65%时,蜂群的采食量、蜂群群势、幼蜂初生重均取得最大值;成蜂蜂体组织内钙含量与人工代用花粉中钙磷含量成正相关,成蜂蜂体组织内磷含量与人工代用花粉中钙磷含量之间没有相关性。     

Effects of pollen availability and the mutation bias on the fixation of mutations disabling the male specificity of self‐incompatibility

The evolution of self‐compatibility (SC) by the loss of self‐incompatibility (SI) is regarded as one of the most frequent transitions in flowering plants. SI systems are generally characterized by specific interactions between the male and female specificity genes encoded at the S‐locus. Recent empirical studies have revealed that the evolution of SC is often driven by male SC‐conferring mutations at the S‐locus rather than by female mutations. In this study, using a forward simulation model, we compared the fixation probabilities of male vs. female SC‐conferring mutations at the S‐locus. We explicitly considered the effects of pollen availability in the population and bias in the occurrence of SC‐conferring mutations on the male and female specificity genes. We found that male SC‐conferring mutations were indeed more likely to be fixed than were female SC‐conferring mutations in a wide range of parameters. This pattern was particularly strong when pollen availability was relatively high. Under such a condition, even if the occurrence of mutations was biased strongly towards the female specificity gene, male SC‐conferring mutations were much more often fixed. Our study demonstrates that fixation probabilities of those two types of mutation vary strongly depending on ecological and genetic conditions, although both types result in the same evolutionary consequence—the loss of SI.     

Apple MdABCF assists in the transportation of S‐RNase into pollen tubes

Self‐incompatibility (SI) is a reproductive isolation mechanism in flowering plants. Plants in the Solanaceae, Rosaceae and Plantaginaceae belong to the gametophytic self‐incompatibility type. S‐RNase, which is encoded by a female‐specific gene located at the S locus, degrades RNA in the pollen tube and causes SI. Recent studies have provided evidence that S‐RNase is transported non‐selectively into the pollen tube, but have not specified how this transportation is accomplished. We show here that the apple (Malus domestica) MdABCF protein, which belongs to group F of the ABC transporter family, assists in transportation of S‐RNase into the pollen tube. MdABCF is located in the pollen tube membrane and interacts with S‐RNase. S‐RNase was unable to enter the pollen tube when MdABCF was silenced by antisense oligonucleotide transfection. Our results show that MdABCF assists in transportation of either self or non‐self S‐RNase into the pollen tube. Moreover, MdABCF coordinates with the cytoskeleton to transport S‐RNase. Blockage of S‐RNase transport disrupts self‐incompatibility in this system.     

Reproductive system and fitness of Vriesea friburgensis,a self‐sterile bromeliad species

Reproductive biology and plant fertility are directly related to many aspects of plant evolution and conservation biology. Vriesea friburgensis is an epiphytic and terrestrial bromeliad endemic to the Brazilian Atlantic rainforest. Hand‐pollination experiments were used to examine the reproductive system in a wild population of V. friburgensis. Plant fertility was assigned considering flower production, fruit and seed set, seed germination, and pollen viability. Self‐sterility observed from spontaneous selfing and manual self‐pollination treatments may be the consequence of late‐acting self‐incompatibility. Hand‐pollination results indicated no pollen limitation in the population studied. Floral biology features such as a few daily open flowers, nectar production, and sugar concentration corroborate hummingbirds as effective pollinators, although bees were also documented as pollinators. Components of fitness such as high flower, fruit, and seed production together with high seed and pollen viability indicate that this wild population is viable. From a conservation point of view, we highlight that this self‐sterile species depends on pollinator services to maintain its population fitness and viability through cross‐pollination. Currently, pollinators are not limited in this population of V. friburgensis. Conversely, the maintenance and continuous conservation of this community is essential for preserving this plant–pollinator mutualism.     

Sequence divergence and loss-of-function phenotypes of S locus F-box brothers genes are consistent with non-self recognition by multiple pollen determinants in self-incompatibility of Japanese pear (Pyrus pyrifolia)

The S-RNase-based gametophytic self-incompatibility (SI) of Rosaceae, Solanaceae, and Plantaginaceae is controlled by at least two tightly linked genes located at the complex S locus; the highly polymorphic S-RNase for pistil specificity and the F-box gene (SFB/SLF) for pollen. Self-incompatibility in Prunus (Rosaceae) is considered to represent a 'self recognition by a single factor' system, because loss-of-function of SFB is associated with self-compatibility, and allelic divergence of SFB is high and comparable to that of S-RNase. In contrast, Petunia (Solanaceae) exhibits 'non-self recognition by multiple factors'. However, the distribution of 'self recognition' and 'non-self recognition' SI systems in different taxa is not clear. In addition, in 'non-self recognition' systems, a loss-of-function phenotype of pollen S is unknown. Here we analyze the divergence of SFBB genes, the multiple pollen S candidates, of a rosaceous plant Japanese pear (Pyrus pyrifolia) and show that intrahaplotypic divergence is high and comparable to the allelic diversity of S-RNase while interhaplotypic divergence is very low. Next, we analyzed loss-of-function of the SFBB1 type gene. Genetic analysis showed that pollen with the mutant haplotype S(4sm) lacking SFBB1-S(4) is rejected by pistils with an otherwise compatible S(1) while it is accepted by other non-self pistils. We found that the S(5) haplotype encodes a truncated SFBB1 protein, even though S(5) pollen is accepted normally by pistils with S(1) and other non-self haplotypes. These findings suggest that Japanese pear has a 'non-self recognition by multiple factors' SI system, although it is a species of Rosaceae to which Prunus also belongs.     

Secondary pollen presentation and the temporal dynamics of stylar hair retraction and style elongation in Campanula trachelium (Campanulaceae)

To increase the accuracy of pollen capture and transfer by pollinators some plant species have developed secondary pollen presentation structures. Because the presence of secondary pollen presentation structures at the pistil may reduce the spatial separation between the sexual functions and increase the risk of self‐interference and selfing, temporal segregation of the sexual organs, triggered by visiting insects, can be expected to occur. We investigated secondary pollen presentation and the temporal dynamics of the sexual phases in combination with the physiological self‐incompatibility system in Campanula trachelium, a protandrous insect‐pollinated herb. Stylar hair retraction (male function) and curling of the stigmatic lobes (female function) were modelled using Gompertz growth functions. Finally, we performed pollination experiments in the lab and field to assess seed set and pollen limitation under natural conditions. About 68% of the total pollen load was captured by stylar hairs. Manual manipulation of the stylar hairs, mimicking pollinator visitation, significantly shortened the male phase and accelerated the female phase, resulting in a significant decline in temporal overlap between the two sexual functions. Conversely, when pollinators and/or manual manipulations were lacking, the male phase was substantially prolonged and sexual overlap was maximal. This suggests that spreading of the sexual phases and thus the risk of sexual interference are largely determined by the interaction between stylar hairs and visiting pollinators. Natural seed set was high and not pollinator limited. Overall, these results indicate that secondary pollen presentation and partial protandry resulted in efficient pollen capture, transfer and deposition.     

Effect of smoke derivatives on in vitro pollen germination and pollen tube elongation of species from different plant families

Plant‐derived smoke stimulates seed germination in numerous plant species. Smoke also has a positive stimulatory effect on pollen germination and pollen tube growth. The range of plant families affected my smoke still needs to be established since the initial study was restricted to only three species from the Amaryllidaceae. The effects of smoke‐water (SW) and the smoke‐derived compounds, karrikinolide (KAR₁) and trimethylbutenolide (TMB) on pollen growth characteristics were evaluated in seven different plant families. Smoke‐water (1:1000 and 1:2000 v:v) combined with either Brewbaker and Kwack's (BWK) medium or sucrose and boric acid (SB) medium significantly improved pollen germination and pollen tube growth in Aloe maculata All., Kniphofia uvaria Oken, Lachenalia aloides (L.f.) Engl. var. aloides and Tulbaghia simmleri P. Beauv. Karrikinolide (10^?6 and 10^?7 m ) treatment significantly improved pollen tube growth in A. maculata, K. uvaria, L. aloides and Nematanthus crassifolius (Schott) Wiehle compared to the controls. BWK or SB medium containing TMB (10^?3 m ) produced significantly longer pollen tubes in A. maculata, K. uvaria and N. crassifolius. These results indicate that plant‐derived smoke and the smoke‐isolated compounds may stimulate pollen growth in a wide range of plant species.     

Relative impact of mate versus pollinator availability on pollen limitation and outcrossing rates in a mass‐flowering species

Plant mating systems are driven by several pre‐pollination factors, including pollinator availability, mate availability and reproductive traits. We investigated the relative contributions of these factors to pollination and to realized outcrossing rates in the patchily distributed mass‐flowering shrub Rhododendron ferrugineum. We jointly monitored pollen limitation (comparing seed set from intact and pollen‐supplemented flowers), reproductive traits (herkogamy, flower size and autofertility) and mating patterns (progeny array analysis) in 28 natural patches varying in the level of pollinator availability (flower visitation rates) and of mate availability (patch floral display estimated as the total number of inflorescences per patch). Our results showed that patch floral display was the strongest determinant of pollination and of the realized outcrossing rates in this mass‐flowering species. We found an increase in pollen limitation and in outcrossing rates with increasing patch floral display. Reproductive traits were not significantly related to patch floral display, while autofertility was negatively correlated to outcrossing rates. These findings suggest that mate limitation, arising from high flower visitation rates in small plant patches, resulted in low pollen limitation and high selfing rates, while pollinator limitation, arising from low flower visitation rates in large plant patches, resulted in higher pollen limitation and outcrossing rates. Pollinator‐mediated selfing and geitonogamy likely alleviates pollen limitation in the case of reduced mate availability, while reduced pollinator availability (intraspecific competition for pollinator services) may result in the maintenance of high outcrossing rates despite reduced seed production.     

Genetic rescue in a plant polyploid complex: Case study on the importance of genetic and trait data for conservation management

Knowledge of the biology of rare plant species is indispensable to aid their survival and to inform efficient conservation actions, but in many cases relevant data are lacking. In addition, while studies of conservation genetics have provided a wealth of information on the considerations arising from inbreeding, mate limitation, or local adaptation, the impact of intraspecific polyploidy remains understudied. In this study, we examined the breeding system of the rare Australian daisy Rutidosis lanata (Asteraceae) and screened ten of its populations for their ploidy level to develop recommendations for management actions, in particular, with regard to seed sourcing and genetic rescue. We found R. lanata to represent a polyploid complex, with tetraploid, pentaploid and hexaploid individuals coexisting in the same species. Crossing experiments confirmed R. lanata to be self‐incompatible. Mate availability varied from c. 49% to c. 76% across populations. Most populations showed mate availability of c. 50%–70%, suggesting that mate limitation resulting from a lack of local genetic diversity may cause or at least contribute to reduced seed set. Crossing between populations resulted in significantly higher reproductive success for all populations except one, suggesting the possibility of genetic rescue through population mixing. However, the crossing experiments also showed that pentaploids suffer from a severely reduced paternal reproductive fitness. Any additional hybrids between tetraploids and pentaploids, as would be created by mixing populations with different genome copy numbers during conservation work, would consequently exacerbate mate limitation and thus reduce population viability. We conclude that seed set and thus population viability can be maximized by mixing populations with the same number of genome copies, but that populations with different numbers should be kept spatially separated. The case of Rutidosis lanata provides an example and a potential template for examining the conservation genetics of other species that may constitute polyploid complexes.     

Limiting inbreeding in disjunct and isolated populations of a woody shrub

Pollen movements and mating patterns are key features that influence population genetic structure. When gene flow is low, small populations are prone to increased genetic drift and inbreeding, but naturally disjunct species may have features that reduce inbreeding and contribute to their persistence despite genetic isolation. Using microsatellite loci, we investigated outcrossing levels, family mating parameters, pollen dispersal, and spatial genetic structure in three populations of Hakea oldfieldii, a fire‐sensitive shrub with naturally disjunct, isolated populations prone to reduction in size and extinction following fires. We mapped and genotyped a sample of 102 plants from a large population, and all plants from two smaller populations (28 and 20 individuals), and genotyped 158–210 progeny from each population. We found high outcrossing despite the possibility of geitonogamous pollination, small amounts of biparental inbreeding, a limited number of successful pollen parents within populations, and significant correlated paternity. The number of pollen parents for each seed parent was moderate. There was low but significant spatial genetic structure up to 10 m around plants, but the majority of successful pollen came from outside this area including substantial proportions from distant plants within populations. Seed production varied among seven populations investigated but was not correlated with census population size. We suggest there may be a mechanism to prevent self‐pollination in H. oldfieldii and that high outcrossing and pollen dispersal within populations would promote genetic diversity among the relatively small amount of seed stored in the canopy. These features of the mating system would contribute to the persistence of genetically isolated populations prone to fluctuations in size.     

Pollen transfer in fragmented plant populations: insight from the pollen loads of pollinators and stigmas in a mass‐flowering species

Pollinator and/or mate scarcity affects pollen transfer, with important ecological and evolutionary consequences for plant reproduction. However, the way in which the pollen loads transported by pollinators and deposited on stigmas are affected by pollination context has been little studied. We investigated the impacts of plant mate and visiting insect availabilities on pollen transport and receipt in a mass‐flowering and facultative autogamous shrub (Rhododendron ferrugineum). First, we recorded insect visits to R. ferrugineum in plant patches of diverse densities and sizes. Second, we analyzed the pollen loads transported by R. ferrugineum pollinators and deposited on stigmas of emasculated and intact flowers, in the same patches. Overall, pollinators (bumblebees) transported much larger pollen loads than the ones found on stigmas, and the pollen deposited on stigmas included a high proportion of conspecific pollen. However, comparing pollen loads of emasculated and intact flowers indicated that pollinators contributed only half the conspecific pollen present on the stigma. At low plant density, we found the highest visitation rate and the lowest proportion of conspecific pollen transported and deposited by pollinators. By contrast, at higher plant density and lower visitation rate, pollinators deposited larger proportion of conspecific pollen, although still far from sufficient to ensure that all the ovules were fertilized. Finally, self‐pollen completely buffered the detrimental effects on pollination of patch fragmentation and pollinator failure. Our results indicate that pollen loads from pollinators and emasculated flowers should be quantified for an accurate understanding of the relative impacts of pollinator and mate limitation on pollen transfer in facultative autogamous species.     

Effects of male fecundity, interindividual distance and anisotropic pollen dispersal on mating success in a Scots pine (Pinus sylvestris) seed orchard

Quantifying the effect of pollen dispersal and flowering traits on mating success is essential for understanding evolutionary responses to changing environments and establishing strategies for forest tree breeding. This study examined, quantitatively, the effects of male fecundity, interindividual distance and anisotropic pollen dispersal on the mating success of Scots pine (Pinus sylvestris), utilizing a well-mapped Scots pine seed orchard. Paternity analysis of 1021 seeds sampled from 87 trees representing 28 clones showed that 53% of the seeds had at least one potential pollen parent within the orchard. Pronounced variation in paternal contribution was observed among clones. Variations in pollen production explained up to 78% of the variation in mating success, which was 11.2 times greater for clones producing the largest amount of pollen than for clones producing the least pollen. Mating success also varied with intertree distance and direction, which explained up to 28% of the variance. Fertilization between neighboring trees 2.3 m apart was 2.4 times more frequent than between trees 4.6 m apart, and up to 12.4 times higher for trees downwind of the presumed prevailing wind direction than for upwind trees. The effective number of pollen donors recorded in the seed orchard (12.2) was smaller than the theoretical expectation (19.7). Based on the empirical observations, a mating model that best describes the gene dispersal pattern in clonal seed orchards was constructed.     

Evaluation of pollinator effectiveness based on pollen deposition and seed production in a gynodieocious alpine plant,Cyananthus delavayi

Examining variations in pollinator effectiveness can enhance our understanding of how pollinators and plants interact. Pollen deposition and seed production after a single visit by a pollinator are often used to estimate pollinator effectiveness. However, seed production is not always directly related to pollen deposition because not all pollen grains that are deposited on a stigma are compatible or conspecific. In the field, we tested pollinator effectiveness based on pollen deposition and the resulting seed production after single visits by different pollinator groups in a gynodieocious alpine plant Cyananthus delavayi (Campanulaceae). Our results showed that mean pollen deposition was generally inconsistent with mean seed production when comparisons were performed among different pollinator groups and sexes. In general, the correlations were not significant between pollen deposition and seed production in both perfect and female flowers after single visits by halictid bees, bumble bees, and hoverflies. We suggest seed set of virgin flowers after single visits is a more reliable indicator of pollinator effectiveness than pollen deposition and would be a better indicator of pollinator effectiveness for future studies.