Identification Of Pollen Grains Research Articles

A primer on pollen assignment by nanopore-based DNA sequencing

The possibility to identify plants based on the taxonomic information coming from their pollen grains offers many applications within various biological disciplines. In the past and depending on the application or research in question, pollen origin was analyzed by microscopy, usually preceded by chemical treatment methods. This procedure for identification of pollen grains is both time-consuming and requires expert knowledge of morphological features. Additionally, these microscopically recognizable features usually have a low resolution at species-level. Since a few decades, DNA has been used for the identification of pollen taxa, as sequencing technologies evolved both in their handling and affordability. We discuss advantages and challenges of pollen DNA analyses compared to traditional methods. With readers with little experience in this field in mind, we present a hands-on primer for genetic pollen analysis by nanopore sequencing. As our lab mainly works with pollen collected within agroecological research projects, we focus on pollen collected by pollinating insects. We briefly consider sample collection, storage and processing in the laboratory as well as bioinformatic aspects. Currently, pollen metabarcoding is mostly conducted with next-generation sequencing methods that generate short sequence reads (<1 kb). Increasingly, however, pollen DNA analysis is carried out using the long-read generating (several kb), low-budget and mobile MinION nanopore sequencing platform by Oxford Nanopore Technologies. Therefore, we are focusing on aspects for palynology with the MinION DNA sequencing device.

Automatic Classification of Pollen Grain Microscope Images Using a Multi-Scale Classifier with SRGAN Deblurring

Pollen allergies are seasonal epidemic diseases that are accompanied by high incidence rates, especially in Beijing, China. With the development of deep learning, key progress has been made in the task of automatic pollen grain classification, which could replace the time-consuming and laborious manual identification process using a microscope. In China, few pioneering works have made significant progress in automatic pollen grain classification. Therefore, we first constructed a multi-class and large-scale pollen grain dataset for the Beijing area in preparation for the task of pollen classification. Then, a deblurring pipeline was designed to enhance the quality of the pollen grain images selectively. Moreover, as pollen grains vary greatly in size and shape, we proposed an easy-to-implement and efficient multi-scale deep learning architecture. Our experimental results showed that our architecture achieved a 97.7% accuracy, based on the Resnet-50 backbone network, which proved that the proposed method could be applied successfully to the automatic identification of pollen grains in Beijing.

NEW APPROACH TO SEPARATE AND DATE SMALL SPORES AND POLLEN FROM LAKE SEDIMENTS IN SEMI-ARID CLIMATES

ABSTRACTThe dating of pollen grains is emerging as the method of choice for lacustrine climate archives that contain few datable macrofossils. Due to the need for high-purity pollen concentrates, new methods are constantly being developed to precisely separate pollen grains. Flow cytometry represents a promising alternative to conventional approaches, enabling the identification of pollen grains through fluorescence and rapid separation for radiocarbon analysis using accelerator mass spectrometry, which has so far been limited to sediments with a high proportion of conifer pollen. We present a revised method for processing large sediment samples, resulting in high-purity pollen and spore concentrates. Using this approach small- to medium-sized pollen and bryophyte spores were isolated from Lake Van sediment samples (Eastern Anatolia, Turkey) in sufficient purity for radiocarbon dating. However, a systematic age discrepancy between pollen and bryophyte spore concentrates was noted. By adapting the chemical and cytometric methods, pure pollen concentrates can be created for sediments with low organic content enabling age determination of climate archives with a low proportion of large pollen or low pollen concentration.

MELISSOPALINOLOGICAL STUDY OF SOUTHERN BESSARABIA HONEY

Different natural and climatic zones, directions of agricultural production, ethnic and cultural features of agriculture, and reliefs of the regions of Ukraine are the result of a wide variety of vegetation. Ukraine occupies less than 6% of Europe’s area but owns about 35% of all its biodiversity. This leads to a considerable list of plants that are sources of nectar for the production of honey of different varieties. Ukraine produces classic monofloral varieties of honey, such as white acacia, rapeseed, linden, buckwheat, sunflower, as well as less common monofloral, such as mustard, clover, raspberry, bird’s-foot, phacelia, fireweed, goldenrod, sophora, honeydew. Today, regional polyfloral varieties of honey are also produced, such as Carpathian, steppe, Crimean, and other locally produced honey from reserves (for example, from Gomilshansky forests, Mezynsky reserve, Askanian meadows). A common way to identify the botanical and geographical origin of honey and identify its species is to study the pollen composition. This work was aimed to determine the pollen profile of spring honeys of Southern Bessarabia using the method of melissopalynology. Bessarabia is an ethnographic region with its own culture of agricultural production and natural and climatic conditions. The material of the study was samples of honey obtained in the apiaries of the Center for Ethnographic, Green, Rural Tourism, and Family Recreation Frumushika-Nova (Odessa region) in 2021. The pollen profile of honey was investigated using the author’s improved method of melissopalynological analysis. Identification of pollen grains was performed using the online database of pollen grains PalDat using a model nutrient medium. The pollen profile of spring honeys of Southern Bessarabia is represented by 30 morphotypes of pollen grains of different plant species, contains honeydew of natural origin and natural impurities (plant particles, wax, bee bodies). In the pollen profile of honey, no dominant (45% or more) pollen grains were detected, secondary – 6, insignificant – 21, inclusions – 3 morphotypes. This allows us to assert the polyflorality of the studied honey with the exception of white acacia, the dominant pollen of which may be 20% or more. Pollen grains of endemic species of steppe plants of Ukraine were found in the pollen profile of spring honeys of Southern Bessarabia (tamarix ramosissima, lathyrus pannonicus, rosa tesquicola, bifora radians, ailanthus altissima, ultus pumila, prunus tenella, viola occulta). The obtained results can be further used to identify the geographical origin of honey from this region. At the next stages of the research, it will be promising to determine the pollen profiles of summer honeys of Southern Bessarabia and other ethnographic regions of Ukraine.

Mangrove distribution and diversity during three Cenozoic thermal maxima in the Northern Hemisphere (pollen records from the Arctic–North Atlantic–Mediterranean regions)

AbstractAimPast pollen records reveal the changes in latitudinal distribution of plants in relation to climate, particularly their expansion in response to global warming. The maximum northward expansion of the mangrove genus Avicennia since the Early Eocene is known, but this information is missing for other mangrove taxa. Here, we evaluate the diversity of past mangroves with respect to latitude during three Cenozoic thermal maxima (PETM: 56 Ma; EECO: 54–49 Ma; MMCO: 17–14 Ma).LocationNorth Atlantic, Mediterranean.TaxaAvicennia, other mangrove taxa (Rhizophoraceae, Nypa, Xylocarpus, Pelliciera, etc.).MethodWe collected well‐dated marine sediments along a Northern Hemisphere latitudinal transect and we analysed their pollen content in order to compare the past distribution of mangrove taxa with the present. The analysis of 89 samples (PETM: 13; EECO: 31; MMCO: 45) was performed and interpreted using a robust botanical background for identification of pollen grains and their representativeness in marine sediments.ResultsDuring the Early Eocene, two palaeolatitudinal thresholds at 65–70°N and 35°N, respectively, delimited the Avicennia‐only mangrove from a diversified but scrawny mangrove and finally from a diversified and well‐developed mangrove. The Avicennia threshold was selective at 40°N during the Mid‐Miocene. The Avicennia range limit was up to 10–15° poleward of the limit for other mangrove taxa during the Early Eocene and the Mid‐Miocene compared with 9° at present.Main conclusionsA buffer zone characterised by a diversified but scrawny mangrove co‐occurring with a few megathermal plants occurred in the Early Eocene between 35°N and 65–70°N. This finding questions the relative influence of a more ‘equable’ climate and/or the ability of some taxa to expand towards areas with cooler conditions in the past. Mangrove provincialism, which was established progressively after the Early Eocene, was probably forced by plate tectonics. The taxonomic impoverishment of the Atlantic East Pacific province was probably caused by successive periods of global cooling. These results support the Tethyan origin of the mangroves.

Applying deep learning to pollen identification using metabolic fingerprints

The daily pollen counts are typically measured by visual identification by trained experts. Automated visual identification of pollen grains using deep learning algorithms has been demonstrated. An alternative would be to identify the pollens via their metabolite signature, also known as metabolic fingerprinting.

Late Quaternary vegetation and climate of SE Europe–NW Asia according to pollen records in three offshore cores from the Black and Marmara seas

High-resolution pollen analyses were performed on two cores from the western Black Sea and one core from the Marmara Sea, covering the Late Glacial-Holocene transition using 14C chronology. Particular effort was invested in the botanical identification of pollen grains thereby significantly improving our knowledge of regional flora. When interpreted with respect to modern vegetation, pollen records revealed all the major changes caused by climatic fluctuations over the last 20,000 years. The results of this study provide evidence for the occurrence of relict thermophilous-hygrophilous trees (papillate Cupressaceae, Carya, Liquidambar, Zelkova) in certain refugia up to the Holocene. Vegetation dynamics is specified for some taxa (e.g. Cupressus–Juniperus, Fagus, Cedrus) and some ecosystems (e.g. mesophilous forests, Mediterranean sclerophyllous populations, steppes). Pollen data enabled palaeoclimatic reconstructions which were compared with available estimates in the region. The use of a powerful pollen ratio between ‘thermophilous and steppe taxa’ led to fruitful climatostratigraphic relationships with the oxygen isotope curve from the NGRIP core. The Younger Dryas and cooling at 8.2 ka are among the most obvious climatic phases identified in the three cores studied here.

ПОШИРЕННЯ ДЕЯКИХ ПРЕДСТАВНИКІВ SCROPHULARIACEAE sensu lato НА РІВНИННІЙ ЧАСТИНІ УКРАЇНИ ВПРОДОВЖ ГОЛОЦЕНУ: ПАЛЕОФЛОРИСТИЧНИЙ І ПАЛІНОМОРФОЛОГІЧНИЙ АСПЕКТИ

Results of the analysis of participation of pollen of some representatives of the fami­ly Scrophulariaceae sensu lato in palynofloras of the Holocene deposits of the plain part of Ukraine are considered. It has been demonstrated that fossil pollen of Scrophulariaceae occurs sporadically, in small quantities and, in most cases, its identification was reported at the family level, occasionally also at the genus level. Our main attention was focused on the generalization of our paleofloristic materials, with species-level identification of pollen grains of Scrophulariaceae, whenever possible. The data available at present can justify the participation of four species, Scrophularia nodosa , Verbascum phoeniceum ( Scrophularia­ceae s. str.), Veronica incana ( Plantaginaceae ), Melampyrum arvense ( Orobanchaceae ) in fossil palynofloras of the Ukrainian Holocene deposits. The data obtained for these types of paleofloristic data are consistent with the changes in the current taxonomy of Scrophularia­ceae , which must be taken into account when compiling specific lists of fossil palynofloras. Quantitative and qualitative diagnostic characters for these four species are generalized; these characters are recommended to be used in the practice of spore-pollen analysis for specific identifications of fossil pollen. Permanent pollen preparations on slides are deposited in the reference palynotheca of the National Herbarium of Ukraine ( KW-P ).

Pollen morphology of selected crop plants from southern China and testing pollen morphological data in an archaeobotanical study

Identification of pollen grains of cultivated plants is essential in archaeobotanical studies. In this study, we investigated the pollen morphology of 30 species which are representatives of most of the crop plants in southern China, using a light microscope. Our results show that the pollen grains of these species or genera can generally be identified by their size, aperture(s) and exine sculpture. We found that: (1) some cultivated cereals can be distinguished from wild species of Poaceae according to their size frequency combined with their morphological features; (2) the lengths of the equatorial diameter (E), polar axis (P) and the greatest dimension of the lumina (the size of the network sculpturing) of the exine reticulum may be diagnostic features to distinguish some brassicaceous vegetables. There are significant differences between the E and P values among Brassica campestris (B. rapa, oilseed rape, Chinese cabbage), B. alboglabra (B. oleracea var. alboglabra, gai lan, Chinese kale), B. parachinensis (B. rapa var. parachinensis, choy sum, Chinese flowering cabbage) and B. chinensis (B. rapa ssp. chinensis, pak choi), but moderate differences in the longer axis length of the reticulum lumina, which provide potential for identifying species on the basis of pollen grains. We compared the P values and the longer axis length of the lumina of modern specimens of Brassicaceae pollen grains with those of fossil pollen extracted from the Ming-Qing cultural layer in the Fuqikou site at Chongqing, China, and found that the fossil pollen grains of Brassicaceae probably represent vegetable plants related to B. parachinensis. Moreover, we measured the diameters of rice pollen grains from modern paddy fields to assess the pollen size frequency and found that the size range from ~ 34 to 38 µm is closely associated with rice pollen in southern China, which can be used to detect pollen signals of human activities in archaeobotanical investigations.

Pollination implications of the diverse diet of tropical nectar-feeding bats roosting in an urban cave.

BackgroundIntense landscaping often alters the plant composition in urban areas. Knowing which plant species that pollinators are visiting in urban areas is necessary for understanding how landscaping impacts biodiversity and associated ecosystem services. The cave nectar bat, Eonycteris spelaea, is an important pollinator for many plants and is often recorded in human-dominated habitats. Previous studies of the diet of E. spelaea relied on morphological identification of pollen grains found in faeces and on the body of bats and by necessity disregarded other forms of digested plant material present in the faeces (i.e., plant juice and remnants). The main objective of this study was to examine the diet of the nectarivorous bat, E. spelaea, roosting in an urban cave at Batu Caves, Peninsular Malaysia by identifying the plant material present in the faeces of bats using DNA metabarcoding.MethodsFaeces were collected under the roost of E. spelaea once a week from December 2015 to March 2016. Plant DNA was extracted from the faeces, Polymerase chain reaction (PCR) amplified at ITS2 and rbcL regions and mass sequenced. The resultant plant operational taxonomic units were searched against NCBI GenBank for identification.ResultsA total of 55 species of plants were detected from faeces of E. spelaea including Artocarpus heterophyllus, Duabanga grandiflora and Musa spp. which are likely to be important food resources for the cave nectar bat.DiscussionMany native plant species that had not been reported in previous dietary studies of E. spelaea were detected in this study including Bauhinia strychnoidea and Urophyllum leucophlaeum, suggesting that E. spelaea remains a crucial pollinator for these plants even in highly disturbed habitats. The detection of many introduced plant species in the bat faeces indicates that E. spelaea are exploiting them, particularly Xanthostemon chrysanthus, as food resources in urban area. Commercial food crops were detected from all of the faecal samples, suggesting that E. spelaea feed predominantly on the crops particularly jackfruit and banana and play a significant role in pollination of economically important plants. Ferns and figs were also detected in the faeces of E. spelaea suggesting future research avenues to determine whether the ‘specialised nectarivorous’ E. spelaea feed opportunistically on other parts of plants.

Reconstruction of Mediterranean flora, vegetation and climate for the last 23 million years based on an extensive pollen dataset

In the last 46 years extensive effort has been devoted to the botanical identification of pollen grains from Neogene deposits. This robust approach is the only reliable way to reconstruct past flora and vegetation from pollen archives, additionally allowing fruitful comparisons with plant macroremains. Rich peri-Mediterranean fossil pollen data provide a comprehensive history of plant ecosystems during the last 23 million years. Successive disappearance of thermophilous-hygrophilous taxa has occurred. Severe changes affected the North Mediterranean vegetation which varied from prevalent forests to dominant open environments. Avicennia mangrove progressively perished from North to South and was replaced by Glyptostrobus swamps which in turn disappeared too in a diachronous way. Subtropical evergreen forests left room for Mediterranean sclerophyllous communities, and finally to Artemisia steppes which alternated with mesophilous forests during glacial-interglacial cycles. The South Mediterranean plant ecosystems contrasted with open subdesertic associations where Avicennia persisted up to the early Pliocene before the steppes invaded the lowlands. Neogene climate changes benefited the Mediterranean sclerophyllous plants that were already present in the early Miocene. Their subsequent fluctuations relate not only to temperature variations but maybe also to phases with high instability in seasonality and low variability in warmth. When present, Microtropis fallax is a serious candidate for identifying the primary Mediterranean sclerophyllous assemblages. Interpretation and climate quantification of the pollen data show a well-marked latitudinal contrast in the Mediterranean area both in temperature and humidity/ dryness. The thermic latitudinal gradient is characterized by a significant increase in the late Miocene towards its modern value. In the late Pliocene, climate evolved from overall warm temperatures and dry seasons to cooler winters and dry summers. General dryness and colder conditions developed during glacial periods, alternating with moister and warmer conditions during interglacials. Present refuges of Zelkova and Pterocarya are discussed. Desiccation of the Mediterranean led subdesertic plants and cedar to migrate in opposite ways. The present distribution of Cedrus illustrates its weakness against coolings although it seems to have been saved by geodynamic events.

The Global Pollen Project: a new tool for pollen identification and the dissemination of physical reference collections

Summary The study of fossil and modern pollen assemblages provides essential information about vegetation dynamics in space and time. A major methodological component of these studies is the identification of pollen grains to plant family, genus and species. This identification is achieved through the use of pollen keys and reference collections of physical specimens, which are regional in scope, disparate and incomplete, slowing the identification process. Reference material is also held in museums and research institutions, where access can be limited. Identification is particularly challenging for those new to the field, such as graduate students. To aid the identification of pollen grains, and provide virtual access to reference material, we present a new online tool: the Global Pollen Project (https://globalpollenproject.org). The project aims to enable people to share and identify pollen grains and through this, will create an open, free and accessible reference library for pollen identification. The online tool has been developed as an open, peer‐reviewed database of global pollen, where content and expertise is crowdsourced from across the world. The tool enables: (i) the submission and identification of unknown pollen grains; (ii) the submission and digitisation of existing ‘physical’ reference collections and (iii) the availability of a free public database of pollen images and their metadata, for use in scientific research and education. The tool connects to external services, including the Global Biodiversity Information Facility and Neotoma Palaeoecology Database, to provide botanical descriptions and occurrence data for each taxon, alongside pollen images and metadata. The database currently holds information for over 1500 species, from Europe, the Americas and Asia. As the collection grows, we envision easier pollen identification, and greater use of the database for novel research on pollen morphology and other characteristics, especially when linked to other palaeoecological databases, such as Neotoma.

Food habits of some Costa Rican bats.

Dietary habits of 42 species of Costa Rican bats were studied by examination of feces, by stomach analysis and by identification of pollen grains adhering to the fur. Food items were identified to a finer level than offered in previous literature and not merely as "plant material", "insects" or "vertebrate food" . Although the sample size for some species prohibits generalizations, the analysis reveals that many genera, supposed to be nectarivorous, eat only insects, primarily Lepidoptera, during April, May and June and that although there may be some overlap in particular food items in the eliets of closely related bats, the major items sought by each bat are different. Generic competition, and in some cases, specific competition for food is low. Because bats are of considerable usefulness in controlling insects, in pollination, ancl in seed dispersal, a plea is made for bat conservation by using the technique of guano analysis rather than sacrificing bats for stomach analysis. The former technique gives equivalent results and proves easier for the investigator.

Atmospheric solids analysis probe mass spectrometry for the rapid identification of pollens and semi-quantification of flavonoid fingerprints.

From allergies to plant reproduction, pollens have important impacts on the health of human and plant populations, yet identification of pollen grains remains difficult and time-consuming. Low-volatility flavonoids generated from pollens cannot be easily characterized and quantified with current analytical techniques. Here we present the novel use of atmospheric solids analysis probe mass spectrometry (ASAP-MS) for the characterization of flavonoids in pollens. Flavonoid patterns were generated for pollens collected from different plant types (trees and bushes) in addition to bee pollens from distinct geographic regions. Standard flavonoids (kaempferol and rhamnazin) and those produced from pollens were compared and assessed with ASAP-MS using low-energy collision MS/MS. Results for a semi-quantitative method for assessing the amount of a flavonoid in pollens are also presented. Flavonoid patterns for pollen samples were distinct with variability in the number and relative abundance of flavonoids in each sample. Pollens contained 2-5 flavonoids, and all but Kochia scoparia contained kaempferol or kaempferol isomers. We establish this method as a reliable and applicable technique for analyzing low-volatility compounds with minimal sample preparation. Standard curves were generated using 0.2-5μg of kaempferol; from these experiments, it was estimated that there is approximately 2mg of kaempferol present in 1g of P. nigra italica pollen. Pollens can be characterized with a simple flavonoid pattern rather than analyzing the whole product pattern or the products-temperature profiles. ASAP-MS is a rapid analytical technique that can be used to distinguish between plant pollens and between bee pollens originating from different regions. Copyright © 2016 John Wiley & Sons, Ltd.

Fungi identify the geographic origin of dust samples.

There is a long history of archaeologists and forensic scientists using pollen found in a dust sample to identify its geographic origin or history. Such palynological approaches have important limitations as they require time-consuming identification of pollen grains, a priori knowledge of plant species distributions, and a sufficient diversity of pollen types to permit spatial or temporal identification. We demonstrate an alternative approach based on DNA sequencing analyses of the fungal diversity found in dust samples. Using nearly 1,000 dust samples collected from across the continental U.S., our analyses identify up to 40,000 fungal taxa from these samples, many of which exhibit a high degree of geographic endemism. We develop a statistical learning algorithm via discriminant analysis that exploits this geographic endemicity in the fungal diversity to correctly identify samples to within a few hundred kilometers of their geographic origin with high probability. In addition, our statistical approach provides a measure of certainty for each prediction, in contrast with current palynology methods that are almost always based on expert opinion and devoid of statistical inference. Fungal taxa found in dust samples can therefore be used to identify the origin of that dust and, more importantly, we can quantify our degree of certainty that a sample originated in a particular place. This work opens up a new approach to forensic biology that could be used by scientists to identify the origin of dust or soil samples found on objects, clothing, or archaeological artifacts.

Identification of Onopordum pollen using the extreme learning machine, a type of artificial neural network

Pollen grains are complex three-dimensional structures, and are identified using specific distinctive morphological characteristics. An efficient automatic system for the accurate and rapid identification of pollen grains would significantly enhance the consistency, objectivity, speed and perhaps accuracy of pollen analysis. This study describes the development and testing of an expert system for the identification of pollen grains based on their respective morphologies. The extreme learning machine (ELM) is a type of artificial neural network, and has been used for automatic pollen identification. To test the equipment and the method, pollen grains from 10 species of Onopordum (a thistle genus) from Turkey were used. In total, 30 different images were acquired for each of the 10 species studied. The images were then used to measure 11 morphological parameters; these were the colpus length, the colpus width, the equatorial axis (E), the polar axis (P), the P/E ratio, the columellae length, the echinae length, and the thicknesses of the exine, intine, nexine and tectum. Pollen recognition was performed using the ELM for the 50–50%, 70–30% and 80–20% training-test partitions of the overall dataset. The classification accuracies of these three training-test partitions of were 84.67%, 91.11% and 95.00%, respectively. Therefore, the ELM exhibited a very high success rate for identifying the pollen types considered here. The use of computer-based systems for pollen recognition has great potential in all areas of palynology for the accurate and rapid accumulation of data.

Grãos de Pólen de Sedimentos Superficiais da Baía de Guanabara, Rio de Janeiro, Brasil

The study presents the description and identification of pollen grains obtained in the top sediments of Guanabara Bay, Rio de Janeiro, Brazil. Pollen analysis enabled to identify 91 pollen types, with five of gymnosperms and 86 of angiosperms. The pollen and the ecological data obtained in the present work will contribute to the recognition of the vegetal formations and the characterization of the vegetation throughout the Holocene.

An automatic identification method for the comparison of plant and honey pollen based on GLCM texture features and artificial neural network

Pollen grains vary in colour and shape and can be detected in honey used as a way of identifying nectar sources. Accurate differentiation between pollen grains record is hampered by the combination of poor taxonomic resolution in pollen identification and the high species diversity of many families. Pollen identification determines the origin and the quality of the honey product, but this indefiniteness is also a big challenge for the beekeepers. This study aimed to develop effective, accurate, rapid and non-destructive analysis methods for pollen classification in honey. Ten different pollen grains of plant species were used for the estimation. GLCM (grey level co-occurrence matrix) texture features and ANN (artificial neural network) were used for the identification of pollen grains in honey by the reference of plant species pollen. GLCM has been calculated in four different angles and offsets for the pollen of the plant and the honey samples. Each angle and offset pair includes five features. At the final step, features were classified using the ANN method; the success of estimation with ANN was 88.00%. These findings suggest that the texture parameters can be useful in identification of the pollen types in honey products.

Three species of native Thai honey bees exploit overlapping pollen resources: identification of bee flora from pollen loads and midguts from Apis cerana, A. dorsata and A. florea

SummaryIn this study, we investigated the bee flora utilised by the Thai honey bees, Apis cerana, A. dorsata and A. florea in Nan province, northern Thailand, through the identification of pollen grains from their pollen loads and midguts. We compared the pollen grains morphologically to match with local flowering plants and determined their protein concentration using a Bradford assay. The results showed that 8 families and 15 species were found by pollen load analysis whilst 12 families and 25 species were found from pollen grains of the bee midguts. The greatest number of bee flora was found from pollen loads of A. cerana, (11 species), while there were fewer for A. florea (10 species) and the fewest with A. dorsata (6 species). The highest number of bee flora species identified from pollen was found in the midgut of A. cerana, with 19 species, while A. florea had 13 and A. dorsata with only 11. The results show that the major pollen source plants of the three native honey bee species of Thailand were Mimosa pudica L., M. pigra, Celosia argentea L., Zea mays L., Wedelia trilobata L. and Syzygium malaccense L. The protein content ranged from 31.85 ± 0.83 to 48.44 ± 0.81 mg/100 mg pollen. The most abundant pollen source was from M. pudica L., perhaps because of the flower structure, shape, size, long blooming season, wide distribution, high protein concentration (43.31 ± 0.79 mg/100 mg pollen) or a combination of these characteristics.

An expert classification system of pollen of Onopordum using a rough set approach

Although pollen grains have a complicated 3-dimensional structure, they can be distinguished from one another by their specific and distinctive characteristics. Using microscopic differences between the pollen grains, it may be possible to identify them by family or even at the genus level. However for the identification of pollen grains at the taxon level, we require expert computer systems. For this purpose, we used 20 different pollen types, obtained from the genus Onopordum L. (Asteraceae). For each pollen grain, 30 different images were photographed by microscope system and 11 different characteristic features (polar axis, equatorial axis, P/E ratio, colpus length, colpus weight, exine, intine, tectum, nexine, columellea, and echinae length) were measured for the analysis. The data set was formed from 600 samples, obtained from 20 different taxa, with 30 different images. The 440 samples were used for training and the remaining 160 samples were used for testing. The proposed method, a rough set-based expert system, has properly identified 145 of 160 pollen grains correctly. The success of the method for the identification of pollen grains was obtained at 90.625% (145/160). We can expect to achieve more efficient results with different genuses and families, considering the successful results in the same genus. Moreover, using computer-based systems in revision studies will lead us to more accurate and efficient results, and will identify which characters will be more effective for pollen identification. According to the literature, this is the first study for the identification and comparison of pollen of the same genus by using the measurements of distinctive characteristics with computer systems.