Characterization of microsatellite loci in the greater horseshoe bat Rhinolophus ferrumequinum

A full understanding of colony organization and life histories in social insects can only be achieved by investigating the genetic structure of colonies and populations (Pamilo et al . 1997). Although most genetic studies on ants have been performed on species with complex colony organization (but see Tay et al . 1997), the study of morphologically primitive ants, characterized by limited caste dimorphism and small colony size, could provide new insights into the evolution of sociality (Peeters 1997). In some morphologically primitive species of Ponerine ants, the queen caste has been lost and one worker mates (the gamergate) and produces diploid offspring (Peeters 1991). In these queenless species the foundation of new colonies occurs only by fission. Even in such a relatively simple social organization, polymorphic genetic markers will be required to estimate the rate of gamergate turnover and colony fission, the extent of population viscosity and their effects on the evolution of sociality. In this note, we characterize eight polymorphic microsatellite markers from the queenless species Diacamma cyaneiventre . Microsatellite markers have been characterized in a number of groups of ants (e.g. Chapuisat 1996; Herbers & Mouser 1998), but none are available for ponerine ants. We also report results of cross-amplification on 11 Ponerine species (seven Diacamma species and four belonging to other genera of Ponerinae). Genomic DNA from nine Diacamma cyaneiventre larvae was extracted by a high-salt procedure using NaCl and digested with the BSP 143I restriction enzyme. Fragments between 300 and 600 bp were selected and ligated to a Bam HIdigested pBluescript II KS+ vector (Stratagene) and cloned in Escherichia coli SL-1 Blue cells (Stratagene). Synthetic oligonucleotides (TC) 10 and (TG) 10 , labelled with the DIG system (Boehringer Mannheim) were used to screen about 1200 recombinant colonies. Out of 146 positive clones, 78 were purified with Qiaprep (Qiagen) and 24 were sequenced either manually using the T7-sequencing kit (Pharmacia) or an ALF express automatic sequencer (Pharmacia). Primers flanking microsatellite repeats were designed for 10 loci using Primer 3 software (Rozen & Skaletsky 1996). Genomic DNA was prepared following either a classic phenol/ chloroform or high-salt extraction and diluted to 1/5 before amplification. PCR reaction mixtures (10 μ L final volume) contained 50 ng of template DNA, 75 μ m dCTP, GTP and dTTP, 7.5 μ m dATP, 0.025 μ Ci 33 P-dATP (Amersham), 4 pmoles each primer, 1 × Taq buffer (containing 1.5 m m MgCl 2 ) and 0.25 U Taq DNA polymerase (Qiagen). For the D18 locus, the amount of dNTP, 33 P-dATP and Taq DNA polymerase was doubled to allow a better amplification of the longest allele in heterozygote individuals. Amplifications were performed in a PTC-100 (MJ Research) thermal cycler using the following parameters: 3 min at 94 ° C followed by 30 cycles with 30 s at 94 ° C, 30 s at the annealing temperature (see Table 1) and 1 min at 72 ° C, and a final elongation step of 10 min at 72 ° C. Amplification products were run on 6% polyacrylamide sequencing gels using an M13 sequence as a size marker. All 10 microsatellite loci gave repeatable and scorable patterns. An estimate of the variability of these loci was determined using a sample of 45 unrelated workers. Eight loci were polymorphic (Table 1). The expected heterozygosity ranged from 0.39 to 0.95, the highest diversity being observed for an uninterrupted microsatellite showing the largest number of repeats (29). No significant deviations from Hardy– Weinberg proportions were detected using a subsample of 33 individuals collected in a locality near Kotigehara (Table 1). Results of the cross-amplification are shown in Table 2. PCR conditions were as described above except that the annealing temperature was 52 ° C for all primers. Where only one individual was extracted, DNA quality was confirmed using mitochondrial DNA primers known to amplify on these species (data not shown). Within the genus Diacamma , cross-species amplification was successful with an average of six pairs of primers amplifying per species. For D. ceylonense and D. sp. (nilgiri) from India, nine individuals were screened to check for polymorphism. Four microsatellites were polymorphic in D. ceylonense and three in D. sp. (nilgiri) . When used on different genera, the efficiency of amplification was low even though these genera belong to the same tribe as Diacamma . Our results may therefore indicate a recent common ancestor for the Diacamma species studied. In conclusion, given their level of polymorphism, the primers presented here should prove to be very useful for investigating population and colony genetic structure of different Diacamma species.