Rapid and reliable method for identification of three medium-sized horseshoe bat species in Europe

Accurate species identification is crucial for ecological research and effective wildlife management. Advances in molecular genetic tools enable this, even for cryptic species complexes that are often morphologically confusing or indistinguishable. However, the costs of these methods and sequencing remain prohibitive for many researchers, particularly in less developed regions. The aim of this study was to test whether ISSR-PCR markers can be used to distinguish three similar horseshoe bat species that are often misidentified and occur in sympatry in Serbia. Samples from 64 Rhinolophus euryale, R. blasii, and R. mehelyi bats were identified using this ISSR-PCR approach, and species identifications were additionally confirmed by sequencing D-loop fragment of mitochondrial DNA. ISSR-PCR yielded species-specific band patterns on the agarose gel that allowed the differentiation of three medium-sized horseshoe bat species. This approach does not require sequencing, making it a quick and inexpensive tool for the genetic identification of these species, and complements already existing methods. There is potential to scale up this method to other cryptic species complexes, reducing misidentifications that lead to inaccurate population trend assessments and have knock-on effects on our ability to monitor and conserve wildlife, especially rare and endangered species.