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Publication ahead of print
Journal
Apidologie
DOI http://dx.doi.org/10.1051/apido/2010055
Published online 24 September 2010

© INRA/DIB-AGIB/EDP Sciences, 2010

1. INTRODUCTION

Euglossini bees, or “orchid bees” (Hymenoptera: Apidae: Euglossini), have an exclusively Neotropical distribution (Búrquez, 1997) and are important pollinators of many angiosperm species (Cancine and Damon, 2007; Guimarães et al., 2008). More than 200 species were described before 2002 (Ramírez et al., 2002) and new species have recently been identified (Oliveira and Nemésio, 2003; Oliveira, 2006).

Some Euglossini species are common in urban habitats (López-Uribe et al., 2008), where gardens and parks provide resources like nectar, pollen and nesting sites during the entire year. Natural habitats have suffered drastic disturbances in the last years and, consequently, cities have increasingly been used as a refuge by some species of bees. Therefore, it is important to verify the genetic health and viability of the populations established in these environments (Grixti et al., 2009).

Euglossini males and females have great flight capacity (Roubik and Hanson, 2004). Adults travel for more than 50 km in a search for nectar or floral fragrances that they use for sexual attraction (Janzen, 1971; Dressler, 1982; Eltz et al., 2003). This ability to fly over long distances allows high dispersal of these bees, which avoids inbred mating. However, some studies revealed that Euglossa cordata Linnaeus 1758 females collect nectar (López-Uribe et al., 2008) and reuse the natal nest or a nest next to it (Garófalo, 1992), suggesting that these females have philopatric behaviour.

Although Euglossini encompass a large number of species, information on them is scarce and based mostly on data obtained from males collected with artificial baits (Sandino, 2004; Farias et al., 2008; Rasmussen, 2009). Because such compounds do not equally attract different species, this sampling strategy can produce biased inferences about species distribution because the samples do not reflect the abundance and species richness in the studied sites (López-Uribe et al., 2008). By analysing only males of a population, we may generate erroneous inferences about its genetic structure. A viable alternative would be to collect adults in flowers, a procedure that allows sampling of both males and females, as all adult bees search for nectar as a food supply. Due to the ease of collecting males with artificial baits and the difficulties of locating nests in nature, female and nesting biology are unknown in 30% (Ramírez et al., 2002) and 80% (Cameron, 2004), respectively, of all Euglossini species.

Even though the biological data point to a high degree of dispersion, literature about the occurrence of diploid males in natural populations of euglossine bees appears contradictory. High frequencies of diploid males have been described in populations of different species in Panama (Roubik et al., 1996; Zayed et al., 2004) and Colombia (López-Uribe et al., 2007), but not in samples from Brazil (Takahashi et al., 2001). The presence of 2n males has been associated with reduced population size caused by habitat fragmentation due to human activities (Zayed and Packer, 2005). Because of their inviability or sterility, diploid males represent a heavy genetic burden for their populations. For this reason, the occurrence of diploid males has been identified as a cause of the simpler social organisation found in this tribe compared to other eusocial corbiculate bees (Roubik et al., 1996). In addition, the high frequency of euglossine diploid males has conferred credibility to the proposition of an extinction vortex for Hymenoptera populations because of their peculiar sex determination system (Zayed et al., 2004; Zayed and Packer, 2005).

Considering the increasing importance of cities as refuges for bee populations, this work aimed to determine the genetic structure of Euglossa cordata urban populations, analysing males and females collected in Thevetia peruviana (Apocynaceae) flowers in 11 cities over a north-south transect in São Paulo state. The analysis of 15 allozyme and 9 microsatellite loci showed that genetic differentiation among populations is low, indicating a high degree of dispersion in this bee species.

2. MATERIAL AND METHODS

2.1. Sampling

A total of 705 individuals of E. cordata were collected from urban areas of 11 cities in São Paulo state (Brazil) (Fig. 1 and Tab. I).

thumbnail Figure 1

Map of the state of São Paulo showing the localities inside urban areas where males and females of Euglossa cordata were sampled. The sampling sites (codes) were described in the legend of Table I.

Table I

Data on samples of Euglossa cordata, including sampling sites and their geographical coordinates and codes, number (N) of bees (M = males and F = females) captured, and number of individuals analyzed for allozymes (alloz) or microsatellites (micrst) loci.

Males and females of E. cordata were captured with a plastic bag while visiting flowers of Thevetia peruviana (Apocynaceae) to collect nectar. In Bertioga, the samples were collected with attractive baits (cineole and vanillin), and therefore, only males were sampled. Captured individuals were identified, placed in plastic tubes on ice and then kept at –20 °C until the analyses were performed.

Individuals from Rifaina, Pedregulho, São Carlos and some individuals from Jaboticabal were collected between 2004 and 2006. Samples from Franca, Ribeirão Preto, Araraquara, Leme, Rio Claro, Piracicaba, Bertioga and part of the sample from Jaboticabal were obtained between 2007 and 2009. In each city, at least five urban sites were sampled to maintain a better representation of the population living there.

2.2. Genetic analysis

Genomic DNA was extracted from the third pair of legs from each individual, using a 10% Chelex protocol (Walsh et al., 1991) or the phenol-chloroform protocol (Sheppard and McPheron, 1991). Both procedures gave the same results in microsatellite analysis, but we preferred to use the latter method when the samples were used for mtDNA analysis too.

Analysis of the allozyme loci was performed primarily to differentiate E. cordata individuals from morphologically similar species, Euglossa securigera and Euglossa townsendi, according to patterns described by López-Uribe and Del Lama (2007). In addition, this method was used to estimate the intra- and interpopulational variation. Electrophoretic analysis was conducted for the following enzymes: acid phosphatase (Acp), esterase-1 and esterase-4 (Est-1 and Est-4, respectively), isocitric dehydrogenase (Icd), superoxide dismutase (Sod), fumarase (Fum), glucose phosphate isomerase (Gpi) and 6-phosphogluconate dehydrogenase (6Pgd) in tris-citric acid buffer, pH 7.5. Tris-citric acid buffer, pH 8.0, was used for electrophoretic analysis of cytosolic and mitochondrial malate dehydrogenases (cMdh and mMdh), phosphoglucomutase (Pgm), α-glycerophosphate dehydrogenase (αGpdh), β-hydroxybutyrate dehydrogenase (βHbdh), hexokinase (Hk) and malic enzyme (Me).

For the microsatellite analysis, nine pairs of species-specific Euglossa cordata oligos (17, 18, 30b, 51, 35, 24, 26, 30a and 37), designed by Souza et al. (2007), were used. Amplification by polymerase chain reaction (PCR) was performed according to the protocol described by these authors, and the products generated were resolved in a MegaBACE-750 (GE) fragment analyser, using the program MegaBACE fragment profiler.

2.3. Data analysis

Genetic variability of each population was determined: (i) through the number of alleles (A), considering data from males and females; (ii) the observed (Ho) and expected (He) heterozygosities and (iii) by the inbreeding coefficient (Fis), estimated from female data only. These parameters were calculated using FSTAT 2.9.3.2 (Goudet, 1995).

To assess the genetic differentiation levels among populations, the software Arlequin 3.1 was used (Excoffier et al., 2005) to estimate the Fst parameter of the F-statistics, according to Weir and Cockerham (1984), and the partition of the genetic variation within and among populations by Analysis of Molecular Variance (AMOVA).

As microsatellites are highly polymorphic markers, Fst and AMOVA values were separately estimated from the loci with higher (loci 17, 18, 24, 26 and 37) and lower (loci 35 and 51) heterozygosity levels and compared with their values when all loci were considered together to see if the sample size used in the microsatellite analysis was suitable.

For all markers, one locus was considered polymorphic when the frequency of the most common allele was smaller than 95%. Statistical tests were performed using a significance level of 5%.

3. RESULTS

3.1. Gene variation

Allozyme phenotypes were determined for 626 individuals from the 11 sampling sites, and all were identified as described by López-Uribe and Del Lama (2007). Of the 15 loci analysed, with 2% missing data, five were monomorphic, and 10 showed electrophoretic enzyme variants, but only two loci exhibited polymorphism at the 95% criterion, Est-1 and cMdh.

A total of 705 individuals from the 11 sampling sites were genotyped for nine microsatellite loci. All loci showed polymorphism, with an average of 16.56 alleles per locus. The absence of amplification or a poor amplification in some individuals and at some loci generated a rate of 4.7% of missing data.

Population number of alleles (A), obtained from allozyme and microsatellite loci analysis, are presented in Table II. The estimation of inbreeding was made by comparing observed and expected heterozygosities from female samples (Tab. II).

Table II

Number of alleles (A) and observed heterozygosity (Ho) at allozyme and microsatellite loci in Euglossa cordata populations from Rifaina (Rif), Pedregulho (Ped), Franca (Fra), Ribeirão Preto (Rpr), Jaboticabal (Jab), Araraquara (Ara), São Carlos (Sca), Leme (Lem), Rio Claro (Rcl), Piracicaba (Pir) and Bertioga (Ber). N = number of individuals (females + males). Values in bold indicate populations with significant inbreeding coefficient (Fis).

3.2. Population differentiation

Differentiation among populations was investigated first by the Fst values obtained from allozyme polymorphic loci, Est-1 and cMdh (Tab. III). The analysis revealed significant genetic differentiation for Est-1 (Fst = 0.04), cMdh (Fst = 0.02) and the mean Fst averaged over all loci (Fst = 0.03), but when we excluded the sample from Bertioga from this analysis, significant genetic differentiation was observed only for the locus Est-1 (Fst = 0.04).

The same analysis was performed for the microsatellite loci (Tab. III). Considering the eleven populations studied, the average Fst was not significant. Excluding the sample from Bertioga from the analysis, some Fst values were no longer significant for two of the analysed loci (loci 18 and 26), confirming the higher genetic homogeneity among the populations of the transect.

Data from microsatellite loci were also used for the Analysis of Molecular Variance (AMOVA). As shown in Table IV, the partition of the variation among the three hierarchical levels showed that the populations are homogeneous with each other (Fst = 0.0031, P > 0.05), although there was considerable variation among individuals within population (Fst = 0.056, P < 0.05) and within individuals (Fst = 0.941, P < 0.05).

The AMOVA analysis gave non-significant results when loci with higher (Fst = 0.0041; P = 0.73) or lower (Fst = –0.00072; P = 1.0) heterozygosities are considered separately, strengthening the accuracy of the Fst averaged over all loci (Tab. IV).

3.3. Diploid male analysis

Allozyme and microsatellite genotypes allowed the identification of the presence of diploid males in these E. cordata populations. 139 males were analysed for the allozyme loci, and for the microsatellite loci, 177 males were analysed. Only one diploid male from Rifaina was detected by its heterozygous condition in 7 of 9 microsatellite loci analysed. The allozyme analysis did not reveal the presence of diploid males.

4. DISCUSSION

This is the first effort to assess the genetic structure of euglossine bee populations sampled inside urban environments using allozyme and microsatellite analysis. It is also the first female-based study of euglossine population genetics. As expected, the number of alleles, the intralocus heterozygosity values and the heterozygosity averaged over all loci were higher with microsatellite loci than with allozymes.

Most of the loci in the studied populations were consistent with genetic equilibrium. However, for some loci and some localities, there was a significant deficit of heterozygotes. This reduction has usually been attributed to null alleles, selection, population subdivision (Wahlund effect) or inbreeding, among other factors (Hartl and Clark, 2007).

Table III

Genetic differentiation (Fst) among populations measured by the distance method (Weir and Cockerham, 1984) viewed through the Est-1 and cMdh loci or through nine microsatellite loci. Average values over all allozyme (Mean A) or microsatellite loci (Mean M) are also estimated considering all populations (A) or excluding the samples from Bertioga (B). Significant values of Fst are denoted with bold characters.

Table IV

Analysis of Molecular Variance (AMOVA) using data from microsatellite loci with higher (A) or lower (B) heterozygosity levels and all loci considered together, showing the distribution of the variation within and among populations. Significant values (P < 0.05) are denoted with bold characters.

All but one male submitted to analysis showed one allele per locus, which means that the presence of null alleles was not the cause of the observed deficit of heterozygotes. As the microsatellite loci are considered neutral markers (McKay and Latta, 2002; Porcher et al., 2006), it is expected that this lack would not be due to strong selective pressures. The observed genetic homogeneity among populations also excludes population subdivision as its cause. High levels of inbreeding are not expected when we consider the low number of detected diploid males in the samples; otherwise, inbreeding effects are expected over all loci and populations (Hartl and Clark, 2007). Further analyses are required to find an alternative explanation for this deficit.

As the Fst and AMOVA values obtained when all loci were considered together were similar to those values when the microsatellite loci were grouped according to their heterozygosity level, we can conclude that our results were not limited or strongly biased by our sample size.

The interpopulational differentiation analysis through allozyme and microsatellite loci revealed that populations are genetically homogeneous (Fst values are not significant when averaged over all loci). However, significant pairwise Fst values were found (data not shown) in most of the comparisons between Bertioga and the other populations, which was expected because this sample was different in several ways. It was collected using attractive compounds, so only males were captured. This site was in an area situated in the Atlantic forest edges, not an urban environment, and was situated outside the transect. Although the sample from the Bertioga population was included only as a reference to the populations of the transect, its inclusion did not produce significant population structuring, which confirms the strong gene flow due to the high dispersion of euglossine bees (Janzen, 1971; Dressler, 1982; Dick et al., 2004; Roubik and Hanson, 2004).

Allozyme and microsatellite data also showed that E. cordata diploid males were rare within the populations sampled. The data described here and in our previous studies (Takahashi et al., 2001; Souza et al., unpubl. data) identified only one diploid among about 300 males of E. cordata analysed and only three diploid males out 1500 males of 26 species of four Euglossini genera analysed. Our results are very different from data reported in other allozyme studies (Roubik et al., 1996; Zayed et al., 2004; López-Uribe et al., 2007). If these differences are real, the diploid male frequencies found must be attributed to particular biological and genetic traits and historical and demographical features of each population studied and further research is required to identify them.

The few studies on population genetics of Euglossini populations have been performed on samples collected from forests or forest fragments (Zayed et al., 2004; Sofia et al., 2005; Suzuki et al., 2010); this is the first one in which urban populations were analysed. As urbanisation causes environmental fragmentation (Niemelä, 2000; Cane, 2005; Zanette et al., 2005), it is expected that urban populations would be under a stronger genetic drift and exhibit reduced population effective size, conditions that favour matings between relatives, allelic loss in the sexual locus (csd) and diploid male production (Cook and Crozier, 1995; van Wilgenburg et al., 2006). Our data indicate that, although E. cordata populations are subject to the damaging effects of human actions, their genetic structure suggests an unexpected degree of genetic health and resistance to these effects.

Data shown here give a positive diagnosis about the genetic health of these E. cordata populations. This does not mean that environmental issues relating to conservation concerns over the decline of pollinators do not deserve the attention that has been given to them. If this is the genetic condition of other Euglossini species, this issue will certainly be addressed when similar studies are conducted with other euglossine species.

There are at least three reasons to believe that our analysis is robust: (i) the number of individuals and samples analysed; (ii) the number of microsatellite loci under analysis and (iii) the similar results obtained when microsatellite data were analysed together or separately in groups of loci with greater or lesser heterozygosity levels. Therefore, we can say that these E. cordata populations have high genetic diversity, usually are in genetic equilibrium, have low population structuring and have rare diploid males; consequently, high gene flow and effective population size (Ne) can be inferred.

Our results are in agreement with reports that these bees fly long distances (Janzen, 1971) and with phylogeographic evidences of long-distance dispersal of South American Euglossini species (Dick et al., 2004), and they reveal a different picture than what has been described by other authors (Zayed et al., 2004; Zayed and Packer, 2005). The observed low differentiation among populations may be primarily dependent on the male ability to disperse, if we take into account the suggested philopatric behaviour of females (Garófalo, 1992; Santos and Garófalo, 1994; Cameron and Ramírez, 2001; Augusto and Garófalo, 2009). Sex-asymmetrical dispersal in Euglossini species is a matter that should be investigated by comparing genetic differentiation among populations from nuclear genes (inherited from both parents) and mitochondrial genes (inherited from one parent).

Acknowledgments

We thank the FAPESP agency for the fellowship to NCMC (2006/59387-8) and the financial support to this project (2004/15801-0). Thanks are also due to Isabel C. Godoy for technical help, to Norma Mortari for laboratory setting courtesy and to Camilla H. da Silva, Juliano C. Almeida and Otávio L. e Silva for help in sampling. We gratefully acknowledge two anonymous referees for their comments and suggestions.

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All Tables

Table I

Data on samples of Euglossa cordata, including sampling sites and their geographical coordinates and codes, number (N) of bees (M = males and F = females) captured, and number of individuals analyzed for allozymes (alloz) or microsatellites (micrst) loci.

Table II

Number of alleles (A) and observed heterozygosity (Ho) at allozyme and microsatellite loci in Euglossa cordata populations from Rifaina (Rif), Pedregulho (Ped), Franca (Fra), Ribeirão Preto (Rpr), Jaboticabal (Jab), Araraquara (Ara), São Carlos (Sca), Leme (Lem), Rio Claro (Rcl), Piracicaba (Pir) and Bertioga (Ber). N = number of individuals (females + males). Values in bold indicate populations with significant inbreeding coefficient (Fis).

Table III

Genetic differentiation (Fst) among populations measured by the distance method (Weir and Cockerham, 1984) viewed through the Est-1 and cMdh loci or through nine microsatellite loci. Average values over all allozyme (Mean A) or microsatellite loci (Mean M) are also estimated considering all populations (A) or excluding the samples from Bertioga (B). Significant values of Fst are denoted with bold characters.

Table IV

Analysis of Molecular Variance (AMOVA) using data from microsatellite loci with higher (A) or lower (B) heterozygosity levels and all loci considered together, showing the distribution of the variation within and among populations. Significant values (P < 0.05) are denoted with bold characters.

All Figures

thumbnail Figure 1

Map of the state of São Paulo showing the localities inside urban areas where males and females of Euglossa cordata were sampled. The sampling sites (codes) were described in the legend of Table I.

In the text