Parsing a plethora of pollen: the role of pollen size and shape in the evolution of Boraginaceae.

Pollen, the microgametophyte of seed plants, has an important role in plant reproduction and, therefore, evolution. Pollen is variable in, for example, size, shape, aperture number; these features are particularly diverse in some plant taxa and can be diagnostic. In one family, Boraginaceae, the range of pollen diversity suggests the potential utility of this family as a model for integrative studies of pollen development, evolution and molecular biology. In the present study, a comprehensive survey of the diversity and evolution of pollen from 538 species belonging to 72 genera was made using data from the literature and additional scanning electron microscopy examination. Shifts in diversification rates and the evolution of various quantitative characters were detected, and the results revealed remarkable differences in size, shape and number of apertures. The pollen of one subfamily, Boraginoideae, is larger than that in Cynoglossoideae. The diversity of pollen shapes and aperture numbers in one tribe, Lithospermeae, is greater than that in the other tribes. Ancestral pollen for the family was resolved as small, prolate grains that bear three apertures and are iso-aperturate. Of all the tribes, the greatest number of changes in pollen size and aperture number were observed in Lithospermeae and Boragineae, and the number of apertures was found to be stable throughout all tribes of Cynoglossoideae. In addition, the present study showed that diversification of Boraginaceae cannot be assigned to a single factor, such as pollen size, and the increased rate of diversification for species-rich groups (e.g. Cynoglossum) is not correlated with pollen size or shape evolution. The palynological data and patterns of character evolution presented in the study provide better resolution of the roles of geographical and ecological factors in the diversity and evolution of pollen grains of Boraginaceae, and provide suggestions for future palynological research across the family.

Effects of heterospecific pollen on stigma behavior in Campsis radicans: Causes and consequences.

PREMISE: Pollinator sharing of co-flowering plants may result in interspecific pollen receipt with a fitness cost. However, the underlying factors that determine the effects of heterospecific pollen (HP) are not fully understood. Moreover, the cost of stigma closure induced by HP may be more severe for plants with special touch-sensitive stigmas than for plants with non-touch-sensitive stigmas. Very few studies have assessed HP effects on stigma behavior. METHODS: We conducted hand-pollination experiments with 10 HP donors to estimate HP effects on stigma behavior and stigmatic pollen germination in Campsis radicans (Bignoniaceae) at low and high pollen loads. We assessed the role of phylogenetic distance between donor and recipient, pollen size, and pollen aperture number in mediating HP effects. Additionally, we observed pollen tube growth to determine the conspecific pollen-tube-growth advantage. RESULTS: Stigma behavior differed significantly with HP of different species. Pollen load increased, while pollen size decreased, the percentage of permanent closure and stigmatic germination of HP. Stigmatic HP germination increased with increasing aperture number. However, HP effects did not depend on phylogenetic distance. In addition, conspecific pollen had a pollen-tube-growth advantage over HP. CONCLUSIONS: Our results provide a good basis for understanding the stigma-pollen recognition process of plant taxa with touch-sensitive stigmas. We concluded that certain flowering traits drive the HP effects on the post-pollination period. To better understand the impact of pollinator sharing and interspecific pollen transfer on plant evolution, we highlight the importance of evaluating more factors that determine HP effects at the community level.

Evolutionary history and genetic diversity of apomictic allopolyploids in Hieracium s.str.: morphological versus genomic features.

PREMISE: The origin of allopolyploids is believed to shape their evolutionary potential, ecology, and geographical ranges. Morphologically distinct apomictic types sharing the same parental species belong to the most challenging groups of polyploids. We evaluated the origins and variation of two triploid taxa (Hieracium pallidiflorum, H. picroides) presumably derived from the same diploid parental pair (H. intybaceum, H. prenanthoides). METHODS: We used a suite of approaches ranging from morphological, phylogenetic (three unlinked molecular markers), and cytogenetic analyses (in situ hybridization) to genome size screening and genome skimming. RESULTS: Genotyping proved the expected parentage of all analyzed accessions of H. pallidiflorum and H. picroides and revealed that nearly all of them originated independently. Genome sizes and genome dosage largely corresponded to morphology, whereas the maternal origin of the allopolyploids had no discernable effect. Polyploid accessions of both parental species usually contained genetic material from other species. Given the phylogenetic distance of the parents, their chromosomes appeared only weakly differentiated in genomic in situ hybridization (GISH), as well as in overall comparisons of the repetitive fraction of their genomes. Furthermore, the repeatome of a phylogenetically more closely related species (H. umbellatum) differed significantly more. CONCLUSIONS: We proved (1) multiple origins of hybridogeneous apomicts from the same diploid parental taxa, and (2) allopolyploid origins of polyploid accessions of the parental species. We also showed that the evolutionary dynamics of very fast evolving markers such as satellite DNA or transposable elements does not necessarily follow patterns of speciation.

Water influences how seed production responds to conspecific and heterospecific pollen.

PREMISE: Outcrossing species depend on pollen from conspecific individuals that may not be exposed to the same abiotic conditions as maternal plants. Additionally, many flowers receive heterospecific pollen, which can also influence seed production. Studies aimed to understand how abiotic conditions influence seed production tend to focus on maternal conditions and leave unexplored the effect of abiotic conditions experienced by pollen donors. We tested how water availability to pollen donors, both conspecific and heterospecific, influenced the seed production of recipient plants exposed to different water availability regimes. METHODS: In a greenhouse setting we manipulated the water availability (low- or high-water treatment) to potted recipient plants (Phacelia parryi), to conspecific pollen donors, and to heterospecific pollen donors (Brassica nigra). We hand pollinated recipient plants with different pollen mixes that represented all combinations of conspecific pollen mixed with heterospecific pollen. From these hand pollinations we determined the amount of pollen that was transferred, pollen volume, pollen shape, and seed production. RESULTS: Higher water availability to conspecific pollen donors led to higher seed production. Under low water availability to heterospecific pollen donors, seed production was unaffected by recipient or conspecific pollen donor treatment. Under high water availability to heterospecific pollen donors, seed production was highest when conspecific pollen donors and pollen recipients also received the high-water treatment. CONCLUSIONS: Environmental conditions of pollen donors can influence the seed production of maternal plants. These results illustrate potential impacts of environmental heterogeneity on post-pollination events that lead to seed production and thus impact a pollinator's contribution to plant fitness.

Interactive effects between donor and recipient species mediate fitness costs of heterospecific pollen receipt in a co-flowering community.

Evaluation of pollen transfer in wild plant communities revealing heterospecific pollen receipt is common, yet experimental hand pollinations have revealed high among-species variation in the magnitude of its effect on recipient fitness. The causes of this among-species variation are unknown, however, prompting the investigation of underlying factors. Here, we conducted a hand-pollination experiment with ten co-flowering species to determine whether the effects of heterospecific pollen receipt are mediated by the pollen donor or recipient species alone, or whether the effects are determined by the interaction between them. We further assessed species traits potentially mediating interactive effects in heterospecific pollen receipt by evaluating the relationship between heterospecific pollen effect size and three different predictors reflecting a unique combination of pollen donor and recipient characteristics. Our results show, for the first time, that the magnitude of the heterospecific pollen receipt effect is determined by the specific combination of donor and recipient species (i.e., interactive effects). However, we were unable to uncover the specific combination of traits mediating these effects. Overall, our study provides strong evidence that an understanding of heterospecific pollen receipt effects based on recipient or donor characteristics alone may be insufficient. This study is an important step toward an understanding of consequences of heterospecific pollen receipt in co-flowering communities.

Transitions from distyly to homostyly are associated with floral evolution in the buckwheat genus (Fagopyrum).

PREMISE OF THE STUDY: Documenting trait transitions among species with dimorphic flowers can help to test whether similar patterns of selection are responsible for divergence in floral traits among different species. Heterostyly is thought to promote outcrossing. Theory suggests that the evolutionary transition from heterostylous to homostylous flowers should be accompanied by a reduction in floral size in which pollen size and style length are expected to covary. Patterns of such correlated floral trait evolution have not, however, been widely examined. METHODS: The evolutionary history of heterostyly and homostyly was reconstructed from a molecular phylogeny of 13 species of Fagopyrum and two outgroups, based on one nuclear gene (nrITS) and three chloroplast regions (matK, atpB-rbcL, and psbA-trnH spacer). Floral traits of nine Fagopyrum species including pollen number and size, as well as stigma depth (length of the capitate stigma), were measured and ancestral characters of the herkogamic condition were reconstructed. KEY RESULTS: Three transitions from distyly to homostyly were observed. In two transitions, flower size (corolla diameter, pedicel length), herkogamy (stigma-anther distance) and pollen production decreased, but stigma depth and pollen size did not. Changes of anther height and style length were inconsistent. The predicted positive relationship between style length and pollen size in the two transitions to homostyly was not observed. CONCLUSIONS: Floral evolution accompanying transitions to homostyly in Fagopyrum were found to be consistent with predictions of mating system evolution theory, and the correlation of traits in distylous vs. homostylous species revealed that pollen size generally correlates with stigma depth rather than style length.

Spines vs. microspines: an overview of the sculpture exine in selected basal and derived Asteraceae with focus on Asteroideae.

This study presents a detailed examination of the echinate and microechinate sculpturing in relation to the size of pollen grains in 31 selected species of Asteraceae belonging to the subfamilies Barnadesioideae, Mutisioideae, Carduoideae and Asteroideae. The aims were to recognize sculpturing patterns, under LM and SEM, within large and small pollen of both basal and derived species and to explore the features that could have taxonomic value to apply in palynological disciplines. The detailed examination of the exine surface showed both the relevance and limits of sculptural patterns for taxonomy. Under LM, the microechinate sculpture gave little taxonomic information, whereas in the echinate sculpture, three exine types and two subtypes were recognized. Type I included microechinate exine, which is commonly present in large pollen grains of the basal lineages. Types II (subtypes IIa and IIb) and III included echinate and smaller pollen grains. In these types, spines were always regularly arranged and, were characterized by the length, shape, tip, perforations and distribution. Type IIa included more or less conical spines usually with a distended base, less than 4 µm in length, present in species of different tribes like Astereae, Eupatorieae, Helenieae, Gnaphalieae, Senecioideae and Heliantheae to a lesser extent. Type IIb includes slender spines with narrower bases, longer than 4 µm, present in species of Coreopsideae, Heliantheae, Tageteae and Eupatorieae to a lesser extent. Type III included spines with swollen base, blunt tip and perforations over their entire surface. This type was present in only one of the basal species-Carduus thoermeri-and in one species of the derived tribe Helenieae, Gaillardia megapotamica. Probably, this is due to evolutionary convergence.

Pollen competition in style: Effects of pollen size on siring success in the hermaphroditic common morning glory, Ipomoea purpurea.

PREMISE OF THE STUDY: Pollen size varies greatly among flowering plant species and has been shown to influence the delivery of sperm cells to the eggs. Relatively little is known, however, about the functional significance of within-species genetic variation in pollen size. This study tests whether pollen size influences the relative siring success of a pollen donor during in vivo pollen competition experiments. METHODS: We used two groups of Ipomoea purpurea plants genetically divergent in their pollen sizes and applied equal number of pollen grains from one large-pollen and one small-pollen donor onto the same stigma. Using microsatellite genetic markers, we identified the pollen parent of each of the resulting progeny to determine the relative siring success of the competing donors. Competitions between donors of equal-sized pollen served as a control. KEY RESULTS: Differences in pollen size significantly affected the relative siring success of a pollen donor; larger-grained individuals outcompeted smaller-grained competitors but not equal-sized competitors. Relative siring success, however, sometimes varied across different pollen recipients. CONCLUSIONS: Pollen size can influence the relative siring success of different individuals competing on the same stigma during postpollination processes. However, other factors, such as pollen-pistil interaction and environmental conditions, are likely to influence these competitions as well.

Study of the Expected Impact of Palm Pollen on Human Respiratory Tract Allergy.

<b>Background and Objective:</b> Date-Palm trees (<i>Phoenix dactylifera</i> L.) are the most abundant crop in Saudi Arabia. Date Palm Pollen (DPP) are considered an important allergens. The reasons for the increase in susceptibility to allergy are not clear. This investigation aimed to link the size, shape, moisture contents and biogenic contents of the collected pollens to susceptibility. <b>Materials and Methods:</b> Pollens were collected from three different regions in the Kingdom. Date palm pollen sizes were determined in wet and dry states by Microtrac. A microscope with a camera was used to image the forms of date palm pollens. The biogenic amines were determined after extraction using HPLC. Data were analyzed by one-way analysis of variance (ANOVA), using SPSS version 16.0. <b>Results:</b> There was a variation in the sizes of pollens from different regions ranging between 3.3-704 µm. Most pollen grains are spherical. The concentrations of six biogenic amines were detected in all samples but with different concentrations. They included B-phenyl ethylamine, Putrescine, Cadaverine, Histamine, Tyramine and Spermidine. <b>Conclusion:</b> The shape and size of the pollen grains studied were variable and will not facilitate deep penetration into the respiratory system but their biogenic contents were very high and suggested to cause allergy.

Consequences of whole genome duplication for 2n pollen performance.

The vegetative cell of the angiosperm male gametophyte (pollen) functions as a free-living, single-celled organism that both produces and transports sperm to egg. Whole-genome duplication (WGD) should have strong effects on pollen because of the haploid to diploid transition and because of both genetic and epigenetic effects on cell-level phenotypes. To disentangle historical effects of WGD on pollen performance, studies can compare 1n pollen from diploids to neo-2n pollen from diploids and synthetic autotetraploids to older 2n pollen from established neo-autotetraploids. WGD doubles both gene number and bulk nuclear DNA mass, and a substantial proportion of diploid and autotetraploid heterozygosity can be transmitted to 2n pollen. Relative to 1n pollen, 2n pollen can exhibit heterosis due to higher gene dosage, higher heterozygosity and new allelic interactions. Doubled genome size also has consequences for gene regulation and expression as well as epigenetic effects on cell architecture. Pollen volume doubling is a universal effect of WGD, whereas an increase in aperture number is common among taxa with simultaneous microsporogenesis and pored apertures, mostly eudicots. WGD instantly affects numerous evolved compromises among mature pollen functional traits and these are rapidly shaped by highly diverse tissue interactions and pollen competitive environments in the early post-WGD generations. 2n pollen phenotypes generally incur higher performance costs, and the degree to which these are met or evolve by scaling up provisioning and metabolic vigor needs further study.

An improved pollen number counting method using a cell counter and mesh columns.

BACKGROUND: The determination of pollen number is important in evolutionary, agricultural, and medical studies. Tree species of the Cupressaceae family cause serious pollinosis worldwide. Although Japanese cedar (Cryptomeria japonica) is the most important forestry species in Japan, it is also the biggest cause of pollinosis in the country. Japanese cedar trees have been selected for growth speed and superior morphological traits and then cloned. These clones may vary in their pollen production, but there has been little research on how many pollen grains are produced by a single male strobilus (flower). A recently reported method for counting pollen number with a cell counter was applicable to Arabidopsis species and wheat, but was not suitable for Japanese cedar because the strobilus does not open with heating (e.g. 60 °C, overnight). RESULTS: Here, we report an improved pollen counting method for Japanese cedar using a precise and rapid cell counter in combination with home-made mesh columns. The male strobilus was gently crushed using a pestle. Large and small debris were then removed using 100- and 20-µm mesh columns, respectively. We successfully detected pollen sizes and numbers that differed between two clones using this method. CONCLUSIONS: This improved method is not only suitable for counting pollen from Japanese cedar, but could also be applied to other species of the Cupressaceae family with hard scale tissue covering the pollen. Moreover, this method could be applied to a broader range of plant species, such as wheat, because there is no need to wait for anthesis and debris can be removed efficiently.

Pollen Grain Counting Using a Cell Counter.

The number of pollen grains is a critical part of the reproductive strategies in plants and varies greatly between and within species. In agriculture, pollen viability is important for crop breeding. It is a laborious work to count pollen tubes using a counting chamber under a microscope. Here, we present a method of counting the number of pollen grains using a cell counter. In this method, the counting step is shortened to 3 min per flower, which, in our setting, is more than five times faster than the counting chamber method. This technique is applicable to species with a lower and higher number of pollen grains, as it can count particles in a wide range, from 0 to 20,000 particles, in one measurement. The cell counter also estimates the size of the particles together with the number. Because aborted pollen shows abnormal membrane characteristics and/or a distorted or smaller shape, a cell counter can quantify the number of normal and aborted pollen separately. We explain how to count the number of pollen grains and measure pollen size in Arabidopsis thaliana, Arabidopsis kamchatica, and wheat (Triticum aestivum).

Change in nuclear DNA content and pollen size with polyploidisation in the sweet potato (Ipomoea batatas, Convolvulaceae) complex.

Genome size evolution and its relationship with pollen grain size has been investigated in sweet potato (Ipomoea batatas), an economically important crop which is closely related to diploid and tetraploid species, assessing the nuclear DNA content of 22 accessions from five Ipomoea species, ten sweet potato varieties and two outgroup taxa. Nuclear DNA amounts were determined using flow cytometry. Pollen grains were studied using scanning and transmission electron microscopy. 2C DNA content of hexaploid I. batatas ranged between 3.12-3.29 pg; the mean monoploid genome size being 0.539 pg (527 Mbp), similar to the related diploid accessions. In tetraploid species I. trifida and I. tabascana, 2C DNA content was, respectively, 2.07 and 2.03 pg. In the diploid species closely related to sweet potato e.g. I. ×leucantha, I. tiliacea, I. trifida and I. triloba, 2C DNA content was 1.01-1.12 pg. However, two diploid outgroup species, I. setosa and I. purpurea, were clearly different from the other diploid species, with 2C of 1.47-1.49 pg; they also have larger chromosomes. The I. batatas genome presents 60.0% AT bases. DNA content and ploidy level were positively correlated within this complex. In I. batatas and the more closely related species I. trifida, the genome size and ploidy levels were correlated with pollen size. Our results allow us to propose alternative or complementary hypotheses to that currently proposed for the formation of hexaploid Ipomoea batatas.

Pollen and stigma size changes during the transition from tristyly to distyly in Oxalis alpina (Oxalidaceae).

Pollen and stigma size have the potential to influence male fitness of hermaphroditic plants, particularly in species presenting floral polymorphisms characterised by marked differences in these traits among floral morphs. In this study, we take advantage of the evolutionary transition from tristyly to distyly experienced by Oxalis alpina (Oxalidaceae), and examined whether modifications in the ancillary traits (pollen and stigma size) respond to allometric changes in other floral traits. Also, we tested whether these modifications are in accordance with what would be expected under the hypothesis that novel competitive scenarios (as in distylous-derived reproductive system) exert morph- and whorl-specific selective pressures to match the available stigmas. We measure pollen and stigma size in five populations of O. alpina representing the tristyly-distyly transition. A general reduction in pollen and stigma size occurred along the tristyly-distyly transition, and pollen size from the two anther levels within each morph converged to a similar size that was characterised by whorl-specific changes (increases or decreases) in pollen size of different anthers in each floral type. Overall, results from this study show that the evolution of distyly in this species is characterised not only by changes in sexual organ position and flower size, but also by morph-specific changes in pollen and stigma size. This evidence supports the importance of selection on pollen and stigma size, which increase fitness of remaining morphs following the evolution of distyly, and raises questions to explore on the functional value of pollen size in heterostylous systems under pollen competition.

Pathways to polyploidy: indications of a female triploid bridge in the alpine species Ranunculus kuepferi (Ranunculaceae).

Polyploidy is one of the most important evolutionary processes in plants. In natural populations, polyploids usually emerge from unreduced gametes which either fuse with reduced ones, resulting in triploid offspring (triploid bridge), or with other unreduced gametes, resulting in tetraploid embryos. The frequencies of these two pathways, and male versus female gamete contributions, however, are largely unexplored. Ranunculus kuepferi occurs with diploid, triploid and autotetraploid cytotypes in the Alps, whereby diploids are mostly sexual, while tetraploids are facultative apomicts. To test for the occurrence of polyploidization events by triploid bridge, we investigated 551 plants of natural populations via flow cytometric seed screening. We assessed ploidy shifts in the embryo to reconstruct female versus male gamete contributions to polyploid embryo and/or endosperm formation. Seed formation via unreduced egg cells (BIII hybrids) occurred in all three cytotypes, while only in one case both gametes were unreduced. Polyploids further formed seeds with reduced, unfertilized egg cells (polyhaploids and aneuploids). Pollen was highly variable in diameter, but only pollen >27 µm was viable, whereby diploids produced higher proportions of well-developed pollen. Pollen size was not informative for the formation of unreduced pollen. These results suggest that a female triploid bridge via unreduced egg cells is the major pathway toward polyploidization in R. kuepferi, maybe as a consequence of constraints of endosperm development. Triploids resulting from unreduced male gametes were not observed, which explains the lack of obligate sexual tetraploid individuals and populations. Unreduced egg cell formation in diploids represents the first step toward apomixis.

Impedance Flow Cytometry as a Tool to Analyze Microspore and Pollen Quality.

Analyzing pollen quality in an efficient and reliable manner is of great importance to the industries involved in seed and fruit production, plant breeding, and plant research. Pollen quality parameters, viability and germination capacity, are analyzed by various staining methods or by in vitro germination assays, respectively. These methods are time-consuming, species-dependent, and require a lab environment. Furthermore, the obtained viability data are often poorly related to in vivo pollen germination and seed set. Here, we describe a quick, label-free method to analyze pollen using microfluidic chips inserted into an impedance flow cytometer (IFC). Using this approach, pollen quality parameters are determined by a single measurement in a species-independent manner. The advantage of this protocol is that pollen viability and germination can be analyzed quickly by a reliable and standardized method.