MOLECULAR ECOLOGY

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MOLECULAR ECOLOGY

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Characterization of 12 polymorphic microsatellite markers for a facultatively eusocial sweat bee (Megalopta genalis)

KAREN M. KAPHEIM,* JOHN P. POLLINGER,* WILLIAM T. WCISLOt and ROBERT K. WAYNE*

*Department of Ecology and Evolutionary Biology, 621 Charles E. Young Dr. South, University of California, Los Angeles, CA 90095 USA, iSmithsonian Tropical Research Institute, Box 0843-03092, Balboa, Ancon, Republic of Panama

Abstract

We developed a library of twelve polymorphic di- and tri-nucleotide microsatellite markers for Megalopta genalis, a facultatively eusocial sweat bee. We tested each locus in a panel of 23 unrelated females and found 7-20 alleles per locus. Observed and expected heterozygosities ranged from 0.65 to 0.96 and from 0.69 to 0.95 respectively. None of the loci deviated from Hardy-Weinberg equilibrium proportions or was found to be in gametic disequilibrium.

Keywords: eusociality, Halictidae, Megalopta genalis, microsatellite, molecular marker, relatedness Received 24 December 2008; revision accepted 26 February 2009

Megalopta genalis (Family: Halictidae) is a nocturnal, stem-nesting bee found throughout Panama and north- ern Colombia (Moure & Hurd 1987). Approximately, 25-50% of foundresses have eusocial nests, with some of the female progeny staying as workers, while other foundresses remain solitary, with all offspring dispersing (Arneson & Wcislo 2003; Smith et al. 2003, 2007; Wcislo et al. 2004; Wcislo & Gonzalez 2006). Females that remain on their natal nests are usually subordinate, performing most of the foraging (Wcislo & Gonzalez 2006; Smith et al. 2007). As Megalopta express differences in social behaviour at the individual level, they are ideal for inves- tigating the behaviour genetics of eusociality (Schwarz et al. 2007).

We collected Megalopta genalis from natural nests on Barro Colorado Island, Republic of Panama. Genomic DNA was extracted from the thorax of each bee by first homogenizing tissue in PBS buffer, and using a QiaAmp DNA Mini-Kit (QIAGEN) according to the manufac- turer's tissue extraction protocol.

We isolated microsatellites following the enrichment protocol developed by Bardeleben et al. (2004). We digested 20 ug of DNA from three individuals with Sau3Al (Promega) and then ligated oligo A and B adap- tors (Refseth et al. 1997). DNA in the 0.5-1.5 kb range was excised from a 1% agarose/TAE gel and purified with a Zymoclean kit (Zymogen). We pooled ligated-DNA, and 1 ug was hybridized overnight to biotin-labelled oligo probes (CT or CTT motifs, 5'-[Motif]8GTGA[Biotinyl-T)

Correspondence: Karen M. Kapheim, Fax: (310) 206 3987; E-mail [email protected]

0-3'), as previously described (Bardeleben et al. 2004). The hybridization reaction was incubated with streptavidin- coated magnetic beads (M-280 Dynabeads, Invitrogen).

After washing, DNA was denatured from the beads in 99 °C H20 and PCR amplified using Oligo A as a primer.

We used 25 uL of this PCR product in a second round of hybridization with identical conditions.

We cloned enriched DNA using a TOPO TA pCR 2.1 Cloning Kit (Invitrogen). Plasmids were isolated from transformed colonies using a MiniPrep kit (QIAGEN) and sequenced with M13 primer (Boutin-Ganache et al. 2001), using an Applied Biosystems (ABI) BigDye Terminator 3.1 Cycle Sequencing kit, on an ABI 3730x1 sequencer.

Plasmid sequences were edited in BioEdit 7.0.9.0 (Hall 1999). Out of 104 isolated plasmid sequences, primers were designed for 25, using Primer 3 (http://frodo.wi.

mit.edu/). Each primer set was screened in 18 bees from four family groups. We used an average of 67 ng (7.4-492.3) of genomic DNA in a 10 uL PCR reaction containing 2 x Multiplex Mix (QIAGEN), 0.4 mg/mL BSA, 0.1 |JM forward primer with M13 sequence added to the 5' end, 0.1 UM 6-FAM dye-labelled M13 primer (ABI) and 2 UM reverse primer. The step-down PCR program began with denaturing at 95 °C for 15 min, followed by 25 cycles of 94 °C for 30 s, Tal (Table 1) for 1.5 min, and 72 °C for 1 min. This was followed by 20 cycles of 94 °C for 30 s, Ta2 for 1.5 min, and 72 °C for 1 min, and a final extension at 60 °C for 30 min. PCR products were geno- typed on an ABI 3730XL sequencer with LIZ500 size stan- dard. Alleles were scored using GeneMapper 4.0 (ABI).

Twelve loci successfully amplified and were polymorphic within and across family groups. These were screened in a set of 23 unrelated females. Imperfect, but

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PERMANENT GENETIC RESOURCES NOTE 1529 reliable alleles were detected at three loci (mg22, mg29,

mg56). We inspected each locus for deviation from Hardy-Weinberg equilibrium proportions in GENEPOP 4.0 (Rousset 2008), using a Bonferroni correction for multiple comparisons (Rice 1989). Expected heterozygosity and observed heterozygosity were determined with Cervus 3.0 (Kalinowski et ah 2007) and were found to range from 0.69 to 0.95 and 0.65 to 0.96, respectively. Gametic disequilibrium or deviation from Hardy-Weinberg equilibrium proportions was not detected for any loci (Table 1). These primers are currently being used in a sociogenetic study of Megalopta genalis.

Acknowledgements

We thank Adam Smith, Margarita Lopez-Uribe, Julian Medina- Gutierrez, Dyana LaRosa, Ricardo Cossio, & Michael Reiser for field assistance, and the Autoridad Nacional del Ambiente of the Republic of Panama for permission to export specimens.

Partial field work support was provided by general research funds from the Smithsonian Tropical Research Institute (WTW), a Smithsonian Tropical Research Institute Short-term Fellowship (KMK), a U. S. Department of Education GAANN fellowship (KMK), and a Holmes O. Miller Fellowship from UCLA EEB (KMK).

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doi:10.1111/j.l755-0998.2009.02693.x