Floral display size and spatial distribution of potential mates affect pollen deposition and female reproductive success in distylous Pulmonaria officinalis (Boraginaceae)

In animal‐pollinated plants, both the spatial distribution of flowering individuals and the number of flowers that an individual displays affect pollen deposition rates and female reproductive success. Heterostylous species are likely to be particularly sensitive to the contingencies of spatial distribution, as they are reproductively subdivided into distinct mating groups, which usually exhibit self‐ and intra‐morph incompatibility and differ in floral morphology. In this paper, we explore the joint effects of both spatial distribution of potential mates and floral display size on morph‐specific pollen deposition rates and seed set patterns in two natural populations of Pulmonaria officinalis, a distylous species with a weak self‐incompatibility system. Both total stigmatic pollen load and the proportion of legitimate pollen decreased with increasing spatial isolation. Legitimate (intermorph) pollen transfer was, however, asymmetric and decreased more rapidly with decreasing proximity to a compatible legitimate mating partner in the S‐morph than in the L‐morph. Total stigmatic pollen loads per flower increased with increasing floral display size, indicating that large plants are disproportionately more visited than smaller individuals. However, because legitimate pollen deposition decreased with increasing floral display size, these results also suggest that larger numbers of flowers increase the degree of geitonogamous pollination. In both the L‐ and S‐morph, seed set significantly decreased with increasing isolation from a legitimate mating partner, but in the L‐morph seed set was less dependent on the spatial distribution of the S‐morph. In addition, seed set significantly increased with floral display size in the L‐morph, but not in the S‐morph. These findings indicate that the spatial distribution of potential mates and variation in floral display size may cause morph‐specific differences in pollen deposition rates and female reproductive success.     

Biased morph ratios and skewed mating success contribute to loss of genetic diversity in the distylous Pulmonaria officinalis

Background and Aims

In heterostylous plant species, skewed morph ratios are not uncommon and may arise from a range of factors. Despite the recognized importance of skewed morph ratios on overall reproductive success within populations, little is known about the impact of skewed morph ratios on population genetic diversity and differentiation in heterostylous species. This study specifically aimed to clarify the effect of population size and morph bias on population genetic diversity and differentiation in the temperate forest herb Pulmonaria officinalis. This species is characterized by a distylous breeding system and shows morph-specific differences in reproductive success.

Methods

Genetic diversity was determined for 27 P. officinalis populations in northern Belgium by using eight recently developed microsatellite markers. Multiple regressions were used to assess the relationship between genetic diversity, morph bias and population size, and F_ST-values were calculated for short- and long-styled morphs separately to study genetic differentiation as a function of morph type.

Key Results

For all genetic measures used, morph bias was more important in explaining patterns of genetic diversity than population size, and in all cases patterns of population genetic diversity followed a quadratic function, which showed a symmetrical decrease in genetic diversity with increasing morph bias. However, probably due to the reproductive advantage of L-morphs relative to S-morphs, maximum genetic diversity was found in populations showing an excess of L-morphs (60·7 % L-morph). On the other hand, no significant difference in pairwise genetic distances between populations was observed between L- (0·107) and S-morphs (0·106).

Conclusions

Our results indicate that significant deviations from equal morph ratios not only affect plant reproductive success but also population genetic diversity of heterostylous plant species. Hence, when defining conservation measures for populations of heterostylous plant species, morph ratios should be considered as an important trait affecting their long-term population viability.     

Influence of asymmetrical mating patterns and male reproductive success on the maintenance of sexual polymorphism in Acer pictum subsp. mono (Aceraceae)

Populations of Acer species often contain more than three sex phenotypes with complex sexual polymorphism including duodichogamy, protandry and protogyny. We identified the mechanisms that maintain sexual polymorphism in Acer pictum subsp. mono, a temperate tree from northern China, by investigating maternal mating patterns and male reproductive success. We used paternity analyses to estimate rates of outcrossing and disassortative mating, as well as male outcrossed siring success, in a population of A. pictum subsp. mono with uneven sex phenotype ratios (duodichogamous 69.1%, protandrous 19.6%, protogynous 11.3%). We used a pollen‐transfer model to investigate whether the unequal ratios of sex phenotypes could be explained by the observed patterns of mating. Most progeny resulted from outcrossing, particularly disassortative among the sex phenotypes. Although the duodichogamous phenotype showed a significant amount of intraphenotypic mating, the frequency did not exceed that of disassortative mating. We detected no significant differences in male outcrossed siring success among the sex phenotypes. The pollen‐transfer model demonstrated that sex phenotype ratios could be maintained by the observed mating pattern in the population. Our results indicate that disassortative mating among the sex phenotypes can maintain sexual polymorphism in A. pictum subsp. mono and that ratios biased towards duodichogamy can result from frequent intraphenotypic mating in this phenotype.     

Pollen limitation of female reproductive success at fine spatial scale in a gynodioecious and wind-pollinated species, Beta vulgaris ssp. maritima

In sexually polymorphic plants, the spatial distribution of sexes is usually not random. Local variation in phenotype frequencies is expected to affect individual fitness of the different phenotypes. For gynodioecious species, with co-occurrence of hermaphrodites and females, if sexual phenotypes are structured in space and pollen flow is spatially restricted, local pollen availability should vary among patches. Female fitness may thus be low when hermaphrodites are locally rare. To test this hypothesis, we analysed how the reproductive output of females varied among patches within two natural study sites of the gynodioecious wind-pollinated Beta vulgaris ssp. maritima. Plants growing in female-biased areas and experiencing pollen limitation were found to have low fruit and seed sets but did not reallocate resources towards better offspring. Our results show that fine-scale processes influence individual fitness and the evolution of sex ratio in sexually polymorphic plants.     

Heteromorphic incompatibility and efficiency of pollination in two distylous Pentanisia species (Rubiaceae)

BACKGROUND AND AIMS: Distyly has been hypothesized to promote cross-pollination by reducing intrafloral and geitonogamous self-pollination, and enhancing intermorph pollination. Distylous plants typically display both reciprocal herkogamy and a heteromorphic incompatibility system, which allows mating only between morphs. Distyly and its pollination consequences were examined in two Pentanisia species with long-tubed flowers which are pollinated almost exclusively by butterflies. METHODS: Anther and stigma heights were measured to quantify reciprocal herkogamy. The type of incompatibility system was determined by observing pollen tubes and seed production following controlled hand pollination. Pollen loads on pollinators and stigmas were also examined to assess the efficiency of intermorph pollen flow. KEY RESULTS: Pentanisia prunelloides and P. angustifolia exhibit reciprocal herkogamy and a host of ancillary dimorphisms, including pollen colour, exine sculpturing, stigmatic papilla shape and floral-tube pubescence. Controlled hand-pollinations revealed the presence of a strong heteromorphic incompatibility system in both species. The site of incompatibility differed between the morphs; intramorph pollen tubes were blocked in the style of the short-styled morph and on the stigmatic surface of the long-styled morph. Butterflies carried pollen from the short- and long-styled morphs primarily on their head and proboscis, respectively. Natural pollination resulted in a higher proportion of pollen transfer from long- to short-styled plants than vice versa. Nevertheless, fruit set did not differ between morphs. CONCLUSIONS: Both Pentanisia species are fully distylous. Reciprocal herkogamy results in pollen from the two morphs being carried on different locations on pollinators' bodies, which in turn promotes intermorph pollination. Intramorph pollination does not result in fertilization, because of an effective heteromorphic incompatibility system.     

Spatio-temporal variation in pollen limitation and reproductive success of two scape morphs in Primula farinosa

In plant populations where reproductive output is limited by pollinator visitation, plants with attractive floral displays should have a selective advantage. We examined the effect of inflorescence height on pollination success in Primula farinosa, which is dimorphic for scape length. To test the hypothesis that fruit and seed initiation are more strongly pollen-limited in the short-scaped than in the long-scaped morph, and that this difference is affected by spatio-temporal variation in pollen limitation, we conducted a hand-pollination experiment in four populations over 2 yr. Pollen limitation of fruit initiation varied among populations and years, and was stronger in the short-scaped than in the long-scaped morph. The results suggest that interactions with pollinators will need to be considered for a full understanding of the maintenance of this striking polymorphism. The study also shows that, although pollen limitation is likely to vary in space and time in many plant species, such variation is not necessarily associated with variation in selection on floral characters.     

Variation in sex ratio, morph-specific reproductive ecology and an experimental test of frequency-dependence in the gynodioecious Kallstroemia grandiflora (Zygophyllaceae)

An enduring puzzle in gynodioecious species is the great variation in female frequency seen among populations. We quantified sex ratio in 44 populations of gynodioecious Kallstroemia grandiflora. Then, we measured pollinator visitation, pollen deposition, autonomous selfing rate and pollen limitation of females. Finally, using experimental populations, we tested whether female fitness responds to the frequency of female plants. We found broad variability in sex ratio among populations (0-44% female). Hermaphrodite flowers received more pollinator visits and pollen grains than females, and bagged hermaphrodite flowers produced fruits. However, we found no evidence of pollen limitation in females. In experimental populations, female plants showed no evidence of frequency-dependent pollinator visitation, fruit set, seed set or total seed mass. These results do not support frequency-dependent variation in fitness as a major mechanism affecting female frequencies in K. grandiflora. Within the context of this study, pollinators are abundant and pollinator movement appears to operate at a large enough scale to overcome the potential reproductive disadvantages of producing solely female flowers.     

Effects of floral display size on male and female reproductive success in Mimulus ringens

Background and Aims

The number of flowers blooming simultaneously on a plant may have profound consequences for reproductive success. Large floral displays often attract more pollinator visits, increasing outcross pollen receipt. However, pollinators frequently probe more flowers in sequence on large displays, potentially increasing self-pollination and reducing pollen export per flower. To better understand how floral display size influences male and female fitness, we manipulated display phenotypes and then used paternity analysis to quantify siring success and selfing rates.

Methods

To facilitate unambiguous assignment of paternity, we established four replicate (cloned) arrays of Mimulus ringens, each consisting of genets with unique combinations of homozygous marker genotypes. In each array, we trimmed displays to two, four, eight or 16 flowers. When fruits ripened, we counted the number of seeds per fruit and assigned paternity to 1935 progeny.

Key Results

Siring success per flower declined sharply with increasing display size, while female success per flower did not vary with display. The rate of self-fertilization increased for large floral displays, but siring losses due to geitonogamous pollen discounting were much greater than siring gains through increased self-fertilization. As display size increased, each additional seed sired through geitonogamous self-pollination was associated with a loss of 9·7 seeds sired through outcrossing.

Conclusions

Although total fitness increased with floral display size, the marginal return on each additional flower declined steadily as display size increased. Therefore, a plant could maximize fitness by producing small displays over a long flowering period, rather than large displays over a brief flowering period.     

Microgametophyte population sizes and plant reproductive output in the insect-pollinated Prunella grandiflora (Lamiaceae)

Intraspecific variation in pollen deposition and number of pollen tubes per style is rarely quantified, but is essential for assessing the occurrence of pollen limitation and pollen competition and their evolutionary implications. Moreover, pollen deposition, pollen tube growth, and the fate of fertilized ovules are rarely distinguished in field studies. Here we present such a study in eight natural populations of Prunella grandiflora. We quantified microgametophyte population sizes and inferred pollen limitation when the number of fertilizable ovules exceeded pollen tubes, and assessed seed set and fate after open pollination. Two and three populations had on average significantly fewer pollen grains and pollen tubes per flower, respectively, than the fixed number of fertilizable ovules per fruit, while one population experienced significant pollen competition. Style length was positively correlated with the number of pollen tubes. While pollen availability was very variable, seed abortion was significantly less frequent in denser populations, and in one population the proportion of well-developed seeds was significantly, positively correlated with the number of pollen tubes in the style. Less pollen deposition, lower numbers of pollen tubes reaching the base of the style, lower pollen quality and therefore increased abortion of fertilized ovules can all reduce seed set in natural P. grandiflora stands. Substantial intraspecific variability implies that microgametophyte competition also occurs in this species. Finally, style morphology may affect pollen receipt.     

Post‐pollination process in a partially self‐compatible distylous plant,Primula sieboldii (Primulaceae)

Pollen germination and pollen‐tube growth under natural conditions were observed in a population of a distylous species, Primula sieboldii, in which partial self‐compatibility has been demonstrated in some long‐styled genets. We observed post‐pollination processes microscopically in styles collected after self‐morph and inter‐morph hand pollination (with standardized pollen load on the stigmas) in four genets each from the following three ‘genet types’: self‐incompatible long‐styled (SI), partially self‐compatible long‐styled (SC) and self‐incompatible short‐styled morph genets. Irrespective of the genet type, pollen germination began within 24 h after pollination and tubes of pollen reached to the style base with 48–96 h after inter‐morph pollination. Although pollen tubes germinated after self‐pollination in the SC genets, the number of germinated pollen tubes was significantly lower than in the case of inter‐morph pollination. Few pollen tubes germinated after self‐pollination of the SI or short‐styled genets. In SC genets, the rate of pollen‐tube growth did not differ between self‐morph and inter‐morph pollination (～1.9 mm/day). Therefore, differences in self‐compatibility between SC and SI genets in P. sieboldii are likely to be attributable to differential pollen germination rates rather than to differential pollen‐tube growth rates.     

Effective population size,reproductive success and sperm precedence in the butterfly,Bicyclus anynana,in captivity

A pedigree approach is used to estimate the effective population size yn two population cages of the butterfly, Bicyclus anynana. Each cage was founded with 54 individually marked adults of each sex. Matings were recorded over a 3‐day period. Eggs were then collected from each female over a similar period before the numbers of hatching larvae were counted to assess progeny number. The males showed a higher variance in reproductive success than the females. Since about one‐quarter of all females mated more than once, we also examined the pattern of sperm precedence using molecular markers or, in separate crossing experiments, wing pattern mutants. Both instances of complete first and last male sperm precedence, as well as of sperm mixing, were found. In some crosses a ‘leakiness’ was found in which some of the early eggs laid by a female were fertilized by a male partner which was subsequently completely unsuccessful. However, the estimates of effective population size were largely unaffected by the pattern of sperm precedence. Estimates for N_e : N in each cage were close to 0.60. The possibility of obtaining comparable estimates in selected natural populations of butterflies is discussed.     

Fragment size, pollination efficiency and reproductive success in natural populations of wind-pollinated Polylepis australis (Rosaceae) trees

It is well known that habitat fragmentation is likely to negatively affect the reproductive success of a species. Despite this fact, studies on the effects of fragmentation on reproductive success in combination with effects on natural pollination of wind-pollinated tree species are very rare. In this study, we analyzed the relationships between fragment size, components of pollination efficiency, reproductive success and progeny vigour of the highly fragmented populations of the wind-pollinated treeline species Polylepis australis (Rosaceae) in Argentina. We conducted our study in the high mountains of Córdoba, comparing pollination efficiency and reproduction between four fragment sizes. Most of our results indicate that P. australis is currently insensitive to fragmentation. However, our study revealed also a linear increase in leaf area and biomass of 40-day-old seedlings with increasing fragment size. Inbreeding problems through fragmentation as well as decreased habitat quality in small fragments are discussed as possible causes for the impaired progeny vigour.     

Effects of population size, seed predation and plant size on male and female reproductive success in Vincetoxicum hirundinaria (Asclepiadaceae)

Here we present the results of a study on the effects of population size, seed predation, and plant size on male and female reproductive success in ten Vincetoxicum hirundinaria populations in southwestern Finland. We investigated male reproductive success by recording pollinia removal, and studying pollen quality and quantity. Pod and seed production, and the number of ovules per ovary were counted to estimate female reproductive success. Both pollen quality and pollinia removal were higher in small V. hirundinaria populations compared to large populations. In contrast, the quantity of pollen was higher in large populations than in small populations. Pod initiation was higher in large populations. However, large populations had higher abortion rates and proportionally fewer intact pods. Plant size did not affect male reproductive success whereas pod and seed production correlated positively with plant size, although only in large populations. Population size had no clear impact on seed predation intensities. Variation was, however, observed among populations. Larger plant individuals suffered from an increased risk of being attacked only in two populations. This correlation was not clearly attributed to plant population size. The results of this study suggest that reproductive success in V. hirundinaria may be highly variable and this variation is to some extent explained by population size. Further, population size affected male and female reproductive success differently.     

Reproductive ecology of the endangered Alpine species Eryngium alpinum L. (Apiaceae): phenology, gene dispersal and reproductive success

BACKGROUND AND AIMS: Eryngium alpinum (Apiaceae) is an endangered perennial, characteristic of the Alpine flora. Because the breeding system influences both demographic (reproductive success) and genetic (inbreeding depression, evolutionary potential) parameters that are crucial for population maintenance, the reproductive ecology of E. alpinum was investigated. Specifically, the aims of the study were (1) to determine the factors (resources and/or pollen) limiting plant fitness; and (2) to assess the potential for gene flow within a plant, within a patch of plants, and across a whole valley where the species is abundant. METHODS: Field experiments were performed at two sites in the Fournel valley, France, over three consecutive years. Studies included a phenological survey, observations of pollinators (visitation rates and flight distances), dispersal of a fluorescent powder used as a pollen analogue, the use of seed traps, determination of the pollen/ovule ratio, and an experiment to test whether seed production is limited by pollen and/or by resources. KEY RESULTS: E. alpinum is pollinated by generalist pollinators, visitation rates are very high and seed set is resource- rather than pollen-limited. The short flights of honeybees indicate a high potential for geitonogamy, and low pollen and seed dispersals suggest strong genetic structure over short distances. These results are interpreted in the light of previous molecular markers studies, which, in contrast, showed complete outcrossing and high genetic homogeneity. CONCLUSION:S. The study highlights the usefulness of adopting several complementary approaches to understanding the dynamic processes at work in natural populations, and the conservation implications for E. alpinum are emphasized. Although the studied populations do not seem threatened in the near future, long-term monitoring appears necessary to assess the impact of habitat fragmentation. Moreover, this study provides useful baseline data for future investigations in smaller and more isolated populations.     

Pollination ecology and reproductive success in Jack-in-the-pulpit (Arisaema triphyllum) in Québec (Canada)

Pollination ecology and reproductive success of Jack-in-the-pulpit ( Arisaema triphyllum ) were studied in two natural populations in Québec, Canada. Individual A. triphyllum plants can be of three types: male, female or bisexual. In both populations studied, the presence of bisexual inflorescences was not negligible (13%), where 'female' and 'male' bisexual plants were categorised according to the relative number of stamens and ovaries. 'Male bisexual' plants produce only pollen and 'female bisexual' plants produce only fruit. Hence, A. triphyllum is a true dioecious plant, as each plant only reproduces through either the male or the female function. 'Female bisexual' plants were equivalent to female plants in terms of visitation rate by insects, fructification rate and production of berries and seeds. Neither agamospermy in female plants nor self-pollination in 'female bisexual' plants was found, thus A. triphyllum relies on insects for cross-pollination. Despite the long flowering cycle, a low visitation rate was documented: only 20–40% of inflorescences were visited, according to gender, by a mean of 1.5 insects. In this study, Mycetophilidae represented the most generically diversified and abundant family, as well as the most efficient insect pollinator, especially the genera Docosia and Mycetophila .     

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

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

Within/between population crosses reveal genetic basis for siring success in Silene latifolia (Caryophyllaceae)

Divergence at reproductive traits can generate barriers among populations, and may result from several mechanisms, including drift, local selection and co-adaptation between the sexes. Intersexual co-adaptation can arise through sexually antagonistic co-evolution, a timely hypothesis addressed in animals but, to our knowledge, not yet in flowering plants. We investigated whether male and female population of origin affected pollen competition success, offspring fitness and sex ratio in crosses within/between six genetically differentiated populations of the white campion, Silene latifolia. Each female was crossed with pollen from one focus male from the same population, and pollen from two focus males from two distinct populations, both as single-donor and two-donor crosses against a fixed tester male with a 2-h interpollination interval (n = 288 crosses). We analysed paternity with microsatellite DNA. Male populations of origin significantly differed for siring success and in vitro pollen germination rates. In vitro pollen germination rate was heritable. Siring success also depended on sex ratio in the female family of origin, but only in between-population crosses. In some female populations, two-donor crosses produced less female-biased sex ratios compared with single-donor crosses, yet in other female populations the reverse was true. Offspring sex ratio varied with donor number, depending on the female population. Within/between population crosses did not differ significantly in seed set or offspring fitness, nor were siring success and offspring fitness significantly correlated. Altogether this suggests reproductive divergence for traits affecting pollen competition in S. latifolia.