The Pollen Spectrum of Apis mellifera Honey from Reconcavo of Bahia, Brazil

Background: The use of pollen grains to establish the geographical and botanical origin of honey samples, and for supplying information concerning to the floral class of a honey produced in a region, has been employed since 1895. Pollen analysis has relevant significance for the quality control of a honey that may include numerous pollen grains and elements of honeydew. The objective of this work was to determine the botanical origin of pollen from plants contributing in the composition of honey from the region of Reconcavo of Bahia, Brazil. Methodology: Form each honey sample, a 10 g subsample was diluted in 20 mL of distilled warm water (40oC) and centrifuged before the supernatant was drawn out. After centrifugation the pollen sediment was acetolysed following for better observation of the pollen grains. Subsequently, this sediment was mounted in microscopy slides with glycerin jelly for latter pollen grains counting and identification. The identification of the pollen grains found in the samples was determined by comparison with a reference collection from the Palynotheca of the Nucleus of Insect Studies from the Federal University of Reconcavo da Bahia and with descriptions obtained from specialized literature. A total of 70 honey samples of Apis mellifera L. were obtained from beekeepers of 17 Original Research Article Nascimento et al.; JSRR, 6(6): 426-438, 2015; Article no.JSRR.2015.167 427 counties in the Reconcavo of Bahia region, from March 2009 to February 2010, and palynologically analysed. Acetolyzed pollen were count and identified. Results: One hundred and twelve pollen types were identified, distributed within 35 families. Among these pollen types, 67.00% occurred with low frequency (rare) within the samples analyzed. Inside abundance class, 63.00% occurred as minor pollen. Mimosaceae was the richest accounting for 13.00% of the pollen types. There was similarity among the sources of trophic resources used by A. mellifera in the counties studied, with the highest similarity index of trophic resources found between the counties of Cabaceiras do Paraguaçu and São Felipe (Cs = 0.68). Conclusion: Pollen analysis demonstrated that honey produced by A. mellifera in the region of Reconcavo of Bahia, Brazil, is predominantly multifloral, with emphasis the Mimosaceae.


INTRODUCTION
Pollen grains are spontaneously collected by bees while they collect nectar from flowers. As a consequence, pollen is present in honey composition and for this reason it becomes an important marker of the botanical and geographical origin of honey. Those grains also help to determine honey variety [1,2]. A quantitative and qualitative palynological survey of a honey sample constitutes its pollen spectrum. This spectrum identifies the nectar producing plant species, the non producer species, the contaminants, and when compared to known base line data can be used to detect mixtures of honey and falsification in labeling [3]. Trough the quantitative analysis of pollen grains is possible to establish the proportion in which each nectariferous plant contributes in honey composition, thus determining the botanical species that gave origin to such honey [4]. This information is important as it identifies those species that should be conserved or restored into the landscape in order to assist in the establishment of a sustainable beekeeping industry [5,6].
While pollen grains from nectar supplying plants, known as nectariferous plants, are honey components, a percentage of honey pollen may also originate from anemophilous plants. There is also a third category of plants, the polliniferous plants, that in addition to supplying small nectar quantities, supply significant quantities of pollen [7].
Nectariferous plants are usually under represented in pollen spectra as they supply a lot of nectar but little pollen. Thus, the presence of a small number of grains may indicate a great quantity of nectar. In pure honey samples from this category, few pollen grains and sediment are present. On the other hand, polliniferous plants are over represented in pollen spectra, supplying few nectar quantities but great quantities of pollen. The presence of vast amounts of pollen may indicate less nectar percentages. When a honey sample contains more than 98% of pollen from a polliniferous plant, it must be considered a monofloral honey [3,7].
The pollen analysis is a method used to describe floral origin of honey and is useful to estimate the diversity of local flora. The knowledge of honey origin allows to map the regions suitable for apiculture. The decline in floral resources used by bees is considered one of the main causes involved in loss of pollinators, therefore, the pollen analysis has become an important tool to identify apicultural flora that can be propagated to increase food resources for honeybees. Thus, considering the "global collapse of honeybees" and "pollination crisis", studies on the identification of apicultural flora are important. [8].
In the Reconcavo of Bahia region, beekeeping is based on a familiar agricultural system, lacking enough information about floral diversity to sustain efficiently such activity. In this context, the objective of the present study was to determine the botanical origin of pollen contained in honey samples of Apis mellifera L. (Hymenoptera: Apidae), contributing to the knowledge of the regional honey flora.  , 2004). Form each honey sample, a 10 g subsample was diluted in 20 mL of distilled water and centrifuged before the supernatant was drawn out. After centrifugation the pollen sediment was acetolysed following Erdtman [11] for better observation of the pollen grains. Subsequently, this sediment was mounted in microscopy slides with glycerin jelly for latter pollen grains counting and identification. The identification of the pollen grains found in the samples was determined by comparison with a reference collection from the Palynotheca of the Nucleus of Insect Studies from the Federal University of Reconcavo da Bahia and with descriptions obtained from specialized literature as Barth [3,12], Barth et al. [13], Moreti et al. [14].

MATERIALS AND METHODS
After the determination of botanical origin of pollen samples, at least 1000 pollen grains/sample were counted consecutively [15]. The relative frequency of each pollen type was established trough the formula: f = (n i /N) x100, where f = relative frequency of the pollen type i within the sample; n i = number of pollen grains of the i type within the sample; N = total number of pollen grains within sample [16].
Subsequently, values of the mean and the confidence interval were calculated for each pollen type (i), with its respective Superior Limit (LS) and Inferior Limit (LI) at 1% and 5% significance level, and the frequency (rare = n i  LI 5% ; Frequent = LI 5% < n i < LS 5% ; and Very Frequent = n i  LS 5% ) and abundance classes (predominant pollen = n i  LS 1% ; secondary pollen = LI 5%  n i < LS 1% ; Important minor pollen = LI 1%  n i < LI 5% ; minor pollen = n i < LI 1% ) acquired [17].
The pollen similarity among honey samples of A. mellifera by county from the Reconcavo of Bahia region was obtained by the use of the similarity coefficient of Sörensen, expressed as the formula: Cs = 2c / (s1 + s2), where: s1 is the number of pollen types within the county 1 samples, s2 is the number of pollen types within the county 2 samples and c is the number of common pollen types for both counties.

RESULTS
By means of pollen analysis of the samples, the botanical origin of 112 pollen types was determined, distributed in 35 families (Table 1). Among families found in the present study, Mimosaceae had the largest number of pollen types, with 13.00% of the total, followed by Asteraceae and Fabaceae, both with 9.00% and Myrtaceae, Caesalpiniaceae and Rubiaceae with 8.00%, 7.00% and 5.00%, respectively.

DISCUSSION
According to various authors, the Mimosaceae has significant potential for beekeeping due to its abundant distribution in the distinct regions in Brazil and as a source of trophic resources (pollen and nectar) for bees [16,[18][19][20]. Moreti et al. [21] showed the importance of the participation of various species of Mimosaceae in the constitution of honey within the State of Bahia, Brazil.
The Asteraceae is considered one of the richest in number of species visited by social bees in different regions of Brazil [22]. In the present study this family was one of the most diverse in pollen types and some of these types were classified as predominant and secondary (Table 1). Locatelli and Machado [23] suggest that this is probably due to the fact this family is one of the largest and greater geographical distribution among angiosperms.
Novais et al. [24] identified 46 pollen types from 22 botanical families in honey samples of A. mellifera from the semi-arid region in Bahia, Brazil, with the Fabaceae having an exceptional number of pollen types, followed by Malvaceae, Asteraceae, Euphorbiaceae, Rubiaceae and Lamiaceae. This is similar to the results found in this study.   According to Melo [25], pollen type of M. pudica was the most frequent in honey samples of Africanized bees in Mundo Novo, Bahia, being predominant pollen in the majority of the samples where it was observed.
Costa [17] working with bee pollen in Cruz das Almas, Bahia, observed that 60.00% of the pollen types were rare and 61.00% minor pollen.
In the county of Castro Alves, Carvalho and Marchini [16] observed that 58.33% of the plant species visited by bees were rare versus 41.67% considered frequent. The greatest quantity of pollen types identified by these authors were rare, as occurred also in the present work.
Some pollen types that occurred as predominant and very frequent pollen belongs to the Mimosaceae and the Mimosa, having their species classified as polliniferous plants.
Members of the Anacardiaceae, Asteraceae, Euphorbiaceae and Sapindaceae were observed as secondary and frequent pollen, with species classified as nectariferous plants (Table 1).
Oliveira [26]  Nascimento et al. [9] conducted a study in the Recôncavo Baiano and identified 240 plant species visited by A. mellifera. The authors observed that most plants are herbaceous (44%), followed by arboreal (26%) and shrubby (18%). Lianas (4%), vines (4%) and palm trees (1%) had less representation. The greatest preference for bee visitation to the flowers of herbaceous plants may be associated with the abundance of these plants in the region, the flowering period with a higher concentration of species between March and June, and the rainy season in the region.
Additional plant species have also important participation in A. mellifera honey, as for example those considered as weeds, possessing honey production potential. Among these species, Almeida et al. [31] highlights Commelina benghalensis, Croton campestri and Portulaca sp. Similar to the results found by these authors, pollen types with affinity with those species were identified in the Reconcavo da Bahia samples, as for example Borreria verticillata, Commelina benghalensis, Crotalaria incana, Croton and Portulaca oleracea (Table 1), stressing the provable potential of these plants for beekeeping in the region.
Samples from the counties of Amargosa, Cachoeira, Cruz das Almas, Governador Mangabeira and Sapeaçu, had M. arenosa, M. pudica and Syagrus coronata as common types. This region had a mean of 27 pollen types among samples. Oliveira [26], working with honey form this region observed Borreria verticillata, Eupatorium, M. sensitiva, M. tenuiflora, S. coronata and Vernonia types in all the samples, with predominant pollen types found in samples from Amargosa (Schinus), Cruz das Almas (M. tenuiflora) and Sapeaçu (S. coronata).
Santos Jr. and Santos [32] demonstrated that S. coronata pollen type was the most frequent, being observed in the monosulcate and tricotomosulcate pollen forms in the counties of the micro-region of the river Paraguassu, in Bahia, Brazil. In the present study, species of the Arecaceae, Asteraceae and Fabaceae families had relevant pollen contribution in honey samples of the same micro-region. The S. coronata type was also preponderant in samples collected in the present work, being observed in both pollen forms (Fig. 2).
Due to its diversified flora, honey classification as monofloral is not common in Brazil. According to Zander and Maurizio [33] honey of Citrus and Lavandula (10-20% of the total pollen grains), are considered pure honey subrepresented in pollen. Considering the percentages referred by these authors, two samples originated from the county of Cruz das Almas, with accessory pollen of Citrus may be classified as being monofloral. In this county, the installation of apiaries next to orange tree orchards is common, contributing for the occurrence of such pollen type.
Pollen types of anemophilous species found in the honey pollen spectrum are considered not important for its composition, but significant for the geographical identification of the honey and for protein supplying for the colonies [7]. Among the three pollen types of anemophilous species identified in the samples of the Reconcavo da Bahia region, the type Cecropia was prominent.

CONCLUSION
Honey produced by Apis mellifera in the Reconcavo of Bahia, Brazil, has a multi-floral pattern with great contribution of the Mimosaceae species. In that sense, programs of implementation or expansion for beekeeping foraging in the region must consider members of this family in the floral composition.