Molecular Ecology Notes (2007) 7, 144–146 doi: 10.1111/j.1471-8286.2006.01557.x © 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd Blackwell Publishing Ltd PRIMER NOTE Characterization of 17 polymorphic microsatellite loci in the Anise swallowtail, Papilio zelicaon (Lepidoptera: Papilionidae), and their amplification in related species EVGUENI V. ZAKHAROV and JESSICA J. HELLMANN Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA Abstract Fifteen polymorphic dinucleotide and two trinucleotide microsatellite loci were identified in the Anise swallowtail, Papilio zelicaon, from DNA genomic libraries enriched for simple sequence repeats. Allele numbers varied from eight to 29, with an excess of homozygotes observed for nine loci. This homozygosity is a feature of other lepidopteran microsatellites and is probably due to null alleles. Sixteen markers were amplified successfully in other representatives of Papilio with 11 loci retaining polymorphism in at least one species. These results suggest that the microsatellites reported here may be appropriate for measuring population genetic structure in a number of Papilio species. Keywords: codominant marker, microsatellite DNA, Papilio, simple sequence repeat, swallowtail butterflies Received 6 June 2006; revision received 7 July 2006; accepted 8 August 2006 Simple sequence repeats (SSR), or microsatellite DNA, have become the marker of choice in population genetic studies because they are polymerase chain reaction (PCR)-based, highly reproducible, very polymorphic, generally codominant, and mostly neutral (Goldstein & Schlötterer 1999). Here we present a number of polymorphic microsatellite loci in Papilio zelicaon that are suitable for population genetic analysis in this species and in other Papilio . These markers are the first to be reported for this widely distributed genus, a group that has been used extensively in ecological and evolutionary studies (Scriber et al . 1995). We developed these microsatellites to examine population structure and describe gene flow patterns across the geographical distribution of P. zelicaon . The development of SSR markers in Lepidoptera has been reported to be rather challenging (Zhang 2004) due either to a low frequency of microsatellites in lepidopteran genomes ( Ji et al . 2003; but see Prasad et al . 2005) or to high similarity between flanking regions of different microsatellite loci (Meglécz et al . 2004). Given this challenge, however, the total number of identified microsatellites in Lepidoptera is growing steadily. We are aware of markers that have been produced for over 40 species in 34 genera from 15 families of butterflies and moths (e.g. Prasad et al . 2005; Zeisset et al . 2005; Mavárez & González 2006). Genomic libraries enriched for microsatellite motifs were constructed by the Genetic Identification Services (GIS, http://www.genetic-id-servicies.com). Libraries were built using a pooled sample containing 100 μ g of genomic DNA extracted from the thorax muscles of six males following a standard organic procedure (Sambrook et al . 1989). Enrich- ments were performed for four types of repeats, and the resulting libraries yielded microsatellites as follows: (i) 49 out of 57 sequences contained a microsatellite for the CA motif; (ii) 32 out of 46 for the GA motif; (iii) 30 out of 45 for the CAG motif; and (iv) one out of nine for the ACC motif. From a total of 112 microsatellite-containing clones sequenced by GIS using universal M-13 primers (forward: 5 ′ -AGGAAACAGCTATGACCATG-3 ′ and reverse: 5 ′ - ACGACGTTGTAAAACGACGG-3 ′ ), only 68 had flanking sequences of length sufficient for primer design using designerpcr version 1.03 (Research Genetics). Amplifica- tion reactions were carried out in a total volume of 20 μ L containing 2 μ L of 10 × PCR buffer, 2–2.5 m m MgCl 2 , 0.2 nm dNTP, 0.5 U Taq DNA polymerase (Promega), 5–10 pm of each primer, and approximately 2 ng of template DNA. Application was performed using the following temperature profile: 3 min at 94 ° C; followed by 40 cycles of 40 s at Correspondence: Jessica J. Hellmann, Fax: (574) 631-7413; E-mail: hellmann.3@nd.edu