Trinucleotide microsatellite loci for a social wasp, Polistes

that build open-faced paper nests in which offspring are reared. The lack of distinct morphological castes and great plasticity in social behaviour among females, combined with the ease of observing individually paintmarked adults, has made this a model genus for behavioural studies (Reeve 1991; Turillazzi & WestEberhard 1996). All species are eusocial, but at a relatively rudimentary level: colonies are relatively small (Reeve 1991), workers are not consistently different from queens (Haggard & Gamboa 1980), social relations are often characterized by strife (West-Eberhard 1969; Strassmann 1981a; Noonan 1981), and the queen in some species must act as the pacemaker, physically manipulating workers into working (Reeve & Gamboa 1983). Polistes has served as a model system for a wide variety of topics including dominance hierarchies (WestEberhard 1969), advantages of group living (Strassmann et al. 1988; Strassmann & Queller 1989; Strassmann 1991), kin selection (Noonan 1981; Strassmann 1981b; Queller & Strassmann 1988), kin recognition (Gamboa et al. 1987), usurpation (Klahn 1988), and social control by queens (Reeve & Gamboa 1983). To date, it has been difficult to combine detailed studies of behavioural interactions with studies of genetic relatedness because of the difficulty in obtaining precise estimates of relatedness for individual pairs of interactants with the available genetic markers (e.g. Strassmann et al. 1989). Clearly, a more polymorphic set of markers is needed. Microsatellite loci are highly polymorphic, codominant, and can be genotyped from many sources of DNA including alcohol-preserved tissues and sperm in a female’s spermatheca (Evans 1993; Queller et al. 1993; Peters et al. 1995). Here we present 18 new microsatellite loci derived from Polistes bellicosus and eight new loci from Polistes annularis. All of these loci contain trinucleotide repeat regions which are much easier to score unambiguously than are dinucleotide repeats. To obtain these loci we constructed very large partial genomic libraries in plasmids with inserts (not enriched for microsatellites) averaging 500 bp (Hughes & Queller 1993; see Strassmann et al. 1996, for the rationale of our approach and specific protocols). We separately probed replicate membranes with synthesized oligonucleotides containing 10 or 12 repeats of either AAT, AAC, AAG, CAT or TAG, five of the 10 possible trinucleotide repeats. We picked up hundreds of potential positives. Reprobing a Southern blot of the inserts indicated that most of these sequences contained repeats. We sequenced part or all of about 80 clones of P. bellicosus. Of these, 59 contained at least one repeat region. We designed polymerase chain reaction (PCR) primers for clones containing uninterrupted repeats, five or more repeats long for which we could read the flanking regions (Table 1). We did not design PCR primers around all repeats for reasons including the nature of the flanks, absence of sufficient flanking sequence, or because they were dinucleotide repeats, not the trinucleotides we were after. We evaluated these primers for heterozygosity on 3–24 unrelated individuals. Genomic DNA was prepared as described in Hughes & Queller (1993) or using protocol Strassmann.1 in Strassmann et al. (1996). PCR was carried out under oil in a 10 μL volume made up of 2 μL diluted genomic DNA (about a nanogram), 2 μL of primer mix (2.5 μM), 0.1 μL 10 mM dNTP mix, 1 μL 10× buffer (provided with Taq), 4.08 μL dH2O, 0.62 μl 25 mM MgCl2, 0.05 μL Taq polymerase (5 units/μL, Promega), 0.15 μL 35S dATP (12.5 μCi/μL). After an initial denaturing for 5 min at 95 °C, we carried out 30–35 cycles of 60-s denaturing at 92 °C, 60-s annealing (at a temperature optimized for the primers used: see Table 1) and 45-s extension at 72 °C. After that, 5 extra minutes at 72 °C allowed for the completion of the extension. PCR products were run on 6% denaturing acrylamide gels (Strassmann et al. 1996). All of the 18 microsatellite loci proved to be polymorphic in P. bellicosus though heterozygosity varied from 0.05 to 1 (Table 1). Seven of the eight loci from P. annularis proved to be polymorphic, with heterozygosities ranging from 0.17 to 1 (Table 1). We have published other microsatellites for Polistes annularis and for another social wasp, Parachartergus PRIMER NOTE