JHR 30: 7-11 (2013) ore JOURNAL OF | *0eerrrieved opencoss ural doi: 10.3897/JHR.30.3870 SHORT COMMUNICATION MEE Hymenoptera www.pensoft.net/journals/jhr The iternatonl Scie of Hymenopeariss, RESEARCH Characterization of microsatellite loci for the stingless bee Scaura latitarsis (Hymenoptera, Apidae, Meliponini) Flavio O. Francisco', Alayne M. T. Domingues-Yamada', Paulo Henrique P. Gongalves', Rute M. Brito'”, Fabiana C. Pioker?, Sidnei Mateus*, Maria C. Arias! | Departamento de Genética e Biologia Evolutiva, Instituto de Biociéncias, Universidade de Séo Paulo, Rua do Matio 277 — sala 320, CEP 05508-090, Sto Paulo, SP Brazil2 Instituto de Genética e Bioquimica, Uni- versidade Federal de Uberlindia, Av. Paré 1720 — 2E, sala 246, CEP 38400-902, Uberlandia, MG, Brazil 3 Departamento de Ecologia, Instituto de Biociéncias, Universidade de Séo Paulo, Rua do Matio, travessa 14, n° 321, CEP 05508-900, Sao Paulo, SP Brazil 4 Departamento de Biologia, Faculdade de Filosofia Ciéncias e Letras de Ribeiriio Preto, Universidade de Sto Paulo, Av. Bandeirantes 3900 - Bloco 7, CEP 14040-901, Ribeiriio Preto, SP Brazil Corresponding author: Maria C. Arias (mcarias@ib.usp.br) Academic editor: Jack Neff | Received 18 August 2012 | Accepted 5 November 2012 | Published 30 January 2013 Citation: Francisco FO, Domingues-Yamada AMT, Goncalves PHP, Brito RM, Pioker FC, Mateus S, Arias MC (2013) Characterization of microsatellite loci for the stingless bee Scaura latitarsis (Hymenoptera, Apidae, Meliponini). Journal of Hymenoptera Research 30: 7-11. doi: 10.3897/JHR.30.3870 Abstract 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. Keywords Microsatellites, Scaura latitarsis, genetic variability, Meliponini Introduction 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, Copyright Flavio O. Francisco et al. 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. 8 Flavio O. Francisco et al. / Journal of Hymenoptera Research 30: 7-11 (2013) 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 S. /atitarsis populations should be highly differentiated. In general, population studies apply combined molecular markers of nuclear and cytoplasm origins. Mito- chondrial 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 contribu- tion, especially from males when mitochondrial data are also available (e.g. Estoup et al. 1996, Franck et al. 2001, Quezada-Euan 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 S. 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 trans- formed Escherichia coli DH5« lineage, 48 were sequenced and 25 contained micros- atellite 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 S. /atitarsis from seven dif- ferent locations in Brazil: Ribeirao Preto, SP (n = 2), Cajuru, SP (n = 2), Pedregulho, SP (n = 2), Ribeirao 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 wl containing 2 uL of the PCR reaction buffer (Invitrogen), 1 pL of dNTP 2 mM each, 0.6 pL of MgCl, 50 mM, 0.4 uL of each primer 20 uM, 15 ng of template DNA and 0.3 pL of Tag DNA polymerase 5U/puL (Invitrogen). PCR condi- tions 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% polyacryla- mide gels and stained with silver nitrate. Allelic richness (&), observed (H/,) and expect- ed (H/,) 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 H, ranged from 0.000 to 0.750 (mean = 0.405) and #7, 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 disequi- librium was detected after Bonferroni correction between SLAT 16/SLAT 24, SLAT 24/SLAT 43, and SLAT 40/SLAT 43. Deviations from HWE and linkage disequilib- rium are expected when individuals are sampled from genetically differentiated popula- tions (Brito et al. 2009). Characterization of microsatellite loci for the stingless bee Scaura latitarsis... 9 Using the same conditions aforementioned we tested the primers for the polymor- phic 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 S. Jongula and S. 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 S. /atitarsis 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 I. Summary of general information for the microsatellite loci isolated from Scaura latitarsis. k: number of alleles; ASR: allele size range (bp); H,: observed heterozygosity; H,,: expected heterozygosity. Genbank Locus | Repeat motif | accession Primer sequences (5’-3”) H, | A, number F: TGAATTAACAGGCCGGAATC REL TCG TEGCELCeCrerrrar F: AAACGAAGGACAGACGTTGG SEAT 6)’ €AG)N. | I QOOIG 7. |e see ect CACCOCETCTTE |. (Taoat72| 01000 10:736 F: GGATCGTCGAACGGAATATC SLAT 18] (AG), TQOOTAO8. | re reat CMT COTCACLEG. (0 [eo et 0200 (ODS Be CGAACCAGT GTEC IGAA LEG T SLAT 24 (GAA), JQ001769 Re GCCTCGAACTCTCGCGCTAT 6 | 184-202] 0.500 | 0.715 F: GY TCCACACCGGAAGACC R: GTGAACGAGCCTCTGCAATC F: GCGGAATTAACAACGGACAT SEATS) (AG) | TQOOUZZ2:) co capa ORA TAMA: foi Eeo in| 0:83" O19 F: GGCATAGGTTACGATTTGGAG SLAT 44] (GA),,(GAA), | JQ001773 R: CTGGCACTCAACGTGTTAGG 3 |222—230| 0.750 | 0.601 Table 2. Cross-species amplification of 7 microsatellite loci from Scaura latitarsis in S. atlantica, S. lon- SLAT 10] (AG), ,TA(AG), | JQ001766 183-197] 0.417 | 0.694 SLAT 40) (GA),AA(GA),, | JQ001771 200-214] 0.333 | 0.653 gula, S. tenuis, Schwarzula timida, Schwarziana quadripunctata, Plebeia droryana, and Plebeia remota. (+): successful amplification; (-): no product or multiple bands. ee Scaura Scaura Scaura Schwarzula Schwarziana Plebeia Plebeia atlantica | longula tenuis timida | quadripunctata| droryana | remota SLAT 10 + + + SLAT 16 + - - SLAT 18 - + + SLAT 24 + + + SLAT 40 - + + SLAT 43 + + - SLAT 44 + + + 10 Flavio O. 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