From single species to communities: microsatellite amplicon sequencing to monitor felids using Feliplex

Abstract The current biodiversity crisis demands a shift from single-species to multispecies approaches in conservation, particularly for rare and endangered species. However, this transition requires tools optimised for multispecies research, which are currently limited. Recent advances in high-throughput sequencing (HTS) technologies and bioinformatics have enabled efficient and robust acquisition of genetic data. Amplicon sequencing approaches, in particular, have demonstrated potential for enhancing non-invasive genetic studies of endangered species, but their application has been mostly limited to single species. To enable multispecies genetic research, we introduce a cost-effective and robust HTS-based amplicon sequencing approach for genotyping multiple species simultaneously, designed for population monitoring, including individual identification and ascertaining patterns of population structure. We developed Feliplex, a multiplex panel of 85 co-amplifying tetranucleotide microsatellite markers for cross-genotyping Felidae species, to demonstrate the utility of our approach. Feliplex was validated on known samples from nine Indian felid species across the genera Panthera, Prionailurus, and Felis. We applied it to invasive (blood and tissue) and non-invasive (hair and faeces) DNA extracts from 173 wild individuals obtaining respectively 70% and 56% multilocus genotyping success rates. The panel accurately identified known population clusters in tigers (Panthera tigris, n=19) and revealed hitherto unknown genetic structure in fishing cats (Prionailurus viverrinus, n=40). Feliplex’s wide applicability across Felidae allows reliable multispecies genotyping from low-quality/quantity samples, while supporting cost-effective genetic studies and conservation monitoring of lesser-known species like small cats. Our approach has a broad applicability and can be adapted to develop similar multispecies panels for closely related species groups. Competing Interest Statement The authors have declared no competing interest. Footnotes FB frederic.boyer{at}univ-grenoble-alpes.fr MDB marta.debarba{at}gmail.com PT pierre.taberlet{at}univ-grenoble-alpes.fr Author Contributions DR and UR conceived the ideas, and together with MDB and PT, designed the methodology. DR collected the data, while DR and FB analysed it. DR led the manuscript writing with assistance from MDB. All authors critically contributed to the drafts and approved the final version for publication. Our study led by researchers from the country where the study was conducted, brings together collaboration of scientists across multiple countries. All authors were engaged in the study from the beginning to provide a broad and holistic perspective to the designing and execution of the study. The authors declare no conflicting interests. Data Accessibility Genotype data, data processing scripts and supplementary data are available on Dryad.