The endangered Iris atropurpurea (Iridaceae) in Israel: honey-bees,night-sheltering male bees and female solitary bees as pollinators

Background and Aims

The coastal plain of Israel hosts the last few remaining populations of the endemic Iris atropurpurea (Iridaceae), a Red List species of high conservation priority. The flowers offer no nectar reward. Here the role of night-sheltering male solitary bees, honey-bees and female solitary bees as pollinators of I. atropurpurea is documented.

Methods

Breeding system, floral longevity, stigma receptivity, visitation rates, pollen loads, pollen deposition and removal and fruit- and seed-set were investigated.

Key Results

The main wild pollinators of this plant are male eucerine bees, and to a lesser extent, but with the potential to transfer pollen, female solitary bees. Honey-bees were found to be frequent diurnal visitors; they removed large quantities of pollen and were as effective as male sheltering bees at pollinating this species. The low density of pollen carried by male solitary bees was attributed to grooming activities, pollen displacement when bees aggregated together in flowers and pollen depletion by honey-bees. In the population free of honey-bee hives, male bees carried significantly more pollen grains on their bodies. Results from pollen analysis and pollen deposited on stigmas suggest that inadequate pollination may be an important factor limiting fruit-set. In the presence of honey-bees, eucerine bees were low removal–low deposition pollinators, whereas honey-bees were high removal–low deposition pollinators, because they removed large amounts into corbiculae and deposited relatively little onto receptive stigmas.

Conclusions

Even though overall, both bee taxa were equally effective pollinators, we suggest that honey-bees have the potential to reduce the amount of pollen available for plant reproduction, and to reduce the amount of resources available to solitary bee communities. The results of this study have potential implications for the conservation of this highly endangered plant species if hives are permitted inside reserves, where the bulk of Oncocyclus iris species are protected.     

Using flow cytometry to estimate pollen DNA content: improved methodology and applications

Background and Aims

Flow cytometry has been used to measure nuclear DNA content in pollen, mostly to understand pollen development and detect unreduced gametes. Published data have not always met the high-quality standards required for some applications, in part due to difficulties inherent in the extraction of nuclei. Here we describe a simple and relatively novel method for extracting pollen nuclei, involving the bursting of pollen through a nylon mesh, compare it with other methods and demonstrate its broad applicability and utility.

Methods

The method was tested across 80 species, 64 genera and 33 families, and the data were evaluated using established criteria for estimating genome size and analysing cell cycle. Filter bursting was directly compared with chopping in five species, yields were compared with published values for sonicated samples, and the method was applied by comparing genome size estimates for leaf and pollen nuclei in six species.

Key Results

Data quality met generally applied standards for estimating genome size in 81 % of species and the higher best practice standards for cell cycle analysis in 51 %. In 41 % of species we met the most stringent criterion of screening 10 000 pollen grains per sample. In direct comparison with two chopping techniques, our method produced better quality histograms with consistently higher nuclei yields, and yields were higher than previously published results for sonication. In three binucleate and three trinucleate species we found that pollen-based genome size estimates differed from leaf tissue estimates by 1·5 % or less when 1C pollen nuclei were used, while estimates from 2C generative nuclei differed from leaf estimates by up to 2·5 %.

Conclusions

The high success rate, ease of use and wide applicability of the filter bursting method show that this method can facilitate the use of pollen for estimating genome size and dramatically improve unreduced pollen production estimation with flow cytometry.     

Counting pollen grains using readily available, free image processing and analysis software

Background and Aims

Although many methods exist for quantifying the number of pollen grains in a sample, there are few standard methods that are user-friendly, inexpensive and reliable. The present contribution describes a new method of counting pollen using readily available, free image processing and analysis software.

Methods

Pollen was collected from anthers of two species, Carduus acanthoides and C. nutans (Asteraceae), then illuminated on slides and digitally photographed through a stereomicroscope. Using ImageJ (NIH), these digital images were processed to remove noise and sharpen individual pollen grains, then analysed to obtain a reliable total count of the number of grains present in the image. A macro was developed to analyse multiple images together. To assess the accuracy and consistency of pollen counting by ImageJ analysis, counts were compared with those made by the human eye.

Key Results and Conclusions

Image analysis produced pollen counts in 60 s or less per image, considerably faster than counting with the human eye (5–68 min). In addition, counts produced with the ImageJ procedure were similar to those obtained by eye. Because count parameters are adjustable, this image analysis protocol may be used for many other plant species. Thus, the method provides a quick, inexpensive and reliable solution to counting pollen from digital images, not only reducing the chance of error but also substantially lowering labour requirements.     

Insights into a hydration regulating system in Cupressus pollen grains

Background and Aims

Hydration, rupture and exine opening due to the sudden and large expansion of intine are typical of taxoid-type pollen grains. A hemispheric outgrowth external to the exine was observed on Cupressus and Juniperus pollen grains before the intine swelling and exine release. However, the actual existence of this permanent or temporary structure and its precise role in pollen hydration is still being debated. The aim of this paper is to collect information on the actual presence of this peculiar outgrowth on the surface of the Cupressus pollen grain, its structure, composition and function.

Methods

Pollen grains of several Cupressus species were observed using various techniques and methodologies, under light and fluorescence microscopy, phase-contrast microscopy, confocal microscopy, scanning electron microscopy, and an environmental scanning electron microscope. Observations were also performed on other species with taxoid-type pollen grains.

Key Results

A temporary structure located just above the pore was observed on Cupressus pollen grains, as well as on other taxoid-type pollens. It is hemispheric, layered, and consists of polysaccharides and proteins. The latter are confined to its inner part. Its presence seems to regulate the entrance of water into the grains at the beginning of pollen hydration.

Conclusions

The presence of a temporary structure over the pore of taxoid-type pollen grains was confirmed and its structure was resolved using several stains and observation techniques. This structure plays a role in the first phases of pollen hydration.     

Successive microsporogenesis affects pollen aperture pattern in the tam mutant of Arabidopsis thaliana

Background and Aims

The tam (tardy asynchronous meiosis) mutant of Arabidopsis thaliana, which exhibits a modified cytokinesis with a switch from simultaneous to successive cytokinesis, was used to perform a direct test of the implication of cytokinesis in aperture-pattern ontogeny of angiosperm pollen grains. The aperture pattern corresponds to the number and arrangement of apertures (areas of the pollen wall permitting pollen tube germination) on the surface of the pollen grain.

Methods

A comparative analysis of meiosis and aperture distribution was performed in two mutant strains of arabidopsis: quartet and quartet-tam.

Key Results

While the number of apertures is not affected in the quartet-tam mutant, the arrangement of the three apertures is modified compared with the quartet, resulting in a different aperture pattern.

Conclusions

These results directly demonstrate the relationship between the type of sporocytic cytokinesis and pollen aperture-pattern ontogeny.     

Pollination in a patchily distributed lousewort is facilitated by presence of a co-flowering plant due to enhancement of quantity and quality of pollinator visits

Background and Aims

Plants surrounded by individuals of other co-flowering species may suffer a reproductive cost from interspecific pollen transfer (IPT). However, differences in floral architecture may reduce or eliminate IPT.

Methods

A study was made of Pedicularis densispica (lousewort) and its common co-flowering species, Astragalus pastorius, to compare reproductive and pollination success of lousewort plants from pure and mixed patches. Floral architecture and pollinator behaviour on flowers of the two plants were compared along with the composition of stigmatic pollen load of the louseworts. The extent of pollen limitation of plants from pure and mixed patches was also explored through supplemental pollination with self- and outcross pollen (PLs and PLx).

Key Results

Mixed patches attracted many more nectar-searching individuals of Bombus richardsi. These bumble-bees moved frequently between flowers of the two species. However, they pollinated P. densispica with their dorsum and A. pastorius with their abdomen. This difference in handling almost completely eliminated IPT. Lousewort plants from mixed patches yielded more seeds, and seeds of higher mass and germinability, than those from pure patches. Moreover, louseworts from mixed patches had lower PLs and PLx compared with those from pure patches.

Conclusions

Differences in floral architecture induced differences in pollinator behaviour that minimized IPT, such that co-flowering plants significantly enhanced quantity and quality of pollinator visits for the lousewort plants in patchy habitat. These findings add to our understanding of the mechanisms of pollination facilitation.     

Desiccation tolerance,longevity and seed-siring ability of entomophilous pollen from UK native orchid species

Background and Aims

Pollinator-limited seed-set in some terrestrial orchids is compensated for by the presence of long-lived flowers. This study tests the hypothesis that pollen from these insect-pollinated orchids should be desiccation tolerant and relatively long lived using four closely related UK terrestrial species; Anacamptis morio, Dactylorhiza fuchsii, D. maculata and Orchis mascula.

Methods

Pollen from the four species was harvested from inflorescences and germinated in vitro, both immediately and also after drying to simulate interflower transit. Their tolerance to desiccation and short-term survival was additionally assessed after 3 d equilibration at a range of relative humidities (RHs), and related to constructed sorption isotherms (RH vs. moisture content, MC). Ageing of D. fuchsii pollen was further tested over 2 months against temperature and RH, and the resultant survival curves were subjected to probit analysis, and the distribution of pollen death in time (σ) was determined. The viability and siring ability, following artificial pollinations, were determined in D. fuchsii pollen following storage for 6 years at –20 °C.

Key Results

The pollen from all four species exhibited systematic increases in germinability and desiccation tolerance as anthesis approached, and pollen from open flowers generally retained high germinability. Short-term storage revealed sensitivity to low RH, whilst optimum survival occurred at comparable RHs in all species. Similarly, estimated pollen life spans (σ) at differing temperatures were longest under the dry conditions. Despite a reduction in germination and seeds per capsule, long-term storage of D. fuchsii pollen did not impact on subsequent seed germination in vitro.

Conclusions

Substantial pollen desiccation tolerance and life span of the four entomophilous orchids reflects a resilient survival strategy in response to unpredictable pollinator visitation, and presents an alternative approach to germplasm conservation.     

Electrophoretic profiling and immunocytochemical detection of pectins and arabinogalactan proteins in olive pollen during germination and pollen tube growth

Background and Aims

Cell wall pectins and arabinogalactan proteins (AGPs) are important for pollen tube growth. The aim of this work was to study the temporal and spatial dynamics of these compounds in olive pollen during germination.

Methods

Immunoblot profiling analyses combined with confocal and transmission electron microscopy immunocytochemical detection techniques were carried out using four anti-pectin (JIM7, JIM5, LM5 and LM6) and two anti-AGP (JIM13 and JIM14) monoclonal antibodies.

Key Results

Pectin and AGP levels increased during olive pollen in vitro germination. (1 → 4)-β-d-Galactans localized in the cytoplasm of the vegetative cell, the pollen wall and the apertural intine. After the pollen tube emerged, galactans localized in the pollen tube wall, particularly at the tip, and formed a collar-like structure around the germinative aperture. (1 → 5)-α-l-Arabinans were mainly present in the pollen tube cell wall, forming characteristic ring-shaped deposits at regular intervals in the sub-apical zone. As expected, the pollen tube wall was rich in highly esterified pectic compounds at the apex, while the cell wall mainly contained de-esterified pectins in the shank. The wall of the generative cell was specifically labelled with arabinans, highly methyl-esterified homogalacturonans and JIM13 epitopes. In addition, the extracellular material that coated the outer exine layer was rich in arabinans, de-esterified pectins and JIM13 epitopes.

Conclusions

Pectins and AGPs are newly synthesized in the pollen tube during pollen germination. The synthesis and secretion of these compounds are temporally and spatially regulated. Galactans might provide mechanical stability to the pollen tube, reinforcing those regions that are particularly sensitive to tension stress (the pollen tube–pollen grain joint site) and mechanical damage (the tip). Arabinans and AGPs might be important in recognition and adhesion phenomena of the pollen tube and the stylar transmitting cells, as well as the egg and sperm cells.     

Differences in pollen viability in relation to different deceptive pollination strategies in Mediterranean orchids

Background and Aims

To date, current research involving pollen viability has been evaluated in a relatively low number of orchid species. In the present study, we focused on five related Mediterranean orchid genera (Anacamptis, Orchis, Dactylorhiza, Ophrys and Serapias) that are characterized by different types of deceptive pollination.

Methods

The in vitro germination ability of increasingly aged pollinaria of eight food-, seven sexually and two shelter-deceptive species was evaluated. Pollination experiments on two food-, one sexually and one shelter-deceptive species were also performed and the percentage of embryonate seeds derived from the increasingly aged pollinaria was checked.

Key Results

All of the examined species showed long-term viabilities (=50 % pollen tube growth) that ranged from 8 to 35 d. Species with the same deceptive pollination strategies exhibited the same pollen viability trends. Interestingly, pollen viabilities of species groups with different deception types have shown significant differences, with sexually and shelter- deceptive species exhibiting a shorter life span than food-deceptive species.

Conclusions

This study confirms the prolonged germination and fertilization capacities of orchid pollinaria, and to our knowledge is the first report demonstrating a clear relationship between pollen viability and pollination system. It is proposed that this relationship is attributed to the different types of reproductive barriers, pre- or post-zygotic, that characterixe Ophrys and Serapias and the food-deceptive species, respectively.     

Do specialized flowers promote reproductive isolation? Realized pollination accuracy of three sympatric Pedicularis species

Background and Aims

Interest in pollinator-mediated evolutionary divergence of flower phenotype and speciation in plants has been at the core of plant evolutionary studies since Darwin. Specialized pollination is predicted to lead to reproductive isolation and promote speciation among sympatric species by promoting partitioning of (1) the species of pollinators used, (2) when pollinators are used, or (3) the sites of pollen placement. Here this last mechanism is investigated by observing the pollination accuracy of sympatric Pedicularis species (Orobanchacae).

Methods

Pollinator behaviour was observed on three species of Pedicularis (P. densispica, P. tricolor and P. dichotoma) in the Hengduan Mountains, south-west China. Using fluorescent powder and dyed pollen, the accuracy was assessed of stigma contact with, and pollen deposition on, pollinating bumble-bees, respectively.

Key Results

All three species of Pedicularis were pollinated by bumble-bees. It was found that the adaptive accuracy of female function was much higher than that of male function in all three flower species. Although peak pollen deposition corresponded to the optimal location on the pollinator (i.e. the site of stigma contact) for each species, substantial amounts of pollen were scattered over much of the bees'' bodies.

Conclusions

The Pedicularis species studied in the eastern Himalayan region did not conform with Grant''s ‘Pedicularis Model’ of mechanical reproductive isolation. The specialized flowers of this diverse group of plants seem unlikely to have increased the potential for reproductive isolation or influenced rates of speciation. It is suggested instead that the extreme species richness of the Pedicularis clade was generated in other ways and that specialized flowers and substantial pollination accuracy evolved as a response to selection generated by the diversity of co-occurring congeners.     

Paternity analysis-based inference of pollen dispersal patterns, male fecundity variation, and influence of flowering tree density and general flowering magnitude in two dipterocarp species

Background and Aims

Knowledge of pollen dispersal patterns and variation of fecundity is essential to understanding plant evolutionary processes and to formulating strategies to conserve forest genetic resources. Nevertheless, the pollen dispersal pattern of dipterocarp, main canopy tree species in palaeo-tropical forest remains unclear, and flowering intensity variation in the field suggests heterogeneity of fecundity.

Methods

Pollen dispersal patterns and male fecundity variation of Shorea leprosula and Shorea parvifolia ssp. parvifolia on Peninsular Malaysian were investigated during two general flowering seasons (2001 and 2002), using a neighbourhood model modified by including terms accounting for variation in male fecundity among individual trees to express heterogeneity in flowering.

Key Results

The pollen dispersal patterns of the two dipterocarp species were affected by differences in conspecific tree flowering density, and reductions in conspecific tree flowering density led to an increased selfing rate. Active pollen dispersal and a larger number of effective paternal parents were observed for both species in the season of greater magnitude of general flowering (2002).

Conclusions

The magnitude of general flowering, male fecundity variation, and distance between pollen donors and mother trees should be taken into account when attempting to predict the effects of management practices on the self-fertilization and genetic structure of key tree species in tropical forest, and also the sustainability of possible management strategies, especially selective logging regimes.     

New views of tapetum ultrastructure and pollen exine development in Arabidopsis thaliana

Background and Aims

The Arabidopsis thaliana pollen cell wall is a complex structure consisting of an outer sporopollenin framework and lipid-rich coat, as well as an inner cellulosic wall. Although mutant analysis has been a useful tool to study pollen cell walls, the ultrastructure of the arabidopsis anther has proved to be challenging to preserve for electron microscopy.

Methods

In this work, high-pressure freezing/freeze substitution and transmission electron microscopy were used to examine the sequence of developmental events in the anther that lead to sporopollenin deposition to form the exine and the dramatic differentiation and death of the tapetum, which produces the pollen coat.

Key Results

Cryo-fixation revealed a new view of the interplay between sporophytic anther tissues and gametophytic microspores over the course of pollen development, especially with respect to the intact microspore/pollen wall and the continuous tapetum epithelium. These data reveal the ultrastructure of tapetosomes and elaioplasts, highly specialized tapetum organelles that accumulate pollen coat components. The tapetum and middle layer of the anther also remain intact into the tricellular pollen and late uninucleate microspore stages, respectively.

Conclusions

This high-quality structural information, interpreted in the context of recent functional studies, provides the groundwork for future mutant studies where tapetum and microspore ultrastructure is assessed.     

The influence of tetrad shape and intersporal callose wall formation on pollen aperture pattern ontogeny in two eudicot species

Background and Aims

In flowering plants, microsporogenesis is accompanied by various types of cytoplasmic partitioning (cytokinesis). Patterns of male cytokinesis are suspected to play a role in the diversity of aperture patterns found in pollen grains of angiosperms. The relationships between intersporal wall formation, tetrad shape and pollen aperture pattern ontogeny are studied.

Methods

A comparative analysis of meiosis and aperture distribution was performed within tetrads in two triporate eudicot species with contrasting aperture arrangements within their tetrads [Epilobium roseum (Onagraceae) and Paranomus reflexus (Proteaceae)].

Key Results and Conclusions

Intersporal wall formation is a two-step process in both species. Cytokinesis is first achieved by the formation of naked centripetal cell plates. These naked cell plates are then covered by additional thick, localized callose deposits that differ in location between the two species. Apertures are finally formed in areas in which additional callose is deposited on the cell plates. The recorded variation in tetrad shape is correlated with variations in aperture pattern, demonstrating the role of cell partitioning in aperture pattern ontogeny.     

The cell wall pectic polymer rhamnogalacturonan-II is required for proper pollen tube elongation: implications of a putative sialyltransferase-like protein

Background and Aims

Rhamnogalacturonan-II (RG-II) is one of the pectin motifs found in the cell wall of all land plants. It contains sugars such as 2-keto-3-deoxy-d-lyxo-heptulosaric acid (Dha) and 2-keto-3-deoxy-d-manno-octulosonic acid (Kdo), and within the wall RG-II is mostly found as a dimer via a borate diester cross-link. To date, little is known regarding the biosynthesis of this motif. Here, after a brief review of our current knowledge on RG-II structure, biosynthesis and function in plants, this study explores the implications of the presence of a Golgi-localized sialyltransferase-like 2 (SIA2) protein that is possibly involved in the transfer of Dha or Kdo in the RG-II of Arabidopsis thaliana pollen tubes, a fast-growing cell type used as a model for the study of cell elongation.

Methods

Two heterozygous mutant lines of arabidopsis (sia2-1+/– and qrt1 × sia2-2+/–) were investigated. sia2-2+/– was in a quartet1 background and the inserted T-DNA contained the reporter gene β-glucuronidase (GUS) under the pollen-specific promoter LAT52. Pollen germination and pollen tube phenotype and growth were analysed both in vitro and in vivo by microscopy.

Key Results

Self-pollination of heterozygous lines produced no homozygous plants in the progeny, which may suggest that the mutation could be lethal. Heterozygous mutants displayed a much lower germination rate overall and exhibited a substantial delay in germination (20 h of delay to reach 30 % of pollen grain germination compared with the wild type). In both lines, mutant pollen grains that were able to produce a tube had tubes that were either bursting, abnormal (swollen or dichotomous branching tip) or much shorter compared with wild-type pollen tubes. In vivo, mutant pollen tubes were restricted to the style, whereas the wild-type pollen tubes were detected at the base of the ovary.

Conclusions

This study highlights that the mutation in arabidopsis SIA2 encoding a sialyltransferase-like protein that may transfer Dha or Kdo on the RG-II motif has a dramatic effect on the stability of the pollen tube cell wall.     

Using the biomass-ratio and idiosyncratic hypotheses to predict mixed-species litter decomposition

Background and Aims

Microsporogenesis leading to monosulcate pollen grains has already been described for a wide range of monocot species. However, a detailed study of additional callose deposition after the completion of the cleavage walls has been neglected so far. The study of additional callose deposition in monosulcate pollen grain has gained importance since a correlation between additional callose deposition and aperture location has recently been revealed.

Methods

Microsporogenesis is described for 30 species belonging to eight families of the monocots: Acoraceae, Amaryllidaceae, Alstroemeriaceae, Asparagaceae, Butomaceae, Commelinaceae, Liliaceae and Xanthorrhoeaceae.

Key Results

Five different microsporogenesis pathways are associated with monosulcate pollen grain. They differ in the type of cytokinesis, tetrad shape, and the presence and shape of additional callose deposition. Four of them present additional callose deposition.

Conclusions

In all these different microsporogenesis pathways, aperture location seems to be linked to the last point of callose deposition.     

Floral display size,conspecific density and florivory affect fruit set in natural populations of Phlox hirsuta,an endangered species

Background and Aims

Natural variation in fruit and seed set may be explained by factors that affect the composition of pollen grains on stigmas. Self-incompatible species require compatible outcross pollen grains to produce seeds. The siring success of outcross pollen grains, however, can be hindered if self (or other incompatible) pollen grains co-occur on stigmas. This study identifies factors that determine fruit set in Phlox hirsuta, a self-sterile endangered species that is prone to self-pollination, and its associated fitness costs.

Methods

Multiple linear regressions were used to identify factors that explain variation in percentage fruit set within three of the five known populations of this endangered species. Florivorous beetle density, petal colour, floral display size, local conspecific density and pre-dispersal seed predation were quantified and their effects on the ability of flowers to produce fruits were assessed.

Key Results

In all three populations, percentage fruit set decreased as florivorous beetle density increased and as floral display size increased. The effect of floral display size on fruit set, however, often depended on the density of nearby conspecific plants. High local conspecific densities offset – even reversed – the negative effects of floral display size on percentage fruit set. Seed predation by mammals decreased fruit set in one population.

Conclusions

The results indicate that seed production in P. hirsuta can be maximized by selectively augmenting populations in areas containing isolated large plants, by reducing the population sizes of florivorous beetles and by excluding mammals that consume unripe fruits.     

Profiling and functional classification of esterases in olive (Olea europaea) pollen during germination

Background and Aims

A pollen grain contains a number of esterases, many of which are released upon contact with the stigma surface. However, the identity and function of most of these esterases remain unknown. In this work, esterases from olive pollen during its germination were identifided and functionally characterized.

Methods

The esterolytic capacity of olive (Olea europaea) pollen was examined using in vitro and in-gel enzymatic assays with different enzyme substrates. The functional analysis of pollen esterases was achieved by inhibition assays by using specific inhibitors. The cellular localization of esterase activities was performed using histochemical methods.

Key Results

Olive pollen showed high levels of non-specific esterase activity, which remained steady after hydration and germination. Up to 20 esterolytic bands were identified on polyacrylamide gels. All the inhibitors decreased pollen germinability, but only diisopropyl fluorophosphate (DIFP) hampered pollen tube growth. Non-specific esterase activity is localized on the surface of oil bodies (OBs) and small vesicles, in the pollen intine and in the callose layer of the pollen tube wall. Acetylcholinesterase (AChE) activity was mostly observed in the apertures, exine and pollen coat, and attached to the pollen tube wall surface and to small cytoplasmic vesicles.

Conclusions

In this work, for the first time a systematic functional characterization of esterase enzymes in pollen from a plant species with wet stigma has been carried out. Olive pollen esterases belong to four different functional groups: carboxylesterases, acetylesterases, AChEs and lipases. The cellular localization of esterase activity indicates that the intine is a putative storage site for esterolytic enzymes in olive pollen. Based on inhibition assays and cellular localization of enzymatic activities, it can be concluded that these enzymes are likely to be involved in pollen germination, and pollen tube growth and penetration of the stigma.     

Water status and associated processes mark critical stages in pollen development and functioning

Background

The male gametophyte developmental programme can be divided into five phases which differ in relation to the environment and pollen hydration state: (1) pollen develops inside the anther immersed in locular fluid, which conveys substances from the mother plant – the microsporogenesis phase; (2) locular fluid disappears by reabsorption and/or evaporation before the anther opens and the maturing pollen grains undergo dehydration – the dehydration phase; (3) the anther opens and pollen may be dispersed immediately, or be held by, for example, pollenkitt (as occurs in almost all entomophilous species) for later dispersion – the presentation phase; (4) pollen is dispersed by different agents, remaining exposed to the environment for different periods – the dispersal phase; and (5) pollen lands on a stigma and, in the case of a compatible stigma and suitable conditions, undergoes rehydration and starts germination – the pollen–stigma interaction phase.

Scope

This review highlights the issue of pollen water status and indicates the various mechanisms used by pollen grains during their five developmental phases to adjust to changes in water content and maintain internal stability.

Conclusions

Pollen water status is co-ordinated through structural, physiological and molecular mechanisms. The structural components participating in regulation of the pollen water level, during both dehydration and rehydration, include the exine (the outer wall of the pollen grain) and the vacuole. Recent data suggest the involvement of water channels in pollen water transport and the existence of several molecular mechanisms for pollen osmoregulation and to protect cellular components (proteins and membranes) under water stress. It is suggested that pollen grains will use these mechanisms, which have a developmental role, to cope with environmental stress conditions.     

Insects, birds and lizards as pollinators of the largest-flowered Scrophularia of Europe and Macaronesia

Background and Aims

It has traditionally been considered that the flowers of Scrophularia are mainly pollinated by wasps. We studied the pollination system of four species which stand out for their large and showy flowers: S. sambucifolia and S. grandiflora (endemics of the western Mediterranean region), S. trifoliata (an endemic of the Tyrrhenian islands) and S. calliantha (an endemic of the Canary Islands). Our principal aim was to test whether these species were pollinated by birds or showed a mixed pollination system between insects and birds.

Methods

Censuses and captures of insects and birds were performed to obtain pollen load transported and deposited on the stigmas. Also, a qualitative and quantitative analysis of the flowers and inflorescences was carried out.

Key Results

Flowers were visited by Hymenoptera and by passerine birds. The Canarian species was the most visited by birds, especially by Phylloscopus canariensis, and its flowers were also accessed by juveniles of the lizard Gallotia stehlini. The most important birds in the other three species were Sylvia melanocephala and S. atricapilla. The most important insect-functional groups in the mixed pollination system were: honey-bees and wasps in S. sambucifolia; bumble-bees and wasps in S. grandiflora; wasps in S. trifoliata; and a small bee in S. calliantha.

Conclusions

The species studied show a mixed pollination system between insects and passerine birds. In S. calliantha there is, in addition, a third agent (juveniles of Gallotia stehlini). The participation of birds in this mixed pollination system presents varying degrees of importance because, while in S. calliantha they are the main pollinators, in the other species they interact to complement the insects which are the main pollinators. A review of different florae showed that the large showy floral morphotypes of Scrophularia are concentrated in the western and central Mediterranean region, Macaronesia and USA (New Mexico).