High-quality carnivore genomes from roadkill samples enable species delimitation in aardwolf and bat-eared fox

In a context of ongoing biodiversity erosion, obtaining genomic resources from wildlife is becoming essential for conservation. The thousands of yearly mammalian roadkill could potentially provide a useful source material for genomic surveys. To illustrate the potential of this underexploited resource, we used roadkill samples to sequence reference genomes and study the genomic diversity of the bat-eared fox ( Otocyon megalotis ) and the aardwolf ( Proteles cristata ) for which subspecies have been defined based on similar disjunct distributions in Eastern and Southern Africa. By developing an optimized DNA extraction protocol, we successfully obtained long reads using the Oxford Nanopore Technologies (ONT) MinION device. For the first time in mammals, we obtained two reference genomes with high contiguity and gene completeness by combining ONT long reads with Illumina short reads using hybrid assembly. Based on re-sequencing data from few other roakill samples, the comparison of the genetic differentiation between our two pairs of subspecies to that of pairs of well-defined species across Carnivora showed that the two subspecies of aardwolf might warrant species status ( P. cristata and P. septentrionalis ), whereas the two subspecies of bat-eared fox might not. Moreover, using these data, we conducted demographic analyses that revealed similar trajectories between Eastern and Southern populations of both species, suggesting that their population sizes have been shaped by similar environmental fluctuations. Finally, we obtained a well resolved genome-scale phylogeny for Carnivora with evidence for incomplete lineage sorting among the three main arctoid lineages. Overall, our cost-effective strategy opens the way for large-scale population genomic studies and phylogenomics of mammalian wildlife using roadkill.