Development of species-specific real-time PCR assays for the identification of five European Rhinolophus bats

The detection and monitoring of bat species using non-invasive sampling and molecular techniques has become increasingly popular in recent years. In Europe, these approaches have been applied to identify horseshoe bats of the genus Rhinolophus , which includes five species: R. hipposideros, R. ferrumequinum, R. euryale, R. mehelyi and R. blasii . While species-specific real-time PCR assays exist for R. ferrumequinum and R. hipposideros , no unified panel of real-time PCR assays currently enables the identification of all five European Rhinolophus species from non-invasively collected samples. Here, we developed five species-specific real-time PCR assays, each targeting interspecies nucleotide variation within the mitochondrial cytochrome b gene. To enhance single-base discrimination, RNase H-dependent PCR (rhPCR) primers were employed, incorporating cleavable blocked primers that require perfect complementarity for extension. The assays were applied to droppings non-invasively collected from 18 caves and one church in Serbia and Romania. Of the 149 samples analysed, 131 (88%) yielded successful amplification of Rhinolophus DNA. Detection probabilities for the three species identified in the field ranged from 0.49 to 0.82. Occupancy estimates varied, with R. euryale showing the highest (0.86; UI: 0.69–0.97) and R. mehelyi the lowest (0.23; UI: 0.08–0.43). The assays were capable of detecting up to three species concurrently within a single pooled sample (approximately 15 droppings). These assays are especially valuable for detecting R. mehelyi , given its rarity and uncertain distribution, and offer a robust tool for monitoring Rhinolophus populations across Europe.