MELISSOPALYNOLOGY IN BRAZIL : A REVIEW OF POLLEN ANALYSIS OF HONEYS , PROPOLIS AND POLLEN LOADS OF BEES

This paper reviews current knowledge on the occurrence of several types of pollen grains in the sediments of honey samples, propolis and bee loads of Apiinae and Meliponinae in Brazil. After a short historical introduction about research activities in Melissopalynology using Brazilian samples, bee products were analyzed in respect to the greater Brazilian regions (South, Southeast, Northeast and North), emphasizing monofloral honeys and the green propolis. Numerous bibliographic references and a short glossary of the technical terms used is presented.


INTRODUCTION
Scientific curiosity about the origin of products elaborated by bees, as well as commercial advantages in determining their quality, stimulated research activities that use the knowledge of pollen grain morphology as a tool for investigation.Pollen grains occur unexpected in honeys when prepared by bees.While collecting nectar from flowers in the field, pollen grains are occasionally swallowed by bees and carried into the hive.There they are transferred together with the nectar from bee to bee into the storage cells to dry and turn to honey.In separate flights, bees collect pollen grains from flowers and carry them in the baskets of their behind legs into the hive for storage in other than honey cells.This is known as the "bee bread", and used for protein nutrition of larvae.
Monofloral honeys, also named unifloral honeys, are undoubtedly the most attractive.They are originated from only one plant species.In Brazil they are produced by imported bees, Apis mellifera L., or by native stingless bees, the Meliponinae.These honeys maintain always the same physico-chemical and organoleptic characteristics and are well appreciated for commerce.It is possible to determine their origin from flowers by the recognition of the dominant pollen grains.
In contraposition, bifloral honeys are coming from two species of bee plants and the heterofloral honeys, also named plurifloral and wild honeys, from the nectar of several plant species.They may be produced by either Apis or native bees.The properties of these honeys are much more variable, in relation to bee species, respective flowering and climatic factors.
Knowledge about pollen is important also to caracterize honeydew honeys, originated from exudates of Aphidae (plant louses) sucking plant sap.These liquids from insects are processed like floral nectars.Pollen grains transported by wind or sticking on the bee coats adhere to these substances.Nevertheless, the pollen grains Sci.Agric.(Piracicaba, Braz.), v.61, n.3, p.342-350, May/June 2004 of these sweet extrafloral liquids are only indicative of the phytogeographical origin of honeydews.
There is in Brazil another product, named cane honey, that is an extrafloral honey elaborated by bees from the sugar sap that sprouds from the sugar cane thatches after be cutted.In this case also, the pollen grains are only indicative of the phytogeographical origin.
To fulfill holes and to embalm predators died inside the hives, Apis bees prepare a product named propolis, composed of plant resin, bee wax and secretions of the head glands of worker bees.When elaborated by stingless bees, this product is named geopropolis and contains in addition clay, earth or soil material.Pollen grains are introduced into these products accidentally.They indicate the geographical origin of the propolis and geopropolis.
The diversity and the frequency of pollen grains are considered at first during the analysis of honey sediments, and constitute the pollen analysis of honey.Therefore, the pollen analysis of bee products (honey, pollen and propolis) consider the pollen grains, their morphological characteristics which lead to the indication of the species or the botanical taxa of their origin, as well as the quantity that may be an indicative of the quality.
Nevertheless, in relation to honeydew honeys and cane honeys, propolis and geopropolis present other structured elements in their sediments that require a palynological analysis.Therefore, the palynological analysis of the bee products consider, besides pollen grains, other structured elements as spores and fungi hyphae, algae, plant hairs, insect bristles, silica, clay and earth, starch and wax, that enrich data and knowledge of the quality of the product, from its origin to its processing.
Citations and lists of bee plants as possible food sources are not considered in the present review of Melissopalynology.They are related to blooming and bee calendars based only upon field observations, without laboratorial confirmation.Summaries of papers presented during congresses and scientific meetings were not considered.

Historical remarks
Pioneer, traditional and standard studies in Melissopalynology (Louveaux et al. 1970;1978) consolidated the development of reports on the occurrence of pollen grains in honeys, propolis and bee loads in Europe (France, Germany and Switzerland).The first investigations in Brazil were made by Santos (1961a;1961b;1963;1964) on pollen grains of bee plants and honeys collected in the region of Piracicaba, SP, followed by studies of Barth (1969;1970a;1970b;1970c;1971a;1971b;1973;1996) in different regions of the country.The position of Palynology in Brazil was presented during the First Latin-American Congress of Botany by Barth (1972), including extensive references and all the data of Melissopalynology available at the time.Subsequently, Barth (1989;1990a;1996), Barth & Luz (1998), Barth & Coré-Guedes (1999), and Barth & Dutra (2000), studied mainly the Southeast region.
Botanical studies in Melissopalynology by Absy & Kerr (1977) were carried at the Instituto Nacional de Pesquisas da Amazônia (INPA), Manaus, with the aim of studying the Meliponinae or stingless bees in relation to bee plant resources in the Amazon region, and the palynological analyses of the respective honey samples and pollen loads.These investigations were followed by Marques-Souza (1993;1996;1999), Marques-Souza et al. (1993;1995;1996;2002), andMarques-Souza &Kerr (2003).
More recently, in the state of Pará, Carreira et al. (1986), andCarreira &Jardim (1994) studied honey samples of Apis.In the state of São Paulo, Carvalho & Marchini (1999), Carvalho et al. (2001), Marchini et al. (2000) and Moreti et al. (2000;2002) analyzed honeys of Apis and Meliponinae, as well as pollen loads and the corresponding flowering for bees.The trophic resources for Apis in the state of Roraima were investigated by Silva (1998) and Silva & Absy (2000), and in the state of Rondônia, by Marques-Souza et al. (1993).
There are a few more publications on this matter, but no other group of investigators was consolidated.Condensed abstracts in several Congresses and extensive lists of regional bee floras and pastures were presented.Commonly, scientific corroboration was not present.Finally, chemists and pharmacists analyzed the proprieties and chemical characteristics of honeys, propolis and pollen using palynological analysis to obtain an exact identification and characterization of the samples (Costa et al., 1999).
The melissopalynological knowledge has been used uncommonly for qualification and market of bee products in Brazil by cooperative and beekeeper associations in several states.It would be desirable to have a more rigorous technical-scientific inspection.

Regional considerations 1. Honey analyses
As a consequence of the great extension of the country there is in Brazil a great variety of honeys, so that it is not possible to come to generalized conclusions.This matter is always of regional concern, with the tendency to address "micro" regions of honey production.The following results are based upon publications in scientific periodicals and books.They do not consider summarized data presented in congresses and scientific meetings, nor notes of journals or newspapers.
The relation between bees and pollen morphology of plant species of Loasaceae (Wittmann & Schlindwein, 1995), Cactaceae (Schlindwein, 1995) and Pontederiaceae (Santos, 1997) were studied in the state of Rio Grande do Sul.The state of Santa Catarina is also an important producer of honey and bee products.Knowledge on pollen grains in honeys, of pollen loads and in propolis are scarce.Bracatinga (Mimosa scabrella) honey containing honeydew from the state of Santa Catarina was analyzed by Campos (1999).The pollen spectrum of samples of honeys from the state of Paraná (Ramalho et al., 1991) can be summarized in Allophylus, Baccharis, Campomanesia, Cecropia, Citrus, Eucalyptus, Matayba, Mimosa scabrella, Paspalum and Vernonia, therefore strongly heterofloral, but with the major occurrence of Eucalyptus.

Southeast region
The first honey analyses of Apis were performed by Santos (1961b;1964;1978) in Piracicaba, State of São Paulo.The pollen spectrum was composed mainly by Eucalyptus, added by Agave sisalana, Baccharis, Dombeya and Persea.Ramalho et al. (1991) mentioned as the most important pollen types of honeys in the state of São Paulo the taxa Cecropia, Citrus, Eucalyptus, Paspalum and Syagrus.The pollen in honeys of Meliponinae presented a very rich and heterogeneous spectrum of plant taxa, in special Alchornea, Eucalyptus, Petroselinum and Schinus (Iwama & Melhem, 1979;Ramalho, 1990).
Melissopalynological data from the state of Espírito Santo were not found in the scientific literature.The analysis of a single honey sample (data not published) reflected the presence of the widespread coffee (Coffea sp.) plantations in the region, and their pollen grains were associated to wild plant species, mainly Asteraceae.
Honeys, pollen and propolis of the state of Minas Gerais were extensively examined by Bastos (1993;1995), Bastos & Brandão (1994), Bastos et al. (1991;1995;1996;2000), Brandão & Bastos (1995) and Brandão et al. (1993).The honey samples were obtained in the regions of Barão dos Cocais, Bom Jesus, Caraça, São Gonçalo and Serra da Piedade, and were considered in relation to dry and humid climatic conditions during several years.The pollen of Eucalyptus dominated over several species widely cultivated in the state, and in one particular case, it was reached by bees over a distance of 10 km.Significant quantity of pollen grains of Alternanthera, Antigonon, Baccharis, Borreria, Croton, Eupatorium, Hyptis, Schinus, Serjania, Terminalia, Trichogonia and Vernonia were also detected.Barth (1970a) reported a monofloral honey with pollen of the Mimosa scabrella type obtained also in the state of Minas Gerais.Barth (1969) found the dominant pollen of Astronium in a honey sample of the Northeast region.The major number of Apis honey samples proceeded from the state of Bahia (Barth, 1970a) with a pollen association of Acacia, Mimosa scabrella type, Eupatorium, Hyptis and Montanoa, the first two taxa of dominant and the other of accessory pollen grains.The pollen of Piptadenia moniliformis, a plant species with a large dispersion in the Northeast and that provides the famous "marmeleiro" honey (the honey sold under this name is in general a mixture of nectars from Piptadenia moniliformis and Croton sp.) dominated another sample.Moreti et al. (2000) found as dominant the pollen grains of Bauhinia sp. and Eucalyptus sp., and the pollen types of Mimosa scabrella and Mimosa verrucosa, and as accessory pollen, Alternanthera, Asteraceae and Cecropia.Carvalho et al. (2001) identified in honeys of Meliponinae the dominant pollen of Eucalyptus and Psidium.Sodré et al. (2001) identified 27 pollen types in 36 samples of honey from Apis, and as dominant pollen, the taxa of Arecaceae, Citrus, Cordia, Eucalyptus, Lithrea, Mimosa scabrella, Mimosa verrucata and Psidium.In the state of Ceará, Barth (1971a) found the association of Borreria verticillata and Piptadenia moniliformis as dominant, together with the accessory pollen types of Mimosa, Alternanthera, Copaifera and Salvia.Some honey samples of Ceará were analyzed by Freitas (2001), three of them were monofloral of Cocos nucifera, Alternanthera tenella and Eucalyptus sp., respectively, and predominantly species of Mimosaceae in heterofloral honeys.Honey samples of Meliponinae in the state of Maranhão (Kerr et al., 1986/ 87) presented variable pollen spectra, without dominance of any taxon.

North region
Extensive pollen studies of honey (Absy et al., 1980;Marques-Souza & Kerr, 2003) and pollen from inside hives of Meliponinae and of pollen loads (Absy & Kerr, 1977;Absy et al., 1984;Marques-Souza, 1993, 1996, 1999;Marques-Souza et al., 1995, 1996;2002;Santos, 1991) were carried out mainly in areas next to Manaus, state of Amazonas.A large variety of pollen grains was identified in these samples, depending upon blooming.These bees visit many plant species, and explore only few resources and turn to be selective in accordance to the botanical offer (Marques-Souza, 1996).The most frequent taxa identified between more than 120 recognized in pot samples of pollen grains were Artocarpus, Bellucia, Carica, Cassia, Cocos, Leucaena, Maximiliana, Miconia, Myrtaceae, Stachytarpheta and Triplaris, with emphasis on Protium, both in honeys and in pots.

Unspecified regions
Various scientific papers refer to pollen analysis of honey samples from several regions of Brazil, frequently without any indication of origin.Barth (1969;1970a;1970b;1970c;1970d;1970e;1971a;1971b;1973;1976;1989;1990a;1996), Barth & Dutra (2000), Barth et al. (1999) and Moreti et al. (2002), in relation to a collection of honey samples from various regions of the South, Southeast and Northeast of Brazil, identified monofloral honeys emphasizing those of Borreria verticillata, Croton, Eucalyptus, Mimosa scabrella type, Piptadenia moniliformis and Vernonia.Honeys of Citrus, from the states of Rio de Janeiro and São Paulo were evaluated in relation to the respective pollen spectra by Barth & Coré-Guedes (1999).Considering the pollen concentration, that is the quantity of pollen grains comprised in one gram of honey, 17 samples of honey of Citrus and Eucalyptus obtained in the states of Minas Gerais, São Paulo and Rio de Janeiro were examined by Barth & Dutra (2000).The absolute number of pollen grains per gram in monofloral honeys of Citrus varied from 776 to 2432, while in honeys of Eucalyptus, varied from 2588 to 17511.
Regarding honey and pollen stored in pots by Meliponinae, Bazlen (2000) analyzed ten samples, most from the state of Bahia, and detected great diversity of botanical families visited by the bees, confirming the trend of bees to be generalist, but having preference for a few plant families.Some data were compared to some honey samples of more selective Apis bees.After extensive chemical analyses it was concluded that the properties and characteristics of Meliponinae honeys depend on the bee species and not on the blooming, as for Apis honey.
Chemical analyses of honey samples based upon the botanical diagnosis of origin, were made by Costa et al. (1999), Da Costa Leite et al. (2000), Duran et al. (1996), Horn (1997), andAzeredo et al. (2003).In all of them the importance of Eucalyptus honey standed out.

Sugar cane honey
Referred by Barth (1970e), sugar cane honey is characterized by a low incidence of nectariferous pollen grains, elevated incidence of anemophilous pollen grains (Poaceae, Cyperaceae, Cecropia), and by the prevailing in sediment volume of a granulous and colorless to light grey paste, originated from the cutted stems of sugar cane.

Propolis
Propolis samples from the states of Minas Gerais, Rio de Janeiro and Rio Grande do Sul were analyzed regarding the presence of morphological elements in their sediments by Barth (1998) and Barth et al. (1999).The main pollen types detected, besides a great quantity of trichomes, were Eucalyptus, Eupatorium and Mimosa caesalpiniaefolia.The high content of anemophilous pollen, in special of Cecropia, was highlighted.
Green propolis from six municipalities in the state of Minas Gerais were analyzed by Bastos (2001) and Bastos et al. (2000).The analysis pointed out the overall dominance of pollen of Eucalyptus, followed by pollen of Astronium, considered as characteristic of the "cerrado" (savanna) vegetation.The presence, however isolated, of Vellozia sp., should be indicative for the occurrence of a natural field vegetation in the area of propolis obtention.The pollen spectrum of these propolis samples from the state of Minas Gerais comprise Astronium sp., Baccharis dracunculifolia, Eucalyptus sp., Hyptis sp. and Vernonia polyanthes.The resin may be collected from leave buttons, mainly from Baccharis dracunculifolia and Vernonia polyanthes.Absy & Kerr (1977) observed that the Meliponinae, collected not only pollen grains but also resin, clay and, in separate loads, the latex of fruits of Vismia, for geopropolis confection.The presence of silica and clay and the absence of trichomes was used, besides pollen grains, to differentiate geopropolis of Meliponinae from propolis of Apis (Barth & Luz, 2003).

Honey and pollen of Meliponinae
Several studies on the nutrition habits of the Meliponinae were made in the vicinity of Manaus, state of Amazonas, in the state of Bahia, and mainly in the state of São Paulo.Even though only few pollen analyses of honey sediments were performed, it was possible to identify more than 60 pollen types in a comparative study of the nutrition preferences of some species of Meliponinae in the region of Manaus (Absy et al., 1980), with prevalence of Protium, and in addition Alchornea, Alternanthera, Borreria, Cassia, Cecropia, Eugenia, Miconia, Mimosa scabrella type, Tapirira and Vismia.In a honey sample of the "uruçu" Meliponidae from Bahia (Carvalho et al., 2001), dominant pollen grains came from Eucalyptus and Psidium, together with Bauhinia, Caesalpiniaceae and the Mimosa verrucosa type.Several studies of honey samples of Meliponinae in the state of São Paulo (Guibu et al., 1988;Iwama & Melhem, 1979;Ramalho, 1990;Ramalho et al., 1985) reported great number of flowering species visited by the bees, but selectively, Alchornea, Arecaceae, Eucalyptus, Moraceae, Myrtaceae, Petroselinum and Schinus.

Analyses of pollen loads
Pollen loads of bee baskets, as well as pollen harvested in the cells or pots inside beehives, were object of several studies.Researchers looked either for the trophic preferences or for pasture or local and regional bee flora.

Analysis of royal jelly
Similarly to propolis and geopropils, royal jelly contains pollen grains in the same proportion (circa 5%), and coming from hives in which pollen was used and harvested by the bees, and is a result of the respective bloomings (data from O.M. Barth, unpublished).

Summary of the current knowledge
Considering the Apis bees, the pollen found in dominant and accessory quantities in honey samples comes from regional bloomings and from a few plant species.In the South of Brazil predominates the pollen of Senecio; in the Southeast Citrus and Eucalyptus; in Minas Gerais, wild plants; in the Northeast, Asteraceae Croton, Eucalyptus, several species of Mimosa and Piptadenia moniliformis; in Amazon, Arecaceae, Mimosa, Protium and Tapirira.Regarding pollen loads, the variety of plant species is bigger, and frequently the pollen of anemophilous species like Cecropia and Poaceae are present.There are no published data for the Central-West region of Brazil.
Meliponinae, in general, visit a larger number of plant species.They may be also selective in this respect, in accordance to the flowering, for nectar and pollen obtention.In all these studies there is a lack of indices of pollen production by plant species, mainly for monofloral honeys and pollen loads.

Future perspectives
With forest devastation in progress, burning and extensive use of agrotoxic products, Brazilian apiculture faces great trouble in future.Another factor of impact, mainly for the small producer, is the theft and the vandalism of beehives.The production of monofloral honeys and pollen will be always of preference and related to migratory activities.They can follow the rational and economically viable natural resources and may by related to the polinization of crops (apples, cashew, Eucalyptus, guarana, coffee, oranges, etc.)

Glossary of botanical taxa and palynological terms used in the present paper
Accessory pollen.Pollen that occurs in honey sediments between 15% and 45% of the total of pollen grains (counting comprises at least 300 pollen grains per sample).
Apis.Genus of Hymenoptera, Apidae.The species Apis mellifera L. is widely exploited by humans since prehistorical times for honey, pollen, propolis, royal jelly and wax.It was introduced in Brazil by European immigrants, mainly germans, in the beginning of the 19 th century in the South of the country.Their hives are constructed with honeycombs that shelter the young, the honey and the pollen.These bees are important polinizators of introduced plants in Brazil, like Eucalyptus, apple and orange.
Bifloral honey.Honey with dominance of pollen of only two plant species.Dominant pollen.Pollen that occurs in honey sediments in excess of 45% of the total of pollen grains (counting comprises at least 300 pollen grains per sample).
Family.Group of botanical genera of morphological and genetical affinities, with names ending always in "aceae".Exemples: Myrtaceae, Asteraceae, Arecaceae.Geopropolis.Product elaborated by Meliponinae bees, starting with wax, resin or vegetal latex, and clay or soil particles, to construct the entrance and coat of hives and to close openings.
Heterofloral or plurifloral or wild honey.Honey with no dominance of pollen of any plant species.
Honey.Product elaborated by bees starting out of flower nectar or other sweet plant exudations.
Honeydew.Product elaborated by bees starting out of exudations of plant sucking Aphidae, presenting always a reduced number of pollen grains.
Isolated pollen.Occurs in honey sediments with less than 15% of the total of pollen grains (counting comprises at least 300 pollen grains per sample).
Meliponinae.A group of genera of Hymenoptera, native of Brazil, named as stingless bees.Their hives are constructed as honeycombs for the young and as pots for honey and pollen.They are important and well adapted polinizators of our native vegetation.
Melissopalynology.Science that deals with pollen grains in relation to bees and bee products.
Monofloral or unifloral honey.Honey with dominance of pollen of a single plant species.
Palynological analysis.Study of pollen grains and other figurated elements as hyphae and spores of fungi, algae cells, starch grains, plant trichomes (glands) in honey, propolis and pollen load, resulting in the palynological spectra.
Pollen.A collective word that means a group of pollen grains.Exemple: the pollen of an anther, a flower, a tree, a local or regional vegetation.
Pollen analysis.Study of pollen grains in honey, propolis and pollen load of bees, resulting in the pollen spectra.
Pollen grain.The masculine unity of plant fertilization found in flower anthers.
Pollen type.Name given after analyzing botanical taxa or sediments, in which frequently appear morphologically equal or similar pollen grains, that may be related to an unique plant species, several species of a same taxon or to several taxa.
Propolis.Product made by honeybees of the genus Apis out of wax and vegetal resin for hive coating.
Royal jelly.Milk-like product secreted by head glands of young worker bees to nourish larvae which will become workers at just the third day of live, and to be queens and drones during all life.
Sugar cane honey.Honey that starts with liquids exuded of cut stalks of sugar cane, very rich in saccharose, containing a large number of anemophilous pollen grains.
Taxon, plural taxa.Taxonomical unit used in Systematic Botany.
Trichomes.Glandular hairs or tectors of plants, localized mainly on leaf surfaces.