Variation of a floral polymorphism at different spatial scales in the Mediterranean geophyte Narcissus assoanus

Aims Habitat fragmentation impacts the spatial extent and isolation of local populations and communities. Although the biological consequences of these impacts have been well studied at the site level, effects directly related to changes in the spatial configuration of populations in the landscape remain less clear. The objective of this study is to examine how changes in the spatial-scale configuration of populations are associated with variability in morph rations in the floral polymorphic Mediterranean geophyte Narcissus assoanus .Methods We performed a nested analysis of morph ratio variation at three spatial scales: a 50×50 km regional scale in SE France, in fourteen 1×1 km landscapes located in the same region, and within 12 spatially extensive population patches. We also quantified variation in the behaviour of pollinators in population patches of contrasting spatial configuration.Important findings At a regional scale, morph ratios show a geographic pattern of increasing isoplethy (1:1 morph ratio) away from centres of human population development and in upland pastures. At the landscape scale, isoplethy of local population patches is more correlated with decreased isolation among patches than with patch size. Finally, within local isoplethic populations, small patches can show significantly biased morph ratios. In small isolated patches, pollinators perform shorter flight distances among consecutive flower visits than in spatially extensive patches. This study of variation in a genetic polymorphism at multiple spatial scales provides new insights into the scale-dependent effects of habitat fragmentation and the potential occurrence of metapopulation dynamics in natural plant populations.     

Effects of pollen quality and quantity on pollen limitation in Crataegus monogyna (Rosaceae) in NW Spain

Pollen limitation occurs when plants produce less fruits and/or seeds than they would with adequate pollen receipt. If the addition of cross-pollen to stigmas increases fruit/seed production, it is interpreted as an evidence of pollen limitation. Much of the limitation may be associated with the quality rather than quantity of pollen; however, most studies do not discriminate between the two, which may lead to misinterpretation of the results. We studied the effects of quality and quantity of pollen on the reproduction of a northern Spanish population of Crataegus monogyna. The treatments included self- and cross-pollination, and supplementation to open and bagged flowers. The response variables considered were number of pollen grains per stigma, pollen tubes per style, and initial and final fruit set. In the Cantabrian range, C. monogyna requires insect pollinators to set fruit and is partially self-incompatible. We found that the number of pollen tubes did not differ between cross- and self-pollination treatments; however, self-pollinated flowers set less fruits than flowers that received pure cross-pollen or were supplemented with both cross- and self-pollen. The experimental design allowed us to infer qualitative rather than quantitative pollen limitation. Comparison of the number of pollen grains and tubes, and initial and final fruit set among pollination treatments suggested post-zygotic embryo selection against selfed progeny.     

Effects of pollen competitive environment on pollen performance in Mirabilis jalapa (Nyctaginaceae)

 We examined the influence of pollen competitive environment on pollen performance in Mirabilis jalapa. We used the number of pollen grains and the number of pollen tubes per pistil as measures of pollen competition. Pollen germination, pollen tube penetration into the style, and pollen tube growth rates were used as measures of pollen performance. All three measures of pollen performance were affected by the competitive environment. Pollen germination was greatest at intermediate pollen load sizes. The percentage of germinated pollen grains that penetrated the stigma and grew into the style decreased with pollen load size. Pollen tube growth rate in the style was greater and more variable with larger numbers of pollen tubes in the style. Controlling for the degree of selection at the stigma indicated that pollen-pollen or pollen-style interactions were the likely causes of increased growth rates. Received: 28 October 1996 / Revision accepted: 24 January 1997     

Discovery of distyly in Narcissus (Amaryllidaceae)

There has been disagreement concerning the nature of sexual polymorphisms in Narcissus, a genus of insect-pollinated geophytes native to the Mediterranean. The existence of trimorphic heterostyly in the genus has recently been confirmed, but the occurrence of distyly remains enigmatic. All sexually dimorphic species previously investigated possess two distinct style lengths but anthers of similar height. Stigma-height dimorphism does not qualify as true distyly because of the absence of a reciprocal correspondence in stigma and anther position in the floral morphs. Such reciprocal herkogamy is generally regarded as the defining feature of heterostyly. Here we report on distyly in N. albimarginatus (section Apodanthae), a rare species confined to a single mountain in northwestern Morocco. A population composed of equal numbers of long- and short-styled plants exhibited reciprocal herkogamy with lower anthers of the long-styled morph and upper anthers of the short-styled morph corresponding in height to stigmas of short- and long-styled plants, respectively. The presence of both stigma-height dimorphism and distyly in Narcissus is of general significance to theoretical models of the evolution of heterostyly.     

Differential ovule development following self- and cross-pollination: the basis of self-sterility in Narcissus triandrus (Amaryllidaceae)

Self-pollination results in significantly lower seed set than cross-pollination in tristylous Narcissus triandrus. We investigated structural and functional aspects of pollen–pistil interactions and ovule–seed development following cross- and self-pollination to assess the timing and mechanism of self-sterility. Ovule development within an ovary was asynchronous at anthesis. There were no significant differences in pollen tube behavior following cross- vs. self-pollination during the first 6 d of growth, regardless of style morph type. Double fertilization was significantly higher following cross- vs. self-pollination. Aborted embryo development was not detected following either pollination type up to seed maturity. Prior to pollen tube entry, a significantly greater number of ovules ceased to develop following self- vs. cross-pollination. These results indicate that self-sterility in N. triandrus operates prezygotically but does not involve differential pollen tube growth typical of many self-incompatibility (SI) systems. Instead, low seed set following self-pollination is caused by a reduction in ovule availability resulting from embryo sac degeneration. We hypothesize that this is due to the absence of a required stimulus for normal ovule development. If this is correct, current concepts of SI may need to be broadened to include a wider range of pollen–pistil interactions.     

Physiology and genetics of self-incompatibility in Echinopsis chamaecereus (Cactaceae)

The self-incompatibility (SI) system of a geophytic cactus (Echinopsis chamaecereus Friedrich & G. Rowley) was examined in a series of experiments. Pollination tests indicated that E. chamaecereus is an obligate outbreeding species with a functional SI system. Incompatible matings were characterized by stylar inhibition of pollen tube growth and lack of fruit set. Two S₁ seedlings were recovered when plants of one clone were exposed to 42°C for 16 h and flowers were selfed immediately after incubation. The two S₁ seedlings and the parental (S₀) clone were crossed in a full diallel. Results were consistent with a one-locus, gametophytic SI system with two different S alleles. Disturbed segregation at isozyme locus Lap-1 was attributed to close linkage with the S locus (recombination frequency = 11±8%). This is the second report of close linkage between Lap-1 and S in the Cactaceae. Recevied: 1 February 2001 / Revision accepted: 13 March 2001     

Selective fruit filling in relation to pollen load size in Alstroemeria aurea (Alstroemeriaceae)

 We conducted an experiment in a natural population of Alstroemeria aurea, a clonal perennial, to determine (1) if reproduction was resource limited, and (2) if fruits would be selectively filled based on differences in pollination intensity when pollen loads were adequate for full seed set. Under these conditions, differences in pollination intensity are unlikely to affect seed number, but could affect seed quality, providing an interesting test of the gametophytic competition hypothesis. To test for resource limitation, percent fruit maturation, number of seeds per fruit and average seed weight were compared to paired controls for ramets in which all but one fruit was removed. To test the effect of pollination intensity on selective resource allocation, three types of pollination treatments were performed: (1) all flowers of the single inflorescence received a low pollen load, (2) all flowers received a high pollen load, (3) alternate flowers of the inflorescence received either a high or a low pollen load. We determined the percentage of fruit that reached maturity, counted the number of seeds and ovules and calculated the average seed weight for all capsules in each treatment. Resources appeared to limit reproduction in this population since seed number and weight were significantly higher than in controls when competing capsules were removed. At the whole ramet level, a four fold difference in pollen loads had no significant effect on any of the parameters measured. However, when pollination intensity varied within an inflorescence, the number of seeds per fruit increased by about 10% in flowers that received the higher pollen load. We observed the same trend in each of 2 years, but the increase was significant in only 1 year. The differences, although not great, were only slightly smaller than when all competing fruits were removed, and were consistent with selective resource allocation based on pollination intensity independent of seed set. Received: 28 September 1997 / Accepted: 30 April 1998     

Evidence for widespread pollen limitation of fruiting success in Cape wildflowers

We used spot checks of stigmatic pollen deposition and hand-pollination experiments to test whether fruit production in Cape wildflower populations is limited by pollen availability. Natural levels of stigmatic pollen deposition were very low (median = 30.0% of flowers) in populations of 33 orchid species. We found similarly low levels of fruit set (median = 32% of flowers per plant) in six Orchidaceae and four Amaryllidaceae species. Experimental hand pollination at the whole plant level caused significant increases in fruit production in 11 of the 12 study populations. These results indicate that pollen limitation of fruit set may occur frequently among some plant families in the Cape flora. Received: 15 April 1996 / Accepted: 10 November 1996     

Isozyme evidence for the allotriploid origin of Lycoris flavescens (Amaryllidaceae)

 Variation in isozyme patterns from ten populations of the Korean endemic Lycoris species was used to test the hypothesis that L. flavescens originated from natural hybridization between diploid L. chinensis and L. sanguinea var. koreana. Lycoris sanguinea var. koreana shows fixed heterozygosity at four of nine loci assayed, suggesting that this species is an allotetraploid instead of a diploid. Electrophoretic data suggest that Lycoris flavescens is an allotriploid species derived from the hybridization between diploid L. chinensis and tetraploid L. sanguinea var. koreana. The patterns of allelic distribution in populations of L. flavescens suggest multiple origins of the allotriploid. Within the L. flavescens complex, our isozyme data support the recognition of two taxa, L. flavescens and a recently recognized species, L. uydoensis. Received August 28, 2000 Accepted December 27, 2000     

Variation of pollination and resource limitation in a low seed-set tree, Liriodendron chinense (Magnoliaceae)

Pollen limitation and resource limitation have been documented as the major factors responsible for plants commonly producing more ovules than seeds, but few studies have examined pollen deposition directly in natural populations at different sites and times. We investigated the causes of low seed set in four populations of Liriodendron chinense (Magnoliaceae), an insect‐pollinated endangered tree endemic to southern China, over 2–3 years. One pistil potentially produces two ovules. The number of pistils per flower varies among populations, but in three of the four populations the variation in a given population was not significantly different among years. Overall, populations with higher pistil numbers tend to set more seeds per flower, but a positive correlation between pistil numbers and seed production per flower was observed in only one of the four populations. The numbers of pollen grains deposited per stigma varied from 0 to 60. The proportion of pollinated stigmas per flower ranged from 44% to 88% among populations and years. The numbers of pollen grains deposited per stigma and the percentages of pollinated stigmas were significantly different between populations, and two populations showed significant differences between years. A positive correlation between stigmatic pollen load and seed set was sought in ten population‐by‐year combinations but, in a given population, high stigmatic pollen loads did not always result in high seed set. Examination of pollen deposition, pistil and seed production over several sites and years showed that in addition to pollination, other factors such as resource or genetic loads were likely to limit the (lower than 10%) seed set in L. chinense. It appears that small, isolated populations experience severe pollination limitation; one population studied had seed/ovule ratios of 0.84% and 1.88% in 1995 and 1996. © 2002 The Linnean Society of London, Botanical Journal of the Linnean Society, 2002, 140 , 31–38.     

Ecophysiology of seed dormancy and the control of germination in early spring‐flowering Galanthus nivalis and Narcissus pseudonarcissus (Amaryllidaceae)

Seed dormancy induction and alleviation in the winter‐flowering, moist temperate woodland species Galanthus nivalis and Narcissus pseudonarcissus are complex and poorly understood. Temperature, light and desiccation were investigated to elucidate their role in the germination ecophysiology of these species. The effect of different seasonal temperatures, seasonal durations, temperature fluctuations, the presence of light during different seasons and intermittent drying (during the summer period) over several ‘years’ on seed germination was investigated with outdoor and laboratory experiments. Warm summer‐like temperatures (20 °C) were necessary for germination at subsequent cooler autumn‐like temperatures (greatest at 15 °C in G. nivalis and 10 °C in N. pseudonarcissus). As the warm temperature duration increased, so did germination at subsequent cooler temperatures; further germination occurred in subsequent ‘years’ at cooler temperatures following a second, and also third, warm period. Germination was significantly greater in darkness, particularly in G. nivalis. Dormancy increased with seed maturation period in G. nivalis, because seeds extracted from green capsules germinated more readily than those from yellow capsules. Desiccation increased dormancy in an increasing proportion of N. pseudonarcissus seeds the later they were dried in ‘summer’. Seed viability was only slightly reduced by desiccation in N. pseudonarcissus, but was poor and variable in G. nivalis. Shoot formation occurred both at the temperature at which germination was greatest and also if 5 °C cooler. In summary, continuous hydration of seeds of both species during warm summer‐like temperatures results in the gradual release of seed dormancy; thereafter, darkness and cooler temperatures promote germination. Cold temperatures, increased seed maturity (G. nivalis) and desiccation (N. pseudonarcissus) increase dormancy, and light inhibits germination. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 177 , 246–262.     

The development of eight microsatellite loci in the wild daffodil Narcissus triandrus (Amaryllidaceae)

We report microsatellite primer pairs for the wild tristylous daffodil, Narcissus triandrus (Amaryllidaceae). From enriched libraries, we identified 58 unique microsatellite loci. We designed primer pairs for 27 of these loci and screened genomic DNA from 38 to 40 adults from a single population. For eight polymorphic loci, the number of alleles per locus ranged from five to 17. As six primers also amplified loci in three other Narcissus species, including two horticultural varieties, we expect that some of these markers will be transferable to other Narcissus species.     

Biology of the polyploid geophyte Allium oleraceum (Amaryllidaceae): Variation in size,sexual and asexual reproduction and germination within and between tetra-, penta- and hexaploid cytotypes

It has been proposed that plant species cytotypes commonly exhibit altered morphology, reproduction, geographic and ecological distributions. We studied phenotypic variation in height, sexual (flowers, seeds) and asexual (aerial bulbils) reproduction in natural populations and in the conditions of a common garden of three cytotypes (2n = 4x, 5x, 6x) of the bulbous geophyte Allium oleraceum in the Czech Republic. Additionally, we compared the germination and dormancy of seeds and bulbils to determine whether propagules have different ecological roles. The pattern of morphological differentiation observed between cytotypes under natural conditions was similar to that observed under common garden conditions, suggesting that variability in morphological characteristics appears to be associated with ploidy levels. We revealed differences in size and sexual and asexual production between A. oleraceum cytotypes, but with wide overlap among cytotypes, suggesting a limited possibility of the studied traits to reliably distinguish between cytotypes. Tetraploids and pentaploids were rather similar; they were taller and produced more flowers than hexaploids, which were mostly flowerless (mean <0.7 flower/plant). All cytotypes were able to produce viable seeds, but their numbers were extremely low, usually less than 3 seeds per inflorescence; clonal reproduction via aerial bulbils dominates in all cytotypes (flower:bulbil ratio <0.5), with tetraploids producing more but lighter bulbils than other cytotypes. The seed: ovule ratio was low (<0.1) in all cytotypes, although hexaploids reached higher values than other cytotypes. Bulbils germinated better (means >80%) than seeds in all cytotypes, with pentaploid bulbils showing the highest germination (mean 90.5%). The cytotypes did not differ in seed germination (range of means 73.4%–76.3%). About 6% of seeds did not germinate but were still viable at the end of the experiment, while all non-germinated bulbils of all cytotypes had rotten away. Seeds, but not bulbils, can likely form a short-term persistent propagule soil bank. We found no evidence of a phenotypic trade-off between the production of flowers (seeds) and the production of bulbils within the inflorescence of all studied cytotypes. Together, inter-cytotypic differences in fitness-related traits cannot completely explain the different geographic and ecological distributions of cytotypes in the Czech Republic found by previous research.     

Calcium signalling in pollen of Papaver rhoeas undergoing the self-incompatibility (SI) response

Self-incompatibility (SI) is a genetically controlled system used by many flowering plants to prevent self-pollination. We established, using calcium imaging, that the SI response in Papaver rhoeas L. (poppy) pollen involves a Ca²⁺-mediated intracellular signalling pathway. Here we review what is known about the signalling components and cascades implicated in the SI response in poppy pollen. We present some studies using calcium green (CG-1) that show SI-induced alterations in CG-1 fluorescence and localization. We have begun to examine potential sources of Ca²⁺ involved in the responses induced by SI. This work presents preliminary data showing that influx of extracellular Ca²⁺ at the ”shank” of the pollen tube is possible. This is the first evidence suggesting that influx at this localization may play a role in the SI response. We also describe preliminary studies that begin to investigate whether the phosphoinositide signalling pathway is implicated in the SI response. Received: 12 December 2000 / Revision Accepted: 22 June 2001     

Effect of pollen competition and stigmatic receptivity on seed set in white clover (Trifolium repens L.)

The effect of competition between incompatible and compatible pollen grains on the seed production of white clover was studied. Stigmatic receptivity was also studied. A selection line of white clover that has red leaves was used as a pollen donor and as a genetic marker to allow determination of the pollen donor responsible for ovule fertilisation. Results show that incompatible pollen did not inhibit compatible pollen grains from fertilising ovules and producing seeds, although it did slightly impair seed yield. At temperatures of 20/10°C (day/night) stigmas of white clover remained receptive to additional pollination up to 32 h after an initial compatible pollination and 40 h after an incompatible pollination. The results imply that factors other than inadequate pollination and the self-incompatibility system are responsible for failure of white clover to attain its potential seed yield. Received: 31 August 1999 / Revision accepted: 28 February 2000     

Transient expression of the green fluorescent protein in pollen

 ZM13 is a pollen-specific maize gene which is expressed in the late stages of pollen development. We wished to utilize the ZM13 promoter to examine the expression of a synthetic green fluorescent protein (SGFP) in germinating pollen. The usefulness of the SGFP expression product is that its appearance and distribution can be monitored non-destructively in vivo. A plasmid containing the SGFP coding region under the control of the ZM13 promoter was constructed and then transiently transformed into pollen of Tradescantia paludosa and Nicotiana tabacum by the use of microprojectile bombardment. The expression of the green fluorescent protein was analyzed by fluorescence microscopy using a fluorescein filter. Expression began about 3 h post-bombardment, and all parts of the pollen grain and tube fluoresced. High levels of fluorescence were observed for several days following treatment. Received: 15 February 1998 / Revision accepted: 22 April 1998     

Behavior of storage lipids during development and germination of olive ( Olea europaea L.) pollen

Summary.  The presence of abundant oil bodies in the mature olive pollen grain has led us to focus on the behavior of these lipid bodies during pollen development and in vitro pollen germination. The appearance, increase, and accumulation of lipid bodies have been determined by following the sequential development of the pollen grain. Semithin slices of anthers and pollen grains were stained with Sudan Black B in order to identify neutral lipids. Ultrastructural studies were also carried out. Our results show a notable increase in lipid bodies between the young-pollen-grain stage and the mature-pollen-grain stage. Substantial polarization of lipid bodies was observed after 1 or 2 h of pollen incubation in germination medium. During pollen tube growth, the lipid bodies are located near the germinative aperture after 3 h of incubation, as well as inside the pollen tube, thus suggesting that the lipid bodies move from the pollen grain to the pollen tube. After 7 h of germination the presence of lipid bodies inside the pollen tube is no longer substantial. Our results support the idea that lipid bodies are involved in pollen germination, stigma penetration, and pollen tube growth. These results are discussed in connection with their implications for the pollen germination process. Received June 4, 2002; accepted October 29, 2002; published online April 8, 2003 RID="*" ID="*" Correspondence and reprints: Departamento de Bioquímica, Biología Celular y Molecular de Plantas, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Profesor Albareda 1, 18008 Granada, Spain.     

Influence of wind direction on pollen concentration in the atmosphere

The daily pollen concentration in the atmosphere of Badajoz (SW Spain) was analysed over a 6-year period (1993–1998) using a volumetric aerobiological trap. The results for the main pollination period are compared with the number of hours of wind each day in the four quadrants: 1 (NE), 2 (SE), 3 (SW) and 4 (NW). The pollen source distribution allowed 16 pollen types to be analysed as a function of their distribution in the four quadrants with respect to the location of the trap. Four of them correspond to species growing in an irrigated farmland environment (Amaranthaceae-Chenopodiaceae, Plantago, Scirpus, and Typha), five to riparian and woodland species (Salix, Fraxinus, Alnus, Populus, and Eucalyptus), four to urban ornamentals (Ulmus, Arecaceae, Cupressaceae, and Casuarina), and three which include the most frequent pollen grains of widely distributed species (Poaceae, Quercus, and Olea). The results show that the distribution of the sources and the wind direction play a very major role in determining the pollen concentration in the atmosphere when these sources are located in certain quadrants, and that the widely distributed pollen sources show no relationship with wind direction. In some years the values of the correlations were not maintained, which leads one to presume that, in order to draw significant conclusions and establish clear patterns of the influence of wind direction, a continuous and more prolonged study will be required. Received: 6 May 1999 / Revised: 30 March 2000 / Accepted: 31 March 2000     

Geographic and population variation in pollinating seed-consuming interactions between senita cacti (Lophocereus schottii) and senita moths (Upiga virescens)

Interspecific interactions can vary within and among populations and geographic locations. This variation can subsequently influence the evolution and coevolution of species interactions. We investigated population and geographic variation in traits important to pollinating seed-consuming interactions between the senita cactus (Lophocereus schottii) and its obligate pollinating moth (Upiga virescens), both of which are geographically restricted to the Sonoran Desert. Female moths actively pollinate senita flowers and oviposit onto flowers. Their larvae consume developing seeds and fruit of flowers pollinated by females. Traits important to this interaction include fruit set from moth pollination, fruit survivorship, and costs of fruit consumption by larvae. We studied these traits for five populations at two widely separated geographic locations. On average, 37% of flowers set fruit, 22% of flowers produced mature fruit, and larvae consumed 25% of immature fruit pollinated by female senita moths. Senita cactus and senita moth interactions were strongly mutualistic in all populations that we studied. Although one population had statistically lower fruit set and fruit production than the other four, all five populations were qualitatively similar in fruit production, costs, and patterns of fruit survivorship. Hand-pollination experiments suggested that fruit set was resource-limited in all but this one population. Apparent pollen limitation in the one population explains the quantitative differences in fruit set and fruit survivorship among the populations. As predicted by theory and exemplified by the senita mutualism, specialized and/or obligate interactions vary little among populations and geographic locations. Received: 5 March 1999 / Accepted: 25 June 1999