Novel Microsatellite Loci, Cross-Species Validation of Multiplex Assays, and By-Catch Mitochondrial Genomes on Ochthebius Beetles from Supratidal Rockpools

SIMPLE SUMMARY: Supratidal rockpools stand out as some of the most extreme environments on the planet. These pools are exposed to the wave’s action, fluctuations in sea level, dramatic shifts in water temperature, high salinity levels, desiccation, and intense sunshine. As a result, only a handful of species can survive there, with aquatic beetles of the genus Ochthebius being the insects that dominate this kind of habitat on the Mediterranean coast. In this work, we genetically analysed four species (O. lejolisii, O. subinteger, O. celatus, and O. quadricollis) found along the Mediterranean coasts of Spain and Malta to develop microsatellite markers, which are repetitive DNA motifs, for the first time for this genus. Additionally, we obtained the complete mitochondrial genome of three species (O. lejolisii, O. subinteger, and O. quadricollis). These newly developed microsatellite markers and mitochondrial genomes for Ochthebius will be valuable in future studies for evolutionary and ecological research on the diversity of this genus: identification, genetic structure, population connectivity, etc. ABSTRACT: Here we focus on designing, for the first time, microsatellite markers for evolutionary and ecological research on aquatic beetles from the genus Ochthebius (Coleoptera, Hydraenidae). Some of these non-model species, with high cryptic diversity, exclusively inhabit supratidal rockpools, extreme and highly dynamic habitats with important anthropogenic threats. We analysed 15 individuals of four species (O. lejolisii, O. subinteger, O. celatus, and O. quadricollis) across 10 localities from the Mediterranean coasts of Spain and Malta. Using next-generation sequencing technology, two libraries were constructed to interpret the species of the two subgenera present consistently (Ochthebius s. str., O. quadricollis; and Cobalius, the rest of the species). Finally, 20 markers (10 for each subgenus) were obtained and successfully tested by cross-validation in the four species under study. As a by-catch, we could retrieve the complete mitochondrial genomes of O. lejolisii, O. quadricollis, and O. subinteger. Interestingly, the mitochondrial genome of O. quadricollis exhibited high genetic variability compared to already published data. The novel SSR panels and mitochondrial genomes for Ochthebius will be valuable in future research on species identification, diversity, genetic structure, and population connectivity in highly dynamic and threatened habitats such as supratidal coastal rockpools.