High-quality carnivoran genomes from roadkill samples enable comparative species delineation 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.

在生物多样性持续丧失的背景下，获取野生动物的基因组资源已成为物种保护工作的核心需求。每年产生的数千具哺乳动物道路致死标本，可为基因组学调查提供极具价值的未被充分利用的研究材料。为阐明这一资源的应用潜力，本研究利用道路致死标本，对大耳狐（Otocyon megalotis）和土狼（Proteles cristata）开展参考基因组测序与基因组多样性分析；这两个物种均根据其在东非与南非的间断分布格局被划定了亚种。通过优化DNA提取方案，我们借助牛津纳米孔科技（Oxford Nanopore Technologies, ONT）的MinION测序平台成功获取了长读长测序数据。本研究在哺乳动物研究中首次采用混合组装策略，结合ONT长读长数据与Illumina短读长数据，成功获得了两个具有高连续性与基因完整性的参考基因组。基于另外少量道路致死标本的重测序数据，本研究将两对亚种的遗传分化水平与食肉目（Carnivora）内多对已明确界定的物种种群进行比较，结果显示土狼的两个亚种或许应被提升为独立物种（P. cristata与P. septentrionalis），而大耳狐的两个亚种则不具备该分类学地位。此外，基于上述测序数据开展的群体历史动态分析显示，两个物种的东非与南非种群具有相似的群体规模变化轨迹，表明二者的种群大小均受到相似的环境波动影响。最后，本研究构建了分辨率较高的食肉目全基因组尺度系统发育树，结果显示三大犬型亚目（Arctoidea）支系间存在不完全谱系分选现象。总体而言，本研究采用的低成本高效策略，为利用道路致死标本开展大规模哺乳动物野生动物种群基因组学与系统基因组学研究开辟了新路径。