The presence of Fabaceae in the pollen profile of propolis produced in northeastern Brazil

Propolis is a resin-like substance composed mainly of resin, wax, essential oils, pollen grains and specific plant parts collected by the honeybee Apis mellifera, which mixes them with fluids they secrete. The components and chemical properties of propolis vary among regions. Therefore, in order assess variation in the botanical composition of propolis, 26 samples of propolis (brown, red, and green) produced throughout northeastern Brazil were analyzed by acetolysis specifically adapted for propolis. In total, 196 pollen types were recorded, representing 123 genera and 47 families, with types of Fabaceae and Rubiaceae being present in 100 % of the samples. Fabaceae was the richest group with 49 pollen types, followed by Malvaceae (10 types), particularly related to the high frequencies of Mimosa pudica (84.62 %), Alternanthera, Borreria verticillata, and Myrcia (80.77 %). Remarkably, 34 % of the pollen types with frequencies above 50 % belonged to Fabaceae, even though this family has been traditionally regarded as less important with regard to propolis production, given that most of its included taxa are classified as polliniferous or nectariferous. Similarity analyses revealed clusters of propolis samples that share pollen types associated with plants having apiculture potential.

The presence of pollen grains in propolis is usually related to their transportation via wind and by their adherence to vegetal resins that represent the major component of propolis.Therefore, the identification of the pollen grains in propolis is regarded as an indirect indicator of both its botanical and geographic origin (Borges et al. 2006).
Brazil is the third largest producer of propolis worldwide, reaching up to 150 tons/year.About 75 % of this total is exported mainly to Japan, followed by USA, Germany, and China, creating an income of around R$300 million per year (Braga 2009).Indeed, the demand for propolis in the international market has been increasing annually as a result of its popularization as a natural medicine by several studies that have attributed to it antioxidant, antitumor, antimicrobial, and antiulcer properties (Braga 2009).
Determining the geographic and botanical origin and the chemical composition of propolis is important inasmuch as these parameters assure quality control of Brazilian propolis in the marketplace (Alencar 2002;Teixeira et al. 2003).The propolis produced in Brazil is very diverse, putatively because of the presence of distinct biomes with unique and diverse flora, according to regional samples.Therefore, 13 types of propolis are recognized according to their physicochemical traits and biological properties (Park et al. 2002;Alencar et al. 2005).
Nonetheless, beekeepers discriminate only three basic types of propolis based on their coloration.Green propolis is produced mainly from Baccharis dracunculifolia (Asteraceae), and is rarely found in northeastern Brazil.Red propolis is mainly associated with Dalbergia ecastophylum (Fabaceae), and is produced mostly in the states of Sergipe and Alagoas in northeastern Brazil.Brown propolis is the most common type, and is produced from several vegetal species (Alencar et al. 2005).Both brown and red propolis account for most of the propolis productivity in northeastern Brazil (Park et al. 2000).
Several recent studies have employed distinct techniques with the aim of effectively identifying the origin of propolis produced in tropical areas.Pollen analysis has found species of the family Fabaceae to represent a major and ubiquitous component of pollen grains (Matos et al. 2014;Matos & Santos 2017), since this family is well represented in regional ecosystems.
Given the significant presence of the family Fabaceae in the flora of northeastern Brazil, particularly throughout the semiarid region (Queiroz 1999), and the abundance of pollen types associated with this family in propolis samples, the goal of the present study was the analyze the pollen grains in propolis samples produced in northeastern Brazil, with particular emphasis on the family Fabaceae, which includes several species useful to indicate vegetation.

Materials and methods
A total of 26 propolis samples produced from June 2016 to July 2017 by small producers and apiculture cooperatives were analyzed.These samples comprised the main types of propolis produced in northeastern Brazil and include 22 samples of brown propolis, three of red propolis and one of green propolis (Fig. 1).
The main criterion for the collection of propolis samples was the location of apiaries in areas of distinct phytophysiognomies that comprise the biomes of northeastern Brazil, with a minimum distance of 100 km between areas.
The samples were collected by shaving the internal walls of boxes, borders, nests, and roofs of beehives, and then stored hermetically in tagged containers for further analyses at the Plant Micromorphology Laboratory (LAMIV) at Universidade Estadual de Feira de Santana (UEFS).
Pollen analysis followed the procedure reported by Matos et al. (2014), in which a subsample of 0.5 grams of propolis was separated for dilution for at least 24 hours in 10 ml of absolute ethanol.The preparations were then centrifuged (2500 rpm for 10 min) and the resulting pellets boiled in 10 % KOH for 5 minutes in a moist chamber, followed by another centrifugation and a wash in distilled water.The material was then filtered twice by discarding the water and placed in 5 ml of glacial acetic acid were it was kept for 2 to 12 hours.The pellets were subsequently submitted to acetolysis in a moist chamber at up to 80 ºC for nearly 2 min (Erdtman 1960).The preparations were then washed in distilled water, centrifuged and stored for 1 to 24 hours in a 50 % glycerin aqueous solution.The pollen sediment was mounted on slides (five per sample), with glycerin jelly, for microscopic analysis.
In order to determine the frequency distribution of pollen types (taxa) in the collected samples, we followed the parameters established by Jones & Bryant (1996), as follows: >50 % = "very frequent"; 20-50 % = "frequent"; 10-20 % = "slightly frequent", and <10 % = "rare".The relative frequency of pollen types in each sample was based on counts of at least 500 grains of pollen per sample, according to Santos (2011).
The pollen grains were identified by comparisons with slides stored in the pollen collection of the Plant Micromorphology Laboratory (LAMIV), pollen atlases and literature reports (Roubik & Moreno 1991;Colinvaux et al. 1999;Carreira & Barth 2003).The pollen types followed those recommended by Santos (2011).
Analysis of floristic similarity among samples was performed by constructing a dendrogram using the software PAST-Palaeontological Statistics, ver.1.89 (Hammer et al. 2001) with Jaccard's similarity coefficient, since it disregards shared absences as similarities.
The taxonomic affinities of pollen types could not be determined for 12 of the propolis samples (Tabs.1, 2).These undetermined types (n = 24) exhibited low frequencies in each sample, and were most conspicuous in propolis from Mombaça -CE (MOM), which had a high frequency (5.4 %) of three distinct pollen types (Tab.1).
The families with the greatest number of pollen types found simultaneously in the three propolis types were Asteraceae (Eupatorium, Mikania, and Vernonia), Fabaceae (Acacia, Caesalpinia I and M. pudica), and Rubiaceae (B.verticillata, Borreria I and Mitracarpus), with three pollen types each.These were followed by the families Anacardiaceae (S. terebinthifolius and S. tuberosa), Arecaceae (C.nucifera and Elaeis oleifera), Myrtaceae (Eucalyptus and Myrcia), and Sapindaceae (Cupania and Serjania) with two pollen types each.The families Euphorbiaceae (Croton), Poaceae (type Poaceae), Rhamnaceae (Ziziphus joazeiro) and Urticaceae (Cecropia) were represented by a single pollen type in all propolis types.
Considering the 172 pollen types identified, 109 belong to groups that offer pollen as floral resources (Tab.3).Other pollen types belonging to groups that produce resins or oils were also found in analyzed samples (Tab.4), with remarkable representation of species of the family Anacardiaceae, as expected since they play a major role in the production of the resin that is the main constituent of propolis.
Regarding the vegetation used for foraging by Apis mellifera, tree-like plants are highlighted in the production of propolis since 36 % of the pollen types were of arboreal species while 34 % were of shrub-like groups.Herbaceous Acta Botanica Brasilica -32(4): 602-614.October-December 2018    one from São Domingos (SAD) characterized by pollen types of Anacardiaceae, Catopsis, Caesalpinia pyramidalis, Krameria, Monocoyledoneae, Nymphaea, Senna II and Zanthoxylum.

Discussion
The pollen types of the studied samples of propolis were mostly from species that play a key role in the maintenance of bee colonies.Thus, pollen types such as Alternanthera, Mimosa pudica, Borreria verticillata, and Myrcia, were distinguished because they are polliniferous and nectariferous plants, and thus important sources of carbohydrates and proteins for colonies.
The most representative pollen type (84.62 %) in the analyzed propolis samples was M. pudica (Fabaceae), a polliniferous and herbaceous species.This pollen type was found in all samples of red and green propolis and in 18 (82 %) of the brown propolis samples.These data are in agreement with previous reports by Matos & Santos (2017), who analyzed 22 pollen samples from nine municipalities along the northern coast of the state of Bahia and found 59 pollen types with M. pudica and Mikania being present in 100 % of the samples.According to beekeepers, M. pudica produces high amounts of pollen, and thus holds great potential for apiculture, particularly in northeastern Brazil where this species is widespread (Queiroz 2009).
The pollen types of B. verticillata (Rubiaceae), which are recognized as herbaceous nectariferous/polliniferous plants, and Myrcia (Myrtaceae), an arboreal nectariferous/ polliniferous group, were also commonly present among the analyzed samples, but in low frequencies in the samples of brown, red, and even green propolis.
In previous studies by Barth & Luz (2009), Borreria-type pollen appeared in all samples of red propolis from the states of Bahia, Alagoas, and Paraíba.Accordingly, Freitas et al. (2010) and Matos & Santos (2017) reported that the pollen type for B. verticillata was widely distributed among the samples analyzed, but at low frequencies, similar to the pattern found in the present study.The taxa related to this pollen type are poor producers of the resin that represents the main component of propolis, and so the presence of these pollen types are likely related to other activities in beehives such as feeding.
Regarding resin production, there are three pollen types that are considered very frequent and thus important for the production of propolis in northeastern Brazil because they represent plant groups that are widely distributed in the region.Among these types are Schinus terebinthifolius (Anacardiaceae), Spondias tuberosa (Anacardiaceae) and Cecropia (Urticaceae).This result indicates that the species related to these pollen types are potential suppliers of resin for the propolis of Apis mellifera, as corroborated by other recent reports (Matos et al. 2014;Matos & Santos 2017).
Considering the habit of the plants associated with the present pollen analysis, tree-like plants represented a large proportion of the pollen types (49.4 %), followed by shrubs (45.3 %); vines represented the least frequent pollen types in propolis samples, accounting for just 1.7 % of the pollen types.According to Pereira et al. (2004), the arboreal stratum is the main supplier of resources (nectar, pollen, oil, and resin) for most bee species.
The pollen types Cecropia and Poaceae were quite relevant, as also reported by Barth & Luz (2009) and Matos & Santos (2017).These genera comprise anemophilous species, which might also explain their conspicuous presence in the studied samples, especially those from more open areas.
The dendrogram based only on the pollen types associated with resinous and oleiferous taxa revealed the formation of groups that differed in pollen types, which might be related to the generalist behavior of foraging by bees.Nonetheless, S. terebinthifolius and S. tuberosa were highly represented in the three types of propolis, reinforcing their importance in the supply of resins for the production of propolis, as previously mentioned.
The pollen types Acacia and M. pudica shared by the samples in group AF are closely related to typical plants of the Atlantic Forest (Freitas et al. 2010;Matos & Santos 2017) that may produce pollen as floral resources.On the other hand, the presence of the types M. pudica and Mimosa tenuiflora in the samples of the CF group are associated with herbaceous and shrubby plants that offer pollen as the main floral resource.The samples from the largest cluster in this analysis (BF, with 19 samples) were not characterized by particular pollen types of Fabaceae, but instead encompassed a large assemblage of pollen types from local flora.
The similarity analysis considering the entire pollen spectrum revealed a close relationship between propolis samples and the physiognomies of each area.For instance, the samples from groups AP, EP, and FP included pollen types belonging to taxa that diverge both in habit and in the types of resources offered to bees.Nevertheless, some sample groups shared pollen types belonging to similar taxa in spite of being derived from physiognomically distinct areas, such as the occurrence of pollen types Croton and Hyptis, which are recognized as nectariferous and shrub-like plants, in all samples of the BP group.It should be pointed out that species of the genera Croton and Hyptis are important resources for the survival of bee colonies because of their production of nectar in semiarid conditions (Santos et al. 2006).The samples from Caetité (CAE) and São Domingos (SAD) were set apart from each other in the dendrogram because of their unique pollen types that appeared at low frequencies, possibly indicating particularities of the local flora, as reported by Freitas et al. (2010).
The present analyses revealed a large representation by the family Fabaceae, which contributed the highest number of pollen types to the production of propolis by Apis mellifera in northeastern Brazil.It is worth mentioning that most taxa of Fabaceae are not directly responsible for the production of propolis, but to the maintenance of colonies since they are mostly polliniferous or nectariferous, and thus assure the health of bees that produce this important product.

Figure 1 .
Figure 1.Map showing the location of the propolis sample collection in Northeastern Brazil.

Figure 3 .
Figure 3. Similarity dendrogram, based on Jaccard's coefficient for resinous and oleiferous pollen types, comparing propolis samples produced in northeastern Brazil.Four sample groups were formed: AR, BR, CR, and DR.See Figure 2 for sample codes.

Figure 4 .
Figure 4. Similarity dendrogram, based on Jaccard's coefficient for pollen types of Fabaceae, comparing propolis samples produced in northeastern Brazil.Three sample groups were formed: AF, BF, and CF.See Figure 2 for sample codes.

Figure 5 .
Figure 5. Similarity dendrogram, based on Jaccard's coefficient for all pollen types, comparing propolis samples produced in northeastern Brazil.Six sample groups were formed: AP, BP, CP, DP, EP, and FP.See Figure 2 for sample codes.

Table 1 .
Frequency (%) of pollen types in samples of brown propolis produced in northeastern Brazil.

Table 2 .
Frequency (%) of pollen types in samples of red and green propolis produced in northeastern Brazil.