Journal of Hymenoptera Research 30: 7–11, doi: 10.3897/JHR.30.3870
Characterization of microsatellite loci for the stingless bee Scaura latitarsis (Hymenoptera,  Apidae,  Meliponini)
Flávio O. Francisco 1, Alayne M. T. Domingues-Yamada 1, Paulo Henrique P. Gonçalves 1, Rute M. Brito 1,2, Fabiana C. Pioker 3,  Sidnei Mateus 4, Maria C. Arias 1
1 Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, Rua do Matão 277 – sala 320, CEP 05508-090, São Paulo, SP, Brazil
2 Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Av. Pará 1720 – 2E, sala 246, CEP 38400-902, Uberlândia, MG, Brazil
3 Departamento de Ecologia, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, travessa 14, nº 321, CEP 05508-900, São Paulo, SP, Brazil
4 Departamento de Biologia, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes 3900 - Bloco 7, CEP 14040-901, Ribeirão Preto, SP, Brazil

Corresponding author: Maria C. Arias (

Academic editor: Jack Neff

received 18 August 2012 | accepted 5 November 2012 | Published 30 January 2013

(C) 2013 Flávio O. Francisco. This is an open access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

For reference, use of the paginated PDF or printed version of this article is recommended.


Seven microsatellite loci were isolated and characterized from a microsatellite enriched genomic library for the stingless bee Scaura latitarsis. Primers were tested in 12 individuals. The number of alleles per locus ranged from 3 to 6 (mean = 4.29) and observed heterozygosity ranged from 0.00 to 0.75 (mean = 0.40). Cross-species tests showed successful amplification for Scaura atlantica, Scaura longula, Scaura tenuis, Schwarzula timida, Schwarziana quadripunctata, Plebeia droryana, and Plebeia remota.


Microsatellites, Scaura latitarsis, genetic variability, Meliponini


Scaura latitarsis is small (4.0 mm) and black stingless bee which nidifies in arboreal termite nests of Nasutitermes (Michener 2007). The species is widely distributed in tropical South America, with records in the following countries: Colombia, Venezuela, Guyana, Suriname, Peru, Bolivia, and Brazil (Camargo and Pedro 2012). The large geographic distribution and the well reported female philopatric behavior in stingless bees (Nogueira-Neto 1954, Brito and Arias 2010, Francisco and Arias 2010) suggest that Scaura latitarsis populations should be highly differentiated. In general, population studies apply combined molecular markers of nuclear and cytoplasm origins. Mitochondrial polymorphism is easily assessed due to universal primers already described (Simon et al. 1994). However, the same is not true for nuclear markers. Microsatellites have been the nuclear marker most used in population genetic studies. As they present Mendelian and biparental inheritance, it is possible to evaluate the parental contribution, especially from males when mitochondrial data are also available (e.g. Estoup et al. 1996, Franck et al. 2001, Quezada-Euán et al. 2011). For future population studies, our first goal was to construct a microsatellite-enriched genomic library to isolate and characterize microsatellite loci for Scaura latitarsis.

Total DNA of 12 workers was extracted using a phenol/chloroform protocol. The microsatellite-enriched genomic library was constructed according to Billotte et al. (1999) with some modifications (Brito et al. 2009). From 96 selected colonies of transformed Escherichia coli DH5α lineage, 48 were sequenced and 25 contained microsatellite sequences. Fifteen of these were chosen for primers design based on sequence quality. The software Primer3 was used (Rozen and Skaletsky 2000) to indicate the best primer sequences. Primers were tested on 12 workers of Scaura latitarsis from seven different locations in Brazil: Ribeirão Preto, SP (n = 2), Cajuru, SP (n = 2), Pedregulho, SP (n = 2), Ribeirão Cascalheira, MT (n = 2), Nova Xavantina, MT (n = 1), Manaus, AM (n = 2), and Londrina, PR (n = 1). Amplification reactions were conducted in a volume of 20 µL containing 2 µL of the PCR reaction buffer (Invitrogen), 1 µL of dNTP 2 mM each, 0.6 µL of MgCl2 50 mM, 0.4 µL of each primer 20 µM, 15 ng of template DNA and 0.3 µL of Taq DNA polymerase 5U/µL (Invitrogen). PCR conditions were the following: 93 °C for 4 min, 35 cycles of denaturation at 93 °C for 40 s, annealing at 60 °C for 50 s and elongation at 72 °C for 40 s. A final extension step of 72 °C for 5 min was performed. PCR products were separated on 5.6% polyacrylamide gels and stained with silver nitrate. Allelic richness (k), observed (HO) and expected (HE) heterozygosity were calculated in FOFPOP 2.0 (Francisco 2009). GENEPOP 4.1.4 (Rousset 2008) was used for Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium tests. Bonferroni corrections were used for multiple comparisons.

Seven out of 15 primer pairs generated fragments of expected size, and they were polymorphic (Table 1). The number of alleles ranged from 3 to 6, with an average of 4.29. Values of HO ranged from 0.000 to 0.750 (mean = 0.405) and HE from 0.531 to 0.736 (mean = 0.664). After Bonferroni correction, only the loci SLAT 16 and SLAT 40 showed significant deviation from Hardy-Weinberg-expectations. Linkage disequilibrium was detected after Bonferroni correction between SLAT 16/SLAT 24, SLAT 24/SLAT 43, and SLAT 40/SLAT 43. Deviations from HWE and linkage disequilibrium are expected when individuals are sampled from genetically differentiated populations (Brito et al. 2009).

Using the same conditions aforementioned we tested the primers for the polymorphic loci in seven stingless bee species: Scaura atlantica, Scaura longula, Scaura tenuis, Schwarzula timida, Schwarziana quadripunctata, Plebeia droryana, and Plebeia remota. Cross-species amplification was 100% efficient for SLAT 10 and SLAT 44 (Table 2). However those loci were the only ones amplified in Scaura longula and Scaura tenuis. At least five out of seven primer pairs were successfully amplified in species from three other genera.

The microsatellite loci described here will be very helpful to gather molecular data of Scaura latitarsis to understand the population structure of this widely distributed species. If queen philopatry is confirmed for this species, these microsatellite loci will allow an evaluation of male’s contributions as gene flow mediators.

Table 1.

Summary of general information for the microsatellite loci isolated from Scaura latitarsis. k: number of alleles; ASR: allele size range (bp); HO: observed heterozygosity; HE: expected heterozygosity.

Locus Repeat motif Genbank accession number Primer sequences (5’-3’) k ASR HO HE
Table 2.

Cross-species amplification of 7 microsatellite loci from Scaura latitarsis in Scaura atlantica, Scaura longula, Scaura tenuis, Schwarzula timida, Schwarziana quadripunctata, Plebeia droryana, and Plebeia remota. (+): successful amplification; (-): no product or multiple bands.

Locus Scaura atlantica Scaura longula Scaura tenuis Schwarzula timida Schwarziana quadripunctata Plebeia droryana Plebeia remota
SLAT 10 + + + + + + +
SLAT 16 + - - - - - -
SLAT 18 - - - + + + +
SLAT 24 + - - + + + +
SLAT 40 - - - + + + +
SLAT 43 + - - - - + -
SLAT 44 + + + + + + +

We thankSusy Coelho for technical support, Dr Anete P. de Souza for helping with the genomic library protocol, Fundação de Amparo à Pesquisa do Estado de São Paulo for PhD scholarships to FOF (2008/08546-4) and FCP, and for financial support (2004/15801-0; 10/50597-5), Conselho Nacional de Desenvolvimento Científico e Tecnológico for a PhD scholarship to AMTDY, and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior for master scholarship to PHPG. This work was developed in the Research Center on Biodiversity and Computing (BioComp) of the Universidade de São Paulo (USP), supported by the USP Provost’s Office for Research.

Billotte N, Lagoda PJR, Risterucci AM, Baurens FC (1999) Microsatellite-enriched libraries: applied methodology for the development of SSR markers in tropical crops. Fruits 54: 277–288.
Brito RM, Arias MC (2010) Genetic structure of Partamona helleri (Apidae, Meliponini) from Neotropical Atlantic rainforest. Insectes Sociaux 57: 413-419. doi: 10.1007/s00040-010-0098-x
Brito RM, Francisco FO, Domingues-Yamada AMT, Gonçalves PHP, Pioker FC, Soares AEE, Arias MC (2009) Characterization of microsatellite loci of Tetragonisca angustula (Hymenoptera, Apidae, Meliponini). Conservation Genetics Resources 1: 183-187. doi: 10.1007/s12686-009-9045-4
Camargo JMF, Pedro SM (2012) Meliponini Lepeletier, 1836. In: Moure JS, Urban D, Melo GAR (Eds) Catalogue of Bees (Hymenoptera, Apoidea) in the Neotropical Region.
Estoup A, Solignac M, Cornuet J-M, Goudet J, Scholl A (1996) Genetic differentiation of continental and island populations of Bombus terrestris (Hymenoptera: Apidae) in Europe. Molecular Ecology 5: 19-31. doi: 10.1111/j.1365-294X.1996.tb00288.x
Francisco FO (2009) FOFpop v.2.0, a pool of Microsoft Excel sheets to analyze genotypic data.
Francisco FO, Arias MC (2010) Inferences of evolutionary and ecological events that influenced the population structure of Plebeia remota, a stingless bee from Brazil. Apidologie 41: 216-224. doi: 10.1051/apido/2009079
Franck P, Garnery L, Loiseau A, Oldroyd BP, Hepburn HR, Solignac M, Cornuet JM (2001) Genetic diversity of the honeybee in Africa: microsatellite and mitochondrial data. Heredity 86: 420-430. doi: 10.1046/j.1365-2540.2001.00842.x
Michener CD (2007) The Bees of the World, 2nd ed. Johns Hopkins University Press, Baltimore, 992 pp.
Nogueira-Neto P (1954) Notas bionômicas sobre meliponíneos: III – Sobre a enxameagem. Arquivos do Museu Nacional 42: 419-451.
Quezada-Euán JJG, May-Itzá WJ, Rincón M, De La Rúa P, Paxton RJ (2011) Genetic and phenotypic differentiation in endemic Scaptotrigona hellwegeri (Apidae: Meliponini): implications for the conservation of stingless bee populations in contrasting environments. Insect Conservation and Diversity. doi: 10.1111/j.1752-4598.2011.00179.x
Rousset F (2008) genepop’007: a complete re-implementation of the genepop software for Windows and Linux. Molecular Ecology Resources 8: 103-106. doi: 10.1111/j.1471-8286.2007.01931.x
Rozen S, Skaletsky HJ (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (Eds). Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa: 365-386.
Simon C, Frati F, Beckenbach A, Crespi B, Liu H, Flook P (1994) Evolution, weighting, and phylogenetic utility of mitochondrial gene-sequences and a compilation of conserved polymerase chain-reaction primers. Annals of the Entomological Society of America 87: 651–701.