A Footworm in the door: Revising Onchocerca phylogeny with previously unknown cryptic species in wild North American ungulates

Onchocerca is an important genus of vector-borne filarial nematodes that infect both humans and animals worldwide. Many Onchocerca species, most of medical and veterinary health relevance, are the focus of a variety of diagnostic and molecular research. However, despite the importance of these parasites, there is growing evidence of previously unexplored genetic diversity of these nematodes, particularly among wild ungulate hosts in North America. These understudied parasites prevent us from comprehending the evolutionary history of the genus Onchocerca, monitoring potential One Health threats, and improving our filarioid diagnostic capabilities. In order to fill these knowledge gaps, we identified five uncharacterized Onchocerca lineages and compared them to other well-known filarioid species using single and concatenated gene regions (i.e., nd5, cox1, 12S, 18S, 28S, hsp70, MyoHC, rbp1). Phylogenetic analyses revealed that the novel Onchocerca lineages of wild North American ungulates segregate into two clades. One clade comprised Onchocerca lineages II, IV, and V and other species found mainly in domestic animals and humans, and the second comprised Onchocerca lineages I and III and other species from a variety of hosts including cervids, bovids, and equids. The formation of two clearly separate clades supports the idea of at least two independent expansion events of ancestral Onchocerca species into the North American continent via the Bering land bridge. Cophylogenetic analysis shows evidence of ancestral Onchocerca species of Bovidae host-switching to wild Cervidae and giving rise to the novel Onchocerca species. Lastly, pairwise analysis confirms informative molecular markers of diagnostic relevance in both mitochondrial and nuclear gene regions of filarioid nematodes. The overall information provides greater context to the genus Onchocerca and emphasizes the need to discover, characterize, and monitor neglected parasites, especially those of wildlife origin.盘尾丝虫属（Onchocerca）是一类重要的经媒介传播的丝虫线虫，在全球范围内可感染人类与多种动物。该属诸多物种兼具医学与兽医学研究价值，是各类诊断学及分子生物学研究的重点对象。然而，尽管这类寄生虫具有重要的研究与应用价值，越来越多的证据表明，这些线虫存在尚未被发掘的遗传多样性，尤其在北美野生有蹄类宿主中更为显著。这些尚未得到充分研究的寄生虫，阻碍了我们对盘尾丝虫属演化历史的认知、对潜在健康一体化（One Health）威胁的监测，以及对丝虫诊断能力的优化提升。为填补这些知识空白，本研究通过单个及串联基因区域（即nd5、cox1、12S、18S、28S、hsp70、MyoHC、rbp1），鉴定出5个未被表征的盘尾丝虫谱系，并将其与其他已知丝虫物种进行比对分析。系统发育分析显示，北美野生有蹄类的新型盘尾丝虫谱系可划分为两个演化支。其中一个演化支包含盘尾丝虫谱系II、IV、V，以及主要寄生于家畜和人类的其他丝虫物种；另一个演化支则包含盘尾丝虫谱系I、III，以及源自鹿科、牛科、马科等多种宿主的其他丝虫物种。这两个明显分化的演化支的形成，佐证了盘尾丝虫祖先物种至少通过白令陆桥发生过两次独立的扩张事件并进入北美大陆这一假说。共系统发育分析结果显示，寄生于牛科动物的盘尾丝虫祖先发生了宿主转换，感染野生鹿科动物并演化出新型盘尾丝虫物种。最后，成对序列比对分析证实，丝虫线虫的线粒体及核基因区域中存在具备诊断应用价值的有效分子标记。本研究的整体结果为盘尾丝虫属的相关研究提供了更全面的背景，并强调了对被忽视的寄生虫——尤其是源自野生动物的寄生虫——进行发现、表征与监测的必要性。