Pollen monitoring: minimum requirements and reproducibility of analysis

Training, quality assurance (QA) and quality control (QC) play an important role in building competence in monitoring and research in aerobiology. The main goals of this paper were to: (a) formulate an updated Minimum Requirements Report for pollen monitoring; (b) carry out a pilot QC exercise of staff involved in pollen counting from various national networks in order to examine between analysts reproducibility and develop a methodology that can be used in future QC exercises. A questionnaire survey was sent to coordinators of participating pollen monitoring networks. In addition, a total of 45 technicians from 15 European countries participated in the pilot QC exercise. All technicians were instructed to analyse two slides containing the following pollen types: (a) Poaceae and Betula pollen grains in the north of Europe; (b) Poaceae and Olea pollen grains in the south of Europe. Minimum Recommendations were produced based on the results of the questionnaire survey, published literature, and the outcomes of a workshop. In the QC exercise, it was noticed that technicians who followed the Minimum Recommendations and examined at least 10 % of the slide tended to have better indicators of precision and accuracy than those technicians who did not follow the Minimum Recommendations. The proposed Minimum Recommendations will help to improve the quality of scientific work, particularly for those who are considering the setting up of new monitoring sites. The results of the pilot QC exercise will help to develop a methodology that can be used again in the future, thereby ensuring data quality.     

Pollen counts statistics and its relevance to precision

In the day to day management of pollen counts from aerobiological samples of national networks, only a small proportion (usually from 12 to 15%) of the daily microscope slide is read. It is generally believed that, otherwise, too much time will be spent reading slides for a minimal increase in precision. Different networks use different slide sampling methods (longitudinal, transverse or at random) and a different number of lines are routinely read. However, the topic of the precision of the different pollen count strategies has seldom been the object of serious investigation. In this study, the precision of different sampling methods of 12 pollen types was investigated by: a) counting pollen grains over the whole slide (3 slides per taxa), b) spatially (i.e. microscope field per microscope field) recording over the 3120 fields found at 400× the location of each pollen grain, c) sub-sampling, by macro procedures, this population by selecting a number of transverse (1 to 48) or longitudinal (1 to 20) lines, or a number of random fields (90 to 2340), so that between 0.96 to 46.15% (transverse), 3 to 66.6% (longitudinal) or 3 to 75% (random) of the whole slide was artificially counted. Between nine and twelve procedures were built per reading strategy. The error found is much higher than what is normally believed, and it was significantly correlated with the abundance of a pollen taxa on the sampled slide. It is only with a total count over 1000 (corresponding to a concentration of above 500 m–3) that the mean error of 4 longitudinal lines (or 13.3% of the slide), the standard protocol of both the Italian Association of Aerobiology (AIA) and the Spanish Aerobiology Network (REA), was always below 30%.     

Factors limiting seed production of Taxus brevifolia (Taxaceae) in Western Oregon

Seed production of Pacific yew (Taxus brevifolia), an understory conifer, was studied at four sites in western Oregon over 2 yr. The effects of pollen supplementation, overstory canopy, and predator exclusion on ovule attrition were examined. Supplemental hand-pollination of ten trees at two sites resulted in significantly increased rates of ovule development and a doubling of seed efficiency (ratio of seeds to ovules). However, seed efficiency still averaged <15% on branches receiving supplemental pollen, so pollination was not a primary factor limiting seed production. The number of developing ovules was positively associated with overstory openness, but seed production was not. Seed efficiency was negatively associated with overstory openness. Branches bagged to exclude vertebrate seed predators had higher seed production than unbagged branches at three of four sites for 2 yr. In contrast to unbagged branches, seed production on bagged branches was positively associated with overstory openness, as was the effectiveness of bagging. Therefore, both vertebrate predation and overstory were important in limiting seed production, and these factors interacted. Factors limiting seed production varied in importance among the four sites and between years, illustrating the importance of examining multiple limiting factors over several sites and years.     

Comparison of two pollen counting methods of slides from a hirst type volumetric trap

Two of the most frequently used methods of pollen counting on slides from Hirst type traps are evaluated in this paper: the transverse traverse method and the longitudinal traverse method. The study was carried out during June–July 1996 and 1997 on slides from a trap at Worcester, UK. Three pollen types were selected for this purpose: Poaceae, Urticaceae and Quercus. The statistical results show that the daily concentrations followed similar trends (p < 0.01, R-values between 0.78–0.96) with both methods during the two years, although the counts were slightly higher using the longitudinal traverses method. Significant differences were observed, however, when the distribution of the concentrations during 24 hour sampling periods was considered. For more detailed analysis, the daily counts obtained with both methods were correlated with the total number of pollen grains for the taxon over the whole slide, in two different situations: high and low concentrations of pollen in the atmosphere. In the case of high concentrations, the counts for all three taxa with both methods are significantly correlated with the total pollen count. In the samples with low concentrations, the Poaceae and Urticaceae counts with both methods are significantly correlated with the total counts, but none of Quercus counts are. Consideration of the results indicates that both methods give a reasonable approximation to the count derived from the slide as a whole. More studies need be done to explore the comparability of counting methods in order to work towards a Universal Methodology in Aeropalynology. This revised version was published online in June 2006 with corrections to the Cover Date.     

Vertical variation in pollen abundance in North-Central London

Summary Pollen data from three samplers located at heights of 0.5m, 10m and 55 m were used to investigate vertical differences in pollen abundance in North-Central London. Weekly accumulative counts for all pollen types were collected from February to September 1988. Distinct variations in abundance between the sites were recorded for some pollen taxa. For example,Gramineae recorded greater abundance at the higher sampling position. Other pollen types, includingPlatanus, were recorded at consistently greater abundance at the 10 m height compared to the 55 m level. Significant differences between the pollen counts at these two heights are discussed in relation to pollen source area, the specific gravity of the pollen grain, airflow patterns of the urban area and the weather conditions affecting pollen dispersal. Tracer experiments using Lycopodium spores were employed to investigate dispersal patterns to all three sampling heights. The results from these trials are used to assist in the interpretation of data from the depositional samplers. The study reported in this paper forms part of a wider survey of 14 sampling sites examining spatial variations in pollen abundance.     

Pollen survey and clinical research in Yunnan, China

This pollen survey includes 7 counties of Yunnan Province, China: Quinjin, Xishuanbanna, Lijiang, Wenshan, Yuxi and Kunming. From 1991–1992, optical and statistical examination of exposed glass slides showed that pollen is air-borne in Yunnan throughout the year. Pollen is also in great quantity and variety with two apparent pollen peaks. This paper also discusses the influences of climate and elevation on the amount of pollen carried by air. A geographical and seasonal variation of pollen level was established. The pollen particles causing possible sensitization were collected in the field and refined into an allergen extract which was applied to clinical tests for patients suffering from allergosis in our hospital with a high rate of pollen allergy. The results show the effectiveness of specific desensitization for pollen allergic patients. This study is therefore of great significance to the diagnosis,treatment and prevention of pollen allergy.     

Types of pollen dispersal units in orchids,and their consequences for germination and fertilization

The various pollen dispersal units (PDU) found in orchids are discussed together with possible evolutionary trends and the consequences for germination and fertilization. Orchids with monad and tetrad pollen form more complex dispersal units by means of pollenkitt, elastoviscin, a callosic wall, common walls or a combination of these. Evolutionary trends include (1) from pollenkitt to elastoviscin; (2) from monad to tetrads and multiples of tetrads; (3) from partially dehydrated (<30 %) to partially hydrated (>30 %) pollen; and (4) from monad pollen to PDUs with many pollen grains. The biological consequences concern both male and female reproductive systems. Some features of the male side are present in all orchids irrespective of the pollen dispersal unit, whereas other characters are found only in orchids with pollinia; the same applies for the female counterpart. Pollen grains of orchids with pollinia germinate at least 24 h after pollination because the pollen grains/tetrads must swell and make space for the growth of pollen tubes.     

Inadequate cervical smears: results of an educational slide exchange scheme

Fifty-six slides, predominantly inadequate and of varying difficulty, were circulated to 12 laboratories as an educationally based slide exchange scheme. Three slides failed to achieve an agreed majority consensus opinion. Seventy percent of participants agreed with the consensus opinion in 80% of slides. Of the slides originally reported as inadequate, the consensus diagnosis was inadequate in 78%, negative in 12% and abnormal in 10%. The latter included two cases of high-grade dyskaryosis. There was good agreement for the two most frequent causes of inadequacy in submitted slides (obscured and poor cellularity). There was poor consistency in reporting the presence or absence of endocervical and immature squamous metaplastic cells, to an extent that questions their use in the assessment of smear adequacy. Three inadequate slides on consensus opinion were associated with subsequent cervical intraepithelial neoplasia (grade III) or invasive squamous cell carcinoma. In the latter case, the slide had originally been reported as negative by the submitting laboratory.     

Possibility of real-time measurement of an airborne Cryptomeria japonica pollen allergen based on the principle of surface plasmon resonance

In order to establish a real-timemeasurement system for the concentration ofairborne pollen allergens, we examined Cry j 1,one of the major allergens of Cryptomeriajaponica pollen, as an example to establishthe system. The feasible system consisted of:collection of airborne pollen allergens usingthe Virtual Impactor or Cyclone sampler,extraction of Cry j 1 using 10mM HEPES buffercontaining 0.125M NH₄HCO₃ andfinally, real-time measurement of Cry j 1 usingthe BIACORE 3000 system. The sensitivity of thesystem was 5 ng/ml, and 0.1 ml sample volumeand at least 500 pg of Cry j 1 were requiredfor each measurement. Quantification of Cry j 1in the air can be determined 30 min aftercollection, i.e. 15 min for extraction, 10 minfor separation from particulate matters and 5min for the measurement.     

Pollen germination and pollen tube growth in Fraxinus pennsylvanica

With regard to adaptation of green ash (Fraxinus pennsylvanica Marshall) to ecological conditions in Croatia, pollen germination and pollen tube length after 2, 4 and 6 hours were examined in vitro at 10, 15, 20 and 25°C during two years 2001 and 2002. Narrow leaved ash (F. angustifolia Vahl) pollen served as a control in 2002. The year, time and temperature, and the interaction between time and temperature were significant for both germination percentage and pollen tube length. Interactions year × temperature and year × time were significant for pollen tube length only. The highest germination percentage (17.86% in 2001 and 19.40% in 2002) of green ash pollen was at 15°C after 6 hours. The pollen tube length was greatest at 20°C (393.46 μm) in 2001 and 25°C (899.50 μm) in 2002 after 6 hours. Narrow leaved ash pollen had the highest germination percentage (19.22%) at 20°C after 6 hours and was significantly reduced at 25°C. The pollen tube length was greatest at 25°C (518.90 μm) after 6 hours. It can be concluded that green ash pollen has satisfactory germination in ecological conditions in Croatia and that the optimum temperature for pollen germination is higher than 20°C.     

Arecaceae potential for production of monofloral bee pollen

Bees often collect pollen from plants that are near their colonies or apiaries. Arecaceae are characterised by having species that flower throughout the year and produce large quantities of pollen. Pollen grains of Arecaceae have two main forms, namely monosulcate and trichotomosulcate. The study area is located in the coastal region of the state of Sergipe, Brazil, where large amounts of bee pollen are produced by Apis mellifera. This study aims to evaluate how Arecaceae contribute to the production of monofloral bee pollen. Samples of bee pollen loads were collected each month for two years (totalling 24 samples), acetolysed, and mounted on permanent slides. At least 500 grains of pollen were counted for each sample, the data was analysed, and figures were made using the Tilia software. The pollen grains of Cocos nucifera were present in 23 samples (96%), and concentrations of this pollen type varied between 1.1 and 46.8% (but were commonly above 20%). The concentration of Arecaceae pollen grains is the result of the large-scale production of coconuts along the coastline. However, the presence of other pollen types, such as Mimosa arenosa, Mimosa pudica, Mikania and Myrcia, compliments the diets of bees in the region. The results show that even when in low frequency, Cocos nucifera pollen grains represent a large proportion of pollen content compared with other pollen types, because of the large size of Arecaceae pollen grains.     

Do gelatin-coated slides increase cellular retention in oral exfoliative cytology?

A minimum number of cells is required for an accurate cytologic assessment of oral lesions; however, the cell yield in some oral smears is reduced, relating to the site from which the cells were harvested and to the lesion examined. The use of gelatin coating to increase the retention of cells in smears from such sites was assessed. Cells were obtained from four sites within the oral cavities of 20 patients (10 male and 10 female) with healthy mouths. Using a wooden spatula, two samples were obtained from each site; one was transferred to a gelatin-coated slide, the other to an uncoated slide. For each slide, the cell yield was rated on a scale of 0 (no cells), 1 (few cells) and 2 (many cells). The results were then analyzed using the Wilcoxon sign-rank test. The coated and uncoated slides yielded similar results for most sites, except that the coated slides seemed to be advantageous for samples from the ventral surface of the tongue.     

Partially hydrated pollen: taxonomic distribution, ecological and evolutionary significance

 The problem of the water content of pollen is reconsidered, especially the distinction between “partially hydrated pollen” (PH pollen), pollen with a water content greater than 30%, and “partially dehydrated pollen” (PD pollen), which has a water content of less than 30%. Both types have been found even in systematically contiguous groups or the same genus. Partially hydrated pollen, encountered in at least 40 families of angiosperms, has the advantage of germinating quickly, normally in a few minutes to less than an hour. Dispersal of highly hydrated pollen also occurs in orchids but for a different reason, i.e. to enable packaging of massulae. The disadvantage of pollen dispersed with a high water content is that water is readily lost and the pollen may desiccate and die unless it has biochemical or anatomical devices to retain water or phenological strategies, such as flowering when temperatures are not too high and when relative humidity is high. Most pollen of Gymnosperms and Angiosperms studied has, however, been found partially dehydrated. Received March 8, 2002; accepted April 8, 2002 Published online: November 14, 2002 Addresses of the authors: G.G. Franchi (e-mail: franchi@unisi.it), Department of Pharmacology, Università di Siena, Via delle Scotte 6, I-53100 Siena, Italy; M. Nepi (e-mail: nepim@unisi.it) and E. Pacini (e-mail: pacini@unisi.it), Department of Environmental Sciences, Università di Siena, Via P.A. Mattioli 4, I-53100 Siena, Italy; A. Dafni (e-mail: adafni@research.haifa.ac.il), Laboratory of Pollination Ecology, Institute of Evolution, University of Haifa, 31905 Israel.     

Airborne pollen flora of Jabalur – the central India

The fortnightly aerobiological survey of pollengrains at five different sites at two differentheights (ground level and at 10 m) was carriedout at Jabalpur city during January toDecember, 1996. Air sampling was done for 30minutes at each sampling point by using aRotorod air sampler. A total of 84 pollen typesbelonging to 34 families of Angiospermae andone family of Gymnospermae were identified. Themaximum mean concentration (1.04/m³ air) ofpollen was contributed by Asteraceae followedby Amaranthaceae/Chenopodiaceae (0.77/m³ air)and the minimum (0.001/m³ air) byCucurbitaceae, Convolvulaceae and Sapotaceae.Maximum numbers of pollen grains werecontributed by herb species (62.07%) followedby shrubs (18.47%) and trees (14.96%). Asmall fraction, i.e., 4.50% of total pollenflora remained unidentified. Morphologicalanalysis revealed maximum concentration(1.81/m³) of triporate pollen grains. There wasa significant difference in the pollenconcentration at ground level and at 10 mheight. The ratio observed was 1.7 : 1.0.There was non-significant negative correlation(r = –0.196) between mean pollen concentrationand mean atmospheric temperature. May, thehottest month, showed minimum mean pollenconcentration (1.65/m³ air), whereas October,with moderate temperature, the maximum(10.86/m³ air).     

High temperature exposure did not affect induced 2n pollen viability in Populus

High temperature exposure is widely used as a physical mutagenic agent to induce 2n gametes in Populus. However, whether high temperature exposure affects induced 2n pollen viability remains unknown. To clarify whether high temperature exposure affected the induced 2n pollen viability, 2n pollen induced by 38 and 41 °C temperatures, pollen morphology, 2n pollen germination in vitro, and crossing induced 2n pollen with normal gametes to produce a triploid was, based on observations of meiosis, conducted in Populus canescens. We found that the dominant meiotic stages (F = 56.6, p < .001) and the treatment duration (F = 21.4, p < .001) significantly affected the occurrence rate of induced 2n pollen. A significant decrease in pollen production and an increase in aborted pollen were observed (p < .001). High temperature sometimes affected in ectexine deposition and some narrow furrows were also analysed via details of ectexine structure. However, no significant difference in 2n pollen germination rate was observed between natural 2n pollen (26.7%) and high‐temperature‐induced 2n pollen (26.2%), and 42 triploids were created by crossing high‐temperature‐induced 2n pollen, suggesting that 38 and 41 °C temperatures exposure will not result in dysfunctional induced 2n pollen.     

Modern pollen rain in mangroves from San Andres Island, Colombian Caribbean

The precise characterisation of present-day mangrove ecosystems from modern pollen rain facilitates the accurate use of fossil pollen data for late Quaternary sea level and environmental reconstructions. Here, we investigate whether the analysis of pollen rain data corroborates existing floristic and structural characterisation of different mangrove types at the Caribbean island of San Andrés, Colombia. At 82 plots along 20 transects of four distinct mangrove types, samples were obtained of (i) surface sediments for pollen analysis, and (ii) a range of environmental parameters (including inundation levels, salinity and pH). This information was compared to previously sampled mangrove composition and tree basal area. In surface sediment samples 82 pollen taxa were found, from which 19 were present in the vegetation plots. However, because pollen may be transported by wind and/or watercourses, the overall floristic composition of the different forest types may not necessarily be reflected by the pollen spectra. Local vegetation (i.e. mangroves and beach) represented > 90% of the pollen spectra, while the regional one (i.e. hinterland forests) represented < 5% of it. Unlike the four mangrove types that were previously described in the vegetation, the analysis of pollen samples suggested only three distinct types of forest.The groups were characterised based on (i) the dominance of at least one of the true mangrove species from pollen data ordination and the presence of associated species, and (ii) their relationship with environmental parameters. Rhizophora was present in all plot samples, but did not contribute to forest type separation. In fact, just three true mangrove species proved reliable indicators of (i) high salinity and fringe mangroves (i.e. Avicennia), (ii) high pH levels and landward mangroves (i.e. Conocarpus), and (iii) natural or anthropogenic caused disturbance of forest stands (Laguncularia and associated Acrostichum fern). Hence our study confirms that mangrove pollen spectra can be accurately used to describe different mangrove environments for fossil based palaeoecological reconstructions.     

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.     

Pollen histochemistry and pollen : ovule ratios in Zingiberaceae

BACKGROUND AND AIMS: Pollen grains of 37 species from 11 genera in the family Zingiberaceae were examined to assess qualitatively starch or lipid contents; the pollen grain and ovule numbers per flower and pollen : ovule ratios were also counted and calculated. Pollen : ovule ratios were studied to reveal patterns of variation in the Zingiberaceae. METHODS: Freshly open flowers with dehiscing anthers were collected at random from plants growing in natural habitats or in botanical gardens. Presence of lipids or starch in pollen grains was tested by Sudan solution and IKI solution, respectively, and examined under a microscope. To estimate the pollen and ovule numbers per flower, one anther from each bud was carefully dissected and all pollen grains were counted; ovaries were carefully dissected out of each flower and counted. Whenever possible, at least 10-15 buds were used in the determination. KEY RESULTS: Thirty-three of all the 37 species examined had starchy pollen. Starch was not found in only four species and lipid was not found in only one species; among the four tribes in subfamily Zingiberoideae, all species of Zingibereae and Globbeae had pollen with no starch, Alpineae and Hedychieae had pollen with and without starch, whereas, all species of subfamily Costoideae had starchy pollen with abundant lipids. The mean pollen : ovule ratios in the members of the Zingiberaceae investigated range from 3.25 +/- 1.56 to 616.52 +/- 117.83. CONCLUSIONS: The pollen nutrition types seemed not related to mating systems. The pollen : ovule ratios in members of the Zingiberaceae with the same breeding system are noticeably lower than those recorded by previous authors. The lower pollen : ovule ratios in this family are presumed to be related to the highly efficient pollination systems, mediated by pollen which can be quite glutinous and the relatively large stigma area. In most of the Alpinia species the anaflexistylous flowers have much larger numbers of pollen grains and higher pollen : ovule ratios than the cataflexistylous flowers. There are significant differences in mean pollen grain numbers and pollen : ovule ratios between different life forms but ovule numbers are approximately the same.