Size-number trade-offs and pollen production by Papilionaceous legumes

The intra- and interspecific relations between the size and number of pollen grains were examined for 21 species of papilionaceous legumes to test for the trade-off expected from the subdivision of limited resources. We observed a tradeoff between pollen grain diameter and the number of grains produced per flower within 17 of the 21 species examined. For 12 of these species, the observed partial regression coefficient for In (pollen grain diameter) equaled -3, as expected. The remaining five species exhibited more negative partial regression coefficients than expected. Relations between pollen grain size and number that are more negative than expected may result from a decelerating relation between pollen grain size and the resource investment per grain. Flower production significantly influenced pollen production for only eight species so that pollen production seems to be determined primarily on a per flower basis. Interspecifically, pollen size also varied inversely with pollen number for the 21 species even after the effect of phylogenetic relatedness was removed. Pollen size varies relatively little compared to the number of pollen grains produced per flower and therefore may be the primary target of natural selection. The presence of a trade-off in pollen production probably constrains the evolutionary options by which flowering plants can maximize male success.     

Morphometric analysis of pollen grains for paleoecological studies: classification of Picea from eastern North America

Little is known about the paleoecological histories of the three spruce species (white spruce, Picea glauca; black spruce, P. mariana; and red spruce P. rubens) in eastern North America, largely because of the difficulty of separating the three species in the pollen record. We describe a novel and effective classification method of distinguishing pollen grains on the basis of quantitative analysis of grain attributes. The method is illustrated by an analysis of a large sample of modern pollen grains (522 grains from 38 collections) of the three Picea species, collected from the region where the three species co-occur today. For each species X we computed a binary regression tree that classified each grain either as X or as not-X; these three determinations for each grain were then combined as Hamming codes in an error/uncertainty detection procedure. The use of Hamming codes to link multiple binary trees for error detection allowed identification and exclusion of problematic specimens, with correspondingly greater classification certainty among the remaining grains. We measured 13 attributes of 419 reference grains of the three species to construct the regression trees and classified 103 other reference grains by testing. Species-specific accuracies among the reliably classified grains were 100, 77, and 76% for P. glauca, P. mariana, and P. rubens, respectively, and 21, 30, and 22% of the grains by species, respectively, were problematic. The method is applicable to any multi-species classification problem for which a large reference sample is available.     

Molecular evidence that most RNAs required for germination and pollen tube growth are stored in the mature pollen grain in petunia

After landing on the stigma, the pollen grain germinates and elongates a tube to deliver its generative nuclei to the egg cell of the ovule. The molecular mechanisms involved in the drastic morphological changes in the pollen grain during this fertilization process remain largely unknown. In this study, the expression of 732 randomly selected genes in petunia pollen and pollen tubes was analyzed by microarray and quantitative PCR analyses. We found no evidence for up-regulation of any of these genes in the pollen tube. Our findings provide support at the gene level for the longstanding hypothesis that pollen germination and tube growth are not dependent on new RNA synthesis and that the large number of RNAs required for germination and tube growth are stored in mature pollen grains.     

凤仙花属棒凤仙亚属(凤仙花科)植物花粉形态的分类学意义

棒凤仙亚属代表着凤仙花属最先分化出的一支,以多年生草本,多于5朵花的总状花序,侧生萼片4枚,3沟花粉,心皮四室且每室具1枚种子,果实棒锤状,种子椭圆形以及三沟花粉其极面观为三角形而区别于其它种类。该亚属主要分布于中国南部,少数种类辐射分布至华中和中南半岛地区。已有报道证实花粉的形态特征对凤仙花属植物的属下分类具有重要意义,但对棒凤仙亚属的种类少有涉及。为了进一步探讨该亚属花粉的形态特征及其分类学价值,该研究应用扫描电镜技术对国产棒凤仙亚属植物的14种以及凤仙花亚属的窄萼凤仙花的花粉进行了观察。结果表明：棒凤仙亚属的花粉以单粒存在,具3条萌发沟,辐射对称,极面观为三角形或三角状圆形,花粉粒外壁具均匀的网状纹饰,网脊平滑或具波状边缘,网眼中具不同密度的颗粒状突起。棒凤仙亚属的花粉形态与水角属的花粉形态相似,表现出了其原始性;其花粉形态特征与花部形态性状等相关性差,对该亚属的组级分类意义较小,但对于该亚属的种间界定具有重要意义;而其花粉的极面观形状、赤道面观形状以及网状纹饰等变异相对稳定,对于界定该亚属具有重要的分类学价值。     

PCR and sequencing from a single pollen grain

In order to eliminate the laborious step of DNA extraction preceding all studies within the field of plant molecular biology we attempted to do PCR amplifications directly on pollen grains. Successful PCR amplification was obtained in reactions including a single pollen grain from Hordeum vulgare or Secale strictum. Both the plastid gene encoding ribulose-1,5-biphosphate carboxylase/oxygenase (rbcL) and the nuclear-encoded internal transcribed spacer regions (ITS) and the 5.8S rDNA region were amplified and sequenced to verify PCR amplification.     

Technical advance: single pollen typing combined with laser-mediated manipulation

We combined single pollen typing with laser-mediated manipulation. After drilling a hole in the wall of a pollen grain from a dioecious plant (Silene latifolia) with a UV-laser microbeam, the single pollen grain was recovered directly in the cap of a PCR tube, using a non-contact method called laser pressure catapulting. The entire genome of the single pollen grain was then amplified with improved primer-extension-preamplification PCR (I-PEP PCR). Nested PCR with sequence tagged site (STS)-specific primers was used to analyze several loci in the haploid genome. The single copy gene MROS1 was detected in most of the single pollen grains analyzed. Bgl10, which is localized on the Y chromosome, was detected in approximately half of the pollen grains. MROS3 is reported to be localized on the X chromosome. Using inverse PCR, we isolated two genomic clones of MROS3: MROS3A and MROS3B. The single pollen analysis using nested PCR showed that MROS3A and MROS3B are derived from different loci that are not located on the X chromosome. Single pollen typing not only reveals sex chromosome-linkage within the haploid genome, but can also discriminate between alleles and different loci. This method should also be useful for measuring recombination frequencies without genetic crossover analysis.     

Old World and New World Cupressus pollen: morphological and cytological remarks

The aim of this work was to collect new information about pollen morphology and pollen wall structure comparing Cupressus species from the Old World and New World. Cupressus is a nonmonophyletic genus that includes species that appear to be divided into Old World and New World clades. Observations in this study on cypress pollen indicate that grain size and composition of intine β-glucans are different between the considered Old World and New World species. Different from all the other American cypress species, pollen of C. macrocarpa reacted to dying in a similar manner to Old World species. Rehydrated pollen grains collected from 20 Asian, Afro-Mediterranean and American cypress species were measured under a light microscope. The size of the pollen grains and the percentage of intine in relation to the pollen grain diameter were significantly different between Old World and New World species. Pollen wall composition was tested after addition of different dyes to the hydration solution, and subsequent observations were carried out by light and fluorescence microscopy. Lugol and calcofluor staining showed differences in composition of the middle and inner intine layers between New World and Old World species.     

玉米花粉粒直接PCR技术研究

利用成熟花粉粒制成悬液作为玉米基因组DNA模板直接进行PCR扩散,研究了不同花粉悬液浓度、花粉上清液对RAPD-PCR的影响,结果表明：花粉悬液浓度在4μg/50μl以上,用10碱基随机引物均能扩增出较清晰的PCR条带,且与叶片按改良Guidet法提取的DNA模板扩增的RAPD带型无明显差异,利用花粉悬液能有效地进行基因组DNA变异的RAPD分析和基因SCAR标记的检测。玉米单株花粉量可用于数百次以上的PCR反应,较叶片等植物组织用常规法提取DNA快速、简便、廉价,可有效地应用于以PCR为基础的植物基因组DNA变异,农艺性状的RAPD标记和分子标记辅助育种的研究。     

Pollen-feeding in thrips (Insecta: Thysanoptera)

Eight species of thrips in three families fed readily on individual grains of pollen. Feeding behaviour is described. Thrips fed on the contents of the grains, but left the pollen wall. In Thrips fuscipennis Halid., the feeding time per grain increased in direct proportion to grain volume up to a grain volume of about 60,000 μm'. but then began to level off. Large grains and tetrads were usually abandoned before all the contents were removed. Mean feeding times ranged from 3 s to 120 s per grain.
Thrips appeared to have alternate periods of feeding and non-feeding. Drops of fluid were sometimes deposited from the end of the abdomen after non-feeding periods. Large amounts of pollen can be consumed; one thrips fed on more than 100 pollen grains in less than 15 min. Pollen-feeding appears to be widespread among flower thrips. The implications for pollination ecology are discussed.     

Genetic mapping of <Emphasis Type="Italic">Echinacea purpurea</Emphasis> via individual pollen DNA fingerprinting

The pollen genome of Echinacea purpurea ‘Magnus’ (purple coneflower) was amplified using a modified primer extension pre-amplification (PEP) procedures followed by AFLP (amplified fragment length polymorphism) analyses of individual pollen grains. Twenty-four AFLP primer pairs were identified that produced 104 scorable markers in eleven linkage groups. This is the first report on the production of AFLP markers from individual pollen grains for genetic mapping in coneflower. Coneflowers are important to both the ornamental and medicinal herb industries.     

Pollen grain size variation in Caryophylloideae: a mixed strategy for pollen deposition along styles with long stigmatic areas?

The literature supports a positive correlation between pollen size and style length and that pollen size may be affected by a trade-off with pollen number. A correlation between pollen size and pollinator type has been hypothesized by some authors but not confirmed. To assess these associations, pollen grain number, pollen grain diameter and style length were measured in 53 species of Caryophylloideae that had been classified beforehand as being diurnal, nocturnal or self-pollinated. Surprisingly, many species showed two, and a few even three, distinct pollen grain size classes per individual anther. Therefore, further analyses were based on the different pollen size classes and respective pollen grain numbers. Firstly, we found no indication of a trade-off between pollen size and number. Secondly, a positive linear correlation between style length and pollen grain diameter was found using conventional analysis methods and phylogenetically independent contrasts. Thirdly, when using conventional analysis methods a strong relationship between style length and pollination mode was found, with nocturnal species having significantly longer styles and larger pollen grains than diurnal and selfing species. These results were not supported, however, when calculating comparisons of phylogenetically independent pairs. We discuss our findings in relation to intra-specific pollen polymorphism and pollen degeneration, both of which have been associated with different pollen grain sizes in Caryophylloideae. Furthermore, we discuss the possibility that pollen polymorphism might be a mixed strategy to optimise the success of pollen grains deposited at different pollen–ovule distances on long styles.     

Gametophytic vs. sporophytic control of pollen aperture number: A generational conflict

In flowering plants, the haploid phase is reduced to the pollen grain and embryo sac. These reproductive tissues (gametophytes) are actually distinct individuals that have a different genome from the plant (sporophyte), and are more or less independent. The morphology of pollen grains, particularly the openings permitting pollen tube germination (apertures), is crucial for determining the outcome of pollen competition. Many species of flowering plants simultaneously produce pollen grains with different aperture numbers in a single individual (heteromorphism). In this paper, we show that the heteromorphic pollen aperture pattern depends on the genetic control of pollen morphogenesis. This points out a conflict of interest between genes expressed in the sporophyte and genes expressed in the gametophyte. More generally, such a conflict should exist whenever heteromorphism is an ESS resulting from a bet-hedging strategy. For pollen aperture, heteromorphism has been observed in about 40% of angiosperm species, suggesting that conflicting situations are the rule. In this context, the sporo-gametophytic conflict could be one of the factors that led to the reduction of the haploid phase in plants.     

Sexual dimorphism in pollen grain size in cryptically dioecious Thalictrum macrostylum

While several plant species are initially described as androdioecious, upon more thorough investigation, many of these are found to be cryptically dioecious with functionally male flowers and perfect flowers that produce inaperturate pollen or are otherwise functionally female on separate plants. The change in function of pollen produced by perfect flowers opens up the possibility for the evolution of sexual dimorphism in pollen grain size. We found that Thalictrum macrostylum (Ranunculaceae) is cryptically dioecious, and produces apparently inaperturate pollen in perfect flowers. In four field sites throughout North Carolina, inaperturate grains are larger than grains from staminate flowers and also show a greater variance in size. We also found substantial variation in pollen grain size among plants. The sites with lower soil nutrient content also had smaller pollen grains of both types, although local adaptation or genetic drift may also be causing among-site variation.     

Identification of genes required for pollen-stigma recognition in Arabidopsis thaliana

In higher plants, cell-cell recognition reactions taking place following pollination allow the selective restriction of self-pollination and/or interspecific pollination. Many of these systems function by regulating the process of water transfer from the cells found at the stigmatic surface to the individual pollen grain. Interspecific pollination studies on the cruciferous weed Arabidopsis thaliana revealed only a broad specificity of pollen recognition such that pollen from all tested members of the crucifer family were recognized, whereas pollen from almost all other species failed to hydrate. Genetic analysis of A. thaliana has identified three genes that are essential for this recognition process. Recessive mutations in any of these genes result in male sterility due to the production of pollen grains that fail to hydrate when placed on the stigma, but that are capable of hydrating and growing a pollen tube in vitro. Results from mixed pollination experiments suggest that the mutant pollen grains specifically lack a functional pollen-stigma recognition system. All three mutations described also result in a defect in the wax layer normally found on stems and leaves, similar to previously described eceriferum (cer) mutations. Genetic complementation and mapping experiments demonstrated that the newly identified mutants are allelic to the previously identified genes cer1, cer3 and cer6. TEM analysis of the ultrastructure of the pollen coating revealed that all of the mutant pollen grains bear coatings of normal thickness and that tryphine lipid droplets are missing in cer1-147, are reduced in size in cer6-2654 and appear normal in cer3-2186. Temperature shift experiments revealed that the block in the recognition step of the mutant pollen grains can be suppressed by pollination at lower temperatures but not by reduced temperatures during pollen development. These results suggest that the lipids which are altered in the cer mutations may be important in regulating some biophysical property of the pollen coating.     

Pollen heteromorphism is pervasive in <Emphasis Type="Italic">Thalictrum</Emphasis> (Ranunculaceae)

Among the angiosperms, features of pollen morphology such as grain size, aperture number and surface ornamentation display striking variation. It is less well appreciated that pollen morphology may vary within and among populations of the same species as well as within individual plants. In some species, individual plants produce multiple types of fertile pollen grains (called pollen heteromorphism). Aspects of pollen morphology, such as aperture number, are likely to affect fertilization success with different morphologies favored in different local competitive and ecological environments. This study surveys variation in pollen grain morphology among species throughout the genus Thalictrum. Pollen from individuals of 36 species was rehydrated from herbarium specimens, and light microscopy was used to quantify pollen grain aperture number and size. I find that pollen aperture-number heteromorphism is present within all Thalictrum species studied, and distributions of aperture-number morphs vary both within and among species. This study provides an example of significant pollen heteromorphism within a genus that also varies widely for pollination mode and sexual system.     

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.     

A non-invasive technique for rapid extraction of DNA from fish scales

DNA markers are being increasingly used in studies related to population genetics and conservation biology of endangered species. DNA isolation for such studies requires a source of biological material that is easy to collect, non-bulky and reliable. Further, the sampling strategies based on non-invasive procedures are desirable, especially for the endangered fish species. In view of above, a rapid DNA extraction method from fish scales has been developed with the use of a modified lysis buffer that require about 2 hr duration. This methodology is non-invasive, less expensive and reproducible with high efficiency of DNA recovery. The DNA extracted by this technique, have been found suitable for performing restriction enzyme digestion and PCR amplification. Therefore, the present DNA extraction procedure can be used as an alternative technique in population genetic studies pertaining to endangered fish species. The technique was also found equally effective for DNA isolation from fresh, dried and ethanol preserved scales.     

POLLEN APERTURE POLYMORPHISM AND GAMETOPHYTE PERFORMANCE IN VIOLA DIVERSIFOLIA

Pollen aperture polymorphism is studied in Viola diversifolia, where all plants produce three- and four-apertured pollen grains. We tested whether there are genetic differences among plants for the proportions of the different pollen morphs, and whether the morphs differ in gametophytic performance. Results show that the more apertures a pollen grain has, the more quickly it germinates but that few-apertured pollen grains have faster growing pollen tubes and longer life expectancies. The proportions of the different pollen morphs, together with pollen tube growth rates, may be inherited traits based on differences among maternal families. These results suggest that the different pollen morphs are favored in different pollination ecology situations. The production of several pollen morphs by the same individual could therefore be evolutionarily advantageous.     

凤仙花属总状花序组的花粉形态及其分类学意义

总状花序组隶属于凤仙花亚属凤仙花属,以具多花的总状花序、侧生萼片2枚、子房5室、果实为线状圆柱形、种子多数、迈卵形、种皮表面具指状突起等特征区别于该亚属的其他类群。前人对凤仙花花粉形态的研究表明凤仙花属的花粉形态对于属下分类及种间界定具有重要的分类学价值。而对于总状花序组来说,有关花粉形态方面的研究少有报道,为了进一步探讨该组花粉S的形态特征及其分类学意义,该研究应用扫描电镜技术对总状花序组的21种植物的花粉进行了观察。结果表明:总状花序组植物的花粉粒均具4条萌发沟,两侧对称,极面观为矩圆形、长矩圆形或近方形;长赤道面观多为椭圆形至矩圆形,短赤道面观为椭圆形;长、短赤道轴比值多在1.4~2.4范围内;表面具网状纹饰,网眼在花粉粒表面均匀分布或赤道面的网眼较极面观的小,网脊边缘光滑或具波状突起,网眼内具不同密度的颗粒状突起。该组的花粉形态与宏观形态性状相关性较小,但花粉极面观形状、长短赤道轴的比值、网眼的分布、网脊边缘形状、网眼内颗粒物密度等特征具有种水平上的稳定性和特异性,对该组植物种间界定具有重要的意义。