Development of microsatellite loci for two Agabus diving beetle species from the pooled DNA and testing their utility in mountain lake populations

Diving beetles (Dytiscidae) are the most speciose water beetle group, occurring on all continents except Antarctica. They inhabit various lotic and lentic habitats and play an important role in ecosystem functioning. In this study, we developed functional polymorphic microsatellites for two widely distributed species from one of the most diversified dytiscid genus Agabus: Agabus bipustulatus (Linnaeus, 1767) and Agabus cf. guttatus from the Agabus guttatus group sensu Foster and Bilton (1997). For microsatellites development, pooled DNA and NGS pyrosequencing were used. Microsatellites are still very useful genetic markers for studying recent population changes, but for dytiscids, highly diversified and ecologically important group of freshwater invertebrates, only 8 microsatellite loci are available for one North American species. To test utility of the developed markers, we used several populations of alpine lakes, situated in the Tatra Mountains (Western Carpathians). From the 60 tested markers, 13 loci for Agabus bipustulatus and 8 loci for A. cf. guttatus showed polymorphism. The number of alleles per locus varied from 2 to 10 and no significant linkage disequilibrium was observed. Observed/expected heterozygosity varied from 0/0.077 to 1/0.834 within populations of A. cf. guttatus and from 0/0.056 to 1/0.837 within populations of A. bipustulatus. The significant deviation from HWE was probably caused by presence of null alleles or undetected biological processes. Bayesian cluster analysis revealed differences in the cluster proportions, confirming applicability of the developed markers for future studies of population structure of both Agabus species.