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

Abstract

O_LIThe 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. C_LIO_LIRecent 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. C_LIO_LITo 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. C_LIO_LIWe 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). C_LIO_LIFeliplexs 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. C_LI