Reproductive versatility in legumes: the case of amphicarpy in Trifolium polymorphum

Amphicarpy is a fascinating reproductive strategy, defined as fruit produced both below the soil surface and as aerial fruit on the same plant. Trifolium polymorphum is a grassland species subject to herbivory that combines amphicarpy with vegetative reproduction through stolons. Underground flowers have been described as obligate autogamous and aerial ones as self‐compatible allogamous, with aerial floral traits favouring cross‐pollination. In the present work we performed different pollination treatments on aerial flowers to analyse rates of pollen tube development and offspring fitness, measured as fruit set, seed production and germination percentage. This last variable was compared to that of seeds produced underground. No significant differences were found between fruit set in self‐ and cross‐pollinations. Seed production was higher in self‐pollinations, which is consistent with the higher rate of pollen tube development observed in self‐crosses. Spontaneous self‐pollination is limited in aerial flowers; thus pollen transfer by means of a vector is required even within the same flower. Germination tests showed that aerial seeds produced after self‐ and cross‐pollination did not differ in fitness, but underground seeds had higher germination percentage than aerial ones. Thus, we conclude that T. polymorphum has a mixed mating system. In grasslands with heavy grazing pressure, clonal propagation and underground seed production ensure persistence in the field. An intermediate level of selfing in aerial flowers ensures offspring, but morphological (herkogamy) and functional (dicogamy) floral traits maintain a window to incorporate genetic variability, allowing the species to tolerate temporal and spatial pressures.     

Ovule pre-emption and pollen limitation in a self-fertile perennial herb (Blandfordia grandiflora,Liliaceae)

The extent, frequency and causes of pollenlimited seed production were examined in partially selffertile populations of Blandfordia grandiflora for 2 years. Percentage seed set of open-pollinated plants (50–57%) did not differ within or between years, and was about 19% less than experimentally cross-pollinated plants (70–75%). Floral visits by honeybees did not differ through the flowering season and the number of pollen grains deposited on stigmas within 12 h of flowers opening exceeded the number of ovules per flower, indicating that the quality rather than the quantity of pollination limited seed set. Pollen limitation was caused by concurrent self- and cross-pollination and the subsequent abortion of some selfed ovules due to inbreeding depression. Natural seed set (55%) was intermediate between selfed (43%) and crossed (75%) flowers and was not increased when flowers that had been available to pollinators for 24 h were hand cross-pollinated, suggesting that ovules were already fertilized. Similarly, experimental pollination with both cross and self pollen within 24 h of flowers opening did not increase seed set relative to natural seed set, indicating that both cross- and self-fertilizations had occurred. In contrast, when selfing followed crossing by 48 h, or vice versa, seed set did not differ from crossed-only or selfed-only flowers, respectively, indicating that ovules were pre-empted by the first pollination. Collectively, these results indicate that under natural conditions self pollen pre-empts ovules, rendering them unavailable for cross-fertilization. This selfing reduces fecundity by 50%, as estimated from the natural production of cross seeds when selfing was prevented. Consequently, selection should favour floral traits, such as increased stigma-anther separation or protandry, that reduce interference between male and female functions that leads to selfing.     

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.     

COSTS OF SELF-POLLINATION IN A SELF-INCOMPATIBLE PLANT,POLEMONIUM VISCOSUM

Plants of Polemonium viscosum are strongly self-incompatible, yet have floral features that enforce spatial and temporal separation of pollen and stigma presentation. In this study, we address the widely held view that such barriers to selfing are maintained because they reduce interference by self pollen in performance of outcross grains. In the first year of the study we found that 24 hr precedence of self pollen on the stigma (24S), reduced outcross pollen germination by 32%, and seed set by 40% in flowers of P. viscosum. In the second year significant negative effects of self pollen on compatible pollen germination were seen when self pollen was applied four hours before (4S) or immediately prior (S×) to outcross pollen. Surprisingly, however, in the second year of study, seed set was not reduced with as much as 50% inhibition of compatible pollen germination. Results suggest that by interfering with normal functioning of outcross grains, incompatible self pollen can increase pollen limitation in P. viscosum. Reduction in seed production as a result of this phenomenon should select for displacement of pollen presentation from stigma receptivity.     

Pollination biology of the hexaploid self‐compatible species Turnera velutina (Passifloraceae)

• The evolution of monomorphisms from heterostylous ancestors has been related to the presence of homostyly and the loss of self‐incompatibility, allowing the occurrence of selfing, which could be advantageous under pollinator limitation. However, flowers of some monomorphic species show herkogamy, attraction and rewarding traits that presumably favour cross‐pollination and/or a mixed mating system. This study evaluated the contributions of pollinators, breeding system and floral traits to the reproduction of Turnera velutina, a herkogamous monomorphic species.
• Floral visitors and frequency of visits were recorded, controlled hand cross‐pollinations were conducted under greenhouse and natural conditions, and individual variation in floral traits was characterised to determine their contribution to seed production.
• Apis mellifera was the most frequent floral visitor. Flowers presented approach herkogamy, high variation in nectar features, and a positive correlation of floral length with nectar volume and sugar concentration. Seed production did not differ between manual self‐ and cross‐pollinations, controls or open cross‐pollinations, but autonomous self‐pollination produced, on average, 82.74% fewer seeds than the other forms, irrespective of the level of herkogamy.
• Differences in seed production among autonomous self‐pollination and other treatments showed that T. velutina flowers depend on insect pollination for reproduction, and that approach herkogamy drastically reduced seed production in the absence of pollen vectors. The lack of differences in seed production from manual cross‐ and self‐pollinations suggests the possible presence of a mixed mating system in the studied population. Overall, this species was possibly derived from a distylous ancestor but appears fully capable of outcrossing despite being monomorphic.

     

Significant differences in outcrossing rate,self‐incompatibility,and inbreeding depression between two widely hybridizing species of Geum

Geum urbanum and Geum rivale are two widely hybridizing perennial herbs. Estimation of the breeding systems of these taxa using nuclear microsatellite markers scored in mother–progeny arrays demonstrated that, in pure populations, G. urbanum is predominantly selfing (outcrossing rate, t = 0.058 to 0.177), whereas G. rivale is predominantly outcrossing (t = 0.686–0.775). Theory suggests that hybridization between inbreeding and outcrossing species can potentially generate novel inbreeding lineages. However, the establishment of such lineages may be restricted either by self‐incompatibility loci or deleterious recessive alleles derived from the outcrossing parent. To assess the likelihood that hybridization between G. urbanum and G. rivale will generate novel inbreeding lineages, self‐incompatibility and inbreeding depression were investigated in the two taxa. Seed set in the absence of pollinators, and after controlled self‐ and cross‐pollination, was measured to study self‐incompatibility. Inbreeding depression was measured by estimating the relative fitness of offspring from controlled self‐and cross‐pollinations. Geum urbanum was fully self‐compatible [self‐compatibility index (SCI) = 1] and bagged flowers showed full seed set. By contrast, only 3% of bagged flowers set seed in G. rivale and controlled self‐pollinations showed a 60–80% reduction in seed set compared to controlled outcross pollinations (SCI = 0.28). There was no evidence for inbreeding depression in G. urbanum, although significant, albeit low levels of inbreeding depression were detected in one of two G. rivale populations (δ = 0.33). The implication of these results is that if genetic material from G. rivale was incorporated into a hybrid with a selfing morphology, the establishment of this selfing lineage could be compromised by self‐incompatibility and inbreeding depression. The wider implications of these results for evolution in hybrid swarms between G. urbanum and G. rivale are discussed. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 101 , 977–990.     

Pollination and fruit‐set of Grevillea robusta in western Kenya

The floral visitors of silky oak, Grevillea robusta A. Cunn. ex R.Br., their foraging behaviour and their effects on fruit‐set were studied at Malava, western Kenya. Grevillea robusta is a popular tree for farm plantings in the eastern and central African highlands. Yield of seed has been disappointingly low in some areas and a lack of appropriate pollinators has been suggested as a possible cause. Investigations involved the monitoring of visitors on active inflorescences, assessment of the rewards available to potential pollinators, and exclusion experiments to establish the effects of various visitors on fruit‐set. The flowers are visited mainly by birds and insects. The likely pollinators of G. robusta are sunbirds (Nectarinia amethystina, N. cyanolaema, N. olivacea, N. superba and N. venusta) and white‐eyes (Zosterops kikuyuensis and Z. senegalensis). Very little aggressive behaviour between birds was recorded. No nocturnal pollinators were observed. Nectar was the major floral reward for pollinators, but is likely depleted by ants and honey bees, the foraging behaviour of which confirmed them to be nectar‐robbers. These insects hardly ever touched stigmas during their visits. Eighty‐nine per cent of bird visits were in the morning (07.00–10.00 hours) when nectar volume was highest. Inflorescences bagged to exclude birds set no fruits, and unmanipulated flowers and flowers bagged with self‐pollen set no fruits, indicating a self‐incompatibility mechanism. Control cross‐pollinated flowers displayed greatly increased fruit‐set (25.1%) compared with natural open‐pollination (0.9%). All these findings confirm the importance of cross‐pollen transfer to flowers and the necessity of pollinators for fruit‐set. Effective seed production requires activity of pollinators for self‐pollen removal and cross‐pollen deposition. Seed production stands for G. robusta should be established where flowering is prolific and bird pollinators are abundant.     

Relatively weak inbreeding depression in selfing but also in outcrossing populations of North American Arabidopsis lyrata

Hermaphroditic plants can potentially self‐fertilize, but most possess adaptations that promote outcrossing. However, evolutionary transitions to higher selfing rates are frequent. Selfing comes with a transmission advantage over outcrossing, but self‐progeny may suffer from inbreeding depression, which forms the main barrier to the evolution of higher selfing rates. Here, we assessed inbreeding depression in the North American herb Arabidopsis lyrata, which is normally self‐incompatible, with a low frequency of self‐compatible plants. However, a few populations have become fixed for self‐compatibility and have high selfing rates. Under greenhouse conditions, we estimated mean inbreeding depression per seed (based on cumulative vegetative performance calculated as the product of germination, survival and aboveground biomass) to be 0.34 for six outcrossing populations, and 0.26 for five selfing populations. Exposing plants to drought and inducing defences with jasmonic acid did not magnify these estimates. For outcrossing populations, however, inbreeding depression per seed may underestimate true levels of inbreeding depression, because self‐incompatible plants showed strong reductions in seed set after (enforced) selfing. Inbreeding‐depression estimates incorporating seed set averaged 0.63 for outcrossing populations (compared to 0.30 for selfing populations). However, this is likely an overestimate because exposing plants to 5% CO₂ to circumvent self‐incompatibility to produce selfed seed might leave residual effects of self‐incompatibility that contribute to reduced seed set. Nevertheless, our estimates of inbreeding depression were clearly lower than previous estimates based on the same performance traits in outcrossing European populations of A. lyrata, which may help explain why selfing could evolve in North American A. lyrata.     

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.

     

Pollination Biology of the Exotic Rattleweed Crotalaria retusa L. (Fabaceae) in NE Brazil1

The rattleweed Crotalaria retusa was introduced in Brazil from Africa, and combines a series of characters that have ensured its establishment in NE Brazil. We focused on its reproductive biology and pollinator behavior to explain its reproductive success. We performed manual pollination and germination experiments, and monitored the behavior of C. retusa's main pollinators in monospecific plots, and in mixed plots where C. retusa occurred together with two congeners, Crotalaria pallida and Crotalaria lanceolata. Crotalaria retusa is self‐compatible and capable of automatic selfing. Inbreeding depression was expressed at the level of percent seed germination, but not seed set. Few insects visited the inflorescences. Legitimate pollinators were two large carpenter bees, Xylocopa frontalis and Xylocopa grisescens which, together, accounted for more than 90 percent of the visits. The former foraged on C. retusa exclusively and has low pollen spread potential. The latter flew longer distances between plants and visited fewer flowers per inflorescence, potentially increasing the extent of pollen carryover, but at the risk of increasing heterospecific pollen transfer, because it visits other Crotalaria species during the same foraging bout. The different foraging strategies, allied to morphological disadvantages represented by pollen overlap on X. grisescens' body, may partially explain the much lower seed germination observed in C. pallida and C. lanceolata. These results are consistent with the hypothesis that a reduction in flower constancy may significantly depress viable seed set by increasing the chances of self‐pollination.     

Mating system in Mexican populations of the annual herb Solanum rostratum Dunal (Solanaceae)

Traditionally, annual colonising species are expected to have high rates of self‐fertilisation, although recent theoretical and empirical studies have shown that cross‐fertilisation can be selected for under heterogeneous pollination environments. Solanum rostratum is a self‐compatible annual herb that colonises disturbed habitats. Despite the lack of physiological mechanisms to prevent self‐fertilisation, pollen transfer between individuals is expected to be favoured because of its complex floral morphology. In previous studies of S. rostratum it has been shown that anther dimorphism within flowers results in precise pollen placement on the pollinator's body, and the presence of mirror‐image floral morphs within plants promotes outcrossing in experimental arrays. However, the mating system of natural populations of S. rostratum has never been assessed, and thus whether it is predominantly selfing or outcrossing remains unknown. We hypothesise that floral and inflorescence morphology of S. rostratum should facilitate cross‐fertilisation, making it a predominantly outcrossing despite its lack of a self‐incompatibility system. To test this hypothesis, we estimated outcrossing rates by genotyping 700 individuals at 13 microsatellite loci, sampled from four populations across a 690‐km transect in the species' native range. We found that populations had mean outcrossing rates of 0.70 ± 0.03, with multiple sires contributing to paternity of each progeny array (average effective number of sires = 8.97 ± 0.57). This indicates that natural populations S. rostratum have relatively high levels of outcrossing, probably facilitated by its floral and inflorescence morphology. We speculate that partial selfing in this species may be an unavoidable consequence of displaying multiple flowers at the same time (geitonogamy), as well as the result of self‐pollen transfer by illegitimate visitors.     

Interspecific pollen transfer as a mechanism of competition: Consequences of foreign pollen contamination for seed set in the alpine wildflower,Polemonium viscosum

Summary Earlier studies showed that under natural conditions foreign pollen receipt is correlated with loss of seed set in populations of P. viscosum. Here, we report on experimental pollinations using foreign pollen from two co-flowering species that share pollinators with P. viscosum. Experiments were designed to ascertain whether improper pollen transfer causes reduced seed set under controlled conditions and, if so, to determine the stage at which interference occurs. In flowers pre-treated by pollination with either Mertensia or Castilleja pollen, adherence of subsequently deposited conspecific pollen grains was unaffected. However, conspecific grains had significantly lower germination and ovule fertilization success in flowers receiving foreign pollen than in controls. These results suggest that pollinator infidelity in high alpine plant communities can reduce female reproductive success of P. viscosum by inhibiting the performance of conspecific pollen grains. Negative effects of Mertensia and Castilleja pollen were comparable despite large interspecific differences in pollen grain morphology. Accordingly, susceptibility to foreign pollen interference appears to be an intrinsic property of P. viscosum that is not dependent on the unique structural properties of pollen produced by competitive species.     

REPRODUCTIVE COSTS OF SELF-POLLINATION IN IPOMOPSIS AGGREGATA (POLEMONIACEAE): ARE OVULES USURPED?

In common with many cosexual angiosperms, the hummingbird-pollinated montane herb Ipomopsis aggregata (Polemoniaceae) is likely to experience self-pollination. Does this incur a fecundity cost even in such a highly self-sterile (presumably self-incompatible) species? Histological studies showed that self pollen germinates, and its tubes penetrate ovules almost as often as those of outcross pollen. Ovules penetrated by self tubes are especially likely to show milky callose occlusion 24–48 hr after pollination, and several observations suggest an association between occlusion and degeneration. Compared to flowers receiving only outcross pollen, seed set was reduced by 42% on average when self pollen was applied along with outcross, either by hand (in emasculated flowers) or by natural autodeposition (in unemasculated flowers). Reductions were statistically indistinguishable whether self pollen was applied 9 hr before outcross pollen, or at the same time. Unemasculated flowers accumulated substantial self pollen loads, and this autodeposition persisted when flowers were probed to mimic hummingbird visitation. Geitonogamy also is substantial, judging from field estimates of pollen transfer. Thus natural self pollen deposition may be sufficient to “usurp” ovules that otherwise could mature. In this light, late-acting self-rejection in I. aggregata seems decidedly less efficient than an early-acting system that would block pollen germination or tube growth.     

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.     

Reproductive biology of Aechmea bracteata (Sw.) Griseb. (Bromelioideae: Bromeliaceae)

• Individuals of Aechmea bracteata show inflorescences with red scape bracts and odourless, yellow, tubular diurnal flowers, with closely arranged sexual organs, producing a large amount of fruits.
• In order to investigate the reproductive system of this species, a suite of characters was assessed: phenology, floral morphology and biology, nectar production dynamics, and fruit and seed production and germination, as a result of controlled pollination crosses. The study was conducted during two flowering seasons in wild populations in Yucatán, Mexico.
• Results suggest an annual flowering pattern with one flowering peak; flowers were diurnal, showing partial dichogamy (protandry)‐herkogamy, anthers and stigma become mature before floral aperture, which could lead to self‐pollination, nectar is produced during anthesis, varying in volume and total sugar concentration during the day; fruits and seeds were produced in all experimental crosses (cross‐pollination, obligated cross‐pollination, assisted and unassisted selfing, geitonogamy and apomixis), as well as high percentage seed germination.
• Several species of Aechmea are reportedly self‐compatible and autogamous, as suggested by results of selfing and non‐assisted selfing crosses, but these results are negated by the presence of apomixis, indicating that the species is apomictic. This is the first report of this breeding system for subgenus Aechmea and the sixth for Bromeliaceae. Polyembryony is here suggested for the first time in this genus and family based on the fact that more seeds were recorded that expected based on ovule numbers. Finally, when performing experimental crosses, estimating reproductive success based on number of seeds is a better approach than number of fruits, due to the effect of pseudogamy.

     

Seed set variation in wild Clarkia populations: teasing apart the effects of seasonal resource depletion,pollen quality,and pollen quantity

In habitats where resource availability declines during the growing season, selection may favor early‐flowering individuals. Under such ephemerally favorable conditions, late‐blooming species (and individuals) may be particularly vulnerable to resource limitation of seed production. In California, a region prone to seasonal drought, members of the annual genus Clarkia are among the last to flower in the spring. We compared pollen limitation (PL) of seed set and outcrossing rates between early‐ and late‐flowering individuals in two mixed‐mating Clarkia taxa to detect whether flowering time is associated with changes in seed set due to resource depletion, PL, or increased selfing. In 2008–2010, we hand‐pollinated one flower on a total of 1855 individual plants either Early (near the onset of flowering) or Late (near the end of flowering) in the flowering season and compared seed set to adjacent, open‐pollinated flowers on the same stem. To assess the contribution of pollen quality to reproduction, we first (2008) used allozymes to estimate outcrossing rates of seeds produced by Early and Late open‐pollinated flowers. Second (2009), we conducted an anther‐removal experiment to estimate self‐pollen deposition. Seed set in Clarkia unguiculata was not pollen‐limited. Clarkia xantiana ssp. xantiana was pollen‐limited in 2008 and 2010, but not 2009. PL did not differ between Early and Late treatments. In both taxa, seed set of Early flowers was greater than Late flowers, but not due to PL in the latter. Reproduction was generally pollinator‐dependent. Most pollen deposition was xenogamous, and outcrossing rates were >0.7 – and similar between Early and Late periods. These results suggest that pollen receipt and pollen quality remain seasonally consistent. By contrast, the resources necessary to provision seeds decline, reducing the fitness benefits associated with resource allocation to ovules.     

Pollinator directionality as a response to nectar gradient: promoting outcrossing while avoiding geitonogamy

Plants with multiple flowers could be prone to autonomous self‐pollination and insect‐mediated geitonogamy, but physiological and ecological features have evolved preventing costs related to autogamy. We studied the rare perennial herb Dictamnus albus as a model plant, with the aim of describing the plant–pollinator system from both plant and pollinator perspectives and analysing features that promote outcrossing in an entomophilous species. The breeding system and reproductive success of D. albus were investigated in experimental and natural conditions, showing that it is potentially self‐compatible, but only intra‐inflorescence insect‐mediated selfing is possible. Nectar analysis showed gender‐biased production towards the female phase, which follows the male phase, and during flowering, full blooming is found in flowers at the bottom of the raceme. Among a wide spectrum of insect visitors, three genera (Bombus, Apis, Megachile) were found to be principal pollinators. A study of insect behaviour showed a tendency towards bottom‐to‐top flights for the most important pollinators Bombus spp. and Apis mellifera: upward movements on the racemes could be explained by foraging behaviour, from more to less rewarding flowers. In accordance with the ‘declining reward hypothesis’, bumblebees and honeybees leave the plant when gain of reward is low, after which few flowers are visited, reducing the chance of self‐pollen transfer among flowers. Intra‐flower self‐pollination is prevented in D. albus by protandry and herkogamy, while the nectar‐induced sequential pattern of pollinator visits avoids geitonogamy and tends to maximise pollen export, promoting outcrossing. All these features for preventing selfing benefit plant fitness and population genetic structure.     

Effects of prior self-pollination on outcrossed seed set in tristylous Pontederia sagittata (Pontederiaceae)

The potential inhibitory effects of incompatible pollen on outcrossed seed set were investigated in mass-flowering, self-incompatible, tristylous Pontederia sagittata. Prior application of self pollen, followed after 2, 4, or 6 h by compatible pollen, was conducted on five genotypes of each of the three style morphs under uniform glasshouse conditions. The greatest reductions in seed set occurred in pollinations of the long-styled (L) morph at the 6 h time interval. Smaller reductions were also found for this treatment in the mid-styled (M) morph. No significant reductions in seed set were observed in the short-styled (S) morph or in the other morphs at shorter time intervals. Observations of pollen germination and pollen tube growth indicated that the lack of inhibitory effects in the S morph may occur because relatively few pollen grains adhered to stigmas in selfpollinations. In the L and M morphs, early germination of self pollen may cause physical clogging of the stigma and style, resulting in a reduced number of compatible pollen tubes in styles. Observations of the structural integrity of styles indicated that prior germination of self pollen resulted in more rapid onset of pistil senescence, particularly in the L morph. These influences may contribute to the morph-specific differences in seed set observed following prior self-pollination of outcrossed flowers. The negative effects of incompatible pollen are likely to be most evident where ecological factors cause delays in the delivery of outcross pollen to stigmas.     

Effects of Inbreeding,Outbreeding, and Supplemental Pollen on the Reproduction of a Hummingbird‐pollinated Clonal Amazonian Herb

Understory herbs are an essential part of tropical rain forests, but little is known about factors limiting their reproduction. Many of these herbs are clonal, patchily distributed, and produce large floral displays of nectar‐rich 1‐d flowers to attract hummingbird pollinators that may transport pollen over long distances. The aim of this study was to investigate the effects of clonality, cross‐proximity, and patchy distribution on the reproduction of the hummingbird‐pollinated Amazonian herb Heliconia metallica. We experimentally pollinated flowers within populations with self‐pollen and with pollen of different diversity, crossed flowers between populations, and added supplemental pollen to ramets growing solitarily or in conspecific patches. Only flowers pollinated early in the morning produced seeds. Selfed flowers produced seeds, but seed number and mass were strongly reduced, suggesting partial sterility and inbreeding depression after selfing. Because of pollen competition, flowers produced more seeds after crosses with several than with single donor plants. Crosses between populations mostly resulted in lower seed production than those within populations, suggesting outbreeding depression. Ramets in patches produced fewer seeds than solitary ramets and were more pollen‐limited, possibly due to geitonogamy and biparental inbreeding in patches. We conclude that high rates of geitonogamy due to clonality and pollen limitation due to the short receptivity of flowers and patchy distribution constrain the reproduction of this clonal herb. Even in unfragmented rain forests with highly mobile pollinators, outbreeding depression may be a widespread phenomenon in plant reproduction.     

Self‐incompatibility and post‐fertilization maternal regulation cause low fecundity in Aegle marmelos (Rutaceae)

Outbreeding confers an evolutionary advantage, and flowering plants have evolved a variety of contrivances for its maximization. However, neither fruit set nor seed set is realized to its fullest potential for a variety of reasons. The causes of low flower to fruit and seed to ovule ratios were investigated in naturally occurring bael trees (Aegle marmelos) at two sites for three seasons. The study established that the mass effect of synchronized flowering attracted a variety of insect pollinators to the generalist flowers; Apis dorsata was the most efficient pollinator. The seed to ovule ratio was low despite high natural pollination efficiency (c. 2400 pollen per stigma). Although pollination‐induced structural and histochemical changes in the style allowed many (9.5 ± 2.1) pollen tubes to grow, only cross‐pollen tubes could grow through the style. Gametophytic self‐incompatibility, manifested in the stylar zone, resulted in a significantly slower growth rate of self‐pollen tubes. The occurrence of obligate self‐incompatibility, coupled with increased self‐pollen deposition (geitonogamy), caused a significant number of flowers to abort. Fruit retention in the trees declined from 40% to 12% as a result of abortion of fruits at different stages of development. The number of mature fruits on a tree was negatively correlated (r = ?0.82) with their size. It is inferred that low natural fecundity in A. marmelos is primarily a result of obligate self‐incompatibility and strong post‐fertilization maternal regulation of allocation of resources to the developing fruits. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172 , 572–585.