Refining species boundaries among velvet worms (Onychophora, Peripatopsidae), with the description of two new species of Opisthopatus from South Africa

In the present study, two new species of velvet worm, Opisthopatus, from the Eastern Cape province of South Africa are described. We used both mitochondrial and nuclear DNA sequence data (cytochrome oxidase subunit I [COI] and 18S rRNA subunit [18S]) to explore the possibility that species boundaries in Opisthopatus have been underestimated. A previous fine-scale phylogeographic study of the velvet worm Opisthopatus amaxhosa demonstrated the presence of two distinct clades, of which the first could be assigned to O. amaxhosa and the second represented a novel as yet undescribed species. In addition, specimens of Opisthopatus from the Valley of Desolation, Camdeboo Nature Reserve, Graaff-Reinet, were not assigned to a new species in the most recent taxonomic revision because of the limited number of specimens. Following recent collection and scanning electron microscopy, we assigned the Graaff-Reinet specimens to Opisthopatus camdebooi sp. nov. and the second lineage in sympatry with O. amaxhosa we described as Opisthopatus baziya sp. nov. The application of four species delimitation methods (Assemble Species by Automatic Partitioning [ASAP], Poisson Tree Processes [PTP], generalized mixed Yule-coalescent model [GMYC], and Species Tree and Classification Estimation, Yarely [STACEY]) generally revealed congruent groupings across clades, suggesting that the alpha taxonomic diversity in Opisthopatus has been underestimated. The continued discovery of new velvet worm species in poorly or unsampled areas suggests that several novel species might still be undescribed in South Africa. Collectively, our results suggest that fine-scale sampling is critical to detect and describe the biodiversity of other saproxylic taxa.