Melliferous flora and pollen characterization of honey samples of Apis mellifera L . , 1758 in apiaries in the counties of Ubiratã and Nova Aurora , PR

The aim of this study was to carry out a survey of the flora with potential for beekeeping in the counties of Ubiratã and Nova Aurora-PR through the collection of plants and pollen analyses in honey samples collected monthly. 208 species of plants were recorded, distributed in 66 families. The families that showed the major richness of pollen types were: Asteraceae, Myrtaceae and Solanaceae. Approximately 80 pollen types were found in honey samples, most of them were characterized as heterofloral. Cultivated plants, such as Glycine max (soybean) and Eucalyptus spp., were representative in some months of the year. Exotic species, such as Ricinus communis and Melia azedarach, were also frequent. However, over than 50% of the pollen types belong to native species of the region, such as Schinus terebinthifolius, Baccharis spp. Alchornea triplinervia, Parapiptadenia rigida, Hexaclamys edulis, Zanthoxylum sp. and Serjania spp., indicating the importance of the native vegetation for the survival of the colonies.


INTRODUCTION
Brazil is a country of great beekeeping potential due to its diverse flowerings and adequate climate conditions making possible the management of beehives during the entire year.Beekeeping is an interesting activity to the rural producer because it requires little initial investment (Nogueira-Couto and Couto 2006).
Plants and their associated insects present interdependence in their evolutionary history and tropical regions have greater diversity of plants with flowers than temperate regions.Therefore, bees from tropics with a rich diversity of food resources demonstrate fast answer to the changes in the availability of the food items and lesser constancy than species of temperate regions (Crane 1990).The mutual benefits between bees and plants depend on the interaction with factors such as phenology and biogeography.In general, these factors are specific of each place and depend on the seasonal patterns (Hill and Webster 1995).
As the honey production and the parameters of honey depend on the vegetal species available to the beekeeping use, many studies have been carried out for the identification of the plants used by A. mellifera in different regions of the country.The authors employ mainly techniques based on the analysis of the pollen content of honey samples and/or analysis of pollen loads from corbicula of bees (Carvalho and Marchini 1999, Moreti et al. 2000, Silva and Absy 2000, Bastos et al. 2003, Viana et al. 2006, Luz et al. 2007, Mendonça et al. 2008, Sodré et al. 2008) and analysis of visiting bees in plants (Marchini et al. 2001, Santos et al. 2006, Vieira et al. 2008).
The knowledge of the regional flora with beekeeping potential possibilities the better use of the floral resources and stimulate the utilization of species of plants with apiarist value in the restoration of the vegetation of rural areas.
This research was carried out to obtain a floristic survey in the surroundings areas of apiaries in rural area in the counties of Ubiratã and Nova Aurora (PR) to identify the floral sources used by Apis mellifera for honey production and to evaluate the pollen spectrum of the honey samples during the year.

AREA OF STUDY
The collection areas (Figure 1) are located between 24°20' to 24°40' S and 52°52' to 53°26' W parallels, distributed in three rural areas in the counties of Ubiratã in the Central-Western meso-region of Paraná State and Nova Aurora in the Occidental-Centre meso region of Paraná State.An Acad Bras Cienc (2013) 85 (1) 309 MELLIFEROUS FLORA AND POLLEN CHARACTERIZATION OF HONEY These counties are located in the phytogeographical domains of Submontane Semidecidous Tropical Forest (IBGE 1992).The predominant soil is Red Latosol (Brazilian classification: Latossolo Vermelho distroférrico, Embrapa 1999) and the region presents a softly waved relief, located in the third plateau of Paraná State at 550 m of height above sea level.
The climate in the region is CwB, humid subtropical, with noticeable dry winters and very rainy summers by Köppen's classification.The mean annual temperature is higher than 22°C in the warmest month and lower than 18°C in the coldest months.The pluviometric precipitation varies from 1,600 mm to 1,900 mm and the relative humidity of the air remains close to 80% (IAPAR 2009).

LOCATION OF THE APIARIES
The collections were carried out in three apiaries, one in the county of Nova Aurora (24°31'50" S and 53°11'50" W -Apiary A) and two in the county of Ubiratã -PR (24°29'41" S and 53°02'43" W -Apiary B and 24°36'40.8"S and 53°00'52.6"W -Apiary C).The sampled areas embrace forest remnants, gardens, pastures, agricultural cultivations (predominance of soybean and corn) and reforestation areas (predominance of Eucalyptus).Apiary A has a legal reserve area of 13.65 ha, Apiary B of 2.4 ha and Apiary C of 15.64 ha.

MATERIAL
The collections were carried out biweekly from October 2008 to November 2009, totalizing 13 months.Considering that the radius of the forest remnants in the surrounding area of the apiaries did not exceed 1,000 meters and the others rural properties were composed by agricultural areas, random collections were determined inside of a radius of approximately 1,000 m around the apiaries.The vegetation survey was conducted with usual techniques of floristic surveys (Fidalgo andBononi 1989, IBGE 1992).
Five samples of each vegetal specie in blooming were collected and then herborized and identified (Cronquist 1981) at Herbarium of the Universidade Tecnológica Federal do Paraná (HCF) in Campo Mourão.When necessary, these samples were also sent to the Botanical Museum of Curitiba (MBM) and to the Herbarium of the Botanical Department of the Universidade Federal do Paraná (UFPR) in Curitiba.The material was recorded using a digital camera Sony-DSC-H9 (8.1MP-15x of optical zoom).
The classification of the species by the habit was adopted in accordance with HCF: tree -higher than 5 m of height and small tree -lower than 5 m, both forming frustum (trunk); shrub -with woody tissue and until 5 m of height, without forming frustum; herb -with no woody tissue.The classification was done in successional stages (Reitz et al. 1978, 1983, SPVS 1996, Roderjan et al. 1998).

COLLECTION OF HONEY SAMPLES
In each apiary, three beehives were marked next to the forest remnant and to the agricultural cultures.Empty frame with beeswax foundation in the super was set up monthly to avoid the mixture of the honey produced in different months.Samples were collected from December 2008 to April 2009 and from May to November 2009 when there was honey stored in the combs, even if it was not mature (with minimum 75% of capped cells).The honeycomb was centrifuged separately in plastic packing to avoid the mixture of the different samples.

REFERENCE SLIDE COLLECTION AND POLLEN ANALYSES
Floral buds were collected and packed separately to avoid the pollen mixture.One pollen slide collection was prepared using the acetolysis method (Erdtman 1952).This method is used to eliminate cell contents and enable observations on pollen size, shape, aperture and exine surface.A camera coupled in a microscope and the Motic images 2000 software captured images of the pollen.ELIZABETE S. SEKINE et al.
To the preparation of the slides of pollen contained in the honey of each month, the samples of the three beehives of each apiary were mixed.To the samples with moisture above 20%, it was used a quantity proportionally higher.An aliquot of 10 g was diluted in 20 mL of distilled water and centrifuged to the acetolysis method.Two slides of each sample were prepared for posterior counting and identification of the pollen types.
The slides were observed in a optical microscope Motic to the qualitative analyses (determination of pollen types) and quantitative (counting of pollen grains).Pollen types were determined by comparing with the reference lamina and also by researches in the specialized bibliography (Barth 1989, Moreti et al. 2002, Melhem et al. 2003, Roubik 2010).To the quantitative analyses, 300 to 500 pollen grains of each slide were counted and the averages of the counting of two slides of each sample were considered.The pollen types were classified in four classes of frequency (Louveaux et al. 1978, Barth 1989): dominant pollen (frequency higher than 45% -D), accessory pollen (16% to 45% -A), important isolated pollen (3% to 15% -I) and occasional isolated pollen (frequency lower than 3% -O).
The Diversity Index of Shannon (H') (Pielou 1975) and Simpson (D) (Poole 1974) were calculated to the pollen data: H' = -Σ pi x ln pi, where: pi = ni/N, ni = number of individuals of the specie i, N = total number of sampled individuals.
It can be observed in Table I that 162 species (76.8%) are native and 34 species (16.1%) are exotic from identified species.Among them, there were introduced species, cultivated in the areas close to the apiaries and with economical importance, such as Eucalyptus spp.and Glycine max (soybean).
Only 14 (17.7%) of the total species showed frequency of more than 15% (accessory or dominant pollen) at least in one sample.
Pollen diversity calculated by the Index of Diversity of Shannon (Table III) indicates that the samples of the apiaries A and B presented higher diversity in relation to the samples of the apiary C. similar to Sakuragui et al. (2011) in a Semidecidous Tropical Forest modified by the anthropic action, in which it was observed predominance of Asteraceae, Rubiaceae, Solanaceae and Fabaceae; and Gasparino et al. (2006) that noted Asteraceae, Leguminosae and Solanaceae among the predominant families in the seeds banks of areas with riparian vegetation in a county of the west region of Paraná State.
From 34 exotic species collected in the area of study, seven were classified as exotic invader species in accordance with the administrative rule 125/2009 of the Environmental Institute of Paraná (PARANÁ 2009) that recognizes the official list of the exotic invader species to Paraná State.These species are: Leucaena leucocephala (leucena), Melia azedarach (santa-bárbara), Psidium guajava (goiaba), Ricinus communis (mamona), Tecoma stans (amarelinho), Eriobotrya japonica (nêspera) and Hovenia dulcis (uva-japão).Some of them, such as R. communis, M. azedarach and L. leucocephala, were representative in the pollen spectrum of the honey collected in the region.
Families with higher number of species in an area are also the main sources of nectar and pollen (Ramalho et al. 1990).Asteraceae and Labiatae species are very numerous in open vegetation areas in South America.Solanaceae, Euphorbiaceae, Palmae and Myrtaceae families are common in neotropical regions.
Asteraceae is one of the families that have the greatest number of species of apiarist importance, as this is one of the families with more species and more widely distributed among the angiosperms (Locatelli and Machado 2001).
The families of plants with better representation in honey samples analysed in this research are similar to Asteraceae, Caesalpiniaceae (Fabaceae), Malvaceae and also Myrtaceae, observed by Carvalho et al. (1999)   Values followed by the same letters, in the same line, did not differ among themselves at the level of 5% of significance.

DISCUSSION
The area of study embraces rural areas that present different types of landscape, including pastures, cultivated areas and forest remnants.The results were MELLIFEROUS FLORA AND POLLEN CHARACTERIZATION OF HONEY Bastos et al. (2003) reported the predominance of pollen of Asteraceae, Euphorbiaceae and Leguminosae (nowadays this one is divided into Caesalpiniaceae, Fabaceae and Mimosaceae) in an area of Cerrado in Minas Gerais State.Luz et al. (2007) reported the predominance of pollen types of Arecaceae, Asteraceae, Mimosaceae and Myrtaceae in honey samples of Rio de Janeiro State.Euphorbiaceae and Myrtaceae were also found as dominant or accessory pollen types in organic honey samples collected in islands located in the frontier of Paraná, São Paulo and Mato Grosso do Sul states, and Mimosaceae, Myrtaceae and Solanaceae in nonorganic honey samples (Sereia et al. 2011).
In general, the most representative families observed in honey samples of this study are similar to those verified in samples collected in other regions of the country that have similar vegetation.
From dominant pollen types, G. max (soybean) and Eucalyptus are from plants cultivated in the area.The two counties are located in agricultural regions where the main crops are corn and soybean.In 2008, the soybean harvest in these counties was 79,950 ha (Ipardes 2010).Chiari et al. (2005Chiari et al. ( , 2008) ) studied the pollination in soybean by A. mellifera and concluded that the visiting of flowers by these honeybees increases the production of this crop in some varieties.
Eucalyptus species are efficient sources to the formation of one type of honey approved by the customers and is the most frequent in the Brazilian market (Komatsu et al. 2002, Sodré et al. 2003).The contribution of Eucalyptus as dominant pollen in honey samples was verified in others researches developed in the southeast region by Bastos et al. (2003) in Cerrado in the State of Minas Gerais; by Barth et al. (2005) in samples of the states of São Paulo and Minas Gerais; by Luz et al. (2007) in samples of the State of Rio de Janeiro and by Mendonça et al. (2008) in samples of the State of São Paulo.Pollen grains of Eucalyptus and Baccharis are among the cited types as geographical indicators of honey of the south of Brazil (Ramalho et al. 1991).
Highly eusocial bees, such as A. mellifera, collect resources preferentially in plants that exhibit a mass-flowering syndrome, with numerous flowers opening in short time (Wilms et al. 1996).This feature can be observed in many cultivated plants and invader species, explaining the occurrence of these pollen types in various samples, even as dominant pollen in some months.
In this research, only 17.7% of the pollen types showed frequency higher than 15% at least in one honey sample.Cortopassi-Laurino and Ramalho (1988), analyzing samples of pollen collected by Apis mellifera on the outskirts of São Paulo city, also observed a low percentage of plants whose pollen accounted for more than 10% in the samples.
Sources of pollen with representativeness between 1% and 10% are resources with little attractiveness, corresponding to potential or secondary sources.These sources supplement the nutritional needs of the colony and may be important in environments where food resources suffer seasonal variations (Ramalho and Kleinert-Giovannini 1986).
The large number of pollen types with low frequency in the samples shows the importance of these plants in total honey production, representing a significant resource for the beekeeping of the region.
Bees have a highly developed sense of smell and they can be trained to look for a flavor or a particular mix of aromas.Once they become conditioned to seek a kind of plant, they will continue to get it (Free 1993).Bees exhibit floral constancy, as a tendency to use fewer species than would be expected considering the number of flowering plants at the site (Cane and Sipes 2006).However, according to Wilms et al. (1996) Africanized bees are also quite general in the exploitation of floral resources, representing a wider niche than other eusocial bees.
The high values verified by the application of the Simpson Index indicate the probability of diversity of the sampled pollen, due to the dominance of one or a few species.ELIZABETE S. SEKINE et al.
Pollen grains of the type Baccharis (Asteraceae), of Euphorbiaceae and Asteraceae and type Eupatorium, were common in samples from Paraná State.Mimosa scabrela is typical from the region of Curitiba and pollen of the type Brassicaceae was also common in the samples (Carpes et al. 2009).
Approximately 52.5% of the pollen types verified in the honey samples are from native species of the region.Native species with bee value can be used in programs of recuperation of degraded areas.Although studies about this theme are not very common, Pegoraro and Ziller (2003) conducted a survey of species with bee value to provide subsidies to the restoration of a legal reserve.These authors reported some genera identified in this research, such as Mimosa, Eugenia and Zanthoxylum, as important to this purpose.Baggio (1988) and Wolff et al. (2007) indicated the application of aroeira, Schinus terebinthifolius, in agroforestry systems for beekeeping.
The use of native species by the bees was confirmed by the diversity of species found in this study, highlighting the importance of preserving native vegetation for the survival of the colonies during periods of supply scarcity of the floral resources of introduced species such as G. max and Eucalyptus spp.

CONCLUSION
In general, the honey collected in the region was heterofloral.Pollen types of cultivated crops were very representative, and the dominant pollen like Glycine max in December and January and Eucalyptus spp. in May and June.From the native species, Parapiptadenia rigida was dominant in November and February and Machaerium stipitatum in April.
Pollen types of other native species, such as Alchornea iricurana, A. triplinervea, Baccharis spp., Casearia sylvestris, Hexaclamys edulis, Schinus terebinthifolius, Serjania spp.and Zanthoxylum sp. were found as accessory pollen.If other pollen types, found as isolated pollen, were considered in group, they were very representative in the samples, highlighting the importance of the native vegetation for the survival of the colonies.

Figure 1 -
Figure 1 -Location of the collection points in the counties of Nova Aurora (Point A) and Ubiratã (Points B and C) in Paraná state.
frequency classes in honey samples of Africanized honeybee Apis mellifera in three apiaries (A; B; C) in Nova Aurora (A) and Ubiratã -PR (B and C) collected from December 2008 to April 2009 and from May to November 2010.*Native species collected in the area.MELLIFEROUS FLORA AND POLLEN CHARACTERIZATION OF HONEY AND POLLEN CHARACTERIZATION OF HONEY Five pollen types identified in the samples are not present in the list of the plants collected in the area: Arecaceae sp. 1, Araceae sp. 1, Stylosanthes sp., Lygustrum sp, Borreria sp., Acacia type and Vernonia sp.Plants of Arecaceae family and of the Herbarium.Microscopic images recorded of same pollen types are show in Figure 2. Dominant pollen types were observed in 14 (47%) of 30 honey samples, Glycine max (soybean) in December/January, Eucalyptus spp. in February/ March, May and June, Machaerium stipitatum in April, Brassicaceae sp1. in July, Melia azedarach (santa-bárbara) in August and Parapiptadenia rigida in November.Only one of the samples of the apiary A, with the largest forest area, presented the dominant pollen type of Eucalyptus in May.
in São Paulo State as the best representatives; and Asteraceae, Myrtaceae, Mimosaceae and Verbenaceae found by Marchini et al. (2001) as the most representative families in São Paulo State.

Figure 3 -
Figure 3 -Number of floristic species collected in the surrounding areas of three apiaries in the counties of Nova Aurora and Ubiratã (PR) from October 2008 to November 2009.

TABLE I (CONTINUATION) HCF Family/Specie Ordinary name Hb O SE B Months J F M A M J J A S O N D
♦ Polypodiaceae Pleopeltis angusta Humb.& Bonpl.Ex Willd.H N S ♦ An Acad Bras Cienc (2013) 85 (1)