Molecular Ecology Notes (2007) 7, 607–609 doi: 10.1111/j.1471-8286.2006.01647.x © 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd Blackwell Publishing Ltd PRIMER NOTE PCR primers for polymorphic microsatellite loci in the plant-ant Azteca ulei cordiae (Formicidae: Dolichoderinae) GABRIEL D. G. DEBOUT,* MARJOLAINE VENTELON-DEBOUT,† BRENT C. EMERSON* and DOUGLAS W. YU* *Evolution and Conservation, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK, †Disease and Stress Biology, John Innes Centre, Colney, Norwich NR4 7UH, UK Abstract Twelve microsatellite loci were isolated from the Peruvian tropical plant-ant, Azteca ulei cordiae (Hymenoptera: Dolichoderinae). High levels of within-population variation were observed at most loci, with number of alleles ranging from four to 18, and heterozygosity from 0.118 to 1 per population sample. Cross-species amplification of these loci was also successfully tested in several other species of the same ant genus Azteca. Keywords: Azteca, Dolichoderinae, microsatellites, plant-ant relationship, social insect Received 30 September 2006; revision accepted 6 November 2006 Azteca ulei cordiae (Dolichoderinae) is the primary ant mutualist of the most abundant ant-plant in southeast Peru, the understorey myrmecophyte Cordia nodosa Lam. (Boraginaceae). Workers of A. u. cordiae protect new leaves and their associated domatia from herbivory, as do workers of several other species in the same genus Azteca that are strictly associated with this particular plant. However, the most frequent occupant of C. nodosa is the castration parasite Allomerus octoarticulatus cf. demerarae (Myrmicinae), whose workers destroy flowers, reducing fruit production to zero in most host plants. Consequently, the C. nodosa-ant system appears to persist only because of the small minority of plants that in each generation are fortunate enough to associate with one of the truly mutualistic Azteca symbionts (Yu & Pierce 1998; Yu et al. 2001). Microsatellite loci were isolated from A. u. cordiae to assess mating system and population genetic structure in the different Azteca species inhabiting the Cordia plants, and also to test predictions emerging from a coexistence model previously proposed to explain the dynamics of this particular system (Yu & Wilson 2001; Yu et al. 2004). Microsatellites were isolated using a fast and cheap pro- tocol, modified from Hamilton et al. (1999) by T.C. Glenn (Savannah River Ecology Laboratory, University of Georgia, USA). The detailed protocol of microsatellite isolation is available at request from us, or from T.C. Glenn (glenn@sre1.edu). Briefly, total genomic DNA was extracted from two pools of four adult queens each using DNeasy Tissue Kit (QIAGEN). A partial enriched genomic library was then constructed by, first, simultaneously digesting DNA with RsaI and ligating 500-bp digested DNA with double stranded SNX linkers (Hamilton et al. 1999). Then, enrichment was made using a mixture (modified from Hamilton) of 3′-biotinylated oligonucleotide probes (AC) 12 (TGTA) 6 (GATA) 6 (ATCT) 6 : ligated DNA was denatured and hybridized to the probes, which were then captured on streptavidin-coated paramagnetic beads (Dynal). DNA unhybridized to the captured DNA was washed away and the remaining DNA eluted from the beads. Enriched DNA was then ligated into a pCR TOPO Vector (Invitrogen) and plasmid ligations were transformed into One Shot TOP 10 Chemically Competent Escherichia coli (Invitrogen), following the procedures of the Invitrogen’s TOPO TA Cloning Kit exactly. A total of 384 white transformant clones were hybridized with (AC) 12 and 192 of these clones (50%) gave a positive signal. The inserted DNA fragment was sequenced in all of these positive clones. Twenty primer pairs were designed using the oligo 3.3 software (Rychlik & Rhoads 1989), among which 12 gave satisfactory amplification patterns [i.e. a polymerase chain reaction (PCR) product of the predicted size, and no supernumerary bands]. PCR amplifications were carried out in a 10-μL final volume, which contained 2 μL extracted DNA (approxi- mately 50 ng of DNA), 0.24 μL 50 mm MgCl 2 , 0.3 mL 2.5 mm dNTP, 1 U BioTaq DNA polymerase (Bioline), 1 μL Correspondence: Gabriel D. G. Debout, Fax: +44 (0)1603 592250; E-mail: g.debout@uea.ac.uk