Pollen profile of bee pollen from semiarid Northeastern Brazil

The Northeastern Brazil has xerical vegetation with different biomes. Its botanical heterogeneity represents shelter for diverse fauna. The region is rich in bees, which demand the resources offered by the plants, and provide pollination services. Bee pollen has been used as a beneficial food source for humans due to its high nutritional content and being a natural antioxidant. Pollen analysis can determine the plant species visited by bees, using them as the source of pollen supply. This study aimed to identify the main pollen types present in the pollen collected by Apis mellifera in the Northeastern Brazil and to define its botanical affinity. Commercial bee pollen samples produced in the region were treated by acetolysis method, mounted on slides, and the frequency of occurrence was estimated for each type of pollen. It was possible to distinguish 113 pollen types belonging to 35 botanical families, distributed in 92 genera. Fabaceae and Asteraceae were the families that most contributed to pollen types. Pollen types of Cocos nucifera and Myrcia were found in more than 50% of the samples. The families Arecaceae and Fabaceae showed high beekeeping potential. There is a striking similarity between the pollen samples from the states of Pernambuco and Rio Grande Norte.


Introduction
The Northeastern Brazil has a territorial area that corresponds to 18.2% of the national territory and includes the states of Bahia, Sergipe, Alagoas, Pernambuco, Paraíba, Rio Grande do Norte, Ceará, Piauí, and Maranhão.It's different geographic and climatic characteristics allow this region to encompass four biomes: Amazonia, Caatinga, Cerrado, and Atlantic Forest (IBGE 2019), showing high botanical variation, with approximately 11,740 plant species belonging to 2,056 genera and 212 families (Flora do Brasil 2020).This high flora diversity reported for the Northeastern region, along with favorable climatic conditions, makes this region highly suitable for beekeeping, with the sustainability of bees helping forest conservation.Therefore, beekeeping is an economic and ecological alternative for the conservation of natural resources (Souza 2007).
In this context, Apis mellifera L. is a species that stands out because of its ability to adapt to different environments, and one of the factors that may contribute to this ability is its generalist behavior.A. mellifera has a broad foraging habit and does not prefer any specific group of plants as food, foraging all the flowering species available in the landscape (Oliveira & Santos 2014), covering a wide foraging area in search of pollen and nectar, and encompassing a variety of plant groups (Proctor et al. 1996;Santos et al. 2006;Köppler et al. 2007;Muniz & Brito 2007).However, the source of pollen directly influences the nutritional value of resources (Andrada & Tellería 2005).
During the collection of pollen grains, honey bees agglutinate and mix them with nectar and salivary enzymes, thus forming bee pollen (Brasil 2001), which is stored in the cells of the honeycombs (Camazine 1993;Brasil 2001;Almeida-Muradian et al. 2005).In the hive, pollen is essential because it is the primary source of proteins necessary for the survival of bees, in addition to having lipids, minerals, and vitamins needed for feeding the larvae and for the development of the bees that have recently emerged (Baldi et al. 2004).
Furthermore, bee pollen is an interesting nutritional source not only for bees but also for humans (Pascoal et al. 2014).It has even been called the "only perfectly complete food" because it contains all the essential amino acids necessary for the human body (Silva et al. 2006).Bee pollen also has bioactive compounds, which positively affect human health; is considered a "functional food" (Mărgăoan et al. 2019), including about 200 different substances; and is significantly rich in natural antioxidants (Komosinska-Vassev et al. 2015).This implies that an increasing number of studies have focused on the composition of bee pollen and its use as a nutritional supplement for the human diet leading to development of a favorable trade system for the consumption of this product and the beekeeping industry (Barreto et al. 2006).
Considering that bee pollen is an essential food for bees and an important nutritional source for man, the importance of studies that contribute to the knowledge of pollen flora throughout the Northeastern Brazil arises.Therefore, this research aimed to evaluate the botanical profile of bee pollen produced in this region, to provide information that contributes to beekeepers in the elaboration of management plans, and to help them in the indication of the most productive sources for the hives.

Study area
The Northeast region of Brazil (Fig. 1) is the third-largest region in the country (1,552,175,412 km 2 ) and incorporates the largest number of states (nine).It is divided into four subregions based on its edaphological characteristics: Meio-Norte, Agreste, Sertão, and Zona da Mata.Furthermore, its vegetation encompasses four biomes: Amazonia, Caatinga, Cerrado, and Atlantic Forest, which allows the region to have a high richness of varied botanical species.

Bee pollen samples
The samples of bee pollen used in this research were produced in several areas of the Northeastern Brazil.For this reason, beekeepers' associations and/or cooperatives established throughout the region and municipal agricultural departments were contacted.In addition, samples were acquired by participating in congresses and meetings focused on beekeeping held in the northeastern states of Brazil, and from markets and natural product stores.A total of 28 samples of dehydrated bee pollen from different Northeastern states were used, distributed in the following way: Alagoas (2), Bahia (12), Ceará (2), Paraíba (2), Pernambuco (3), Piauí (2), Rio Grande do Norte (3), and Sergipe (2).Samples from the state of Maranhão were not included in this study.

Pollen analysis
Bee pollen analysis was performed following the method proposed by Alvarado & Delgado-Rueda (1985), with some modifications Novais & Absy (2013) using the acetolysis method (Erdtman 1960).The sediment was mounted on slides in glycerin jelly and sealed with paraffin wax.To perform qualitative and quantitative analyses of the pollen spectrum, five permanent slides were prepared for each sample.
Our study was based on the morphological characteristics of each pollen type to identify specific taxon (e.g., a species, a group of species, a genus, or a family) to which the bee pollen samples belonged.Thus, pollen type identification was carried out using a light microscope by referring to scientific articles, specialized palynological catalogs (Roubik & Moreno 1991;Carreira et al. 1996;Melhem et al. 2003;Lima et al. 2008;Bauermann et al. 2013;Silva et al. 2016), and the pollen library of the Laboratory of Plant Micromorphology, Department of Biological Sciences, State University of Feira de Santana (LAMIV/DCBio/UEFS), as proposed by Santos (2011).
To estimate the percentage of occurrence of each pollen type in the sample set, the frequency of occurrence (FO) was calculated.For this, a minimum of 500 pollen grains were counted in each sample (Bucher et al. 2004).Based on FO values, pollen types were classified according to the following classes: very frequent (>50%), frequent (21-50%), low frequent (10-20%), and rare (<10%) (Jones & Bryant Jr. 1996).
Through quantitative analysis, only pollen types with an FO value greater than 10% per sample were considered as "important sources" of pollen (Imperatriz-Fonseca et al. 1994), while those with FO value between 1%-10% were regarded as "secondary sources."Furthermore, samples in which the predominance of a particular pollen type exceeded 80% were deemed to be monofloral products (Campos et al. 2008).

Statistical analysis
The similarity between the samples was analyzed using the Jaccard coefficient, as it does not consider the shared absences as evidence of similarity.Analysis was performed using the PAST software -Paleontological Statistics, version 3.15 - (Hammer et al. 2001).
Cocos nucifera pollen type was registered in 23 analyzed samples (88.5%), classified as an important source in 17 samples, and stood out in one sample from Bahia and one from Piauí since it registered representativeness higher than 50% in both samples (65% and 86%, respectively).Furthermore, Myrcia pollen type was present in 21 analyzed samples, representing 80.8% of the total samples, was important in 10 of them, stood out in two samples from Alagoas (54% and 73%), and exhibited the highest participation as a secondary source (11 samples).M. pudica/sensitiva pollen type was observed in 20 samples (76.9%), and it was classified as an important source in 13 of them.In addition, it registered more than 80% of the representativeness of two samples from Bahia.Finally, although the Poaceae pollen type showed an FO value of 57.7%, it was classified as a secondary source in all samples in which it was present (<10% per sample) (Fig. 3).Some pollen types did not exceed 50% of representativeness when considering the total sample set; however, they stood out by showing a percentage value higher than 50% in some specific samples: M. candollei (71%), Baccharis type (59%), and M. caesalpiniifolia (54,6%) were predominant in one sample from Paraíba, Bahia, and Rio Grande do Norte, respectively.
The pollen type richness showed an average value of 16 pollen types for samples from Ceará, 15 from Bahia, 13 for Sergipe and Pernambuco, 12 for Rio Grande do Norte, and 6 for Piauí (Table 1).On the other hand, three samples were considered monofloral, namely, PI1, which was represented by C. nucifera (86.1%), and BA7 and BA10, both represented by M. pudica/sensitiva pollen type (84.8% and 87.6%, respectively).Although some beekeepers affirmed that samples from Sergipe were monofloral with a predominance of Arecaceae, our results did not corroborate this information.
The statistical analysis showed a higher similarity index value between the Pernambuco 1 and Rio Grande do Norte 3 samples (approximately 62%).The presence of C. nucifera, M. caesalpiniifolia, M. pudica/sensitiva, Commelina, and B. verticillata contributed to the similarity between these samples.

Discussion
The pollen richness recorded in this study evidenced the vegetal diversity of the Northeastern Brazil; however, some pollen types indicated that certain plant species are more visited for the collection of pollen grains by honey bees in this region.
The presence of pollen grains belonging to C. nucifera (Arecaceae) is an important characteristic of bee pollens produced in the Northeastern Brazil.Consequently, it is considered a geographical indicator and it is also essential to produce bee pollen in Rio Grande do Norte, Bahia, and Sergipe (Freitas et al. 2013;Alves & Santos 2016;2018).This species is critical in the study area, even in the beekeeping industry.Although it is regarded as an anemophilous species (Alves & Santos 2016), it is highly polliniferous and desirable to honey bees (Conceição et al. 2004).According to Leite & Conceição (2002), C. nucifera flowers all year long, strengthening this species' utilization by honey bees.This fact is of interest from the commercial point of view since, according to Bauermann et al. (2010), Arecaceae pollen grains contribute to texture and taste that please the consumer.
The Fabaceae family also stood out in this study, and it has been highlighted in several studies focused on bee pollen produced in the Northeastern region (Muniz & Brito 2007;Novais et al. 2010;Poderoso et al. 2012;Costa et al. 2015;and Alves & Santos 2018).Carvalho & Marchini (1999) emphasized that within Fabaceae, the Mimosoideae clade includes several species with an important polliniferous potential that are constantly visited by honey bees.In addition to their pollen production, these species bloom almost year-round (Carvalho et al. 2001;Santos Júnior & Santos 2003;Silva et al. 2004).The M. pudica/sensitiva pollen type, which was present in most of the analyzed samples, presented high beekeeping potential (Alves & Santos 2016) owing to the invasive nature and rapid spreading of these species in anthropogenic environments (Queiroz 2009;Dutra et al. 2020).
Myrtaceae family was also highlighted in this work, mainly due to the presence of Myrcia pollen type.According to Queiroz et al. (2006), Myrtaceae is one of the best-

Pollen profile of bee pollen from semiarid Northeastern Brazil
Acta Botanica Brasilica, 2022, 36: e20220205   Brazil.See Table 1 for sample codes.
represented families in the Brazilian semiarid region and is one of the most diverse families in the Caatinga.Furthermore, Myrcia is the second most represented pollen type in melissopalynological studies throughout Brazil and the seventh in the Northeastern (Souza et al. 2018).
According to Freitas et al. (2013) and Alves & Santos (2016;2018), this pollen type stood out in bee pollen samples from Bahia, Sergipe, and Rio Grande do Norte as it was classified as very frequent, which was corroborated in this research.
The Poaceae pollen type analyzed in the bee pollen samples in this study is an important representative of an anemophilous taxon.This pollen type represents plant species that are highly polliniferous and that contribute significantly to bee pollen composition (Alves & Santos 2014).Souza et al. (2018) corroborated this information by reporting that Poaceae is the sixth most represented pollen type in melissopalynological studies in Brazil.Alves & Santos (2016;2018) also identified this pollen type in bee pollen samples from Bahia and Sergipe.Dórea et al. (2010) pointed out that despite the high frequency of the Poaceae pollen type when considering the entire sample set, it was present in low percentages per sample, which indicates its importance as a secondary source, as confirmed by our results.
Although a total of 113 pollen types were registered in the analyzed samples, which reinforces the generalist behavior of A. mellifera (Köppler et al. 2007), some samples were classified as monofloral (PI1, BA7, and BA10).This corroborates the results obtained by Suwannapong et al. (2012), who reported honey bees preference for plants that provide resources in greater quantity, taking advantage of the pollen source until its exhaustion.
Other samples from the state of Sergipe were indicated as monofloral because of the content of C. nucifera pollen grains present at the time of its sale.Nonetheless, this information was not confirmed in this study.In addition to the insufficient presentation of more precise information on the label of commercialized products, beekeepers lack a better understanding of the polliniferous sources of their apiaries and knowledge for developing a management plan for beehives and cultivation of plant species of beekeeping importance.

Conclusion
Based on the analyzed data, the richness of pollen types found in this study provides essential information about the flora visited by A. mellifera in the Northeastern Brazil, highlighting the contribution of some botanical groups to the protein diet.
Fabaceae and Asteraceae were the primary polliniferous sources used by A. mellifera in this region.In contrast, pollen types of C. nucifera, M. pudica/sensitiva, Myrcia, and Poaceae were very frequent in the samples.The first two sources contributed to samples that were considered monofloral bee pollen.This result is excellent for beekeepers because these species are widely distributed in the Northeast region.Cocos nucifera, in addition to contribute significantly to the composition of bee pollen samples in the region, also adds appreciable flavor and texture for human consumption.
The knowledge about the botanical sources that support the pollen (protein) diet of A. mellifera in Northeastern of Brazil contributes to the preservation of plant species of beekeeping importance in the region, as well as favoring the commercialization of pollen from these bees by beekeepers.

Figure 2 .
Figure 2. Pollen type representativeness (%) in commercialized bee pollen samples from states of the Northeastern Brazil.

Figure 4 .
Figure 4. Dendrogram of similarity (Jaccard index) for samples of bee pollen produced and commercialized in the Northeastern