Methodological Perspectives on Cryptic Species Monitoring: Insights from Strictly Protected Lesser Blind Mole Rat.

Cryptic biodiversity presents an ongoing challenge to the accuracy and effectiveness of biodiversity assessment and conservation. To address this, we developed a non-lethal genetic monitoring framework for the subterranean rodent, European lesser blind mole rat (BMR), Nannospalax leucodon species complex, which encompasses multiple chromosomally diversified and reproductively isolated cryptic-species and subspecies. Our workflow combines species and sex detection through: karyotyping, Inter-Simple Sequence Repeat (ISSR) PCR profiling, and Sry-based sex determination of 33 individual samples from five BMR cryptic-species collected at 25 localities in Serbia. As conventional karyotyping protocols for BMR required animal sacrifice, we developed the first non-lethal fibroblast culture-based karyotyping approach from BMR skin biopsy, producing high-quality metaphase chromosomes across five cryptic-species. Among twelve tested ISSR primers, three yielded reproducible, species-specific DNA profiles that resolved four of the five cryptic-species. The Sry assay accurately determined the sex of all examined individuals. Our findings demonstrate that integrating fibroblast culture-based karyotyping, with fast, cost-effective ISSR-PCR species identification, and Sry-based sex determination, provides a reliable approach for identifying and monitoring cryptic BMR species without sacrificing individuals. This framework has potential applications in conservation programmes and contributes to an integrative taxonomy approach essential for the study and protection of other cryptic taxa.