Open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology ISSN: 0361-6525 DOI: 10.13102/sociobiology.v60i4.405-412Sociobiology 60(4): 405-412 (2013) Temporal variation in the Abundance of Orchid Bees (Hymenoptera: Apidae) in a Neotropical Hygrophilous Forest MMN Castro1, CA Garófalo1, JC Serrano1, CI Silva1,2 Introduction Bees of the tribe Euglossini, also known as orchid bees, are characterized by a relatively long tongue, highly modified hind tibias in males where they can store fragrances and, in most cases, a bright metallic tegument (Cruz Landim et al., 1965). Euglossines occur in the Neotropics and are found from southern North America (Minckley & Reyes, 1996) and northern Mexico to northern Argentina (Dressler, 1982), at altitudes that vary from sea level to 1,500 and 1,600 m a.s.l.; euglossines are rare above 2,000 m a.s.l. (Dressler, 1982). The fast flight, characteristic of this bee group and the rarity of finding them at flowers (Nemésio & Silveira, 2007), probably explains why these bees have been underestimated in studies on Apoidea communities in the past. However, the accidental discovery of male attraction (Lopez, 1963) to synthetic terpenoids and aromatic hydrocarbons, mimetic of chemical products found in floral fragrances (Rebêlo & Ca- Abstract Although bees are important pollinators in several ecosystems around the world, studies on bee diversity in hygrophilous forests are scarce. This type of vegetation is restricted to permanently wet soils and, therefore, has particular floristic, structure and physiognomy. The goal of the present study was to inventory and analyze the temporal variation of the euglossine bees that occur in a neotropical hygrophilous forest. In order to sam- ple male bees we used four chemical baits, eucalyptol, eugenol, vanillin, and methyl salicylate. The captures were made once a month, from March 2010 to February 2011, from 9:00 am to 12:00 pm. We captured 113 individuals of three genera and five spe- cies (in decreasing order of abundance): Eulaema nigrita Lepeletier, 1841 (n=52), Eu- glossa pleosticta Dressler, 1982 (34), Exaerete smaragdina (Guérin-Méneville, 1844) (12), Euglossa carolina Nemésio, 2009 (11), and Euglossa fimbriata Rebêlo & Moure, 1968 (4). The most attractive bait was eucalyptol (n=98), followed by vanillin (11), and eugenol (4). Both temperature and rainfall affected significantly the distribution of the number of males throughout the year. The highest number of Euglossini species and individuals was sampled in the warm and rainy season, with activity peaks varying among species. Sociobiology An international journal on social insects 1- Universidade de São Paulo (FFCLRP-USP), Ribeirão Preto, São Paulo, Brazil. 2- Universidade Federal do Ceará (UFCE), Fortaleza, Ceará, Brazil. RESEARCH ARTICLE - BEES Article History Edited by: Candida M L Aguiar, UEFS, Brazil Received 19 June 2013 Initial acceptance 19 July 2013 Final acceptance 29 July 2013 Keywords Richness, abundance, Euglossini, chemical baits, temporal fluctuation. Corresponding author Cláudia Inês da Silva Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto – USP Avenida Bandeirantes 3900 14040-901 Ribeirão Preto, SP, Brazil E-Mail: claudiainess@gmail.com bral, 1997), allowed to quickly advance the knowledge about the ecology of these bees. In addition to floral fragrances, Euglossini males are also attracted to a variety of non-floral fragrances, which are produced in wood, fungi, tree wounds, fruits (Ackerman, 1983; Whitten et al., 1993), and feces (Eltz et al., 2007). The studies carried out by Roubik and Han- son (2004) and Zimmermann et al. (2006) confirmed that the fragrances collected by males attract conspecific males, supporting the idea that those fragrances are analogous to pheromones and probably involved in mate recognition and choice (Zimmermann et al., 2009). Although the development of fragrances in the past 40 years allowed a large increase in the sampling of Euglossini communities in several Brazilian ecosystems, hygrophilous forests have been poorly studied. Also known as seasonal semi-deciduous riparian forests with permanent fluvial in- fluence (Rodrigues, 2004), broadleaf hygrophilous forests, swamp forests (Leitão Filho, 1982), or just hygrophilous forests (Toniato et al., 1998), this vegetation is characterized by MMN Castro, CA Garófalo, JC Serrano, CI Silva - Orchid Bees of a Hygrophilous Forest406 a permanently flooded soil, contrary to periodically flooded riparian forests (Toniato et al., 1998), and has the important function of protecting water sources (Joly, 1992; Marques, 1994; Teixeira et al., 2008). Currently, hygrophilous forests are extremely vulnerable to human impacts (Teixeira et al., 2008). This kind of vegetation has been largely devastated in the State of São Paulo (Torres et al., 1994), and so has progres- sively disappeared before being studied (Gomes et al., 2006). Until the present study no information on Euglossini commu- nities of hygrophilous forests was available. These bees are important pollinators in several Brazilian biomes and have close relationships with several plants (Rocha-Filho et al., 2012; Silva et al., 2012) of different families (Apocynaceae, Bignoniaceae, Clusiaceae, Commelinaceae, Convolvulaceae, Cucurbitaceae, Leguminosae, Melastomataceae, Myrtaceae, Rubiaceae, Solanaceae and Verbenaceae) that also occur in hygrophilous forests (Castro, personal observation). Hence, the objective of the present study was to describe the richness, abundance and the diversity of Euglossini bees that occur in a fragment of hygrophilous forest, as well as to assess the efficiency of odor baits in the attraction of males in this environment and to test for an effect of climate on the distribution and temporal fluctuations in the abundance of Euglossini bees throughout the year. Material and Methods Study area The study was carried out in Reserva Toca da Paca (hereafter RTP), classified as a private reserve of natural heritage by the Brazilian environmental law. This private reserve is within the boundaries of Boa Vista Farm, on the margins of the Mogi-Guaçu River (21º27’30” S - 48º05’12” W) at 510m a.s.l., in the city of Guatapará, São Paulo State, southeastern Brazil (Fig 1). The 187ha of RTP’s area has a predominant vegetation of hygrophilous forest which, despite being situated in a transition area of the cerrado and semi- deciduous forest vegetation types, is currently surrounded by agricultural crops, especially sugarcane. The regional climate type is Aw (Köppen’s classifica- tion), with two well-defined seasons: a dry and cold season, from April to September (autumn-winter), with the average temperature varying between 19.3 and 22.9 ºC and 201.5mm of total rainfall; and a rainy and warm season, from October to March (spring-summer), with the average temperature var- ying between 23.8 and 25.2ºC and 982.1mm of total rainfall (data from Centro de Pesquisas Meteorológicas e Climáticas Aplicadas a Agricultura - UNICAMP). Sampling of male bees We sampled bees monthly from March 2010 to February 2011 on a 50m long transect, inside the fragment and parallel to its edge. The distance between transect and the edge of the fragment was 80m and the nearest crop sugarcane was 200m (Fig 1). We placed on this transect four odor baits with euca- lyptol, eugenol, vanillin, and methyl salicylate, which proved to be efficient for attracting euglossine male bees (Rebêlo & Garófalo, 1991; Sofia & Suzuki, 2004). We prepared the baits using a wad of paper soaked with only one fragrance, which was tied with a string and attached to the vegetation on Fig 1. Location of the area where the Euglossini bees were sampled in Guatapará, São Paulo State, Brazil. The thicker gray line marks the edge of Reserva Toca da Paca natural vegetation. A: Distance of 80m from the sample point to the edge of Reserva Toca da Paca’s natural vegetation. B: Distance of 200m from the sample point to the edge the sugarcane plantation. Sociobiology 60(4): 405-412 (2013) 407 a shaded place 1.5m above the ground and 10m away from other odor baits. Sampling was always carried out at the same sites on a transect and each sampling session lasted three hours, from 9:00am to 12:00pm. This interval corresponds to the period of highest activity of males in the field in northeastern São Paulo State (Rebêlo & Garófalo, 1991). At each half an hour we replaced the essences to compensate the loss of volatile compounds, as suggested by Sofia and Suzuki (2004). We captured the bees attracted to the baits with an in- sect net and stored them individually in 10ml falcon tubes, where they were killed with ethyl acetate vapor released by a paper soaked with the substance and attached to the cap. The specimens were deposited in the Collection of Bees and Soli- tary Wasps at the Department of Biology, FFCLRP-USP. Data analysis Correlations between species richness, the number of individuals collected each month and average temperature and rainfall were made by Pearson coefficient (r), following Zar (1999). The software used for these calculations was pro- gram R version 2.15.2 (R Development Core Team, 2012). To test the monthly distribution of the males activities in the range of one year, we used the Rayleigh test of uni- formity (Z) and ran the calculations in the software Oriana 4.0 (Kovach Computing Services, 2012), in which months were converted to angles, beginning at 30º, which corre- sponds to January, and ending at 330º, which corresponds to December, in intervals of 30º. After the conversion, we cal- culated the mean date (a) of the capture frequency of males and the concentration (r) of this event around the mean angle. In the histogram, the vector indicates the mean angle, which corresponds to the mean date of occurrence of the event. The hypotheses were: H0= the males of each species are evenly distributed throughout the year and, hence, there is no tem- poral variation; H1= the males of each species are unevenly distributed throughout the year and, hence, there is temporal variation. In case H1 is true, i.e., (P<0.05), the concentration of male captures around the mean angle, denoted by (r), may be considered a measurement of temporal variation. Accord- ing to Morellato et al. (2000), r=0 when the distribution is even throughout the year, and r=1 when the distribution is concentrated around a single month. In discussion, to compare the Euglossini richness of RTP with other areas of the same region in northeastern São Paulo State, a rarefaction curve was made to equalize the dif- ferences in sampling effort. The diversity of bees were calcu- lated by Shannon-Wiener index, and the values of the indices were compared using Hutcheson’s t-test (Hutcheson, 1970). Uniformity and dominance were quantified following Pielou Index and Berger-Parker Index (Magurran, 2004) respective- ly. We also applied Sørensen similarity coefficient (Sørensen, 1948) to compare species composition among areas. Since this coefficient does not consider abundance, it reduces the chances of errors caused by studies with different sampling effort. All of the aforementioned statistical analyses were done using the software PAST 2.17c (Hammer et al., 2001). Results We collected 113 males of five species and three gen- era in the studied hygrophilous forest. Among the species studied, the most abundant was Eulaema nigrita Lepeletier, 1941, with 46% of all collected individuals, followed by Euglossa pleosticta Dressler, 1982 (30.1%), Exaerete sma- ragdina (Guérin-Méneville, 1844) (10.6%), Euglossa caro- lina Nemésio, 2009 (9.7%), and Euglossa fimbriata Rebêlo & Moure, 1968 (3.5%) (Table 1). Among the four odor baits used, eucalyptol attracted males of all species and also the highest number of males (n=98). Except for Eg. carolina, and Eg. fimbriata, whose males were attracted exclusively to eucalyptol, the other spe- cies were attracted to at least two different kinds of odor baits (Table 1). Methyl salicylate did not attract any males. During the study, the average monthly temperature Table 1. Number of Euglossini bees collected with Eucalyptol (EC), Vanillin (VA), Eugenol (EG) and Methyl salicylate (MS) in Reser- va Toca da Paca, Guatapará, Brazil, from March 2010 to February 2011. varied from 18 to 26 ºC and monthly rainfall varied from 0 to 296 mm (Fig 2). We observed the highest rainfall and temperature values between November and March, when the highest number of specimens was sampled (Fig 2). Species richness showed a significant correlation with temperature (r=0.85; p<0.01) and rainfall (r=0.83; p<0.01); male abun- dance also showed a significant correlation with temperature (r=0.92; p<0.01) and rainfall (r=0.81; p<0.01). The Rayleigh test indicated a temporal variation in the occurrence of bees in RTP (r=0.50; Z=28.35; P<0.01) (Fig 3A), with concentration of individuals around Janu- ary. We also observed temporal variation in different species separately, except for Eg. fimbriata (r=0.69; Z=1.91; P=1.15) (Fig 3C), which was not sampled in December; this species, though, has a marked occurrence period between October and February. Euglossa carolina (Fig 3B) and Eg. pleosticta (Fig 3D) showed activity peaks in January, and El. nigrita (Fig 3E) was the only species sampled throughout the year, Species N % Aromatic baits EC VA EG MS Eulaema nigrita 52 46 47 5 0 0 Euglossa pleosticta 34 30.1 29 2 3 0 Exaerete smaragdina 12 10.6 7 4 1 0 Euglossa carolina 11 9.7 11 0 0 0 Euglossa fimbriata 4 3.5 4 0 0 0 Abunda nce 113 100 98 11 4 0 Richness 5 - 5 3 2 0 MMN Castro, CA Garófalo, JC Serrano, CI Silva - Orchid Bees of a Hygrophilous Forest408 with a peak in December (Table 2 and Fig 3E). El. nigrita, as well as the other species, had no males attracted to the baits in August. Discussion The euglossine bees sampled in the studied fragment of hygrophilous forest have also been observed in other envi- ronments of northeastern São Paulo State, Brazil. (Table 3). This similarity in bees species occurrence may be explained by the fact that the hygrophilous forest is composed of plant species that are also found in other vegetation types that oc- cur in this region, such as the semi-deciduous forest and the cerrado. In addition, the geographic closeness and the relative similar altitudes of the areas in the northeastern São Paulo State region may influence the share of the species composi- tion of this tribe of bees, as has been observed in other studies (Brosi, 2009; Cordeiro et al., 2013). In contrast, species richness, expressed by the rarefac- Fig 2. Monthly rainfall (mm), average temperature (oC), and number of Euglossini males collected each month in Reserva Toca da Paca, Guatapará, Brazil. From March 2010 to February 2011. Fig 3. Temporal variation of the total abundance and for each Eu- glossini species sampled in Reserva Toca da Paca, Guatapará, Bra- zil, from March 2010 to February 2011. The line at the top of the vector stands for standard deviation. Except for Eg. fimbriata with P=0.151, the remaining species presented P<0.01. The test values corresponding to these distributions are shown in Table 2. E u glo s s a fim bria ta J a nua ry F e brua ry M a rch A pril M a y J uneJ uly A ugus t S e pte mbe r O ctobe r N ov e mbe r De ce mbe r 20 20 20 20 10 10 10 10 E u glo s s a ple o s tic ta J a nua ry F e brua ry M a rch A pril M a y J uneJ uly A ugus t S e pte mbe r O ctobe r N ov e mbe r De ce mbe r 20 20 20 20 10 10 10 10 E u la e m a n igrita J a nua ry F e brua ry M a rch A pril M a y J uneJ uly A ugus t S e pte mbe r O ctobe r N ov e mbe r De ce mbe r 20 20 20 20 10 10 10 10 E x a e re te s m a ra gdin a J a nua ry F e brua ry M a rch A pril M a y J uneJ uly A ugus t S e pte mbe r O ctobe r N ov e mbe r De ce mbe r 20 20 20 20 10 10 10 10 A F B C D E T ota l numbe r of indiv idua ls J a nua ry F e brua ry M a rch A pril M a y J uneJ uly A ugus t S e pte mbe r O ctobe r N ov e mbe r De ce mbe r 20 20 20 20 10 10 10 10 E u glo s s a c a ro lin a J a nua ry F e brua ry M a rch A pril M a y J uneJ uly A ugus t S e pte mbe r O ctobe r N ov e mbe r De ce mbe r 20 20 20 20 10 10 10 10 Table 2. Results of circular statistic analyses testing for the occur- rence of temporal variation in the abundance of Euglossini species sampled in Reserva Toca da Paca, Guatapará, Brazil, from March 2010 to February 2011. Rayleigh test was performed for signifi- cance of the mean angle. Eulaema nigrita Euglossa pleosticta Exaerete smaragdina Euglossa carolina Euglossa fimbriata Total number of individuals Number of males (n) 52 34 12 11 4 113 Mean angle 354.43° 28.62° 41.45° 356.31° 345° 12.47° Mean date (a) December January February December December January Concentration (r) 0.39 0.64 0.68 0.64 0.69 0.50 Rayleigh test (Z) 7.95 14.08 5.56 4.51 1.90 28.35 Rayleigh test (P) <0.01 <0.01 <0.01 <0.01 0.151 < 0.01 tion curves (Fig 4), indicates a lower number of species than observed in other areas of northeastern São Paulo State. This reflects in the Sørensen similarity coefficient presented by cluster analysis in which the RTP is the least similar to other areas of same region (Fig 5). The Euglossini richness in areas of semi-deciduous forest varies from eight to 14 species (Rebêlo & Garófalo, 1997; Jesus & Garófalo, 2000). Also in a semi-deciduous for- est, but very close to the ecotone with cerrado, Silveira et al. (2011) sampled 13 species, whereas Hirotsu et al. (2010) in a cerrado and Rebêlo and Garófalo (1991) in a second-growth area (capoeira) sampled eight species each. The difference in Euglossini richness found between RTP and other areas studied by Rebêlo and Garófalo (1991, 1997) and Jesus and Garófalo (2000) may be due to the size of the areas studied, since the fragments studied by these authors were significant- ly larger than RTP. Another reason for the smaller number of species observed in the present study, compared to the one made by Silveira et al. (2011), could be a difference in sam- pling effort, which in this latter were analyzed two simulta- neous sampling points against one of the RTP. Storck-Tonon et al. (2013) reported that those differences is also caused by differences in the index of edge in the fragment, the connec- tivity and the landscape structure, or as observed by Milet- Pinheiro and Schlindwein (2005) affected by the surrounding matrix of the studied area. The abundance of Euglossini bees in the RTP was also lower than other studied areas in the region, except the area studied by Hirotsu et al. (2010) (Table 3). Eulaema nigrita and Eg. pleostica were by far the most frequent species through- Sociobiology 60(4): 405-412 (2013) 409 out the year and also the most abundant species, represent- ing together 76% of all individuals collected in the present study. This was expected, since these species were also the most common in all other studies carried out in the same re- gion of São Paulo (Rebêlo & Garófalo, 1991, 1997; Jesus & Garófalo, 2000; Hirotsu et al., 2010; Silveira et al., 2011). However, while in RTP and the area studied by Rebêlo and Garófalo (1997) El. nigrita was the most abundant bee, being responsible for a dominance of 0.46 and 0.27 respectively, in the other studied areas Eg. pleosticta was the most numerous species, reaching a 0.72 dominance in Rebêlo and Garófalo (1997) studied area (Table 3). The 1.302 diversity index of RTP was very similar to that obtained by Jesus and Garófalo (2000), not been significantly different (t=0.216; p>0.05) and only higher than the one presented by Rebêlo and Garófalo (1997), which was influenced by its high dominance value (Table 3). Eucalyptol, or 1,8-cineol, was the most attractive bait for Euglossini males in RTP, which corroborates the efficiency of that compound as attractive scent bait for a large number of species and individuals, as emphasized by Dressler (1982) and observed in other studies carried out in several neotropical regions (e.g., Janzen et al., 1982; Acker- man, 1983; Pearson & Dressler, 1985; Rebêlo & Garófalo, 1991, 1997; Cordeiro et al. 2013; Rocha-Filho & Garófalo, 2013). As reported by Alvarenga et al. (2007) and Neves and Viana (1999) in their study areas, methyl salicylate was also not attractive for males in the RTP. Despite the significant efficiency of odor baits for the study of Euglossini communities, two species that nested in the study area, Euglossa townsendi Cockerell, 1904 and Eu- glossa truncata Rebêlo & Moure, 1996, were not sampled with the baits used in the present study (Castro, personal observation). Similar observations were reported by Rebêlo and Garófalo (1991), who found nests of Eufriesea auriceps Friese, 1899 and Eg. townsendi in semi-deciduous forests, but did not collect males of these species with odor baits. Recently, Knoll and Penatti (2012) reported that six fra- grances used as odor baits, for 77 months, did not attract Eg. townsendi males, which were captured only on flowers. These observations reveal a lack of attraction of males of those spe- cies by the baits used in those studies or, in the case of Eg. townsendi, a weak association, because even when they were attracted by the baits they were always in small numbers (Janzen et al., 1982; Ackerman, 1989; Rebêlo & Garófalo, 1997; Nemésio & Silveira, 2007; Abrahamczyk et al., 2011; Silva, 2012). Such occurrences show the importance of using alternative methods to sample euglossine bees, such as trap nests (Garófalo et al., 1993) and direct collection on flowers Fig 4. Rarefaction curve for Euglossini species richness in areas of northeastern São Paulo State, Brazil. A- Guatapará (present study), B- Santa Rita do Passa Quatro (Hirotsu et al. 2010), C- Sertãozinho (Rebêlo & Garófalo, 1997), D- Matão (Jesus & Garófalo 2000), E- Patrocínio Paulista (Silveira et al. 2011), F- Cajuru (Rebêlo & Garófalo, 1991, 1997). with insect nets (Rocha-Filho et al., 2012), in order to obtain representative samples of the species composition. Rebêlo (2001) pointed out that males have different preferences for aromatic fragrances depending on locality and season. Regarding temporal variation in bee abundance, in the present study, temperature and rainfall affected significantly the distribution of males throughout the year. Both the number of species and number of individuals were higher in the warm Table 3. Number of Euglossini bees sampled in areas of the northe- astern São Paulo State. Gua=Guatapará (present study), Sta=Santa Rita do Passa Quatro (Hirotsu et al., 2010), Ser=Sertãzinho (Rebêlo & Garófalo, 1997), Mat=Matão (Jesus & Garófalo, 2000), Pat=Patrocínio Paulista (Sil- veira et al., 2011), Caj=Cajuru (Rebêlo & Garófalo, 1991, 1997). 1Eg. cordata is treated as Eg. carolina by Nemésio, 2009. MMN Castro, CA Garófalo, JC Serrano, CI Silva - Orchid Bees of a Hygrophilous Forest410 and rainy months with activity peaks varying among species. These results are consistent with other studies carried out in different ecosystems (e.g., Rebêlo & Garófalo, 1991, 1997; Rebêlo & Cabral, 1997; Ramalho et al., 2009; Cordeiro et al., 2013; Silva, 2012) suggesting that the peaks in number of individuals and species are probably related to a higher avail- ability of floral resources (Rebêlo & Garófalo, 1997), since the flowering in tropical environments is cued by the rains and many of the orchid bees may also use these rains as a cue larity in species composition with other regional vegetation types contrasts with the very low number of individuals sam- pled and suggests a strong effect of area and spatial isolation by sugarcane culture and deforestation. Other studies should be carried out in other hygrophilous forests areas in order to increase the species list and also to advance the knowledge about community composition of these bees in this kind of vegetation, which is still poorly known. Acknowledgements We thank Daniela de Azevedo Souza Defina, for al- lowing us to sample in her farm; the Guatapará municipal government (through Júlio Yoji Takaki) for providing the meteorological data; FAPESP (process #2010/10285-4) and CAPES-PNPD (process #02958/09-0), for financial support, CAPES-Demanda Social for granted Maurício M. N. Castro a scholarship and Research Center on Biodiversity and Com- puting (BioComp). We thank three anonymous reviewers for comments that helped to improve this article. References Abrahamczyk, S., Gottleuber, P., Matauschek, C. & Kessler, M. (2011). Diversity and community composition of euglos- sine bee assemblages (Hymenoptera: Apidae) in western Am- azonia. Biodivers. Conserv., 20: 2981-3001. doi: 10.1007/ s10531-011-0105-1 Ackerman, J.D. (1983). Specificity and mutual dependency of the orchid-euglossine bee interaction. Biol. J. Linn. Soc., 20: 301-314. doi: 10.1111/j.1095-8312.1983.tb01878.x Ackerman, J.D. (1989). 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Apidologie, 44: 254-267. doi: 10.1007/s13592-012-0176-3 Fig 5. Similarity dendogram based in Sørensen coefficient between areas of northeastern São Paulo State, Brazil. A- Guatapará (present study), B- Santa Rita do Passa Quatro (Hirotsu et al. 2010), C- Sertãozinho (Rebêlo & Garófalo, 1997), D- Matão (Jesus & Garó- falo 2000), E- Patrocínio Paulista (Silveira et al. 2011), F- Cajuru (Rebêlo & Garófalo, 1991, 1997). for emergence (Pearson & Dressler, 1985). However, indi- vidual analyzes of the most abundant species in a community may show temporal variations different from that observed in RTP. In Peru, Pearson and Dressler (1985) observed the occurrence of two peaks in the activities of males, a major peak at the end of the dry season and beginning of the wet season, and another minor peak at the end of the wet season and into the dry season. According to those authors, smaller orchid bee species predominate in the hot/dry season. 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